- Email: [email protected]
Oleanolic acid inhibits mouse spinal cord injury through suppressing inflammation and apoptosis via the blockage of p38 and JNK MAPKs
Biomedicine & Pharmacotherapy 123 (2020) 109752
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Oleanolic acid inhibits mouse spinal cord injury through suppressing inflammation and apoptosis via the blockage of p38 and JNK MAPKs
T
Jiang-Lin Wanga,1, Chang-He Rena,1, Jian Fengb, Ce-Hua Oua,*, Li Liuc,* a
Department of Pain Management, The Affiliated Hospital of Southwest Medical University, Luzhou City, Sichuan Province, 646000, China Department of Cardiology, The Affiliated Hospital of Southwest Medical University, Luzhou City, Sichuan Province, 646000, China c Department of Anesthesiology, The Affiliated Hospital of Southwest Medical University, Luzhou City, Sichuan Province, 646000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Spinal cord injury (SCI) Oleanolic acid (OA) Apoptosis and inflammation NF-κB p38 and JNK
Spinal cord injury (SCI) is reported as a devastating disease, leading to tissue loss and neurologic dysfunction. However, there is no effective therapeutic strategy for SCI treatment. Oleanolic acid (OA), as a triterpenoid, has anti-oxidant, anti-inflammatory, and anti-apoptotic activities. However, its regulatory effects on SCI have little to be elucidated, as well as the underlying molecular mechanisms. In this study, we attempted to explore the role of OA in SCI progression. Behavior tests suggested that OA treatments markedly alleviated motor function in SCI mice. Evans blue contents up-regulated in spinal cords of SCI mice were significantly reduced by OA in a dosedependent manner, demonstrating the improved blood-spinal cord barrier. Moreover, we found that OA treatments significantly reduced the apoptotic cell death in spinal cord samples of SCI mice through decreasing the expression of cleaved Caspase-3. In addition, pro-inflammatory response in SCI mice was significantly attenuated by OA treatments. Furthermore, SCI mice exhibited higher activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways, but these effects were clearly blocked in SCI mice with OA treatments, as evidenced by the down-regulated phosphorylation of p38, c-Jun-NH 2 terminal kinase (JNK), IκB kinase α (IKKα), inhibitor of nuclear factor κB-α (IκBα) and NF-κB. The protective effects of OA against SCI were confirmed in lipopolysaccharide (LPS)-stimulated mouse neurons mainly through the suppression of apoptosis and inflammatory response, which were tightly associated with the blockage of p38 and JNK activation. Together, our data demonstrated that OA treatments could dose-dependently ameliorate spinal cord damage through impeding p38- and JNK-regulated apoptosis and inflammation, and therefore OA might be served as an effective therapeutic agent for SCI treatment.
1. Introduction Spinal cord injury (SCI) affects 2.5–4 million patients in the world. According to the level and extent of the damage, the symptoms range from minimal sensory/motor deficits to complete tetraplegia [1]. In addition, neuropathic pain arises in a variety of cases, which further reduces the quality of life for patients with SCI [2,3]. Currently, the molecular mechanisms revealing the pathophysiology of SCI could be mainly divided into two broad successive phases [4]. On the one, the primary phase includes the initial mechanical injury, which is followed by a secondary phase consisted of some degenerative processes, such as vascular disruption, oxidative stress, ischemia, neuroinflammation and apoptosis [5,6]. The excessive release of pro-inflammatory factors, which is deleterious to neighboring neurons, resulting in the bloodspinal cord barrier disruption, spinal cord edema, as well as neutrophil
influx following SCI [7,8]. Unfortunately, there are no effective therapeutic strategies available to prevent SCI progression. Herein, it is essential to find and develop new treatments to attenuate SCI. Oleanolic acid (OA) is a natural triterpenoid and an aglycone of some saponins [9]. OA is widely existed in fruits of Olea europaea and Ligustrum lucidum, in vegetables, as well as in various kinds of medicinal herbs [10]. OA has been used as an over-the-counter Chinese medicine to alleviate inflammatory diseases and tumor growth [11–13]. For instance, OA could improve type 2 diabetes-related complications, especially inflammation, which is mainly via the suppression of the NFκB signaling pathway [14]. In addition, OA treatment significantly attenuated liver ischemic reperfusion injury partly through suppressing apoptosis [15]. Recently, OA was suggested to keep blood-brain barrier integrity through targeting p38 MAPK signaling pathway [16]. Moreover, OA ameliorated cognitive dysfunction induced by blocking
⁎
Corresponding authors at: The Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street, Luzhou City, Sichuan Province, 646000, China. E-mail addresses: [email protected] (C.-H. Ou), [email protected] (L. Liu). 1 Jiang-Lin Wang and Chang-He Ren are the co-first author. https://doi.org/10.1016/j.biopha.2019.109752 Received 5 September 2019; Received in revised form 7 November 2019; Accepted 29 November 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Aldrich, USA) was injected (45 mg/kg, iv) into the tail vein of mice 1 h before euthanizing. After adequate perfusion with saline under deep anesthesia, the L3-6 segments were removed and soaked in methanide for 24 h at 60 °C. After centrifugation, the evans blue levels were calculated as absorbance of supernatant at 632 nm using a microplate reader and measured as the amount of evans blue each wet tissue weight. As for the fluorescent evaluation, the tissues were fixed in 4 % paraformaldehyde, sectioned (6-μm thickness), sealed in a light-tight container and frozen. Evans blue staining was finally observed using a fluorescent microscope.
endoplasmic reticulum stress and inflammatory pathways [17]. Therefore, we hypothesized that OA might be a promising therapeutic candidate for SCI treatment. However, the effects of OA on SCI are unclear. In the present study, we found that OA treatments significantly improved motor function in SCI mice, accompanied with reduced evans blue contents in spinal cord samples, indicating the alleviated bloodspinal cord barrier disruption. We also suggested that OA administration dose-dependently inhibited the apoptosis and inflammatory response in spinal cord tissues from SCI mice, which were mainly dependent on the blockage of p38 and JNK MAPKs signaling pathway. Together, these findings demonstrated that OA treatment could be considered as an effective therapeutic agent to improve SCI.
2.4. Histological analysis The spinal cord lumbar segments were fixed in 4 % paraformaldehyde overnight, and were then transferred into 30 % sucrose in 4 % paraformaldehyde until reaching the bottom. Then, the spinal cord samples from 30 % sucrose solutions were cut into sections (6-μm thickness) using a cryostat microtome (Leica Microsystems, USA). Sections were then subjected to Nissl staining [26]. The survival condition of neurons in five sections from each mouse was analyzed and quantified with a light microscope by two pathologists blinded to the experimental treatments and outcomes.
2. Materials and methods 2.1. Animals and SCI model The experimental protocols were approved by the Ethics Committee of The Affiliated Hospital of Southwest Medical University (Sichuan, China) and complied with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. The female, 4–5 months old C57BL/6J mice were purchased from the Experimental Animal Center of Sichuan University (Sichuan, China). Guangdong Medical Animal Center (Guangdong, China). Mice used in the study were housed in a temperature- and humidity-controlled room (12 h light/dark) for 7 days before surgery with free access to the standard animal chow and water. All efforts were made to minimize animal suffering in the experiments. SCI animal model was established as previously described [18]. In brief, animals were anesthetized through intraperitoneal injections of 100 mg/kg of ketamine and 5 mg/kg of xylazine. A longitudinal dorsal incision was made to expose T6-T10 spinous processes. Laminectomy was conducted at the T7-T9 level using mouse laminectomy forceps (Heidelberg, Germany). The spinal cord was manually exposed, and then maximally compressed (100 %) with forceps (Fine Science Tools) to cause a robust and reproducible lesion [19]. Then, the muscles and skin were closed with 6-0 nylon stitches (Ethicon, Germany). Following the operation, mice were injected intraperitoneally with 0.9 % saline solution as supplementary body liquid. After operation, animals were maintained on a heated pad (37 °C) for 8 h to prevent hypothermia, and then were singly housed in a temperature-controlled (26 °C) room with free access to food and water. During the post-operative time, the bladders were manually voided as required. 25, 50 and 100 mg/kg of OA (MedChemExpress, USA) were chosen [20,21] and treated to mice via intraperitoneal injection once daily immediately began from trauma until 6 weeks subsequent to SCI. PBS was administered for the vehicle control group. As for the Shamoperated control group, mice underwent a T7-T9 laminectomy in the absence of compression injury and without treatment with OA.
2.5. Immunofluorescent (IF) staining After being transferred to 30 % sucrose solutions, the spinal cords samples were cut into sections (6-μm thickness) with a cryostat microtome. Then, the sections were blocked in 10 % BSA (Sigma Aldrich) for 1 h and incubated with primary antibodies overnight at 4 °C. Sections were rinsed with PBS and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 (#ab150117, 1:300 dilution, Abcam, USA) or 594 (#ab150088, 1:300 dilution, Abcam) for 1 h at room temperature. Subsequently, the sections were washed in PBS and incubated with DAPI (Beyotime) in the dark. Representative images were obtained with a fluorescent microscope. ImageJ 1.46r software (National Institutes of Health, USA) was used for quantification. Primary antibodies used here included anti-NeuN (#ab104224, 1:200 dilution, Abcam), anti-Caspase-3 (#9664, 1:150 dilution, Cell Signaling, USA), anti-NF-κB (#6956, 1:150 dilution, Cell Signaling) and Iba-1 (#MA5-27726, 1:200 dilution, ThermoFisher Scientific, USA). 2.6. Real-time quantitative PCR (RT-qPCR) assays From the long spinal cord segments containing the injury epicenter, total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer’s protocols. For calculation of mRNA expression, the first-strand synthesis was performed using a PrimeScript RT reagent kit (TaKaRa, Japan). RT-qPCR was then conducted on an Applied Biosystems 9700 Thermocycler (Applied Biosystems Life Technologies Corporation, USA) with the SYBR Premix Ex Taq kit (TaKaRa). Primers used in the study were designed and supplied by Sangon Biotech (Shanghai, China). Gene expression changes were quantified through the delta-delta CT method [27]. GAPDH was used as an internal control. Primer sequences of the targeted genes used in this study were listed in Supplementary table S1. All experiments were performed at least three times.
2.2. Motor function analysis The recovery of ground locomotion was measured through the Basso Mouse Scale (BMS) as previously indicated [22]. Moreover, the single-frame motion analysis was used to calculate the foot-stepping angle and rump-height index via the beam walking test [23]. The limb extension-flexion ratio was determined from video from recordings of voluntary movements by the “pencil” test [24]. Measurement was conducted before and at 7, 14, 21, 28, 35 and 42 days after injury. The obtained values for the left and right extremities were averaged.
2.7. Western blot assays After the animals (n = 6 per group) were sacrificed, 5-mm long segments of spinal cord encompassing the injury site were harvested and homogenized in RIPA solution with protease and phosphatase inhibitors (Beyotime) for western blot assays. Protein concentrations were then measured with a bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific). Each protein sample (30 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
2.3. Evans blue content and fluorescence analysis After SCI for 42 days, the evans blue levels and fluorescence were quantified for the calculation of blood-spinal cord barrier (BSCB) disruption following SCI as previously indicated [25]. Evans blue (Sigma 2
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 1. Oleanolic acid improves motor function of SCI mice. (A) BMS score analysis (n = 9 mice per group at each time point). (B) Calculation of foot-stepping angle (n = 9 mice per group at each time point). (C) Rump-height index of SCI mice was determined (n = 9 mice per group at each time point). (D) Quantification of extension/flexion ratio (n = 9 mice per group at each time point). (E) Blood-spinal cord barrier was measured using evans blue fluorescence. (F) The fluorescent density (%) of evans blue (n = 6 mice in each group, with 5 images for each mouse). (G) Evans blue levels in spinal cord samples were measured (n = 6 mice in each group). Data were means ± SEM. ++p < 0.01 vs the Sham group; *p < 0.05, **p < 0.01 vs the SCI group.
