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Large-earthquake rupturing and slipping behavior along the range-front Maidan fault in the southern Tian Shan, northwestern China
Journal Pre-proofs Large-earthquake rupturing and slipping behavior along the range-front Maidan fault in the southern Tian Shan, Northwestern China Chuanyong Wu, Wenjun Zheng, Zhuqi Zhang, Qichao Jia, Huili Yang PII: DOI: Reference:
S1367-9120(19)30545-0 https://doi.org/10.1016/j.jseaes.2019.104193 JAES 104193
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Journal of Asian Earth Sciences
Received Date: Revised Date: Accepted Date:
15 August 2019 29 November 2019 7 December 2019
Please cite this article as: Wu, C., Zheng, W., Zhang, Z., Jia, Q., Yang, H., Large-earthquake rupturing and slipping behavior along the range-front Maidan fault in the southern Tian Shan, Northwestern China, Journal of Asian Earth Sciences (2019), doi: https://doi.org/10.1016/j.jseaes.2019.104193
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Phillyrin protects mice from traumatic brain injury by inhibiting the inflammation of microglia via PPARγ signaling pathway Running title:traumatic brain injury and phillyrin Qian Jiang, Jun Chen , Xiaobing Long, Xiaolong Yao, Xin Zou , Yiping Yang , Guangying Huang , Huaqiu Zhang* 1) Qian Jiang; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected] 2) Jun Chen; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail:[email protected] 3) Xiaobing Long; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail:[email protected] 4) Xiaolong Yao; 1Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China, 2Department of Neurosurgery, Taikang Tongji Hospital, Wuhan, 430050, PR China;E-mail: [email protected] 5) Xin Zou; Department of Traditional Chinese Medicine, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected] 6) Yiping Yang; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail:[email protected] 7) Guangying Huang; Department of Traditional Chinese Medicine, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected] 8) Huaqiu Zhang; corresponding author, Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected]
Abstract The neuroinflammatory response induced by microglia plays a vital role in causing secondary brain damage after traumatic brain injury (TBI). Previous studies have found that the improved regulation of activated microglia could reduce neurological damage post-TBI. Phillyrin (Phi) is one of the main active ingredients extracted from the fruits of the medicinal plant Forsythia suspensa (Thunb.) with anti-inflammatory effects. Our study attempted to investigate the effects of phillyrin on microglial activation and neuron damage after TBI. The TBI model was applied to induce brain injury in mice, and neurological scores, brain water content, hematoxylin and eosin staining and Nissl staining were employed to determine the neuroprotective effects of phillyrin. Immunofluorescent staining and western blot analysis were used to detect nuclear factor-kappa B (NF-κB) and peroxisome proliferator–activated receptor gamma (PPARγ) expression and nuclear translocation, and the inflammation-related proteins and mRNAs were assessed by western blot analysis and quantitative real-time PCR. The results revealed that phillyrin not only inhibited the proinflammatory response induced by activated microglia but also attenuated neurological impairment and brain edema in vivo in a mouse TBI model. Additionally, phillyrin suppressed the phosphorylation of NF-κB in microglia after TBI insult. These effects of phillyrin were mostly abolished by the antagonist of PPARγ. Our results reveal that phillyrin could prominently inhibit the inflammation of microglia via the PPARγ signaling pathway, thus leading to potential neuroprotective treatment after traumatic brain injury. Keywords: microglia; phillyrin; inflammation; traumatic brain injury; PPARγ.
Introduction Traumatic brain injury (TBI) is one of the major causes of death and disability worldwide [1]. The primary impact of TBI is direct neural cell loss following a wave of secondary injury cascades, including excitotoxicity, oxidative stress, blood–brain barrier disruption and inflammation [2-4]. Studies have confirmed that overactive microglia play important roles in the cause of secondary injuries both during the early and late phases of TBI [5, 6]. As the immune cells of the central nervous system, microglia can be activated by molecules such as damage-associated molecular patterns (DAMPs) around the damage lesions [7-9]. Afterwards, superfluous inflammatory mediators including interleukin (IL)-1β, tumor necrosis factor-α (TNFα), and IL-6 are released from the activated microglia; those mediators could not only directly induce neuronal apoptosis and brain edema but also cause the destruction of the blood-brain barrier (BBB), thus further exacerbating CNS injury [10-13]. Targeting the neuroinflammation mediated by microglia is thought to become an effective therapeutic strategy for TBI [14]. During the process of microglial activation, the toll-like receptors (TLRs), which are mainly expressed on the surfaces of microglia, respond rapidly to DAMPs. The downstream signaling pathways of TLRs, including
myeloid differentiation factor 88 (MyD88) and NF-κB, are subsequently activated, accompanied by the upregulation of genes related to inflammation [15, 16]. Additionally, targeting TLRs or their downstream signaling pathways has shown conspicuous effects on regulating microglia-induced neuroinflammation [17, 18]. Drugs such as rosiglitazone [19] and pioglitazone [20], which can act as potent anti-inflammatory agents in the suppression of pro-inflammatory cytokine production, have shown prominent roles in decreasing the neurological deficits caused by TBI. The inhibition of posttraumatic neuroinflammation has been an important approach in the treatment of TBI. Phillyrin (also called forsythin) is one of the main chemical constituents of Forsythia suspensa (Thunb.). In fact, as an important traditional Chinese medicine, F. suspensa (“Lianqiao” in Chinese) has historically been used for the treatment of influenza, inflammation, pyrexia and ulcers [21-24]. Several studies have found that phillyrin can also exert anti-inflammatory effects against neutrophils in an LPS-induced inflammation model [25]. Specifically, phillyrin also exhibits protective effects on the central nervous system. For instance, Wei T [26] found that phillyrin effectively inhibited H2O2-induced oxidative stress and apoptosis in PC12 cells. However, less is known about the effects of phillyrin on traumatic brain injury and posttraumatic neuroinflammation. In the present study, we attempted to study the anti-inflammatory effects of phillyrin on activated microglia as well as the neuroprotective effects in a mouse TBI model. Additionally, the potential mechanisms of phillyrin were also investigated.
