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HO-1 signaling pathways
International Immunopharmacology 45 (2017) 148–155
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effects of morin on lipopolysaccharide/ D-galactosamine-induced acute liver injury by inhibiting TLR4/NF-κB and activating Nrf2/HO-1 signaling pathways Ye Tian 1, Zheng Li 1, Bingyu Shen, Qiaoling Zhang, Haihua Feng ⁎ a
Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China
a r t i c l e
i n f o
Article history: Received 27 September 2016 Received in revised form 4 February 2017 Accepted 7 February 2017 Available online xxxx Keywords: LPS/D-GalN Morin Nrf2 NF-κB
a b s t r a c t Morin, a bioactive flavonoid extracted from the bark of Moraceae plants and many medicinal herbs, has anti-inflammatory and antioxidative effects. In this research, we explored the protective effects of morin against lipopolysaccharide (LPS) and D-galactosamine (D-GalN) induced acute liver injury in mice. Mice were given an intraperitoneal injection of morin before LPS and D-GalN treatment and the HepG2 cells were only given morin to investigate its effects. The results showed that morin markedly inhibited the production of serum alanine transaminase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), tumor necrosis factor (TNFα) and hepatic TNF-α, IL-6, and myeloperoxidase (MPO) induced by LPS/D-GalN. In order to evaluate morin effect in the future, we investigated the expression of nuclear factor E2 related factor 2 (Nrf2), nuclear factorkappaB (NF-κB), toll like receptor 4 (TLR4) on liver injury. Taken together, these results suggested that morin could exert the anti-inflammatory and anti-oxidative effects against LPS/D-GalN-induced acute liver injury by activating Nrf2 signal pathways and inhibiting NF-κB activation. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The occurrence of liver injury is often accompanied by endotoxemia and sepsis and can cause high mortality in animals and human beings due to the lack of effective drugs [1,2]. Therefore, finding an effective drug to treat acute liver injury is in desperate need. LPS and D-GalN-induced acute liver injury in mice model is widely used in human liver damage [3,4]. The mice model has been recognized as a potential treatment of drug research method [5]. D-GalN is a kind of hepatotoxic agent and can cause hepatotoxicity in liver mainly through inhibiting hepatocyte RNA and protein synthesis [6]. LPS is a kind of endotoxin that could activate inflammatory cytokines, which can bring about liver tissue injury [7,8]. Nuclear factor E2 related factor 2 is an important regulatory factor that regulates cellular defense against oxidative stress. It can protect against oxidative damage triggered by liver injury and inflammation by regulating the expression of antioxidant proteins. Nuclear factor κB, a nuclear transcription factor, plays a great role in the regulation of inflammation. The activation of NF-κB is considered to respond to the ⁎ Corresponding author. E-mail address: [email protected] (H. Feng). 1 The authors Ye Tian, Zheng Li, contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2017.02.010 1567-5769/© 2017 Elsevier B.V. All rights reserved.
oxidative stress. Recent study revealed that LPS/D-GalN was capable of activating NF-κB and Nrf2 signaling [9]. However, the molecular basis of morin mitigating the LPS/D-GalN-induced liver damage by induction of Nrf2 remains elusive. This led us to reason whether morin can activate Nrf2 and inhibit NF-κB signaling, which exert protective effect against LPS/D-GalN-mediated oxidant damage. If the above hypothesis is feasible, our results will show that morin may be a potential drug target to mitigate liver damage via inhibition of NF-κB and up-regulation of Nrf2 signal pathways. Morin (20, 3, 40, 5, 7-pentahydroxyflavone), a kind of flavonol, is extracted from herbal medicine which has anti-oxidant [10], anti-hyperglycemic [11] and liver-protective effects [12]. Previous studies showed that morin could inhibit the Fyn kinase, an enzyme linked to Nrf2 degradation [13,14] and influence LPS-BV2 cells by suppressing NF-κB activity and activating HO-1 induction [15]. In addition, morin was also found to inhibit LPS-induced acute lung injury in mice [16]. However, the effects of morin on LPS/D-GalN-induced liver injury remain unclear. In this study, we evaluated whether morin played a positive role in protecting liver tissue against LPS/D-GalN-induced acute liver injury. We demonstrated, for the first time, that morin could show functional and morphological protection against LPS/D-GalN-induced liver injury by alleviating inflammatory and oxidative stress.