κB (p65) (#8242, Cell Signaling), phosphorylated NF-κB (p65) (#3033, Cell Signaling), IKKα (#61294, Cell Signaling), phosphorylated IKKα (#2078, Cell Signaling), IκBα (#4814, Cell Signaling), phosphorylated IκBα (#2859, Cell Signaling), JNK (#9252, Cell Signaling), phosphorylated JNK (#9255, Cell Signaling), p38 (#8690, Cell Signaling), phosphorylated p38 (#4511, Cell Signaling), ERK1/2 (#4695, Cell
PAGE), and transferred to polyvinyl difluoride membranes (PVDF, Millipore, USA). Nonspecific bands were blocked in TBS-T (pH 7.5) containing 5 % non-fat milk for 1.5 h at room temperature. Subsequently, the membranes were incubated with the primary antibodies at 4 °C overnight at a dilution of 1:500 or 1:1000 to calculate Bcl2 (#sc-7382, Santa Cruz, USA), Caspase-3 (#9664, Cell Signaling), NF3
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 2. Oleanolic acid treatment alleviates apoptosis in SCI mice. (A) Nissl staining of spinal cord sections. Quantification of neuron survival rate was exhibited (n = 6 mice in each group, with 6 images for each mouse). (B) IF staining of NeuN (green fluorescence) and Caspase-3 (red fluorescence) in spinal cord sections. The number of Caspase-3-positive cells was analyzed (n = 6 mice in each group, with 5 images for each mouse). (C) Western blot analysis was used to measure Bcl-2 and cleaved Caspase-3 expression levels in spinal cord samples (n = 4 mice in each group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Sham group; *p < 0.05 and **p < 0.01 vs the SCI group.
DMEM (Gibco, USA) supplemented with 10 % (v/v) FBS (Gibco) and 0.1 % penicillin-streptomycin, and incubated at 37 °C under 5 % CO2. To study the protective effects of OA, LPS (Sigma Aldrich) was used to stimulate HT22 cells. The p38 inhibitor SB203580 and JNK inhibitor SP600125 were obtained from MedChemExpress (USA) to block the activation of p38 and JNK, respectively.
Signaling), phosphorylated ERK1/2 (#9101, Cell Signaling), Iba-1 (#ab178847, Abcam) and GAPDH (#ab8245, Abcam). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#A0208 or #A0216, 1:2000 dilution, Beyotime) and detected with ECL substrate (ThermoFisher Scientific). Relative protein expression was normalized to GAPDH and compared with the control group. Images were captured using Quantity One software (BioRad, USA). Protein levels were compared by measuring mean density values. Quantification of target proteins was carried out using Image J 1.46r software (National Institutes of Health, USA).
2.10. Cell viability calculation The cytotoxicity of OA was measured using a microculture 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay (Beyotime) according to the manufacturer’s instructions. Cells were then incubated in 24-well plates at a density of 5 × 105 cells each well. MTT solution (5 μL of 5 mg/mL) was then added to each well. After incubation for 4 h at 37 °C in 5 % CO2, the supernatant was removed. Then, the formazan crystals produced in viable cells were solubilized in 150 μL dimethylsulfoxide (DMSO, Beyotime). The absorbance of each well was read at 570 nm with a microplate reader.
2.8. ELISA analysis for inflammatory cytokines The spinal cord samples were homogenized, and protein concentration was measured using the BCA Protein Assay kit (ThermoFisher Scientific). Samples were then concentrated to 4 mg/ml using MicroCon centrifugation filters (Millipore) to ensure equal amounts of protein. Subsequently, the IL-1β (#MLB00C), IL-6 (#M6000B) and TNF-α (#MTA00B) contents were determined using commercial available kits (R&D System, USA) according to the manufacturer’s instructions.
2.11. Flow cytometry analysis
2.9. Cells and treatments
Apoptotic cell death was conducted using an Annexin-VFITC apoptosis detection kit (BD Biosciences, USA). The treated cells were collected using trypsin, washed with ice-cold PBS and resuspended in 100 μl of flow cytometry binding buffer. Next, the cells were stained
HT22 murine hippocampal neuronal cells were purchased from TongPai Technology (Shanghai, China). Cells were maintained in 4
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 3. Oleanolic acid treatment inhibits inflammatory response in SCI mice. (A) IF staining of Iba-1 in spinal cord sections. Iba-1-positive cells were quantified (n = 6 mice in each group, with 5 images for each mouse). (B) RT-qPCR and (C) western blot analysis were used to determine Iba-1 expression levels in spinal cord samples (n = 3 mice in each group). (D) ELISA analysis was used to measure IL-1β, IL-6 and TNF-α contents in spinal cord tissues (n = 8 mice in each group). The mRNA levels of (E) iNOS, IL-8, IL-12, (F) IL-10, SOCS3 and Arg1 in spinal cord tissues were measured using RT-qPCR analysis (n = 3 mice in each group). Data were means ± SEM. +p < 0.05, ++p < 0.01 and +++p < 0.001 vs the Sham group; *p < 0.05 and **p < 0.01 vs the SCI group.
with 5 μl of Annexin V/FITC followed by 5 μl of PI in the dark for 15 min at room temperature. Then, 400 μl of binding buffer was added to each tube. The percentage of apoptotic cells was analyzed using FACSCalibur flow cytometer (BD Biosciences).
excluded. All in vitro experiments were replicated using different cell cultures. A randomization process was performed in grouping mice. All in vivo and imaging studies were performed in a blinded manner. The experimenters were blinded to the grouping information.
2.12. Statistical analysis
3. Results
The data were expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments unless specifically stated in the figure legends. Data were analyzed using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, USA). Behavioral data were analyzed using two-way repeated measures one-way analysis of variance (ANOVA) with Bonferroni post hoc tests. The other statistical differences between two groups were analyzed using Student’s t-test. P value < 0.05 was considered statistically significant. No data were
3.1. Oleanolic acid improves motor function of SCI mice In order to calculate the effects of OA on SCI, motor function of mice was measured. As shown in Fig. 1A, SCI mice showed the decreased BMS score was markedly rescued by OA treatment in a dose-dependent manner, revealing the better recovery of motor function. Then, we found that the foot-stepping angle was changed from about 30° before injury to approximate 170° after injury in all groups of mice. The 5
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 4. Oleanolic acid treatment blocks MAPKs and NF-κB signaling pathways in SCI mice. (A) Western blot analysis was performed to calculate p-p38, p-ERK1/ 2 and p-JNK protein expression levels in spinal cord samples (n = 4 mice in each group). (B) The protein expression levels of p-IKKα, p-IκBα and p-NF-κB in spinal cord tissues were measured using western blot analysis (n = 3 mice in each group). (C) IF staining for NeuN (green fluorescence) and NF-κB (red fluorescence) in spinal cord sections (n = 6 mice per group, with 5 images for each mouse). The number of NF-κB-positive cells was quantified. Data were means ± SEM. +p < 0.05, ++p < 0.01 and +++p < 0.001 vs the Sham group; *p < 0.05 and **p < 0.01 vs the SCI group.
3.3. Oleanolic acid treatment inhibits inflammatory response in SCI mice
decrease of foot-stepping angle represents the improved recovery of SCI mice [28]. Here, OA treatment dose-dependently decreased the footstepping angle of SCI mice compared to the SCI mice (Fig. 1B). The rump-height index presents the ability to support body weight during ground locomotion [29]. OA treatment significantly up-regulated the rump-height index in mice with SCI in a dose-dependent fashion (Fig. 1C). Additionally, SCI mice treated with OA showed increased extension/flexion ratio especially at the highest concentration of 100 mg/kg (Fig. 1D). To explore if OA treatment could alleviate the blood-spinal cord barrier, evans blue staining was performed. As shown in Fig. 1E–G, SCI mice showed significantly up-regulated evans blue contents in spinal cord samples, which were alleviated by OA treatment in a dose-dependent manner, indicating the alleviated blood-spinal cord barrier permeability. The findings above demonstrated that OA administration could attenuate motor dysfunction in SCI mice with alleviated blood-spinal cord barrier integrity.
Microglia were activated after SCI [30]. Iba-1, a critical marker of microglial cells, was found to be significantly up-regulated in spinal cord samples of SCI mice when compared to the Sham group of mice. However, OA treatments markedly reduced the expression of Iba-1 from mRNA and protein levels in a dose-dependent manner (Fig. 3A–C). Then, ELISA analysis demonstrated that SCI mice had higher contents of pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α in spinal cord samples than that of the Sham mice, which were markedly reduced by OA treatments in a dose-dependent manner (Fig. 3D). Furthermore, the mRNA levels of iNOS, IL-8 and IL-12, representing inflammatory response, increased in SCI mice were significantly reduced by OA (Fig. 3E). In contrast, the expression levels of anti-inflammatory factors including IL-10, SOCS3 and Arg1 decreased in SCI mice were dosedependently restored by OA treatments (Fig. 3F). Together, results in this part demonstrated that OA could alleviate SCI in mice via suppressing pro-inflammatory response.
3.2. Oleanolic acid treatment alleviates apoptosis in SCI mice In this part, Nissl staining indicated that OA-treated mice exhibited significantly improved survival rate of neuron in spinal cord sections of SCI mice (Fig. 2A). IF staining showed higher expression levels of Caspase-3 in SCI mice than that of the Sham mice, but these effects were dose-dependently reduced by OA treatments (Fig. 2B). Moreover, we found that SCI-induced high expression levels of cleaved Caspase-3 were markedly decreased by OA. In contrast, the protein expression levels of anti-apoptotic signal Bcl-2 were down-regulated by SCI, while being restored by OA in a dose-dependent manner (Fig. 2C). Results above elucidated that OA could alleviate SCI in mice through inhibiting apoptosis via the blockage of Caspase-3 activation.
3.4. Oleanolic acid treatment blocks MAPKs and NF-κB signaling pathways in SCI mice MAPKs and NF-κB signaling pathways play critical role in regulating inflammatory response during SCI progression [31,32]. Western blot analysis suggested that SCI mice showed significantly increased expression of phosphorylated p38, ERK1/2 and JNK in spinal cord samples. Notably, OA treatments significantly reduced the activation of p38 and JNK in a concentration-dependent manner. However, ERK1/2 phosphorylation up-regulated in SCI mice was not significantly alleviated by OA (Fig. 4A). Then, we found that SCI mice exhibited the 6
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 5. Oleanolic acid incubation alleviates apoptotic cell death in mouse neuron cells. (A) Mouse neuron HT22 cells were incubated with OA (0, 0.5, 1, 2.5, 5, 10, 20, 40 and 80 μM) for 24 h. Then, cells were collected for cell viability calculation using MTT analysis (n = 4 per group). (B) HT22 cells were treated with OA (40 μM) for the indicated time (0, 4, 8, 16, 24, 48, 72 and 96 h). Then, all cells were collected for cell viability calculation using MTT analysis (n = 4 per group). (C) Morphology of HT22 cells treated with 24 h of LPS (100 ng/ml) in the absence or presence of OA (10, 20 and 40 μM) (n = 3 per group). (D) Flow cytometry analysis for apoptosis rate calculation in LPS (100 ng/ml)-stimulated HT22 cells for 24 h with or without OA (10, 20 and 40 μM) (n = 3 per group). (E) HT22 cells were exposed to 100 ng/ml of LPS for 24 h with or without OA (10, 20 and 40 μM), followed by western blot analysis of Bcl-2 and cleaved Caspase-3 in cells (n = 3 per group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Con group; *p < 0.05 and **p < 0.01 vs the LPS group.
by LPS, were rescued in OA-treated cells (Fig. 5E). Therefore, results here confirmed the effects of OA against apoptosis during SCI progression.
markedly increased expression of p-IKKα, p-IκBα and p-NF-κB in spinal cord tissues compared to the Sham group, while being significantly attenuated by OA treatments also in a dose-dependent manner (Fig. 4B). IF staining demonstrated that nuclear NF-κB expression was obviously up-regulated in neuron of spinal cord sections from SCI mice, which were clearly reduced by OA administration (Fig. 4C). Collectively, the results suggested that OA treatment could inhibit MAPKs, except ERK1/2, and NF-κB signaling pathways in SCI mice.