Materials and methods Animals All animal experiments were approved by the Huazhong University of Science and Technology Committee for the Care of Animals. Adult male mice (C57BL/10ScNJ; 10-12 weeks old, 20–22 g) were obtained from Huazhong Keji Co., housed in a controlled environment (temperature: 22±3°C, under a 12 h light/dark cycle) and provided with a standard rodent diet and water. For the subsequent experiments, the mice were randomly divided into different groups. Controlled cortical impact (CCI) model of TBI Anesthesia was surgically induced with chloral hydrate (400 mg/kg body weight) administered intraperitoneally (i.p.). Surgical procedures and cortical contusions were performed under isoflurane anesthesia (2%) as previously described [17]. Each had 10 mice. Five mice in each group were sacrificed for neurological evaluation and histological studies and the remaining five mice were used for molecular studies. Drug treatment Phillyrin and GW9662 (Sigma-Aldrich, St Louis, MO, USA) were dissolved in DMSO and diluted with 0.9% saline. Then, equal amounts of the solvent, phillyrin (10 mg/kg) and combinations of phillyrin (10 mg/kg) and GW9662 (1 µmol/kg) were given by intraperitoneal injection immediately after the surgery was conducted and once
daily until the 7th day. The concentrations administered were based on previous studies [24, 25, 27 28]. Two researchers blinded to the group assignment performed the neurobehavioral tests, lesion size assessment, and cell counting. For the cells cultured on the plates, phillyrin (10-50 µg/ml) and GW9662(1 µM) were administered at the same time as the other stimulations and incubated for 4 h or 24 h before they were collected for research. Measurement of neurological deficits Modified neurological severity scores (mNSS) were used to evaluate the neurological deficits of mice at pre-injury, 3, 7 ,14 and 28 days after TBI as previously described [29]. The mNSS are determined by tests of motor function, gait, balance, sensation and reflex and are widely used to assess the neurological function of TBI mice. The neurological function was graded as scores of 0–18 (0, no neurological deficit; 18, maximal deficit). The rotarod protocol was modified slightly from that in the previous report [29]. Briefly, mice underwent a 2-day testing phase with rotarod (Unibiolab, Beijing, China), which gradually accelerated from 5 to 45 rpm over 5 min. During the procedure, the latency to fall was recorded as the time before rats fell off the rod or gripped around for two successive revolutions from day 3 after TBI. The assessment was conducted in duplicate and performed at different time points by investigators who were blinded to the treatment and groups. Morris water maze (MWM) test was performed as previously described [30] to detect learning latency and spatial memory. All mice were tested for 5 consecutive days (from the 21st to 24th day after TBI or sham treatment) before sacrifice. At the beginning of a trial, mice were placed randomly at one of the four fixed starting points and allowed to swim for 90 s or until it found the platform within 90 s.The platform was fixed in the NW quadrant 1 cm below the water level towards the middle of the pool. Each animal underwent 4 trials from different directions per day. On the last testing day, all animals underwent one probe trial, in which the platform was removed from the pool. The time latency, the time spent in correct quadrant, and the times traveling across platform were recorded by a computer (SLYWMS, Huaibeizhenghua, China). Assessment of cerebral edema Brain water content (BWC), a sensitive measure of cerebral edema, was quantified using the wet-dry method referred to [31] at 1, 3, and 7 days postinjury. BWC was estimated in 3 mm coronal sections of the ipsilateral cortex (or corresponding contralateral cortex), centered upon the impact site. Tissue was immediately weighed (wet weight) and then dehydrated at 65°C. The sample was reweighed 48 h later to obtain a dry weight. The percentage of water content in the tissue was calculated using the following formula: BWC = [(wet weight)-(dry weight)/wet weight] * 100%. Western blot analysis
Western blotting was performed to detect the expression of different proteins in the cultured cells or the traumatic penumbra area of the injured brain and a similar area from the sham group. Protein concentrations were determined by a BSA kit (Beyotime), and then the protein samples were diluted with 5x sample buffer solution. Next, the samples underwent electrophoresis in a 12% separation gel for 90 min and were blocked with 1x PBS and 5% (w/v) non-fat dried milk (PM) for 1 h at room temperature. The samples were subsequently probed with specific anti-iNOS (1:500, Abcam, UK), anti-IL-1β (1:1000, Abcam, UK), anti-phospho-STAT6 (1:1500, Abcam, UK), antiSTAT6 (1:1500, Abcam, UK), anti-phospho-NK-κB (1:1500, Abcam, UK), anti-NK-κB (1:2000, Abcam, UK), anti-PPAR-γ (1:1000, Abcam, UK) Abs in 1x PBS, 5% w/v non-fat dried milk, and 0.1% Tween-20 (PMT) at 4°C overnight. Membranes were incubated with fluorescently labeled secondary antibodies for 1.5 h at room temperature and then exposed and photographed on the Gene Gnome exposure instrument. Finally, the expression of the proteins was standardized for densitometric analysis to β-actin levels. Quantitative real-time PCR Total cellular RNA was isolated from microglia using TRIzol (Invitrogen, Carlsbad, CA, USA) before being washed with PBS and reverse-transcribed to cDNA with the PrimeScriptTM RT Reagent Kit (Thermo, USA) according to the datasheet from the manufacturer. The gene products of IL-1β, TNF-α, IL6 and iNOS were then amplified by quantitative real-time PCR on an ABI-Prism 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Premix Ex TaqTM II (Takara). All gene-specific PCR products were normalized with the internal standard GAPDH. The following primer sequences were used in the present study: IL-1β, forward, 5’-ggctcatctgggatcctctc-3’, reverse, 5’-tcatcttttggggtccgtca-3’; IL6, forward, 5’-ggagcccaccaagaacgata-3’, reverse, 5’-caggtctgttgggagtggta-3’; TNF-α, forward, 5’-ggattatggctcagggtcca-3’, reverse, 5’-acattcgaggctccagtgaa-3’; iNOS, forward, 5’-gtttgaccagaggacccaga-3’, reverse, 5’-gtgagctggtaggttcctgt-3’; and GAPDH, forward, 5’-aacgaccccttcattgacct-3’, reverse, 5’- atgttagtggggtctcgctc-3’. H&E and Nissl’s staining On the 3rd or 7th day after CCI, mice were deeply anesthetized and perfused transcardially with 0.1 PBS, pH 7.4, followed by 4% paraformaldehyde in PBS. The brains were then removed, post-fixed in 4% paraformaldehyde at 4 °C for 48 h, processed into paraffin blocks, and dissected into sections of 10 µm every 20 µm. Each section then was deparaffinized, hydrated, washed, and stained with Hematoxylin-eosin (H&E) staining. For Nissl’s staining, formaldehyde-fixed specimens were embedded in paraffin and cut into 4-µm-thick sections that were deparaffinized with xylene and rehydrated in a graded series of alcohol. Samples were treated with Nissl staining solution for 5 min. Damaged neurons were shrunken or contained vacuoles, whereas normal neurons had a relatively large, full soma and
round, large nuclei. Average intensities or cell counts were calculated from the same sections in five mice per group with Image J by investigators who were blinded to the experimental groups Immunofluorescence and immunohistochemical staining Mice were perfused with saline and paraformaldehyde 3 or 7 days after TBI. Coronal brain sections at 0–2.0 mm posterior to the bregma were obtained. Cryosections were permeabilized with Triton X-100 and blocked with donkey serum. Permeabilization and blocking were omitted for the IgG immunostaining. The sections were incubated with primary antibodies overnight at 4°C, including anti-Caspase3 (1:100, Abcam, Cambridge，UK), anti-p-NF-κB (1:100, Abcam, UK), anti-Iba-1 (1:200, Abcam, Cambridge，UK), or anti-PPARγ (1:100, Abcam, Cambridge，UK). Then, the sections were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Alexa488 or Alexa647 (1:200; Abcam) for 1 h at 37°C. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). After mounting, the immunofluorescence or immunohistochemistry signals were observed with an Olympus microscope (Olympus, Tokyo, Japan), and the positive cells were counted with the researcher blinded to the treatment conditions using Image J. Statistical analyses All data are expressed as the mean ± SD. For the in vitro experiments, we pooled the samples from three culture wells and repeated the experiments three times. We performed statistical analyses using the Graphpad programs. One-way ANOVA followed by Newman–Keuls post hoc test was used for multiple comparisons. A nonpaired t test was used when two groups were compared. Two-way ANOVA was used to compare the NDs between the three groups at different time points.