Y. Tian et al. / International Immunopharmacology 45 (2017) 148–155
2. Materials and methods 2.1. Materials Morin (purity ≥98%) was purchased from Chengdu Reference Products (Chengdu, China). Dimethylsulfoxide (DMSO), LPS (Escherichia coli lipopolysaccharide, 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CCK8 was purchased from Beyotime Biotechnology (Beijing. China). Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM), penicillin and streptomycin were purchased from Invitrogen-Gibco (Grand Island, NY). We purchased D-galactosamine hydrochloride from Aladdin Industrial Corporation (Shanghai, China). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits were provided by Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Mouse TNF-α, and IL-6 enzymelinked immunosorbent assay (ELISA) kits were provided by BioLegend (CA, USA), and Rabbit Ab Phosphor-NF-κB, Rabbit Ab phosphor-IᴋB and IᴋB were purchased from Abcam (USA). Anti-HO-1 and anti-Nrf2 monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-Lamin B, anti-TLR4 monoclonal antibodies were purchased from Proteintech Group Inc. (Boston, MA, USA), and HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). All other chemicals were of reagent grade. 2.2. Animals C57/BL6 mice (male, 6–8 weeks old, weighing approximately 18 to 22 g each) were purchased from Liaoning Changsheng Biotechnology (Liaoning, China). These mice were given adequate food and water ad libitum and housed in clean cages for 2–3 d. The laboratory temperature was 24 ± 1 °C and relative humidity was 40%–80%. All studies were performed in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health.
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injection (i.p.) of different concentrations of morin. Then PBS was given in the control group. One hour later, the mice were given LPS/DGalN treatment. In addition, others mice were given morin (25, 50, and 100 mg/kg) treatment. Mice blood and liver tissues were collected subsequently for future research.
2.4.2. MPO (myeloperoxidase) assay MPO is a marker of neutrophil activation, oxidative stress and oxidative tissue damage. MPO levels were detected 3 h after LPS/D-GalN injection. Liver tissues were homogenized for further research through using MPO ELISA kit (Jiancheng Company, Nanjing, China). MPO levels were determined by measuring the absorbance under 450 nm using a spectrophotometer.
2.4.3. Cytokine assays The C57/BL6 mice were randomly divided into six groups and were treated as described before. In the end, the blood and liver tissues were collected for measuring TNF-α and IL-6 by using ELISA kits (BioLegend) according to the production directions.
2.4.4. Histopathology evaluation of the liver tissues Livers were collected 3 h after LPS/D-GalN treatment and tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sliced and stained with hematoxylin and eosin (H&E). The pathological changes of liver tissues were observed with a light microscope.
2.4.5. Analysis of liver enzymes Blood were collected from all mice to detect plasma enzyme of ALT and AST changes. The ALT and AST levels marked the degree of Hepatocyte damage. Serum was collected to detect ALT and AST levels with test kits purchased from Jiancheng Bioengineering Institute of Nanjing according to the instructions.
2.3. In vitro study 2.5. Western blot 2.3.1. Cell culture and treatment The HepG2 human hepatoma cell line was purchased from the China Cell Line Bank (Beijing, China). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated Fetal bovine serum, 3 mM Glutamine, antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37 °C and 5% CO2 conduction. All cell experiments were incubated in the presence or absence of various concentrations of morin for 12 h followed by natural culture. 2.3.2. CCK8 assay for cell viability HepG2 cells were plated at a density of 4 × 105 cells/ml onto 96-well plates and incubated in 37 °C and 5% CO2 incubator for 12 h (100 μl/ well) after trypsin treated. Then the cell culture medium was discarded, and the cells were treated with 100 μl morin of distinctive concentrations (0–128 μg/ml). After 12 h, 10 μl CCK8 was added to each well, and the cells were incubated for an extra 3 h at 37 °C with 5% CO2. The optical density was measured at 450 nm on a microplate reader (TECAN, Austria). 2.4. In vivo study 2.4.1. LPS/D-GalN-induced acute liver injury mode The C57/BL6 mice were randomly divided into six groups: the control group, LPS/D-GalN-stimulated group (intraperitoneal injection with 600 mg/kg D-GalN and 10 μg/kg LPS) and morin (25, 50, and 100 mg/kg) groups which were treated with morin 1 h before LPS/DGalN stimulation. The negative control was only given morin (100 mg/kg) treatment. Firstly, mice were given intraperitoneal
HepG2 cells were trypsinized and plated onto 6-well plate. The density of cells was 4 × 105 cells/ml. The cells were incubated in 37 °C and 5% CO2 for 12 h. afterwards, morin in different concentrations (8, 16, and 32 μg/ml) were added into every plate. After 8 h, the cellular proteins were collected. Liver tissues were collected 3 h after LPS/D-GalN injection. Nuclear and cytoplasmic proteins of the liver tissues and cells were extracted from livers by using Nuclear and cytoplasmic protein Extraction Kit (Beyotime). The protein concentrations were assayed by using BCA protein assay kit (Thermo, USA) following the manufacturer's instructions. 30 μg of sample proteins were regularly collected, followed by being fractionated on 12% polyacrylamide-SDS gel and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked in 5% skim milk on the table for 2 h at room temperature and incubated with primary antibody (1:1000) at 4 °C overnight. The membranes were washed by TBST four times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h (1:5000). Then the membranes were washed by TBST four times and the immunoreactive proteins were detected by chemiluminescence (ECL) Western blotting detection kit (Thermo, USA).