3.6. Oleanolic acid suppresses inflammatory response in LPS-treated HT22 cells by blocking MAPKs and NF-κB signaling pathways Western blot analysis demonstrated that OA showed no significant influence on the activation of ERK1/2 induced by LPS (Supplementary Fig. 1A). Consistently, LPS-induced up-regulation of p-p38 and p-JNK was evidently abolished by OA. At the same time, we found that OA functioned as p38 and JNK inhibitors to block p38 and JNK activation in LPS-stimulated HT22 neurons as shown in Fig. 6A and B. Then, the protein expression levels of p-IκBα and p-NF-κB in LPS-treated HT22 cells were considerably diminished by OA, SB203580 or SP600125 treatment (Fig. 6C). Subsequently, LPS-incubated HT22 cells showed higher mRNA levels of IL-1β, IL-6, TNF-α, iNOS, IL-8 and IL-12 than that of the Con group, while being abolished by the co-treatment of OA, SB203580 or SP600125. In contrast, anti-inflammatory molecules IL10, SOCS3 and Arg1 down-regulated by LPS were highly rescued by OA and the suppression of p38 and JNK in HT22 neurons through RT-qPCR analysis (Fig. 6D). Therefore, results here demonstrated that OA-impeded pro-inflammatory response in SCI was at least partly associated with the blockage of p38 and JNK MAPKs, and NF-κB signaling pathways.
3.5. Oleanolic acid incubation alleviates apoptotic cell death in mouse neuron cells The in vivo results suggested that OA showed protective role against SCI development in mice. To confirm its regulatory effects on SCI, the in vitro analysis was then performed in mouse neurons with or without LPS stimulation. At first, MTT analysis suggested that OA treatments ranging from 0 to 80 μM showed no significant difference in the cell viability of HT22 cells (Fig. 5A). In addition, 40 μM of OA treatment from 0 to 72 h also did not reduce the cell viability of HT22 cells (Fig. 5B). Then, the concentrations of OA at 10, 20 and 40 μM were chosen for cell treatment for 24 h. As shown in Fig. 5C, LPS stimulation led to HT22 cell damage, which was, however, improved by OA treatments. Flow cytometry analysis suggested that LPS resulted in significant apoptotic cell death in HT22 cells, and these effects were significantly alleviated by OA treatments in a dose-dependent manner (Fig. 5D). Meanwhile, LPS-enhanced expression of cleaved Caspase-3 was markedly abrogated by OA, while Bcl-2 expression levels, inhibited 7
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 6. Oleanolic acid suppresses inflammatory response in LPS-treated HT22 cells by blocking MAPKs and NF-κB signaling pathways. (A–D) HT22 cells were pre-treated with p38 inhibitor SB203580 and/or JNK inhibitor SP600125 for 2 h, and then were exposed to LPS (100 ng/ml) for another 24 h combined with or without OA (40 μM). All cells were then harvested for the following analysis. The protein expression levels of (A) p-p38, and (B) p-JNK, (C) p-IκBα and p-NF-κB were measured by western blot analysis (n = 4 per group). (D) RT-qPCR analysis was performed to determine IL-1β, IL-6, TNF-α, iNOS, IL-8, IL-12, IL-10, SOCS3 and Arg1 in cells (n = 3 per group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Con group; *p < 0.05 and **p < 0.01 vs the LPS group.
Fig. 7. Oleanolic acid-suppressed apoptosis in HT22 cells is dependent on MAPKs blockage. (A–D) HT22 cells were pre-treated with SB203580 or SP600125 for 2 h at the indicated concentrations, and then were incubated with 100 ng/ml of LPS for another 24 h with or without OA (40 μM). All cells were finally collected for the following studies. (A) Flow cytometry analysis was used to measure apoptosis in cells (n = 3 per group). (B) IF staining of Caspase-3 in the treated cells. (C) Quantification of Caspase-3 expression levels following IF analysis (n = 3 per group, with 3 images for each group). (D) Bcl-2 and cleaved Caspase-3 expression levels in HT22 cells were measured by western blot analysis (n = 4 per group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Con group; *p < 0.05 and **p < 0.01 vs the LPS group.
8
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Targeting of the IKK/NF-κB signaling improved the recovery of locomotor function through decreasing the infiltration of inflammatory cells following SCI [50]. It has been indicated that OA inhibited NF-κB and NF-κB-regulated gene products via direct inhibition of IKK [51]. In the present study, we found that OA treatments significantly reduced the expression of phosphorylated IκBα in the spinal cords of the mice after SCI. We also observed a subsequent reduction in the expression of phosphorylated NF-κB (p65) in spinal cord samples from OA-treated SCI mice, contributing to the decreased release of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, iNOS, IL-8 and IL-12. In contrast, the mRNA levels of signals with anti-inflammatory effects, including IL10, SOCS3 and Arg1, were highly rescued in spinal cord samples of SCI mice. MAPKs, including p38, ERK1/2 and JNK, are known to participate in the signal transduction of neuronal apoptosis and inflammation [33,52]. Previous studies have suggested that MAPKs signaling is activated after SCI in neurons, microglia/macrophages and astrocytes, and is also involved in regulating chronic pain [53,54]. Suppressing MAPKs after SCI have indicated differing results, from slightly alleviated inflammation and decreased apoptosis [55]. OA modulates inflammation and cellular dedifferentiation of chondrocytes through MAPKs signaling pathways [56]. OA could regulate p53-dependent apoptosis also via the ERK/JNK pathway in cancer cell lines [57]. Our data suggested that the activation of p38, ERK and JNK was implicated in SCI. Notably, OA treatment markedly reduced the phosphorylation of p38 and JNK, while showed no significantly inhibitory effects on ERK1/2 activation in spinal cord samples of SCI mice. These effects were confirmed in LPSstimulated neurons in vitro. Importantly, we found that OA functioned as p38 and JNK inhibitors, which could alleviate inflammatory response and apoptotic cell death in neurons with LPS stimulation. Thus, OA treatments showed beneficial effects against SCI partly through blocking MAPKs signaling pathway. In summary, the results in our study demonstrated that OA could significantly alleviate disruption of motor function and blood-spinal cord barrier in SCI mice. Furthermore, OA treatments markedly repressed apoptosis and inflammation in spinal cord tissues from SCI mice, which was largely dependent on the blockage of Caspase-3 and IKK/NF-κB signaling pathways. These effects were mainly through the inhibition of MAPKs (p38 and JNK) by OA. On the basis of our data, OA treatment may have therapeutic utility for the treatment of SCI.
3.7. Oleanolic acid-suppressed apoptosis in HT22 cells is dependent on MAPKs blockage MAPKs signaling pathway has been reported to modulate apoptosis during SCI development [33]. Here, flow cytometry results suggested that OA treatment and the suppression of p38 or JNK significantly reduced the apoptotic cell death in LPS-stimulated HT22 neurons, which was accompanied with reduced expression of Caspase-3 by IF analysis (Fig. 7A–C). We also found that LPS-decreased expression of Bcl-2 was markedly restored by OA, SB203580 or SP600125 treatment. However, the cleaved Caspase-3 protein levels potentiated by LPS were evidently abrogated by OA or the blockage of p38 and JNK activation (Fig. 7D). Taken together, the findings here suggested that OA-alleviated apoptosis triggered by LPS in mouse neurons was also tightly associated with the inhibition of MAPKs signaling pathway. 4. Discussion The results from the present study suggested that OA treatments significantly improved the motor function of SCI mice, which were mainly associated with the reduced apoptosis and inflammatory response. As reported, the disruption of blood-spinal cord barrier subsequent to SCI results in an intensive local inflammation by the infiltration of blood cells including neutrophils and macrophages, which leads to cell death and permanent neurological disability [34]. The integrity of the blood-spinal cord barrier could be examined by evans blue dye extravasation as previously described [35]. Here, we also demonstrated that the evans blue contents in spinal cord samples of SCI mice were markedly reduced by OA treatments in a dose-dependent manner, illustrating that OA could attenuate the disruption of bloodspinal cord barrier after SCI, which was similar to previous results that OA improved blood-brain barrier integrity in animal model with vasogenic edema induced by subarachnoid hemorrhage [36]. Therefore, OA treatments likely contributed to the success in functional recovery. Apoptosis, as indicated by nuclear DNA fragmentation and caspase activation, is a prominent characteristic in the spinal cord following SCI [26,37]. Apoptotic cell death was detected in neurons, and was prominent in the white matter. After SCI, caspase activation could be detected in neurons at the injury site within hours [38,39]. The significant executioners in the apoptotic programme are proteases, which are known as caspases. The caspase family of cysteine proteases regulates the execution of the mammalian apoptotic cell death programme [40]. Caspase-3 could be activated during apoptosis in various types of central nervous system (CNS) cells, and its activation might be a critical apoptotic event in the CNS [41]. In contrast, Bcl-2 has been suggested as an inhibitor of apoptosis [40,42]. Increasing studies have suggested that OA shows significant role in regulating apoptosis to control the growth of various types of tumors [43]. OA could also alleviate renal ischemia reperfusion injury partly through its anti-apoptotic activities [44]. OA also showed anti-apoptotic effects to protect injured podocytes [45]. In this study, we found that OA treatments significantly improved the survival rate of neurons in spinal cord samples of SCI mice via Nissl staining, which was along with the significantly reduced expression of cleaved Caspase-3 and the elevated Bcl-2 expression levels. Therefore, OA-alleviated SCI was at least partly associated with the inhibition of apoptosis via reducing Caspase-3 activation. Inflammatory responses are known as a major component of secondary injury and play an important role in modulating the pathogenesis of acute and chronic SCI, which is likely to be essential for nerve injury, contributing to the process of the regenerative response [46]. At the same time, inflammation may lead to apoptosis in neurons and consequently result in the decrease of neuronal function [47]. Thus, suppressing inflammatory response could down-regulate secondary degeneration and the functional deficits following SCI. Moreover, the IKK/NF-κB signaling pathway plays a predominant role in the meditation of inflammation and apoptosis during SCI progression [48,49].
Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. Acknowledgments This work was supported by (1) Scientific Research Project of Sichuan Provincial Health Commission [No. 19PJ289] and (2) the Science and Technology Strategic Cooperation Programs of Luzhou Municipal People’s Government and Southwest Medical University [No. 2019LZXNYDJ38]. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109752. References [1] M.E.L. Van den Berg, J.M. Castellote, J. de Pedro-Cuesta, et al., Survival after spinal cord injury: a systematic review, J. Neurotrauma 27 (8) (2010) 1517–1528. [2] P.J. Siddall, J.D. Loeser, Pain following spinal cord injury, Spinal Cord 39 (2) (2001) 63.