Results Phillyrin reduced the neurological deficits and brain edema of TBI Neurological deficits are frequent events in TBI. Moreover, brain edema acts as a vital pathophysiological process aggravating TBI [32, 33]. To investigate the neuroprotective effects of phillyrin, we determined the neurological behaviors (including sensorimotor function, spatial learning and memory ability and motor function) as well as brain edema of mice. The results revealed that mice in TBI had significant neurological deficits (higher mNSS score, more latency time of Morris water maze and less time of rotarod to fall) compared with Sham group. Phillyrin improved the neurological functions including sensorimotor function, spatial learning and memory ability and motor function (vs. TBI-veh group) with the lowest concentration of 5 mg/kg body weight, the stronger effects were seen at 10 mg/kg body weight (Fig. 1A-C). Additionally, we performed H&E staining and wet-dry method to detect the histopathology and brain edema of mice. The phillyrin attenuated the brain edema of mice on the 3rd and
7th days induced by TBI and the effects were more significant at a dose of 5 mg/kg and 10 mg/kg(Fig. 1D and E). Phillyrin reduced the apoptotic cells and neuronal loss of TBI lesions Increased apoptosis of neurons belongs to an important cause of TBI-mediated neurological impairment. To further explore the mechanism of Phi on neuroprotection, the apoptotic cells and neurons in the lesions were labeled by Nissl staining and immunohistochemistry, respectively. The results showed that TBI led to the significant augmentation of Nissl+ and Caspase3+ cells on the 7th day of TBI (Fig. 2A-D). While Phi reduced Nissl+ and Caspase3+ cells, moderate at 2.5 mg/kg body weight and achieving maximal effects at10 mg/kg body weight as depicted in Fig. 2 A-D. Phillyrin attenuated the inflammatory response of microglia after TBI Microglia become activated under the stimulation of the complex microenvironment in TBI brain lesions [34]. To verify the anti-inflammatory effects of phillyrin in vivo, the inflammatory responses of microglia on the insult of TBI were detected by RT-PCR, immunofluorescence and western blot analysis. As expected, TBI caused significant activation of microglia (Fig. 3A), expression proinflammatory factors (including IL-1β, IL-6, TNFα and iNOS) (Fig. 3 B and C) and proteins (including MyD88 and phospho-NK-κB) (Fig. 3D). Under the treatment of Phi, the number of microglia in the brain lesions as well as proinflammatory factors and proteins the IL-1β and iNOS proteins were all decreased, meanwhile, the effects were more obvious with the higher concentrations of Phi (5 mg/kg and 10 mg/kg) (Fig. 3A-D). Interestingly, PPARγ，as an important with anti-inflammatory effect, was also up-regulated by Phi with dose efffect(Fig. 3D). Inhibiton of PPARγ attenuated the anti-inflammatory and neuroprotecitve effects induced by phillyrin Signal transducer and activator of transcription 6(STAT6) is another crucial transcriptional meditator that has an anti-inflammatory function targeting via up-regulation of PPARγ[35-37]. To further confirm the role and mechanism of STAT6 and PPARγ in phillyrin-mediated anti-inflammation and neuroprotection, we used GW9662 to inhibit the effect of PPARγ in TBI mice and the expressions of STAT6 and PPARγ were determined by western blot. Our results revealed that administration of GW attenuated the expression of PPARγ but had no significant effect on the phosphorylation expression of STAT6. Additionally, TBI+GW group had more expression of proinflammatory factors(including IL-1β，IL-6, TNF-α and iNOS) and proteins (MyD88 and phosphorylation NF-κB) (p<0.05 vs.TBI-veh group). What’s more, expression of IL-1β, IL-6, TNFα, and iNOS mRNAs in the TBI+Phi+GW group were significantly increased compared with TBI+Phi group, which were the same with the protein levels of MyD88 and phosphorylated NF-κB as well as its nuclear translocation (Fig.4A-C). In addition, the expression and nuclear translocation of PPARγ in Iba1-labled microglia were both inhibited by GW9662 (Fig.4B and D). Next, we conducted experiments to test the neuroprotecitve effects of PPARγ induced by phillyrin. The results showed that compared with TBI+Phi group, mice in TBI+Phi + GW group had worse neurological functions, including higher
mNSS scores, more latency time of Morris water maze and less time of rotarod to fall(Fig. 5A-C). Meanwhile, inhibiton of PPARγ also reversed the effects of phillyrin in reducing brain edema and neuronal apoptosis (Fig. 5D-G).
Discussion Microglia are known to be widely involved in the neuroinflammatory response, which can drive ongoing neurological deficits following TBI even for many years after a single injury. Moreover, overactive microglia are also associated with lesion expansion and neurodegeneration [5, 38]. In this study, we found that phillyrin treatment not only improved functional recovery of mice after TBI but also alleviated brain edema, neuronal apoptosis and neuroinflammation (Fig. 6). As an important kind of traditional Chinese medicine, Forsythia suspensa (Thunb.) exerts a generalized anti-inflammatory effect in curing infectious or inflammatory diseases [21-24]. Phillyrin, as one of the main active ingredients extracted from Forsythia suspensa (Thunb.), has been found to show similar anti-inflammatory effects towards peripheral immune cells. However, less is known about the neuroprotective effects of phillyrin after TBI and the underlying mechanism of these effects. In this study, we firstly established a TBI model in mice and found that TBI caused obvious neurological injuris and histological tissue damage, including cell apoptosis and neuronal loss. In this study, mice subjected to TBI exhibited significant neurological impairment and neuronal apoptosis, which were partially attenuated by phillyrin. In addition, the development of cerebral edema is also central to the development of injury following brain trauma, which reaches its peak within the first week of injury [39]. Here, the results also showed that t phillyrin abated the brain edema of mice on the 3rd day and 7th day after injury. In the brain areas suffered from traumatic injury, microglia could not only release superfluous neuroinflammatory factors and aggravate the secondary injuries but could also establish a neuroprotective environment by mitigating the deleterious consequences of the injury [40]. However, in the subsequent “subacute” and “chronic” phases of TBI, the anti-inflammatory microglia are gradually replaced by the pro-inflammatory cells, thus exacerbating the cerebral pathology [41, 42]. As a result, therapeutics targeting the immune response caused by the overactive microglia reveal a promising future in curing TBI [43]. For instance, DHA administration improved short term cognitive function of rats by modulation of microglial activation toward a less inflammatory profile in the first week after CCI [44]. ω-3 PUFA supplementation inhibited TBI-induced microglial activation and the subsequent inflammatory response by regulating HMGB1/TLR4/NF-κB signaling pathway, thus leading to neuroprotective effects [45].In this study, we detected activated microglia and inflammatory responses on the 7th day of TBI. The results showed that the expression of inflammatory markers inlcuding IL-1β, IL-6, TNFα, and iNOS increased significantly in the brain lesions, which were the same with MyD88/ NF-κB signaling pathway. Phillyrin not only
inhibited the activation of microglia but also downregulated the expression of these inflammatory markers both in vitro and in vivo. Consequently, the neuroprotective function of phillyrin is potentially exerted through inhibiting microglial activation. The activation of the NF-κB signaling pathway is considered to be pivotal in the TBI-induced inflammatory response due to the secretion of pro-inflammatory factors [30]. Our study also showed that there was increased phosphorylation and nuclear translocation in microglia in response to the LPS and TBI insults, which was quite analogous with the changes induced in the expression of IL-1β, iNOS and TNFα. However, these effects were significantly inhibited by phillyrin. Moreover, many factors have been found to be involved in microglial inflammatory responses other than NK-κB. Some studies have demonstrated that PPARγ, a member of the nuclear hormone receptor superfamily, can exhibit potent anti-inflammatory properties on microglia/macrophages as well as neuroprotective effects in TBI, partly due to the inhibition of the phosphorylation and nuclear translocation of the p65 subunit of NF-κB[19, 46, 47]. In addition, STAT6 is a upstream transcription factor of PPARγ and its activation can directly lead to the upregulation of PPARγ[35]. Our detection of PPARγ and STAT6 revealed that phillyrin increased both of the phosphorylation expression of STAT6 and nuclear translocation of PPARγ, indicating that phillyrin might promote PPARγ expression via activating STAT6 pathway. To further confirm the effects of phillyrin on the PPARγ signaling pathway, GW9662, a PPARγ-specific antagonist [29], was administered in mice in vivo. The results showed that GW9662 attenuated the expression of PPARγ and its nuclear translocation in microglia. More importantly, the inhibition of the PPARγ signaling pathway decreased the anti-inflammatory effects of phillyrin on TBI-induced microglia and the neurological function of the TBI+Phi +GW9662 group was also more impaired than that of the TBI+Phi group (Fig. 5A), which was accompanied by an increase in brain edema, neuronal apoptosis.