2.6. Statistical analysis All values were expressed as mean ± SEM. Differences between mean values of normally distributed data were assessed by two-tailed Student's t-test. Statistical significance was accepted at P b 0.05 or P b 0.01.
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Fig. 1. Effects of morin on MPO activities and mortality in the liver of acute liver injury. (A) The chemical structure of Morin. (B) Mice were administered morin (25, 50, and 100 mg/kg) i.p. for 1 h, followed by exposure to a lethal dose of LPS (10 μg/kg) and D-GalN (600 mg/kg). The data are expressed as the percentage of surviving mice at each time point. The values presented are the means ± SEM (n = 10 in each group) of three independent experiments. (C) Effects of morin on MPO activity in liver homogenates. The values presented are the means ± SEM (n = 9 in each group). ##P b 0.01 vs. the control group; ⁎P b 0.05 vs. the LPS/D-GalN group; ⁎⁎P b 0.01 vs. the LPS/D-GalN group.
3. Results 3.1. Effects of morin on mortality in LPS/D-GalN-induced mice As is shown in Fig. 1B, the mortality rate of the LPS (10 μg/kg) and DGalN (600 mg/kg)-treated mice was much higher than that of the LPS/ D-GalN plus morin (25, 50, and 100 mg/kg)-treated mice. In addition, we found that the morin (25, 50, and 100 mg/kg) plus LPS (10 μg/kg)/ D-GalN (600 mg/kg) groups could improve mice survival rate in a dose-dependent manner. The only morin (100 mg/kg)-treated mice group was not found toxic. These results suggested that morin effectively protected against LPS/D-GalN induced hepatotoxicity 3.2. Effects of morin on MPO activity in LPS/D-GalN-induced acute liver injury mice To research the effect of morin on neutrophil accumulation within the LPS/D-GalN-induced hepatic tissue, MPO activity was measured by MPO ELISA kit. According to the protocol of the manufacturer, The MPO activity in LPS/D-GalN group was higher than that in the control group (##P b 0.01). Nevertheless, morin could reduce MPO production after LPS/D-GalN stimulation efficiently (Fig. 1C). 3.3. Effects of morin on the secretion of cytokines The levels of TNF-α and IL-6 in serum and hepatic samples were detected by ELISA. As is shown in Fig. 2A, B, C, and D, the TNF-α and IL-6 levels in serum and hepatic samples increased significantly after LPS (10 μg/kg)/D-GalN (600 mg/kg) treatment. These results showed that LPS/D-GalN could improve inflammatory cytokines release. But the levels of TNF-α and IL-6 showed significant attenuation after morin
(25, 50, and 100 mg/kg) treatment in a dose-dependent manner. Our ELISA results indicated that morin could inhibit the production of LPS/ D-GalN-induced inflammatory cytokines. 3.4. Effects of Morin protects against LPS/D-GalN-induced hepatic damage We histopathologically examined the extent of liver injury to verify the protective effects of morin. As is shown in Fig. 2E (a), the livers of the control group showed normal liver architecture. As is indicated by the arrows in Fig. 2E (b), treatment of LPS/D-GalN led to severe damage to the liver, such as extensive hemorrhage, necrosis and neutrophil infiltration. As is indicated by the arrows in Fig. 2E (c, d, and e), treatment of morin markedly modify liver injury induced by LPS/D-GalN. 3.5. Effects of morin on ALT and AST levels induced by LPS/D-GalN We next assessed the effects of pretreatment with morin on LPS/DGalN-induced hepatotoxicity and found that LPS (10 μg/kg)/D-GalN (600 mg/kg) dramatically increased the serum AST and ALT levels, whereas morin (25, 50, and 100 mg/kg) reduced these increases (Fig. 2F and G). Serum ALT and AST levels were detected 3 h after LPS/DGalN injection. It was found that AST and ALT levels increased significantly in LPS/D-GalN stimulation group. The only morin (100 mg/kg)treated mice group was not found change. These increases were attenuated by morin administration. These results suggested that morin effectively protected against LPS/D-GalN-induced hepatotoxicity. 3.6. Effects of morin on ALT and AST levels in the serum There were no changes in serum levels of ALT and AST among groups treated with morin under different concentrations (0–100 mg/kg)