9
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
[3] P.J. Siddall, M.J. Cousins, A. Otte, et al., Pregabalin in central neuropathic pain associated with spinal cord injury: a placebo-controlled trial, Neurology 67 (10) (2006) 1792–1800. [4] C.S. Ahuja, M. Fehlings, Concise review: bridging the gap: novel neuroregenerative and neuroprotective strategies in spinal cord injury, Stem Cells Transl. Med. 5 (7) (2016) 914–924. [5] M. Cordaro, I. Paterniti, R. Siracusa, et al., KU0063794, a dual mTORC1 and mTORC2 inhibitor, reduces neural tissue damage and locomotor impairment after spinal cord injury in mice, Mol. Neurobiol. 54 (4) (2017) 2415–2427. [6] A.J.E. Combes, L. Matthews, J.S. Lee, et al., Cervical cord myelin water imaging shows degenerative changes over one year in multiple sclerosis but not neuromyelitis optica spectrum disorder, Neuroimage Clin. 16 (2017) 17–22. [7] A.I. Faden, J. Wu, B.A. Stoica, et al., Progressive inflammation‐mediated neurodegeneration after traumatic brain or spinal cord injury, Br. J. Pharmacol. 173 (4) (2016) 681–691. [8] T. Matsushita, K.L. Lankford, E.J. Arroyo, et al., Diffuse and persistent blood–spinal cord barrier disruption after contusive spinal cord injury rapidly recovers following intravenous infusion of bone marrow mesenchymal stem cells, Exp. Neurol. 267 (2015) 152–164. [9] D. Kashyap, A.S. Sharma, H. Tuli, et al., Ursolic acid and oleanolic acid: pentacyclic terpenoids with promising anti-inflammatory activities, Recent Pat. Inflamm. Allergy Drug Discov. 10 (1) (2016) 21–33. [10] J. Pollier, A. Goossens, Oleanolic acid, Phytochemistry 77 (2012) 10–15. [11] W. Lee, E.J. Yang, S.K. Ku, et al., Anti-inflammatory effects of oleanolic acid on LPSinduced inflammation in vitro and in vivo, Inflammation 36 (1) (2013) 94–102. [12] R. Martín, J. Carvalho-Tavares, M. Hernández, et al., Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: a potential therapeutic role, Biochem. Pharmacol. 79 (2) (2010) 198–208. [13] M.K. Shanmugam, X. Dai, A.P. Kumar, et al., Oleanolic acid and its synthetic derivatives for the prevention and therapy of cancer: preclinical and clinical evidence, Cancer Lett. 346 (2) (2014) 206–216. [14] D. Camer, Y. Yu, A. Szabo, et al., The molecular mechanisms underpinning the therapeutic properties of oleanolic acid, its isomer and derivatives for type 2 diabetes and associated complications, Mol. Nutr. Food Res. 58 (8) (2014) 1750–1759. [15] B. Gui, F. Hua, J. Chen, et al., Protective effects of pretreatment with oleanolic acid in rats in the acute phase of hepatic ischemia-reperfusion injury: role of the PI3K/ Akt pathway, Mediators Inflamm. 2014 (2014). [16] R. Martín, J. Carvalho-Tavares, M. Hernández, et al., Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: a potential therapeutic role, Biochem. Pharmacol. 79 (2) (2010) 198–208. [17] J. Lu, D. Wu, Y. Zheng, et al., Ursolic acid improves high fat diet-induced cognitive impairments by blocking endoplasmic reticulum stress and IκB kinase β/nuclear factor-κB-mediated inflammatory pathways in mice, Brain Behav. Immun. 25 (8) (2011) 1658–1667. [18] A. Mehanna, I. Jakovcevski, A. Acar, et al., Polysialic acid glycomimetic promotes functional recovery and plasticity after spinal cord injury in mice, Mol. Ther. 18 (1) (2010) 34–43. [19] R. Curtis, D. Green, R.M. Lindsay, et al., Up-regulation of GAP-43 and growth of axons in rat spinal cord after compression injury, J. Neurocytol. 22 (1) (1993) 51–64. [20] S. Su, G. Wu, X. Cheng, et al., Oleanolic acid attenuates PCBs-induced adiposity and insulin resistance via HNF1b-mediated regulation of redox and PPARγ signaling, Free Radic. Biol. Med. 124 (2018) 122–134. [21] X. Wang, Y.L. Li, H. Wu, et al., Antidiabetic effect of oleanolic acid: a promising use of a traditional pharmacological agent, Phytother. Res. 25 (7) (2011) 1031–1040. [22] D.M. Basso, L.C. Fisher, A.J. Anderson, et al., Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains, J. Neurotrauma 23 (5) (2006) 635–659. [23] D. Lutz, H. Kataria, R. Kleene, et al., Myelin basic protein cleaves cell adhesion molecule L1 and improves regeneration after injury, Mol. Neurobiol. 53 (5) (2016) 3360–3376. [24] I. Apostolova, A. Irintchev, M. Schachner, Tenascin-R restricts posttraumatic remodeling of motoneuron innervation and functional recovery after spinal cord injury in adult mice, J. Neurosci. 26 (30) (2006) 7849–7859. [25] X.Q. Li, H.W. Lv, W.F. Tan, et al., Role of the TLR4 pathway in blood-spinal cord barrier dysfunction during the bimodal stage after ischemia/reperfusion injury in rats, J. Neuroinflammation 11 (1) (2014) 62. [26] H. Zhao, S. Chen, K. Gao, et al., Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway, Neuroscience 348 (2017) 241–251. [27] M.A. Mendieta-Serrano, F.J. Mendez-Cruz, M. Antúnez-Mojica, et al., NADPHOxidase-derived reactive oxygen species are required for cytoskeletal organization, proper localization of E-cadherin and cell motility during zebrafish epiboly, Free Radic. Biol. Med. 130 (2019) 82–98. [28] F. Papastefanaki, I. Jakovcevski, N. Poulia, et al., Intraspinal delivery of polyethylene glycol-coated gold nanoparticles promotes functional recovery after spinal cord injury, Mol. Ther. 23 (6) (2015) 993–1002. [29] S. Sahu, R. Li, P.K. Kadeyala, et al., The human natural killer-1 (HNK-1) glycan mimetic ursolic acid promotes functional recovery after spinal cord injury in mouse, J. Nutr. Biochem. 55 (2018) 219–228. [30] M. Tsuda, Microglia in the spinal cord and neuropathic pain, J. Diabetes Investig. 7 (1) (2016) 17–26. [31] S.J. Huang, J.Q. Yan, H. Luo, et al., IL-33/ST2 signaling contributes to radicular
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47] [48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
10
pain by modulating MAPK and NF-κB activation and inflammatory mediator expression in the spinal cord in rat models of noncompressive lumber disk herniation, J. Neuroinflammation 15 (1) (2018) 12. Y. Su, S. Zong, C. Wei, et al., Salidroside promotes rat spinal cord injury recovery by inhibiting inflammatory cytokine expression and NF‐κB and MAPK signaling pathways, J. Cell. Physiol. 234 (8) (2019) 14259–14269. S. Liu, Y. Yang, M. Jin, et al., Xenon-delayed postconditioning attenuates spinal cord ischemia/reperfusion injury through activation AKT and ERK signaling pathways in rats, J. Neurol. Sci. 368 (2016) 277–284. Z. He, S. Zou, J. Yin, et al., Inhibition of endoplasmic reticulum stress preserves the integrity of blood-spinal cord barrier in diabetic rats subjected to spinal cord injury, Sci. Rep. 7 (1) (2017) 7661. B. Zheng, Y. Zhou, H. Zhang, et al., Dl-3-n-butylphthalide prevents the disruption of blood-spinal cord barrier via inhibiting endoplasmic reticulum stress following spinal cord injury, Int. J. Biol. Sci. 13 (12) (2017) 1520. Y.W. Han, X.J. Liu, Y. Zhao, et al., Role of Oleanolic acid in maintaining BBB integrity by targeting p38MAPK/VEGF/Src signaling pathway in rat model of subarachnoid hemorrhage, Eur. J. Pharmacol. 839 (2018) 12–20. H.T. Li, X.Z. Zhao, X.R. Zhang, et al., Exendin-4 enhances motor function recovery via promotion of autophagy and inhibition of neuronal apoptosis after spinal cord injury in rats, Mol. Neurobiol. 53 (6) (2016) 4073–4082. B. Yuan, S. Pan, W.W. Zhang, Effects of gangliosides on expressions of caspase-3 and NGF in rats with acute spinal cord injury, Eur. Rev. Med. Pharmacol. Sci. 21 (24) (2017) 5843–5849. K. Gao, Z. Shen, Y. Yuan, et al., Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β‐catenin signaling pathway after spinal cord injury, J. Neurochem. 138 (1) (2016) 139–149. D. Zhao, M. Zhang, H. Yuan, et al., Ginsenoside Rb1 protects against spinal cord ischemia-reperfusion injury in rats by downregulating the Bax/Bcl-2 ratio and caspase-3 and p-Ask-1 levels, Exp. Mol. Pathol. 105 (3) (2018) 229–235. B. Spencer, R. Potkar, J. Metcalf, et al., Systemic central nervous system (CNS)targeted delivery of neuropeptide Y (NPY) reduces neurodegeneration and increases neural precursor cell proliferation in a mouse model of Alzheimer disease, J. Biol. Chem. 291 (4) (2016) 1905–1920. Y. Luo, C. Fu, Z. Wang, et al., Mangiferin attenuates contusive spinal cord injury in rats through the regulation of oxidative stress, inflammation and the Bcl-2 and Bax pathway, Mol. Med. Rep. 12 (5) (2015) 7132–7138. J. Wu, C. Yang, C. Guo, et al., SZC015, a synthetic oleanolic acid derivative, induces both apoptosis and autophagy in MCF-7 breast cancer cells, Chem. Biol. Interact. 244 (2016) 94–104. C. Long, J. Yang, H. Yang, et al., Attenuation of renal ischemia/reperfusion injury by oleanolic acid preconditioning via its antioxidant, anti-inflammatory, and antiapoptotic activities, Mol. Med. Rep. 13 (6) (2016) 4697–4704. J. Zheng, S. Zhang, H. Chen, et al., Protosappanin‐A and oleanolic acid protect injured podocytes from apoptosis through inhibition of AKT‐mTOR signaling, Cell Biol. Int. (2019). A.I. Faden, J. Wu, B.A. Stoica, et al., Progressive inflammation‐mediated neurodegeneration after traumatic brain or spinal cord injury, Br. J. Pharmacol. 173 (4) (2016) 681–691. B.N. Jaeger, S.L. Parylak, F.H. Gage, Mechanisms of dietary flavonoid action in neuronal function and neuroinflammation, Mol. Aspects Med. 61 (2018) 50–62. J. Xu, J. He, H. He, et al., TWEAK-Fn14 influences neurogenesis status via modulating NF-κB in mice with spinal cord injury, Mol. Neurobiol. 54 (9) (2017) 7497–7506. H. Wei, X. Ma, Tamoxifen reduces infiltration of inflammatory cells, apoptosis and inhibits IKK/NF-kB pathway after spinal cord injury in rats, Neurol. Sci. 35 (11) (2014) 1763–1768. D.S. Tian, J.L. Liu, M.J. Xie, et al., Tamoxifen attenuates inflammatory‐mediated damage and improves functional outcome after spinal cord injury in rats, J. Neurochem. 109 (6) (2009) 1658–1667. J.Q. Ma, J. Ding, L. Zhang, et al., Ursolic acid protects mouse liver against CCl4induced oxidative stress and inflammation by the MAPK/NF-κB pathway, Environ. Toxicol. Pharmacol. 37 (3) (2014) 975–983. R.B. Guo, G.F. Wang, A.P. Zhao, et al., Paeoniflorin protects against ischemia-induced brain damages in rats via inhibiting MAPKs/NF-κB-mediated inflammatory responses, PLoS One 7 (11) (2012) e49701. Z.Y. Zhuang, Y. Kawasaki, P.H. Tan, et al., Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injuryinduced cleavage of fractalkine, Brain Behav. Immun. 21 (5) (2007) 642–651. Z.G. Piao, I.H. Cho, C.K. Park, et al., Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury, Pain 121 (3) (2006) 219–231. J. Wang, B. Su, H. Zhu, et al., Protective effect of geraniol inhibits inflammatory response, oxidative stress and apoptosis in traumatic injury of the spinal cord through modulation of NF-κB and p38 MAPK, Exp. Ther. Med. 12 (6) (2016) 3607–3613. A.R. Phull, S.H. Eo, S.J. Kim, Oleanolic acid (OA) regulates inflammation and cellular dedifferentiation of chondrocytes via MAPK signaling pathways, Cell Mol. Biol. (Noisy-le-Grand) 63 (2017) 12–17. G.J. Kim, H.J. Jo, K.J. Lee, et al., Oleanolic acid induces p53-dependent apoptosis via the ERK/JNK/AKT pathway in cancer cell lines in prostatic cancer xenografts in mice, Oncotarget 9 (41) (2018) 26370.