Conclusion In summary, the major discovery of this study is that phillyrin exhibits anti-inflammatory effects by inhibiting the phosphorylation of NF-κB as well as inhibiting the release of its downstream pro-inflammatory factors from microglia after insult of TBI. This function showed potent neuroprotective effects in the TBI model. The activation of the STAT6/PPARγ signaling pathway by phillyrin was the potential mechanism involved in the described effect (Fig.6).
Abbreviations BBB: Blood-brain barrier; BWC: Brain water content; CCI: Controlled cortical impact; CCK-8: Cell counting kit-8; DAMP: Damage-associated molecular pattern; ELISA: Enzyme-linked immunosorbent assay; IL: Interleukin; iNOS: inducible nitric oxide synthase; LPS: Lipopolysaccharide; mNSS: Modified neurological severity scores; MyD88:
Myeloid differentiation factor 88; NF-κB: Nuclear factor-kappa B; PPARγ: Peroxisome proliferator–activated receptor gamma; STAT6: Signal transducer and activator of transcription 6; TBI: Traumatic brain injury; TLR: Toll-like receptor; TNF-α: Tumor necrosis factor-alpha.
Funding This study was supported by the National Natural Science Foundation of China (No. 81371381).
Availability of data and materials The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.
Consent for publication Not applicable.
Ethical approval The experimental protocols used in the present study, including all the surgical procedures and animal uses, were approved by the Huazhong University of Science and Technology Committee for the Care of Animals (Wuhan, China).
Competing interests The authors declare no competing interests.
Authors’ contributions QJ designed the study, performed the surgical operation, cultured the primary cells, completed the western blot analysis, performed data analysis and drafted the article. XBL, JC and YPY finished RT-PCR and immunofluorescence. XLY, XZ, GYH participated in the ELISA and in editing the article. HQZ conceived the study, participated in its design and edited the manuscript.
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Titles and legends of each figure
Figure 1. Phillyrin reduced the neurological deficits and brain edema of TBI A. Modified neurological severity score (mNSS) was determined prior injury and after TBI. B. Time latency spent in Morris Water Maze (MWM) test from the 21st to the 24th day after TBI. C. Rotarod test was conducted and the time on the rotarod to fall was calculated. D. Pathological changes of the brain lesions on the 3rd day after CCI were detected by hematoxylin and eosin staining. E. Brain water content on the 3rd and 7th day after TBI. The values are expressed as the mean ± SD. ***p < 0.001 vs. Sham group, &p < 0.05, &&p < 0.01, &&&p < 0.001 vs. TBI+veh group. n = 5/group.
Figure 2. Quantitative changes of apoptotic cells and neurons in the TBI brain lesions. A. Nissl’s staining was used to detect apoptotic neurons. Representative photomicrographs of Nissl-stained neurons are shown; arrows indicate apoptotic neurons. B. Immunohistochemical staining was used to detect apoptotic cells (labeled by caspase3) in the brain lesions. C and D. Quantitative data of the number of Nissl- or caspase3-positive cells per 0.1 mm2.The values are expressed as the mean ± SD: *** p < 0.001 vs. Sham group. &p < 0.05, &&&p < 0.001 vs. TBI+veh group. n = 5/group.
Figure. 3. Phillyrin inhibited the inflammatory response of TBI-activated microglia. A. Immunofluorescence was used to detect Iba1 + microglia in the brain lesions 7 days after TBI. Quantitative data of the number of Iba1+ microglia per 0.01 mm2 are shown. B. The Pro-inflammatory cytokines IL-1β, IL-6, TNFα, and iNOS mRNAs were detected by RT-PCR. C and D. Western blot was conducted to detect expression of proteins. The representative blots of cleaced IL-1β, iNOS, PPARγ， MyD88, NF-κB and the densitometric analyses of the brain lesions are shown. The values are expressed as the means ± SD: ***p < 0.001 vs.Sham group. &p < 0.05, &&p < 0.01, &&&p < 0.001 vs. TBI+veh group. n = 5/group.
Figure 4. Phillyrin inhibited the inflammatory response of TBI-activated microglia via the STAT6/PPARγ signaling pathway. The mice were treated with phillyrin (10 mg/kg body weight ) or GW9662 (1 µmol/kg body weight). A. The Pro-inflammatory cytokines IL-1β, IL-6, TNFα, and iNOS mRNAs were detected by RT-PCR. B. Protein levels of STAT6, PPARγ, MyD88, NF-κB were detected by western blot. C and D. Immunofluorescence was used to detect the expression and translocation of phospho-NK-κB p65 and PPAR-γ in Iba1-labeled microglia, respectively. The values are expressed as the mean ± SD, ***p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. TBI+veh group; &&p < 0.01 vs. TBI+Phi group. n = 5/group.
Figure 5. Phillyrin modulates the inflammatory response of TBI-activated microglia via the PPARγ signaling pathway. A. Modified neurological severity score (mNSS) was determined prior injury and after TBI. B. Time latency spent in Morris Water Maze (MWM) test from the 21st to the 24th day after TBI. C. Rotarod test was conducted and the time on the rotarod to fall was calculated. D. Brain water content on the 3rd and 7th day after TBI. E. Pathological changes of the brain lesions on the 3rd day after CCI were detected by hematoxylin and eosin staining. F. Nissl’s staining was used to detect apoptotic neurons. Representative photomicrographs of Nissl-stained neurons are shown; arrows indicate apoptotic neurons. The values are expressed as the mean ± SD:ns represents p>0.05, *p < 0.01 ** p < 0.01, ***p < 0.001. n = 5/group.
Figure 6. Schematic illustration of the possible neuroprotective mechanisms of phillyrin. As illustrated, microglia become activated into an inflammatory state and release many proinflammatory factors, such as TNF-α, IL-1β, and IL-6, over the course of TBI. At the same time, phospho-NK-κB p65 is upregulated and translocates into the nucleus. Phillyrin, which is extracted from the fruit of the medicinal plant F. suspensa (Thunb.), inhibits the nuclear translocation of phospho-NK-κB p65 via the PPARγ signaling pathway, thus exhibiting favorable anti-inflammatory properties in microglia as well as neuroprotective effects following experimental traumatic brain injury.
Highlights of this article: 1. Phillyrin attenuates TBI-mediated neurological deficits in mice. 2. Phillyrin attenuates TBI-mediated microglial neuroinflammation. 3. Phillyrin mediates microglia induced neuroinflammation through inhibiting NF-κB via activating PPARγ
S1367-9120(19)30545-0 https://doi.org/10.1016/j.jseaes.2019.104193 JAES 104193
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Please cite this article as: Wu, C., Zheng, W., Zhang, Z., Jia, Q., Yang, H., Large-earthquake rupturing and slipping behavior along the range-front Maidan fault in the southern Tian Shan, Northwestern China, Journal of Asian Earth Sciences (2019), doi: https://doi.org/10.1016/j.jseaes.2019.104193
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Phillyrin protects mice from traumatic brain injury by inhibiting the inflammation of microglia via PPARγ signaling pathway Running title:traumatic brain injury and phillyrin Qian Jiang, Jun Chen , Xiaobing Long, Xiaolong Yao, Xin Zou , Yiping Yang , Guangying Huang , Huaqiu Zhang* 1) Qian Jiang; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected] 2) Jun Chen; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail:[email protected] 3) Xiaobing Long; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail:[email protected] 4) Xiaolong Yao; 1Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China, 2Department of Neurosurgery, Taikang Tongji Hospital, Wuhan, 430050, PR China;E-mail: [email protected] 5) Xin Zou; Department of Traditional Chinese Medicine, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected] 6) Yiping Yang; Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail:[email protected] 7) Guangying Huang; Department of Traditional Chinese Medicine, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected] 8) Huaqiu Zhang; corresponding author, Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, 430030, PR China; E-mail: [email protected]
Abstract The neuroinflammatory response induced by microglia plays a vital role in causing secondary brain damage after traumatic brain injury (TBI). Previous studies have found that the improved regulation of activated microglia could reduce neurological damage post-TBI. Phillyrin (Phi) is one of the main active ingredients extracted from the fruits of the medicinal plant Forsythia suspensa (Thunb.) with anti-inflammatory effects. Our study attempted to investigate the effects of phillyrin on microglial activation and neuron damage after TBI. The TBI model was applied to induce brain injury in mice, and neurological scores, brain water content, hematoxylin and eosin staining and Nissl staining were employed to determine the neuroprotective effects of phillyrin. Immunofluorescent staining and western blot analysis were used to detect nuclear factor-kappa B (NF-κB) and peroxisome proliferator–activated receptor gamma (PPARγ) expression and nuclear translocation, and the inflammation-related proteins and mRNAs were assessed by western blot analysis and quantitative real-time PCR. The results revealed that phillyrin not only inhibited the proinflammatory response induced by activated microglia but also attenuated neurological impairment and brain edema in vivo in a mouse TBI model. Additionally, phillyrin suppressed the phosphorylation of NF-κB in microglia after TBI insult. These effects of phillyrin were mostly abolished by the antagonist of PPARγ. Our results reveal that phillyrin could prominently inhibit the inflammation of microglia via the PPARγ signaling pathway, thus leading to potential neuroprotective treatment after traumatic brain injury. Keywords: microglia; phillyrin; inflammation; traumatic brain injury; PPARγ.
Introduction Traumatic brain injury (TBI) is one of the major causes of death and disability worldwide [1]. The primary impact of TBI is direct neural cell loss following a wave of secondary injury cascades, including excitotoxicity, oxidative stress, blood–brain barrier disruption and inflammation [2-4]. Studies have confirmed that overactive microglia play important roles in the cause of secondary injuries both during the early and late phases of TBI [5, 6]. As the immune cells of the central nervous system, microglia can be activated by molecules such as damage-associated molecular patterns (DAMPs) around the damage lesions [7-9]. Afterwards, superfluous inflammatory mediators including interleukin (IL)-1β, tumor necrosis factor-α (TNFα), and IL-6 are released from the activated microglia; those mediators could not only directly induce neuronal apoptosis and brain edema but also cause the destruction of the blood-brain barrier (BBB), thus further exacerbating CNS injury [10-13]. Targeting the neuroinflammation mediated by microglia is thought to become an effective therapeutic strategy for TBI [14]. During the process of microglial activation, the toll-like receptors (TLRs), which are mainly expressed on the surfaces of microglia, respond rapidly to DAMPs. The downstream signaling pathways of TLRs, including
myeloid differentiation factor 88 (MyD88) and NF-κB, are subsequently activated, accompanied by the upregulation of genes related to inflammation [15, 16]. Additionally, targeting TLRs or their downstream signaling pathways has shown conspicuous effects on regulating microglia-induced neuroinflammation [17, 18]. Drugs such as rosiglitazone [19] and pioglitazone [20], which can act as potent anti-inflammatory agents in the suppression of pro-inflammatory cytokine production, have shown prominent roles in decreasing the neurological deficits caused by TBI. The inhibition of posttraumatic neuroinflammation has been an important approach in the treatment of TBI. Phillyrin (also called forsythin) is one of the main chemical constituents of Forsythia suspensa (Thunb.). In fact, as an important traditional Chinese medicine, F. suspensa (“Lianqiao” in Chinese) has historically been used for the treatment of influenza, inflammation, pyrexia and ulcers [21-24]. Several studies have found that phillyrin can also exert anti-inflammatory effects against neutrophils in an LPS-induced inflammation model [25]. Specifically, phillyrin also exhibits protective effects on the central nervous system. For instance, Wei T [26] found that phillyrin effectively inhibited H2O2-induced oxidative stress and apoptosis in PC12 cells. However, less is known about the effects of phillyrin on traumatic brain injury and posttraumatic neuroinflammation. In the present study, we attempted to study the anti-inflammatory effects of phillyrin on activated microglia as well as the neuroprotective effects in a mouse TBI model. Additionally, the potential mechanisms of phillyrin were also investigated.