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Oleanolic acid inhibits mouse spinal cord injury through suppressing inflammation and apoptosis via the blockage of p38 and JNK MAPKs
T
Jiang-Lin Wanga,1, Chang-He Rena,1, Jian Fengb, Ce-Hua Oua,*, Li Liuc,* a
Department of Pain Management, The Affiliated Hospital of Southwest Medical University, Luzhou City, Sichuan Province, 646000, China Department of Cardiology, The Affiliated Hospital of Southwest Medical University, Luzhou City, Sichuan Province, 646000, China c Department of Anesthesiology, The Affiliated Hospital of Southwest Medical University, Luzhou City, Sichuan Province, 646000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Spinal cord injury (SCI) Oleanolic acid (OA) Apoptosis and inflammation NF-κB p38 and JNK
Spinal cord injury (SCI) is reported as a devastating disease, leading to tissue loss and neurologic dysfunction. However, there is no effective therapeutic strategy for SCI treatment. Oleanolic acid (OA), as a triterpenoid, has anti-oxidant, anti-inflammatory, and anti-apoptotic activities. However, its regulatory effects on SCI have little to be elucidated, as well as the underlying molecular mechanisms. In this study, we attempted to explore the role of OA in SCI progression. Behavior tests suggested that OA treatments markedly alleviated motor function in SCI mice. Evans blue contents up-regulated in spinal cords of SCI mice were significantly reduced by OA in a dosedependent manner, demonstrating the improved blood-spinal cord barrier. Moreover, we found that OA treatments significantly reduced the apoptotic cell death in spinal cord samples of SCI mice through decreasing the expression of cleaved Caspase-3. In addition, pro-inflammatory response in SCI mice was significantly attenuated by OA treatments. Furthermore, SCI mice exhibited higher activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways, but these effects were clearly blocked in SCI mice with OA treatments, as evidenced by the down-regulated phosphorylation of p38, c-Jun-NH 2 terminal kinase (JNK), IκB kinase α (IKKα), inhibitor of nuclear factor κB-α (IκBα) and NF-κB. The protective effects of OA against SCI were confirmed in lipopolysaccharide (LPS)-stimulated mouse neurons mainly through the suppression of apoptosis and inflammatory response, which were tightly associated with the blockage of p38 and JNK activation. Together, our data demonstrated that OA treatments could dose-dependently ameliorate spinal cord damage through impeding p38- and JNK-regulated apoptosis and inflammation, and therefore OA might be served as an effective therapeutic agent for SCI treatment.
1. Introduction Spinal cord injury (SCI) affects 2.5–4 million patients in the world. According to the level and extent of the damage, the symptoms range from minimal sensory/motor deficits to complete tetraplegia [1]. In addition, neuropathic pain arises in a variety of cases, which further reduces the quality of life for patients with SCI [2,3]. Currently, the molecular mechanisms revealing the pathophysiology of SCI could be mainly divided into two broad successive phases [4]. On the one, the primary phase includes the initial mechanical injury, which is followed by a secondary phase consisted of some degenerative processes, such as vascular disruption, oxidative stress, ischemia, neuroinflammation and apoptosis [5,6]. The excessive release of pro-inflammatory factors, which is deleterious to neighboring neurons, resulting in the bloodspinal cord barrier disruption, spinal cord edema, as well as neutrophil
influx following SCI [7,8]. Unfortunately, there are no effective therapeutic strategies available to prevent SCI progression. Herein, it is essential to find and develop new treatments to attenuate SCI. Oleanolic acid (OA) is a natural triterpenoid and an aglycone of some saponins [9]. OA is widely existed in fruits of Olea europaea and Ligustrum lucidum, in vegetables, as well as in various kinds of medicinal herbs [10]. OA has been used as an over-the-counter Chinese medicine to alleviate inflammatory diseases and tumor growth [11–13]. For instance, OA could improve type 2 diabetes-related complications, especially inflammation, which is mainly via the suppression of the NFκB signaling pathway [14]. In addition, OA treatment significantly attenuated liver ischemic reperfusion injury partly through suppressing apoptosis [15]. Recently, OA was suggested to keep blood-brain barrier integrity through targeting p38 MAPK signaling pathway [16]. Moreover, OA ameliorated cognitive dysfunction induced by blocking
⁎
Corresponding authors at: The Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street, Luzhou City, Sichuan Province, 646000, China. E-mail addresses: [email protected] (C.-H. Ou), [email protected] (L. Liu). 1 Jiang-Lin Wang and Chang-He Ren are the co-first author. https://doi.org/10.1016/j.biopha.2019.109752 Received 5 September 2019; Received in revised form 7 November 2019; Accepted 29 November 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Aldrich, USA) was injected (45 mg/kg, iv) into the tail vein of mice 1 h before euthanizing. After adequate perfusion with saline under deep anesthesia, the L3-6 segments were removed and soaked in methanide for 24 h at 60 °C. After centrifugation, the evans blue levels were calculated as absorbance of supernatant at 632 nm using a microplate reader and measured as the amount of evans blue each wet tissue weight. As for the fluorescent evaluation, the tissues were fixed in 4 % paraformaldehyde, sectioned (6-μm thickness), sealed in a light-tight container and frozen. Evans blue staining was finally observed using a fluorescent microscope.
endoplasmic reticulum stress and inflammatory pathways [17]. Therefore, we hypothesized that OA might be a promising therapeutic candidate for SCI treatment. However, the effects of OA on SCI are unclear. In the present study, we found that OA treatments significantly improved motor function in SCI mice, accompanied with reduced evans blue contents in spinal cord samples, indicating the alleviated bloodspinal cord barrier disruption. We also suggested that OA administration dose-dependently inhibited the apoptosis and inflammatory response in spinal cord tissues from SCI mice, which were mainly dependent on the blockage of p38 and JNK MAPKs signaling pathway. Together, these findings demonstrated that OA treatment could be considered as an effective therapeutic agent to improve SCI.
2.4. Histological analysis The spinal cord lumbar segments were fixed in 4 % paraformaldehyde overnight, and were then transferred into 30 % sucrose in 4 % paraformaldehyde until reaching the bottom. Then, the spinal cord samples from 30 % sucrose solutions were cut into sections (6-μm thickness) using a cryostat microtome (Leica Microsystems, USA). Sections were then subjected to Nissl staining [26]. The survival condition of neurons in five sections from each mouse was analyzed and quantified with a light microscope by two pathologists blinded to the experimental treatments and outcomes.
2. Materials and methods 2.1. Animals and SCI model The experimental protocols were approved by the Ethics Committee of The Affiliated Hospital of Southwest Medical University (Sichuan, China) and complied with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. The female, 4–5 months old C57BL/6J mice were purchased from the Experimental Animal Center of Sichuan University (Sichuan, China). Guangdong Medical Animal Center (Guangdong, China). Mice used in the study were housed in a temperature- and humidity-controlled room (12 h light/dark) for 7 days before surgery with free access to the standard animal chow and water. All efforts were made to minimize animal suffering in the experiments. SCI animal model was established as previously described [18]. In brief, animals were anesthetized through intraperitoneal injections of 100 mg/kg of ketamine and 5 mg/kg of xylazine. A longitudinal dorsal incision was made to expose T6-T10 spinous processes. Laminectomy was conducted at the T7-T9 level using mouse laminectomy forceps (Heidelberg, Germany). The spinal cord was manually exposed, and then maximally compressed (100 %) with forceps (Fine Science Tools) to cause a robust and reproducible lesion [19]. Then, the muscles and skin were closed with 6-0 nylon stitches (Ethicon, Germany). Following the operation, mice were injected intraperitoneally with 0.9 % saline solution as supplementary body liquid. After operation, animals were maintained on a heated pad (37 °C) for 8 h to prevent hypothermia, and then were singly housed in a temperature-controlled (26 °C) room with free access to food and water. During the post-operative time, the bladders were manually voided as required. 25, 50 and 100 mg/kg of OA (MedChemExpress, USA) were chosen [20,21] and treated to mice via intraperitoneal injection once daily immediately began from trauma until 6 weeks subsequent to SCI. PBS was administered for the vehicle control group. As for the Shamoperated control group, mice underwent a T7-T9 laminectomy in the absence of compression injury and without treatment with OA.
2.5. Immunofluorescent (IF) staining After being transferred to 30 % sucrose solutions, the spinal cords samples were cut into sections (6-μm thickness) with a cryostat microtome. Then, the sections were blocked in 10 % BSA (Sigma Aldrich) for 1 h and incubated with primary antibodies overnight at 4 °C. Sections were rinsed with PBS and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 (#ab150117, 1:300 dilution, Abcam, USA) or 594 (#ab150088, 1:300 dilution, Abcam) for 1 h at room temperature. Subsequently, the sections were washed in PBS and incubated with DAPI (Beyotime) in the dark. Representative images were obtained with a fluorescent microscope. ImageJ 1.46r software (National Institutes of Health, USA) was used for quantification. Primary antibodies used here included anti-NeuN (#ab104224, 1:200 dilution, Abcam), anti-Caspase-3 (#9664, 1:150 dilution, Cell Signaling, USA), anti-NF-κB (#6956, 1:150 dilution, Cell Signaling) and Iba-1 (#MA5-27726, 1:200 dilution, ThermoFisher Scientific, USA). 2.6. Real-time quantitative PCR (RT-qPCR) assays From the long spinal cord segments containing the injury epicenter, total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer’s protocols. For calculation of mRNA expression, the first-strand synthesis was performed using a PrimeScript RT reagent kit (TaKaRa, Japan). RT-qPCR was then conducted on an Applied Biosystems 9700 Thermocycler (Applied Biosystems Life Technologies Corporation, USA) with the SYBR Premix Ex Taq kit (TaKaRa). Primers used in the study were designed and supplied by Sangon Biotech (Shanghai, China). Gene expression changes were quantified through the delta-delta CT method [27]. GAPDH was used as an internal control. Primer sequences of the targeted genes used in this study were listed in Supplementary table S1. All experiments were performed at least three times.
2.2. Motor function analysis The recovery of ground locomotion was measured through the Basso Mouse Scale (BMS) as previously indicated [22]. Moreover, the single-frame motion analysis was used to calculate the foot-stepping angle and rump-height index via the beam walking test [23]. The limb extension-flexion ratio was determined from video from recordings of voluntary movements by the “pencil” test [24]. Measurement was conducted before and at 7, 14, 21, 28, 35 and 42 days after injury. The obtained values for the left and right extremities were averaged.
2.7. Western blot assays After the animals (n = 6 per group) were sacrificed, 5-mm long segments of spinal cord encompassing the injury site were harvested and homogenized in RIPA solution with protease and phosphatase inhibitors (Beyotime) for western blot assays. Protein concentrations were then measured with a bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific). Each protein sample (30 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
2.3. Evans blue content and fluorescence analysis After SCI for 42 days, the evans blue levels and fluorescence were quantified for the calculation of blood-spinal cord barrier (BSCB) disruption following SCI as previously indicated [25]. Evans blue (Sigma 2
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 1. Oleanolic acid improves motor function of SCI mice. (A) BMS score analysis (n = 9 mice per group at each time point). (B) Calculation of foot-stepping angle (n = 9 mice per group at each time point). (C) Rump-height index of SCI mice was determined (n = 9 mice per group at each time point). (D) Quantification of extension/flexion ratio (n = 9 mice per group at each time point). (E) Blood-spinal cord barrier was measured using evans blue fluorescence. (F) The fluorescent density (%) of evans blue (n = 6 mice in each group, with 5 images for each mouse). (G) Evans blue levels in spinal cord samples were measured (n = 6 mice in each group). Data were means ± SEM. ++p < 0.01 vs the Sham group; *p < 0.05, **p < 0.01 vs the SCI group.