Materials and methods Animals All animal experiments were approved by the Huazhong University of Science and Technology Committee for the Care of Animals. Adult male mice (C57BL/10ScNJ; 10-12 weeks old, 20–22 g) were obtained from Huazhong Keji Co., housed in a controlled environment (temperature: 22±3°C, under a 12 h light/dark cycle) and provided with a standard rodent diet and water. For the subsequent experiments, the mice were randomly divided into different groups. Controlled cortical impact (CCI) model of TBI Anesthesia was surgically induced with chloral hydrate (400 mg/kg body weight) administered intraperitoneally (i.p.). Surgical procedures and cortical contusions were performed under isoflurane anesthesia (2%) as previously described [17]. Each had 10 mice. Five mice in each group were sacrificed for neurological evaluation and histological studies and the remaining five mice were used for molecular studies. Drug treatment Phillyrin and GW9662 (Sigma-Aldrich, St Louis, MO, USA) were dissolved in DMSO and diluted with 0.9% saline. Then, equal amounts of the solvent, phillyrin (10 mg/kg) and combinations of phillyrin (10 mg/kg) and GW9662 (1 µmol/kg) were given by intraperitoneal injection immediately after the surgery was conducted and once
daily until the 7th day. The concentrations administered were based on previous studies [24, 25, 27 28]. Two researchers blinded to the group assignment performed the neurobehavioral tests, lesion size assessment, and cell counting. For the cells cultured on the plates, phillyrin (10-50 µg/ml) and GW9662(1 µM) were administered at the same time as the other stimulations and incubated for 4 h or 24 h before they were collected for research. Measurement of neurological deficits Modified neurological severity scores (mNSS) were used to evaluate the neurological deficits of mice at pre-injury, 3, 7 ,14 and 28 days after TBI as previously described [29]. The mNSS are determined by tests of motor function, gait, balance, sensation and reflex and are widely used to assess the neurological function of TBI mice. The neurological function was graded as scores of 0–18 (0, no neurological deficit; 18, maximal deficit). The rotarod protocol was modified slightly from that in the previous report [29]. Briefly, mice underwent a 2-day testing phase with rotarod (Unibiolab, Beijing, China), which gradually accelerated from 5 to 45 rpm over 5 min. During the procedure, the latency to fall was recorded as the time before rats fell off the rod or gripped around for two successive revolutions from day 3 after TBI. The assessment was conducted in duplicate and performed at different time points by investigators who were blinded to the treatment and groups. Morris water maze (MWM) test was performed as previously described [30] to detect learning latency and spatial memory. All mice were tested for 5 consecutive days (from the 21st to 24th day after TBI or sham treatment) before sacrifice. At the beginning of a trial, mice were placed randomly at one of the four fixed starting points and allowed to swim for 90 s or until it found the platform within 90 s.The platform was fixed in the NW quadrant 1 cm below the water level towards the middle of the pool. Each animal underwent 4 trials from different directions per day. On the last testing day, all animals underwent one probe trial, in which the platform was removed from the pool. The time latency, the time spent in correct quadrant, and the times traveling across platform were recorded by a computer (SLYWMS, Huaibeizhenghua, China). Assessment of cerebral edema Brain water content (BWC), a sensitive measure of cerebral edema, was quantified using the wet-dry method referred to [31] at 1, 3, and 7 days postinjury. BWC was estimated in 3 mm coronal sections of the ipsilateral cortex (or corresponding contralateral cortex), centered upon the impact site. Tissue was immediately weighed (wet weight) and then dehydrated at 65°C. The sample was reweighed 48 h later to obtain a dry weight. The percentage of water content in the tissue was calculated using the following formula: BWC = [(wet weight)-(dry weight)/wet weight] * 100%. Western blot analysis
Western blotting was performed to detect the expression of different proteins in the cultured cells or the traumatic penumbra area of the injured brain and a similar area from the sham group. Protein concentrations were determined by a BSA kit (Beyotime), and then the protein samples were diluted with 5x sample buffer solution. Next, the samples underwent electrophoresis in a 12% separation gel for 90 min and were blocked with 1x PBS and 5% (w/v) non-fat dried milk (PM) for 1 h at room temperature. The samples were subsequently probed with specific anti-iNOS (1:500, Abcam, UK), anti-IL-1β (1:1000, Abcam, UK), anti-phospho-STAT6 (1:1500, Abcam, UK), antiSTAT6 (1:1500, Abcam, UK), anti-phospho-NK-κB (1:1500, Abcam, UK), anti-NK-κB (1:2000, Abcam, UK), anti-PPAR-γ (1:1000, Abcam, UK) Abs in 1x PBS, 5% w/v non-fat dried milk, and 0.1% Tween-20 (PMT) at 4°C overnight. Membranes were incubated with fluorescently labeled secondary antibodies for 1.5 h at room temperature and then exposed and photographed on the Gene Gnome exposure instrument. Finally, the expression of the proteins was standardized for densitometric analysis to β-actin levels. Quantitative real-time PCR Total cellular RNA was isolated from microglia using TRIzol (Invitrogen, Carlsbad, CA, USA) before being washed with PBS and reverse-transcribed to cDNA with the PrimeScriptTM RT Reagent Kit (Thermo, USA) according to the datasheet from the manufacturer. The gene products of IL-1β, TNF-α, IL6 and iNOS were then amplified by quantitative real-time PCR on an ABI-Prism 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Premix Ex TaqTM II (Takara). All gene-specific PCR products were normalized with the internal standard GAPDH. The following primer sequences were used in the present study: IL-1β, forward, 5’-ggctcatctgggatcctctc-3’, reverse, 5’-tcatcttttggggtccgtca-3’; IL6, forward, 5’-ggagcccaccaagaacgata-3’, reverse, 5’-caggtctgttgggagtggta-3’; TNF-α, forward, 5’-ggattatggctcagggtcca-3’, reverse, 5’-acattcgaggctccagtgaa-3’; iNOS, forward, 5’-gtttgaccagaggacccaga-3’, reverse, 5’-gtgagctggtaggttcctgt-3’; and GAPDH, forward, 5’-aacgaccccttcattgacct-3’, reverse, 5’- atgttagtggggtctcgctc-3’. H&E and Nissl’s staining On the 3rd or 7th day after CCI, mice were deeply anesthetized and perfused transcardially with 0.1 PBS, pH 7.4, followed by 4% paraformaldehyde in PBS. The brains were then removed, post-fixed in 4% paraformaldehyde at 4 °C for 48 h, processed into paraffin blocks, and dissected into sections of 10 µm every 20 µm. Each section then was deparaffinized, hydrated, washed, and stained with Hematoxylin-eosin (H&E) staining. For Nissl’s staining, formaldehyde-fixed specimens were embedded in paraffin and cut into 4-µm-thick sections that were deparaffinized with xylene and rehydrated in a graded series of alcohol. Samples were treated with Nissl staining solution for 5 min. Damaged neurons were shrunken or contained vacuoles, whereas normal neurons had a relatively large, full soma and
round, large nuclei. Average intensities or cell counts were calculated from the same sections in five mice per group with Image J by investigators who were blinded to the experimental groups Immunofluorescence and immunohistochemical staining Mice were perfused with saline and paraformaldehyde 3 or 7 days after TBI. Coronal brain sections at 0–2.0 mm posterior to the bregma were obtained. Cryosections were permeabilized with Triton X-100 and blocked with donkey serum. Permeabilization and blocking were omitted for the IgG immunostaining. The sections were incubated with primary antibodies overnight at 4°C, including anti-Caspase3 (1:100, Abcam, Cambridge，UK), anti-p-NF-κB (1:100, Abcam, UK), anti-Iba-1 (1:200, Abcam, Cambridge，UK), or anti-PPARγ (1:100, Abcam, Cambridge，UK). Then, the sections were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Alexa488 or Alexa647 (1:200; Abcam) for 1 h at 37°C. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). After mounting, the immunofluorescence or immunohistochemistry signals were observed with an Olympus microscope (Olympus, Tokyo, Japan), and the positive cells were counted with the researcher blinded to the treatment conditions using Image J. Statistical analyses All data are expressed as the mean ± SD. For the in vitro experiments, we pooled the samples from three culture wells and repeated the experiments three times. We performed statistical analyses using the Graphpad programs. One-way ANOVA followed by Newman–Keuls post hoc test was used for multiple comparisons. A nonpaired t test was used when two groups were compared. Two-way ANOVA was used to compare the NDs between the three groups at different time points.