κB (p65) (#8242, Cell Signaling), phosphorylated NF-κB (p65) (#3033, Cell Signaling), IKKα (#61294, Cell Signaling), phosphorylated IKKα (#2078, Cell Signaling), IκBα (#4814, Cell Signaling), phosphorylated IκBα (#2859, Cell Signaling), JNK (#9252, Cell Signaling), phosphorylated JNK (#9255, Cell Signaling), p38 (#8690, Cell Signaling), phosphorylated p38 (#4511, Cell Signaling), ERK1/2 (#4695, Cell
PAGE), and transferred to polyvinyl difluoride membranes (PVDF, Millipore, USA). Nonspecific bands were blocked in TBS-T (pH 7.5) containing 5 % non-fat milk for 1.5 h at room temperature. Subsequently, the membranes were incubated with the primary antibodies at 4 °C overnight at a dilution of 1:500 or 1:1000 to calculate Bcl2 (#sc-7382, Santa Cruz, USA), Caspase-3 (#9664, Cell Signaling), NF3
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 2. Oleanolic acid treatment alleviates apoptosis in SCI mice. (A) Nissl staining of spinal cord sections. Quantification of neuron survival rate was exhibited (n = 6 mice in each group, with 6 images for each mouse). (B) IF staining of NeuN (green fluorescence) and Caspase-3 (red fluorescence) in spinal cord sections. The number of Caspase-3-positive cells was analyzed (n = 6 mice in each group, with 5 images for each mouse). (C) Western blot analysis was used to measure Bcl-2 and cleaved Caspase-3 expression levels in spinal cord samples (n = 4 mice in each group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Sham group; *p < 0.05 and **p < 0.01 vs the SCI group.
DMEM (Gibco, USA) supplemented with 10 % (v/v) FBS (Gibco) and 0.1 % penicillin-streptomycin, and incubated at 37 °C under 5 % CO2. To study the protective effects of OA, LPS (Sigma Aldrich) was used to stimulate HT22 cells. The p38 inhibitor SB203580 and JNK inhibitor SP600125 were obtained from MedChemExpress (USA) to block the activation of p38 and JNK, respectively.
Signaling), phosphorylated ERK1/2 (#9101, Cell Signaling), Iba-1 (#ab178847, Abcam) and GAPDH (#ab8245, Abcam). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#A0208 or #A0216, 1:2000 dilution, Beyotime) and detected with ECL substrate (ThermoFisher Scientific). Relative protein expression was normalized to GAPDH and compared with the control group. Images were captured using Quantity One software (BioRad, USA). Protein levels were compared by measuring mean density values. Quantification of target proteins was carried out using Image J 1.46r software (National Institutes of Health, USA).
2.10. Cell viability calculation The cytotoxicity of OA was measured using a microculture 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay (Beyotime) according to the manufacturer’s instructions. Cells were then incubated in 24-well plates at a density of 5 × 105 cells each well. MTT solution (5 μL of 5 mg/mL) was then added to each well. After incubation for 4 h at 37 °C in 5 % CO2, the supernatant was removed. Then, the formazan crystals produced in viable cells were solubilized in 150 μL dimethylsulfoxide (DMSO, Beyotime). The absorbance of each well was read at 570 nm with a microplate reader.
2.8. ELISA analysis for inflammatory cytokines The spinal cord samples were homogenized, and protein concentration was measured using the BCA Protein Assay kit (ThermoFisher Scientific). Samples were then concentrated to 4 mg/ml using MicroCon centrifugation filters (Millipore) to ensure equal amounts of protein. Subsequently, the IL-1β (#MLB00C), IL-6 (#M6000B) and TNF-α (#MTA00B) contents were determined using commercial available kits (R&D System, USA) according to the manufacturer’s instructions.
2.11. Flow cytometry analysis
2.9. Cells and treatments
Apoptotic cell death was conducted using an Annexin-VFITC apoptosis detection kit (BD Biosciences, USA). The treated cells were collected using trypsin, washed with ice-cold PBS and resuspended in 100 μl of flow cytometry binding buffer. Next, the cells were stained
HT22 murine hippocampal neuronal cells were purchased from TongPai Technology (Shanghai, China). Cells were maintained in 4
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 3. Oleanolic acid treatment inhibits inflammatory response in SCI mice. (A) IF staining of Iba-1 in spinal cord sections. Iba-1-positive cells were quantified (n = 6 mice in each group, with 5 images for each mouse). (B) RT-qPCR and (C) western blot analysis were used to determine Iba-1 expression levels in spinal cord samples (n = 3 mice in each group). (D) ELISA analysis was used to measure IL-1β, IL-6 and TNF-α contents in spinal cord tissues (n = 8 mice in each group). The mRNA levels of (E) iNOS, IL-8, IL-12, (F) IL-10, SOCS3 and Arg1 in spinal cord tissues were measured using RT-qPCR analysis (n = 3 mice in each group). Data were means ± SEM. +p < 0.05, ++p < 0.01 and +++p < 0.001 vs the Sham group; *p < 0.05 and **p < 0.01 vs the SCI group.
with 5 μl of Annexin V/FITC followed by 5 μl of PI in the dark for 15 min at room temperature. Then, 400 μl of binding buffer was added to each tube. The percentage of apoptotic cells was analyzed using FACSCalibur flow cytometer (BD Biosciences).
excluded. All in vitro experiments were replicated using different cell cultures. A randomization process was performed in grouping mice. All in vivo and imaging studies were performed in a blinded manner. The experimenters were blinded to the grouping information.
2.12. Statistical analysis
3. Results
The data were expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments unless specifically stated in the figure legends. Data were analyzed using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, USA). Behavioral data were analyzed using two-way repeated measures one-way analysis of variance (ANOVA) with Bonferroni post hoc tests. The other statistical differences between two groups were analyzed using Student’s t-test. P value < 0.05 was considered statistically significant. No data were
3.1. Oleanolic acid improves motor function of SCI mice In order to calculate the effects of OA on SCI, motor function of mice was measured. As shown in Fig. 1A, SCI mice showed the decreased BMS score was markedly rescued by OA treatment in a dose-dependent manner, revealing the better recovery of motor function. Then, we found that the foot-stepping angle was changed from about 30° before injury to approximate 170° after injury in all groups of mice. The 5
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 4. Oleanolic acid treatment blocks MAPKs and NF-κB signaling pathways in SCI mice. (A) Western blot analysis was performed to calculate p-p38, p-ERK1/ 2 and p-JNK protein expression levels in spinal cord samples (n = 4 mice in each group). (B) The protein expression levels of p-IKKα, p-IκBα and p-NF-κB in spinal cord tissues were measured using western blot analysis (n = 3 mice in each group). (C) IF staining for NeuN (green fluorescence) and NF-κB (red fluorescence) in spinal cord sections (n = 6 mice per group, with 5 images for each mouse). The number of NF-κB-positive cells was quantified. Data were means ± SEM. +p < 0.05, ++p < 0.01 and +++p < 0.001 vs the Sham group; *p < 0.05 and **p < 0.01 vs the SCI group.
3.3. Oleanolic acid treatment inhibits inflammatory response in SCI mice
decrease of foot-stepping angle represents the improved recovery of SCI mice [28]. Here, OA treatment dose-dependently decreased the footstepping angle of SCI mice compared to the SCI mice (Fig. 1B). The rump-height index presents the ability to support body weight during ground locomotion [29]. OA treatment significantly up-regulated the rump-height index in mice with SCI in a dose-dependent fashion (Fig. 1C). Additionally, SCI mice treated with OA showed increased extension/flexion ratio especially at the highest concentration of 100 mg/kg (Fig. 1D). To explore if OA treatment could alleviate the blood-spinal cord barrier, evans blue staining was performed. As shown in Fig. 1E–G, SCI mice showed significantly up-regulated evans blue contents in spinal cord samples, which were alleviated by OA treatment in a dose-dependent manner, indicating the alleviated blood-spinal cord barrier permeability. The findings above demonstrated that OA administration could attenuate motor dysfunction in SCI mice with alleviated blood-spinal cord barrier integrity.
Microglia were activated after SCI [30]. Iba-1, a critical marker of microglial cells, was found to be significantly up-regulated in spinal cord samples of SCI mice when compared to the Sham group of mice. However, OA treatments markedly reduced the expression of Iba-1 from mRNA and protein levels in a dose-dependent manner (Fig. 3A–C). Then, ELISA analysis demonstrated that SCI mice had higher contents of pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α in spinal cord samples than that of the Sham mice, which were markedly reduced by OA treatments in a dose-dependent manner (Fig. 3D). Furthermore, the mRNA levels of iNOS, IL-8 and IL-12, representing inflammatory response, increased in SCI mice were significantly reduced by OA (Fig. 3E). In contrast, the expression levels of anti-inflammatory factors including IL-10, SOCS3 and Arg1 decreased in SCI mice were dosedependently restored by OA treatments (Fig. 3F). Together, results in this part demonstrated that OA could alleviate SCI in mice via suppressing pro-inflammatory response.
3.2. Oleanolic acid treatment alleviates apoptosis in SCI mice In this part, Nissl staining indicated that OA-treated mice exhibited significantly improved survival rate of neuron in spinal cord sections of SCI mice (Fig. 2A). IF staining showed higher expression levels of Caspase-3 in SCI mice than that of the Sham mice, but these effects were dose-dependently reduced by OA treatments (Fig. 2B). Moreover, we found that SCI-induced high expression levels of cleaved Caspase-3 were markedly decreased by OA. In contrast, the protein expression levels of anti-apoptotic signal Bcl-2 were down-regulated by SCI, while being restored by OA in a dose-dependent manner (Fig. 2C). Results above elucidated that OA could alleviate SCI in mice through inhibiting apoptosis via the blockage of Caspase-3 activation.
3.4. Oleanolic acid treatment blocks MAPKs and NF-κB signaling pathways in SCI mice MAPKs and NF-κB signaling pathways play critical role in regulating inflammatory response during SCI progression [31,32]. Western blot analysis suggested that SCI mice showed significantly increased expression of phosphorylated p38, ERK1/2 and JNK in spinal cord samples. Notably, OA treatments significantly reduced the activation of p38 and JNK in a concentration-dependent manner. However, ERK1/2 phosphorylation up-regulated in SCI mice was not significantly alleviated by OA (Fig. 4A). Then, we found that SCI mice exhibited the 6
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 5. Oleanolic acid incubation alleviates apoptotic cell death in mouse neuron cells. (A) Mouse neuron HT22 cells were incubated with OA (0, 0.5, 1, 2.5, 5, 10, 20, 40 and 80 μM) for 24 h. Then, cells were collected for cell viability calculation using MTT analysis (n = 4 per group). (B) HT22 cells were treated with OA (40 μM) for the indicated time (0, 4, 8, 16, 24, 48, 72 and 96 h). Then, all cells were collected for cell viability calculation using MTT analysis (n = 4 per group). (C) Morphology of HT22 cells treated with 24 h of LPS (100 ng/ml) in the absence or presence of OA (10, 20 and 40 μM) (n = 3 per group). (D) Flow cytometry analysis for apoptosis rate calculation in LPS (100 ng/ml)-stimulated HT22 cells for 24 h with or without OA (10, 20 and 40 μM) (n = 3 per group). (E) HT22 cells were exposed to 100 ng/ml of LPS for 24 h with or without OA (10, 20 and 40 μM), followed by western blot analysis of Bcl-2 and cleaved Caspase-3 in cells (n = 3 per group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Con group; *p < 0.05 and **p < 0.01 vs the LPS group.
by LPS, were rescued in OA-treated cells (Fig. 5E). Therefore, results here confirmed the effects of OA against apoptosis during SCI progression.
markedly increased expression of p-IKKα, p-IκBα and p-NF-κB in spinal cord tissues compared to the Sham group, while being significantly attenuated by OA treatments also in a dose-dependent manner (Fig. 4B). IF staining demonstrated that nuclear NF-κB expression was obviously up-regulated in neuron of spinal cord sections from SCI mice, which were clearly reduced by OA administration (Fig. 4C). Collectively, the results suggested that OA treatment could inhibit MAPKs, except ERK1/2, and NF-κB signaling pathways in SCI mice.