Results Phillyrin reduced the neurological deficits and brain edema of TBI Neurological deficits are frequent events in TBI. Moreover, brain edema acts as a vital pathophysiological process aggravating TBI [32, 33]. To investigate the neuroprotective effects of phillyrin, we determined the neurological behaviors (including sensorimotor function, spatial learning and memory ability and motor function) as well as brain edema of mice. The results revealed that mice in TBI had significant neurological deficits (higher mNSS score, more latency time of Morris water maze and less time of rotarod to fall) compared with Sham group. Phillyrin improved the neurological functions including sensorimotor function, spatial learning and memory ability and motor function (vs. TBI-veh group) with the lowest concentration of 5 mg/kg body weight, the stronger effects were seen at 10 mg/kg body weight (Fig. 1A-C). Additionally, we performed H&E staining and wet-dry method to detect the histopathology and brain edema of mice. The phillyrin attenuated the brain edema of mice on the 3rd and
7th days induced by TBI and the effects were more significant at a dose of 5 mg/kg and 10 mg/kg(Fig. 1D and E). Phillyrin reduced the apoptotic cells and neuronal loss of TBI lesions Increased apoptosis of neurons belongs to an important cause of TBI-mediated neurological impairment. To further explore the mechanism of Phi on neuroprotection, the apoptotic cells and neurons in the lesions were labeled by Nissl staining and immunohistochemistry, respectively. The results showed that TBI led to the significant augmentation of Nissl+ and Caspase3+ cells on the 7th day of TBI (Fig. 2A-D). While Phi reduced Nissl+ and Caspase3+ cells, moderate at 2.5 mg/kg body weight and achieving maximal effects at10 mg/kg body weight as depicted in Fig. 2 A-D. Phillyrin attenuated the inflammatory response of microglia after TBI Microglia become activated under the stimulation of the complex microenvironment in TBI brain lesions [34]. To verify the anti-inflammatory effects of phillyrin in vivo, the inflammatory responses of microglia on the insult of TBI were detected by RT-PCR, immunofluorescence and western blot analysis. As expected, TBI caused significant activation of microglia (Fig. 3A), expression proinflammatory factors (including IL-1β, IL-6, TNFα and iNOS) (Fig. 3 B and C) and proteins (including MyD88 and phospho-NK-κB) (Fig. 3D). Under the treatment of Phi, the number of microglia in the brain lesions as well as proinflammatory factors and proteins the IL-1β and iNOS proteins were all decreased, meanwhile, the effects were more obvious with the higher concentrations of Phi (5 mg/kg and 10 mg/kg) (Fig. 3A-D). Interestingly, PPARγ，as an important with anti-inflammatory effect, was also up-regulated by Phi with dose efffect(Fig. 3D). Inhibiton of PPARγ attenuated the anti-inflammatory and neuroprotecitve effects induced by phillyrin Signal transducer and activator of transcription 6(STAT6) is another crucial transcriptional meditator that has an anti-inflammatory function targeting via up-regulation of PPARγ[35-37]. To further confirm the role and mechanism of STAT6 and PPARγ in phillyrin-mediated anti-inflammation and neuroprotection, we used GW9662 to inhibit the effect of PPARγ in TBI mice and the expressions of STAT6 and PPARγ were determined by western blot. Our results revealed that administration of GW attenuated the expression of PPARγ but had no significant effect on the phosphorylation expression of STAT6. Additionally, TBI+GW group had more expression of proinflammatory factors(including IL-1β，IL-6, TNF-α and iNOS) and proteins (MyD88 and phosphorylation NF-κB) (p<0.05 vs.TBI-veh group). What’s more, expression of IL-1β, IL-6, TNFα, and iNOS mRNAs in the TBI+Phi+GW group were significantly increased compared with TBI+Phi group, which were the same with the protein levels of MyD88 and phosphorylated NF-κB as well as its nuclear translocation (Fig.4A-C). In addition, the expression and nuclear translocation of PPARγ in Iba1-labled microglia were both inhibited by GW9662 (Fig.4B and D). Next, we conducted experiments to test the neuroprotecitve effects of PPARγ induced by phillyrin. The results showed that compared with TBI+Phi group, mice in TBI+Phi + GW group had worse neurological functions, including higher
mNSS scores, more latency time of Morris water maze and less time of rotarod to fall(Fig. 5A-C). Meanwhile, inhibiton of PPARγ also reversed the effects of phillyrin in reducing brain edema and neuronal apoptosis (Fig. 5D-G).
Discussion Microglia are known to be widely involved in the neuroinflammatory response, which can drive ongoing neurological deficits following TBI even for many years after a single injury. Moreover, overactive microglia are also associated with lesion expansion and neurodegeneration [5, 38]. In this study, we found that phillyrin treatment not only improved functional recovery of mice after TBI but also alleviated brain edema, neuronal apoptosis and neuroinflammation (Fig. 6). As an important kind of traditional Chinese medicine, Forsythia suspensa (Thunb.) exerts a generalized anti-inflammatory effect in curing infectious or inflammatory diseases [21-24]. Phillyrin, as one of the main active ingredients extracted from Forsythia suspensa (Thunb.), has been found to show similar anti-inflammatory effects towards peripheral immune cells. However, less is known about the neuroprotective effects of phillyrin after TBI and the underlying mechanism of these effects. In this study, we firstly established a TBI model in mice and found that TBI caused obvious neurological injuris and histological tissue damage, including cell apoptosis and neuronal loss. In this study, mice subjected to TBI exhibited significant neurological impairment and neuronal apoptosis, which were partially attenuated by phillyrin. In addition, the development of cerebral edema is also central to the development of injury following brain trauma, which reaches its peak within the first week of injury [39]. Here, the results also showed that t phillyrin abated the brain edema of mice on the 3rd day and 7th day after injury. In the brain areas suffered from traumatic injury, microglia could not only release superfluous neuroinflammatory factors and aggravate the secondary injuries but could also establish a neuroprotective environment by mitigating the deleterious consequences of the injury [40]. However, in the subsequent “subacute” and “chronic” phases of TBI, the anti-inflammatory microglia are gradually replaced by the pro-inflammatory cells, thus exacerbating the cerebral pathology [41, 42]. As a result, therapeutics targeting the immune response caused by the overactive microglia reveal a promising future in curing TBI [43]. For instance, DHA administration improved short term cognitive function of rats by modulation of microglial activation toward a less inflammatory profile in the first week after CCI [44]. ω-3 PUFA supplementation inhibited TBI-induced microglial activation and the subsequent inflammatory response by regulating HMGB1/TLR4/NF-κB signaling pathway, thus leading to neuroprotective effects [45].In this study, we detected activated microglia and inflammatory responses on the 7th day of TBI. The results showed that the expression of inflammatory markers inlcuding IL-1β, IL-6, TNFα, and iNOS increased significantly in the brain lesions, which were the same with MyD88/ NF-κB signaling pathway. Phillyrin not only
inhibited the activation of microglia but also downregulated the expression of these inflammatory markers both in vitro and in vivo. Consequently, the neuroprotective function of phillyrin is potentially exerted through inhibiting microglial activation. The activation of the NF-κB signaling pathway is considered to be pivotal in the TBI-induced inflammatory response due to the secretion of pro-inflammatory factors [30]. Our study also showed that there was increased phosphorylation and nuclear translocation in microglia in response to the LPS and TBI insults, which was quite analogous with the changes induced in the expression of IL-1β, iNOS and TNFα. However, these effects were significantly inhibited by phillyrin. Moreover, many factors have been found to be involved in microglial inflammatory responses other than NK-κB. Some studies have demonstrated that PPARγ, a member of the nuclear hormone receptor superfamily, can exhibit potent anti-inflammatory properties on microglia/macrophages as well as neuroprotective effects in TBI, partly due to the inhibition of the phosphorylation and nuclear translocation of the p65 subunit of NF-κB[19, 46, 47]. In addition, STAT6 is a upstream transcription factor of PPARγ and its activation can directly lead to the upregulation of PPARγ[35]. Our detection of PPARγ and STAT6 revealed that phillyrin increased both of the phosphorylation expression of STAT6 and nuclear translocation of PPARγ, indicating that phillyrin might promote PPARγ expression via activating STAT6 pathway. To further confirm the effects of phillyrin on the PPARγ signaling pathway, GW9662, a PPARγ-specific antagonist [29], was administered in mice in vivo. The results showed that GW9662 attenuated the expression of PPARγ and its nuclear translocation in microglia. More importantly, the inhibition of the PPARγ signaling pathway decreased the anti-inflammatory effects of phillyrin on TBI-induced microglia and the neurological function of the TBI+Phi +GW9662 group was also more impaired than that of the TBI+Phi group (Fig. 5A), which was accompanied by an increase in brain edema, neuronal apoptosis.
Conclusion In summary, the major discovery of this study is that phillyrin exhibits anti-inflammatory effects by inhibiting the phosphorylation of NF-κB as well as inhibiting the release of its downstream pro-inflammatory factors from microglia after insult of TBI. This function showed potent neuroprotective effects in the TBI model. The activation of the STAT6/PPARγ signaling pathway by phillyrin was the potential mechanism involved in the described effect (Fig.6).
Abbreviations BBB: Blood-brain barrier; BWC: Brain water content; CCI: Controlled cortical impact; CCK-8: Cell counting kit-8; DAMP: Damage-associated molecular pattern; ELISA: Enzyme-linked immunosorbent assay; IL: Interleukin; iNOS: inducible nitric oxide synthase; LPS: Lipopolysaccharide; mNSS: Modified neurological severity scores; MyD88:
Myeloid differentiation factor 88; NF-κB: Nuclear factor-kappa B; PPARγ: Peroxisome proliferator–activated receptor gamma; STAT6: Signal transducer and activator of transcription 6; TBI: Traumatic brain injury; TLR: Toll-like receptor; TNF-α: Tumor necrosis factor-alpha.
Funding This study was supported by the National Natural Science Foundation of China (No. 81371381).
Availability of data and materials The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.
Consent for publication Not applicable.
Ethical approval The experimental protocols used in the present study, including all the surgical procedures and animal uses, were approved by the Huazhong University of Science and Technology Committee for the Care of Animals (Wuhan, China).
Competing interests The authors declare no competing interests.
Authors’ contributions QJ designed the study, performed the surgical operation, cultured the primary cells, completed the western blot analysis, performed data analysis and drafted the article. XBL, JC and YPY finished RT-PCR and immunofluorescence. XLY, XZ, GYH participated in the ELISA and in editing the article. HQZ conceived the study, participated in its design and edited the manuscript.
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Titles and legends of each figure
Figure 1. Phillyrin reduced the neurological deficits and brain edema of TBI A. Modified neurological severity score (mNSS) was determined prior injury and after TBI. B. Time latency spent in Morris Water Maze (MWM) test from the 21st to the 24th day after TBI. C. Rotarod test was conducted and the time on the rotarod to fall was calculated. D. Pathological changes of the brain lesions on the 3rd day after CCI were detected by hematoxylin and eosin staining. E. Brain water content on the 3rd and 7th day after TBI. The values are expressed as the mean ± SD. ***p < 0.001 vs. Sham group, &p < 0.05, &&p < 0.01, &&&p < 0.001 vs. TBI+veh group. n = 5/group.
Figure 2. Quantitative changes of apoptotic cells and neurons in the TBI brain lesions. A. Nissl’s staining was used to detect apoptotic neurons. Representative photomicrographs of Nissl-stained neurons are shown; arrows indicate apoptotic neurons. B. Immunohistochemical staining was used to detect apoptotic cells (labeled by caspase3) in the brain lesions. C and D. Quantitative data of the number of Nissl- or caspase3-positive cells per 0.1 mm2.The values are expressed as the mean ± SD: *** p < 0.001 vs. Sham group. &p < 0.05, &&&p < 0.001 vs. TBI+veh group. n = 5/group.
Figure. 3. Phillyrin inhibited the inflammatory response of TBI-activated microglia. A. Immunofluorescence was used to detect Iba1 + microglia in the brain lesions 7 days after TBI. Quantitative data of the number of Iba1+ microglia per 0.01 mm2 are shown. B. The Pro-inflammatory cytokines IL-1β, IL-6, TNFα, and iNOS mRNAs were detected by RT-PCR. C and D. Western blot was conducted to detect expression of proteins. The representative blots of cleaced IL-1β, iNOS, PPARγ， MyD88, NF-κB and the densitometric analyses of the brain lesions are shown. The values are expressed as the means ± SD: ***p < 0.001 vs.Sham group. &p < 0.05, &&p < 0.01, &&&p < 0.001 vs. TBI+veh group. n = 5/group.
Figure 4. Phillyrin inhibited the inflammatory response of TBI-activated microglia via the STAT6/PPARγ signaling pathway. The mice were treated with phillyrin (10 mg/kg body weight ) or GW9662 (1 µmol/kg body weight). A. The Pro-inflammatory cytokines IL-1β, IL-6, TNFα, and iNOS mRNAs were detected by RT-PCR. B. Protein levels of STAT6, PPARγ, MyD88, NF-κB were detected by western blot. C and D. Immunofluorescence was used to detect the expression and translocation of phospho-NK-κB p65 and PPAR-γ in Iba1-labeled microglia, respectively. The values are expressed as the mean ± SD, ***p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. TBI+veh group; &&p < 0.01 vs. TBI+Phi group. n = 5/group.
Figure 5. Phillyrin modulates the inflammatory response of TBI-activated microglia via the PPARγ signaling pathway. A. Modified neurological severity score (mNSS) was determined prior injury and after TBI. B. Time latency spent in Morris Water Maze (MWM) test from the 21st to the 24th day after TBI. C. Rotarod test was conducted and the time on the rotarod to fall was calculated. D. Brain water content on the 3rd and 7th day after TBI. E. Pathological changes of the brain lesions on the 3rd day after CCI were detected by hematoxylin and eosin staining. F. Nissl’s staining was used to detect apoptotic neurons. Representative photomicrographs of Nissl-stained neurons are shown; arrows indicate apoptotic neurons. The values are expressed as the mean ± SD:ns represents p>0.05, *p < 0.01 ** p < 0.01, ***p < 0.001. n = 5/group.
Figure 6. Schematic illustration of the possible neuroprotective mechanisms of phillyrin. As illustrated, microglia become activated into an inflammatory state and release many proinflammatory factors, such as TNF-α, IL-1β, and IL-6, over the course of TBI. At the same time, phospho-NK-κB p65 is upregulated and translocates into the nucleus. Phillyrin, which is extracted from the fruit of the medicinal plant F. suspensa (Thunb.), inhibits the nuclear translocation of phospho-NK-κB p65 via the PPARγ signaling pathway, thus exhibiting favorable anti-inflammatory properties in microglia as well as neuroprotective effects following experimental traumatic brain injury.
Highlights of this article: 1. Phillyrin attenuates TBI-mediated neurological deficits in mice. 2. Phillyrin attenuates TBI-mediated microglial neuroinflammation. 3. Phillyrin mediates microglia induced neuroinflammation through inhibiting NF-κB via activating PPARγ