3.6. Oleanolic acid suppresses inflammatory response in LPS-treated HT22 cells by blocking MAPKs and NF-κB signaling pathways Western blot analysis demonstrated that OA showed no significant influence on the activation of ERK1/2 induced by LPS (Supplementary Fig. 1A). Consistently, LPS-induced up-regulation of p-p38 and p-JNK was evidently abolished by OA. At the same time, we found that OA functioned as p38 and JNK inhibitors to block p38 and JNK activation in LPS-stimulated HT22 neurons as shown in Fig. 6A and B. Then, the protein expression levels of p-IκBα and p-NF-κB in LPS-treated HT22 cells were considerably diminished by OA, SB203580 or SP600125 treatment (Fig. 6C). Subsequently, LPS-incubated HT22 cells showed higher mRNA levels of IL-1β, IL-6, TNF-α, iNOS, IL-8 and IL-12 than that of the Con group, while being abolished by the co-treatment of OA, SB203580 or SP600125. In contrast, anti-inflammatory molecules IL10, SOCS3 and Arg1 down-regulated by LPS were highly rescued by OA and the suppression of p38 and JNK in HT22 neurons through RT-qPCR analysis (Fig. 6D). Therefore, results here demonstrated that OA-impeded pro-inflammatory response in SCI was at least partly associated with the blockage of p38 and JNK MAPKs, and NF-κB signaling pathways.
3.5. Oleanolic acid incubation alleviates apoptotic cell death in mouse neuron cells The in vivo results suggested that OA showed protective role against SCI development in mice. To confirm its regulatory effects on SCI, the in vitro analysis was then performed in mouse neurons with or without LPS stimulation. At first, MTT analysis suggested that OA treatments ranging from 0 to 80 μM showed no significant difference in the cell viability of HT22 cells (Fig. 5A). In addition, 40 μM of OA treatment from 0 to 72 h also did not reduce the cell viability of HT22 cells (Fig. 5B). Then, the concentrations of OA at 10, 20 and 40 μM were chosen for cell treatment for 24 h. As shown in Fig. 5C, LPS stimulation led to HT22 cell damage, which was, however, improved by OA treatments. Flow cytometry analysis suggested that LPS resulted in significant apoptotic cell death in HT22 cells, and these effects were significantly alleviated by OA treatments in a dose-dependent manner (Fig. 5D). Meanwhile, LPS-enhanced expression of cleaved Caspase-3 was markedly abrogated by OA, while Bcl-2 expression levels, inhibited 7
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Fig. 6. Oleanolic acid suppresses inflammatory response in LPS-treated HT22 cells by blocking MAPKs and NF-κB signaling pathways. (A–D) HT22 cells were pre-treated with p38 inhibitor SB203580 and/or JNK inhibitor SP600125 for 2 h, and then were exposed to LPS (100 ng/ml) for another 24 h combined with or without OA (40 μM). All cells were then harvested for the following analysis. The protein expression levels of (A) p-p38, and (B) p-JNK, (C) p-IκBα and p-NF-κB were measured by western blot analysis (n = 4 per group). (D) RT-qPCR analysis was performed to determine IL-1β, IL-6, TNF-α, iNOS, IL-8, IL-12, IL-10, SOCS3 and Arg1 in cells (n = 3 per group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Con group; *p < 0.05 and **p < 0.01 vs the LPS group.
Fig. 7. Oleanolic acid-suppressed apoptosis in HT22 cells is dependent on MAPKs blockage. (A–D) HT22 cells were pre-treated with SB203580 or SP600125 for 2 h at the indicated concentrations, and then were incubated with 100 ng/ml of LPS for another 24 h with or without OA (40 μM). All cells were finally collected for the following studies. (A) Flow cytometry analysis was used to measure apoptosis in cells (n = 3 per group). (B) IF staining of Caspase-3 in the treated cells. (C) Quantification of Caspase-3 expression levels following IF analysis (n = 3 per group, with 3 images for each group). (D) Bcl-2 and cleaved Caspase-3 expression levels in HT22 cells were measured by western blot analysis (n = 4 per group). Data were means ± SEM. ++p < 0.01 and +++p < 0.001 vs the Con group; *p < 0.05 and **p < 0.01 vs the LPS group.
8
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
Targeting of the IKK/NF-κB signaling improved the recovery of locomotor function through decreasing the infiltration of inflammatory cells following SCI [50]. It has been indicated that OA inhibited NF-κB and NF-κB-regulated gene products via direct inhibition of IKK [51]. In the present study, we found that OA treatments significantly reduced the expression of phosphorylated IκBα in the spinal cords of the mice after SCI. We also observed a subsequent reduction in the expression of phosphorylated NF-κB (p65) in spinal cord samples from OA-treated SCI mice, contributing to the decreased release of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, iNOS, IL-8 and IL-12. In contrast, the mRNA levels of signals with anti-inflammatory effects, including IL10, SOCS3 and Arg1, were highly rescued in spinal cord samples of SCI mice. MAPKs, including p38, ERK1/2 and JNK, are known to participate in the signal transduction of neuronal apoptosis and inflammation [33,52]. Previous studies have suggested that MAPKs signaling is activated after SCI in neurons, microglia/macrophages and astrocytes, and is also involved in regulating chronic pain [53,54]. Suppressing MAPKs after SCI have indicated differing results, from slightly alleviated inflammation and decreased apoptosis [55]. OA modulates inflammation and cellular dedifferentiation of chondrocytes through MAPKs signaling pathways [56]. OA could regulate p53-dependent apoptosis also via the ERK/JNK pathway in cancer cell lines [57]. Our data suggested that the activation of p38, ERK and JNK was implicated in SCI. Notably, OA treatment markedly reduced the phosphorylation of p38 and JNK, while showed no significantly inhibitory effects on ERK1/2 activation in spinal cord samples of SCI mice. These effects were confirmed in LPSstimulated neurons in vitro. Importantly, we found that OA functioned as p38 and JNK inhibitors, which could alleviate inflammatory response and apoptotic cell death in neurons with LPS stimulation. Thus, OA treatments showed beneficial effects against SCI partly through blocking MAPKs signaling pathway. In summary, the results in our study demonstrated that OA could significantly alleviate disruption of motor function and blood-spinal cord barrier in SCI mice. Furthermore, OA treatments markedly repressed apoptosis and inflammation in spinal cord tissues from SCI mice, which was largely dependent on the blockage of Caspase-3 and IKK/NF-κB signaling pathways. These effects were mainly through the inhibition of MAPKs (p38 and JNK) by OA. On the basis of our data, OA treatment may have therapeutic utility for the treatment of SCI.
3.7. Oleanolic acid-suppressed apoptosis in HT22 cells is dependent on MAPKs blockage MAPKs signaling pathway has been reported to modulate apoptosis during SCI development [33]. Here, flow cytometry results suggested that OA treatment and the suppression of p38 or JNK significantly reduced the apoptotic cell death in LPS-stimulated HT22 neurons, which was accompanied with reduced expression of Caspase-3 by IF analysis (Fig. 7A–C). We also found that LPS-decreased expression of Bcl-2 was markedly restored by OA, SB203580 or SP600125 treatment. However, the cleaved Caspase-3 protein levels potentiated by LPS were evidently abrogated by OA or the blockage of p38 and JNK activation (Fig. 7D). Taken together, the findings here suggested that OA-alleviated apoptosis triggered by LPS in mouse neurons was also tightly associated with the inhibition of MAPKs signaling pathway. 4. Discussion The results from the present study suggested that OA treatments significantly improved the motor function of SCI mice, which were mainly associated with the reduced apoptosis and inflammatory response. As reported, the disruption of blood-spinal cord barrier subsequent to SCI results in an intensive local inflammation by the infiltration of blood cells including neutrophils and macrophages, which leads to cell death and permanent neurological disability [34]. The integrity of the blood-spinal cord barrier could be examined by evans blue dye extravasation as previously described [35]. Here, we also demonstrated that the evans blue contents in spinal cord samples of SCI mice were markedly reduced by OA treatments in a dose-dependent manner, illustrating that OA could attenuate the disruption of bloodspinal cord barrier after SCI, which was similar to previous results that OA improved blood-brain barrier integrity in animal model with vasogenic edema induced by subarachnoid hemorrhage [36]. Therefore, OA treatments likely contributed to the success in functional recovery. Apoptosis, as indicated by nuclear DNA fragmentation and caspase activation, is a prominent characteristic in the spinal cord following SCI [26,37]. Apoptotic cell death was detected in neurons, and was prominent in the white matter. After SCI, caspase activation could be detected in neurons at the injury site within hours [38,39]. The significant executioners in the apoptotic programme are proteases, which are known as caspases. The caspase family of cysteine proteases regulates the execution of the mammalian apoptotic cell death programme [40]. Caspase-3 could be activated during apoptosis in various types of central nervous system (CNS) cells, and its activation might be a critical apoptotic event in the CNS [41]. In contrast, Bcl-2 has been suggested as an inhibitor of apoptosis [40,42]. Increasing studies have suggested that OA shows significant role in regulating apoptosis to control the growth of various types of tumors [43]. OA could also alleviate renal ischemia reperfusion injury partly through its anti-apoptotic activities [44]. OA also showed anti-apoptotic effects to protect injured podocytes [45]. In this study, we found that OA treatments significantly improved the survival rate of neurons in spinal cord samples of SCI mice via Nissl staining, which was along with the significantly reduced expression of cleaved Caspase-3 and the elevated Bcl-2 expression levels. Therefore, OA-alleviated SCI was at least partly associated with the inhibition of apoptosis via reducing Caspase-3 activation. Inflammatory responses are known as a major component of secondary injury and play an important role in modulating the pathogenesis of acute and chronic SCI, which is likely to be essential for nerve injury, contributing to the process of the regenerative response [46]. At the same time, inflammation may lead to apoptosis in neurons and consequently result in the decrease of neuronal function [47]. Thus, suppressing inflammatory response could down-regulate secondary degeneration and the functional deficits following SCI. Moreover, the IKK/NF-κB signaling pathway plays a predominant role in the meditation of inflammation and apoptosis during SCI progression [48,49].
Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. Acknowledgments This work was supported by (1) Scientific Research Project of Sichuan Provincial Health Commission [No. 19PJ289] and (2) the Science and Technology Strategic Cooperation Programs of Luzhou Municipal People’s Government and Southwest Medical University [No. 2019LZXNYDJ38]. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109752. References [1] M.E.L. Van den Berg, J.M. Castellote, J. de Pedro-Cuesta, et al., Survival after spinal cord injury: a systematic review, J. Neurotrauma 27 (8) (2010) 1517–1528. [2] P.J. Siddall, J.D. Loeser, Pain following spinal cord injury, Spinal Cord 39 (2) (2001) 63.
9
Biomedicine & Pharmacotherapy 123 (2020) 109752
J.-L. Wang, et al.
[3] P.J. Siddall, M.J. Cousins, A. Otte, et al., Pregabalin in central neuropathic pain associated with spinal cord injury: a placebo-controlled trial, Neurology 67 (10) (2006) 1792–1800. [4] C.S. Ahuja, M. Fehlings, Concise review: bridging the gap: novel neuroregenerative and neuroprotective strategies in spinal cord injury, Stem Cells Transl. Med. 5 (7) (2016) 914–924. [5] M. Cordaro, I. Paterniti, R. Siracusa, et al., KU0063794, a dual mTORC1 and mTORC2 inhibitor, reduces neural tissue damage and locomotor impairment after spinal cord injury in mice, Mol. Neurobiol. 54 (4) (2017) 2415–2427. [6] A.J.E. Combes, L. Matthews, J.S. Lee, et al., Cervical cord myelin water imaging shows degenerative changes over one year in multiple sclerosis but not neuromyelitis optica spectrum disorder, Neuroimage Clin. 16 (2017) 17–22. [7] A.I. Faden, J. Wu, B.A. Stoica, et al., Progressive inflammation‐mediated neurodegeneration after traumatic brain or spinal cord injury, Br. J. Pharmacol. 173 (4) (2016) 681–691. [8] T. Matsushita, K.L. Lankford, E.J. Arroyo, et al., Diffuse and persistent blood–spinal cord barrier disruption after contusive spinal cord injury rapidly recovers following intravenous infusion of bone marrow mesenchymal stem cells, Exp. Neurol. 267 (2015) 152–164. [9] D. Kashyap, A.S. Sharma, H. Tuli, et al., Ursolic acid and oleanolic acid: pentacyclic terpenoids with promising anti-inflammatory activities, Recent Pat. Inflamm. Allergy Drug Discov. 10 (1) (2016) 21–33. [10] J. Pollier, A. Goossens, Oleanolic acid, Phytochemistry 77 (2012) 10–15. [11] W. Lee, E.J. Yang, S.K. Ku, et al., Anti-inflammatory effects of oleanolic acid on LPSinduced inflammation in vitro and in vivo, Inflammation 36 (1) (2013) 94–102. [12] R. Martín, J. Carvalho-Tavares, M. Hernández, et al., Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: a potential therapeutic role, Biochem. Pharmacol. 79 (2) (2010) 198–208. [13] M.K. Shanmugam, X. Dai, A.P. Kumar, et al., Oleanolic acid and its synthetic derivatives for the prevention and therapy of cancer: preclinical and clinical evidence, Cancer Lett. 346 (2) (2014) 206–216. [14] D. Camer, Y. Yu, A. Szabo, et al., The molecular mechanisms underpinning the therapeutic properties of oleanolic acid, its isomer and derivatives for type 2 diabetes and associated complications, Mol. Nutr. Food Res. 58 (8) (2014) 1750–1759. [15] B. Gui, F. Hua, J. Chen, et al., Protective effects of pretreatment with oleanolic acid in rats in the acute phase of hepatic ischemia-reperfusion injury: role of the PI3K/ Akt pathway, Mediators Inflamm. 2014 (2014). [16] R. Martín, J. Carvalho-Tavares, M. Hernández, et al., Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: a potential therapeutic role, Biochem. Pharmacol. 79 (2) (2010) 198–208. [17] J. Lu, D. Wu, Y. Zheng, et al., Ursolic acid improves high fat diet-induced cognitive impairments by blocking endoplasmic reticulum stress and IκB kinase β/nuclear factor-κB-mediated inflammatory pathways in mice, Brain Behav. Immun. 25 (8) (2011) 1658–1667. [18] A. Mehanna, I. Jakovcevski, A. Acar, et al., Polysialic acid glycomimetic promotes functional recovery and plasticity after spinal cord injury in mice, Mol. Ther. 18 (1) (2010) 34–43. [19] R. Curtis, D. Green, R.M. Lindsay, et al., Up-regulation of GAP-43 and growth of axons in rat spinal cord after compression injury, J. Neurocytol. 22 (1) (1993) 51–64. [20] S. Su, G. Wu, X. Cheng, et al., Oleanolic acid attenuates PCBs-induced adiposity and insulin resistance via HNF1b-mediated regulation of redox and PPARγ signaling, Free Radic. Biol. Med. 124 (2018) 122–134. [21] X. Wang, Y.L. Li, H. Wu, et al., Antidiabetic effect of oleanolic acid: a promising use of a traditional pharmacological agent, Phytother. Res. 25 (7) (2011) 1031–1040. [22] D.M. Basso, L.C. Fisher, A.J. Anderson, et al., Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains, J. Neurotrauma 23 (5) (2006) 635–659. [23] D. Lutz, H. Kataria, R. Kleene, et al., Myelin basic protein cleaves cell adhesion molecule L1 and improves regeneration after injury, Mol. Neurobiol. 53 (5) (2016) 3360–3376. [24] I. Apostolova, A. Irintchev, M. Schachner, Tenascin-R restricts posttraumatic remodeling of motoneuron innervation and functional recovery after spinal cord injury in adult mice, J. Neurosci. 26 (30) (2006) 7849–7859. [25] X.Q. Li, H.W. Lv, W.F. Tan, et al., Role of the TLR4 pathway in blood-spinal cord barrier dysfunction during the bimodal stage after ischemia/reperfusion injury in rats, J. Neuroinflammation 11 (1) (2014) 62. [26] H. Zhao, S. Chen, K. Gao, et al., Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway, Neuroscience 348 (2017) 241–251. [27] M.A. Mendieta-Serrano, F.J. Mendez-Cruz, M. Antúnez-Mojica, et al., NADPHOxidase-derived reactive oxygen species are required for cytoskeletal organization, proper localization of E-cadherin and cell motility during zebrafish epiboly, Free Radic. Biol. Med. 130 (2019) 82–98. [28] F. Papastefanaki, I. Jakovcevski, N. Poulia, et al., Intraspinal delivery of polyethylene glycol-coated gold nanoparticles promotes functional recovery after spinal cord injury, Mol. Ther. 23 (6) (2015) 993–1002. [29] S. Sahu, R. Li, P.K. Kadeyala, et al., The human natural killer-1 (HNK-1) glycan mimetic ursolic acid promotes functional recovery after spinal cord injury in mouse, J. Nutr. Biochem. 55 (2018) 219–228. [30] M. Tsuda, Microglia in the spinal cord and neuropathic pain, J. Diabetes Investig. 7 (1) (2016) 17–26. [31] S.J. Huang, J.Q. Yan, H. Luo, et al., IL-33/ST2 signaling contributes to radicular
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47] [48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
10
pain by modulating MAPK and NF-κB activation and inflammatory mediator expression in the spinal cord in rat models of noncompressive lumber disk herniation, J. Neuroinflammation 15 (1) (2018) 12. Y. Su, S. Zong, C. Wei, et al., Salidroside promotes rat spinal cord injury recovery by inhibiting inflammatory cytokine expression and NF‐κB and MAPK signaling pathways, J. Cell. Physiol. 234 (8) (2019) 14259–14269. S. Liu, Y. Yang, M. Jin, et al., Xenon-delayed postconditioning attenuates spinal cord ischemia/reperfusion injury through activation AKT and ERK signaling pathways in rats, J. Neurol. Sci. 368 (2016) 277–284. Z. He, S. Zou, J. Yin, et al., Inhibition of endoplasmic reticulum stress preserves the integrity of blood-spinal cord barrier in diabetic rats subjected to spinal cord injury, Sci. Rep. 7 (1) (2017) 7661. B. Zheng, Y. Zhou, H. Zhang, et al., Dl-3-n-butylphthalide prevents the disruption of blood-spinal cord barrier via inhibiting endoplasmic reticulum stress following spinal cord injury, Int. J. Biol. Sci. 13 (12) (2017) 1520. Y.W. Han, X.J. Liu, Y. Zhao, et al., Role of Oleanolic acid in maintaining BBB integrity by targeting p38MAPK/VEGF/Src signaling pathway in rat model of subarachnoid hemorrhage, Eur. J. Pharmacol. 839 (2018) 12–20. H.T. Li, X.Z. Zhao, X.R. Zhang, et al., Exendin-4 enhances motor function recovery via promotion of autophagy and inhibition of neuronal apoptosis after spinal cord injury in rats, Mol. Neurobiol. 53 (6) (2016) 4073–4082. B. Yuan, S. Pan, W.W. Zhang, Effects of gangliosides on expressions of caspase-3 and NGF in rats with acute spinal cord injury, Eur. Rev. Med. Pharmacol. Sci. 21 (24) (2017) 5843–5849. K. Gao, Z. Shen, Y. Yuan, et al., Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β‐catenin signaling pathway after spinal cord injury, J. Neurochem. 138 (1) (2016) 139–149. D. Zhao, M. Zhang, H. Yuan, et al., Ginsenoside Rb1 protects against spinal cord ischemia-reperfusion injury in rats by downregulating the Bax/Bcl-2 ratio and caspase-3 and p-Ask-1 levels, Exp. Mol. Pathol. 105 (3) (2018) 229–235. B. Spencer, R. Potkar, J. Metcalf, et al., Systemic central nervous system (CNS)targeted delivery of neuropeptide Y (NPY) reduces neurodegeneration and increases neural precursor cell proliferation in a mouse model of Alzheimer disease, J. Biol. Chem. 291 (4) (2016) 1905–1920. Y. Luo, C. Fu, Z. Wang, et al., Mangiferin attenuates contusive spinal cord injury in rats through the regulation of oxidative stress, inflammation and the Bcl-2 and Bax pathway, Mol. Med. Rep. 12 (5) (2015) 7132–7138. J. Wu, C. Yang, C. Guo, et al., SZC015, a synthetic oleanolic acid derivative, induces both apoptosis and autophagy in MCF-7 breast cancer cells, Chem. Biol. Interact. 244 (2016) 94–104. C. Long, J. Yang, H. Yang, et al., Attenuation of renal ischemia/reperfusion injury by oleanolic acid preconditioning via its antioxidant, anti-inflammatory, and antiapoptotic activities, Mol. Med. Rep. 13 (6) (2016) 4697–4704. J. Zheng, S. Zhang, H. Chen, et al., Protosappanin‐A and oleanolic acid protect injured podocytes from apoptosis through inhibition of AKT‐mTOR signaling, Cell Biol. Int. (2019). A.I. Faden, J. Wu, B.A. Stoica, et al., Progressive inflammation‐mediated neurodegeneration after traumatic brain or spinal cord injury, Br. J. Pharmacol. 173 (4) (2016) 681–691. B.N. Jaeger, S.L. Parylak, F.H. Gage, Mechanisms of dietary flavonoid action in neuronal function and neuroinflammation, Mol. Aspects Med. 61 (2018) 50–62. J. Xu, J. He, H. He, et al., TWEAK-Fn14 influences neurogenesis status via modulating NF-κB in mice with spinal cord injury, Mol. Neurobiol. 54 (9) (2017) 7497–7506. H. Wei, X. Ma, Tamoxifen reduces infiltration of inflammatory cells, apoptosis and inhibits IKK/NF-kB pathway after spinal cord injury in rats, Neurol. Sci. 35 (11) (2014) 1763–1768. D.S. Tian, J.L. Liu, M.J. Xie, et al., Tamoxifen attenuates inflammatory‐mediated damage and improves functional outcome after spinal cord injury in rats, J. Neurochem. 109 (6) (2009) 1658–1667. J.Q. Ma, J. Ding, L. Zhang, et al., Ursolic acid protects mouse liver against CCl4induced oxidative stress and inflammation by the MAPK/NF-κB pathway, Environ. Toxicol. Pharmacol. 37 (3) (2014) 975–983. R.B. Guo, G.F. Wang, A.P. Zhao, et al., Paeoniflorin protects against ischemia-induced brain damages in rats via inhibiting MAPKs/NF-κB-mediated inflammatory responses, PLoS One 7 (11) (2012) e49701. Z.Y. Zhuang, Y. Kawasaki, P.H. Tan, et al., Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injuryinduced cleavage of fractalkine, Brain Behav. Immun. 21 (5) (2007) 642–651. Z.G. Piao, I.H. Cho, C.K. Park, et al., Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury, Pain 121 (3) (2006) 219–231. J. Wang, B. Su, H. Zhu, et al., Protective effect of geraniol inhibits inflammatory response, oxidative stress and apoptosis in traumatic injury of the spinal cord through modulation of NF-κB and p38 MAPK, Exp. Ther. Med. 12 (6) (2016) 3607–3613. A.R. Phull, S.H. Eo, S.J. Kim, Oleanolic acid (OA) regulates inflammation and cellular dedifferentiation of chondrocytes via MAPK signaling pathways, Cell Mol. Biol. (Noisy-le-Grand) 63 (2017) 12–17. G.J. Kim, H.J. Jo, K.J. Lee, et al., Oleanolic acid induces p53-dependent apoptosis via the ERK/JNK/AKT pathway in cancer cell lines in prostatic cancer xenografts in mice, Oncotarget 9 (41) (2018) 26370.