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Fig. 2. Effects of morin on LPS/D-GalN-induced cytokines and liver injury. (A, B, C, and D) Mice were given an intraperitoneal injection of morin (25, 50, and 100 mg/kg) 1 h prior to administration of LPS/D-GalN. The negative control was only given morin (100 mg/kg) treatment. After 3 h LPS/D-GalN challenge, we harvested serum and hepatic for the analysis of cytokines levels. The values presented are the means ± SEM (n = 9 in each group). (E) Mice were given an intraperitoneal injection of morin (25, 50, and 100 mg/kg) 1 h prior to LPS/D-GalN administration. After 3 h LPS/D-GalN challenge, we processed each group for histological evaluation. (a) Control group: Normal control mouse showing clear liver structure. (b) LPS (10 μg/kg)/D-GalN (600 mg/kg) group: LPS/D-GalN administered mouse showing collapsed liver structure with wide focal area of hemorrhage, inflammatory cell infiltration and necrosis. (c) LPS (10 μg/kg)/D-GalN (600 mg/kg) + morin (25 mg/kg) group, (d) LPS (10 μg/kg)/D-GalN (600 mg/kg) + morin (50 mg/kg) group, (e) LPS (10 μg/kg)/ D-GalN (600 mg/kg) + morin (100 mg/kg) group, (c, d, and e): morin treated mouse showing more obvious liver structure with mild area of focal hemorrhage. (Hematoxylin and eosin staining, magnification 200×). (F, G) At 1 h after the last dose of morin, LPS (10 μg/kg)/D-GalN (600 mg/kg) was administered for 3 h. Then, the serum levels of ALT and AST were measured. The date of ALT and AST are the mean ± SEM (n = 9 in each group) average from three independent experiments. (H) Mice only were given an intraperitoneal injection of morin (0, 25, 50, and 100 mg/kg). After 3 h, we harvested serum for the analysis of ALT and AST levels. Date is presented as mean ± SEM (n = 9 in each group) average from three independent experiments. ##P b 0.01 indicates significant differences from the control group; ⁎⁎P b 0.01 vs. the LPS/D-GalN group; ⁎P b 0.05 vs. the LPS/D-GalN group.
without LPS/D-GalN injection (Fig. 2H). The results confirmed that morin had no toxicity on the liver, and it had no effect on the experiment results.
simulation group, suggesting that morin could activate expression of Nrf2 and HO-1. 3.8. Effects of morin on TLR4 expression and NF-κB activation
3.7. Effects of morin on Nrf2 and HO-1 expression in liver issue We further examined the effects of morin on the activities of Nrf2 and HO-1, which may be responsible for its hepatoprotective effects, and the HO-1 in liver tissues. The effects of morin on LPS/D-GalN-induced Nrf2 and HO-1 expression were detected by Western blotting. Our western results showed that the expression of Nrf2 and HO-1 were up-regulated after morin treatment. As is shown in Fig. 3A and B, morin (25, 50, and 100 mg/kg) administration increased the HO-1 expression and nuclear translocation of Nrf2 better than LPS/D-GalN
To investigate the anti-inflammatory mechanisms by which morin inhibits LPS/D-GalN-induced cytokine production, we observed TLR4 and NF-κB signaling pathways by Western blotting analysis using different phosphor-special antibodies. In order to study the anti-inflammatory mechanism of morin, we detected the expression of NF-κB and TLR4. The results showed that LPS/D-GalN markedly enhanced the expression of TLR4 and the activation of NF-κB. But we found that morin treatment could attenuate LPS/D-GalN-induced TLR4 expression and NF-κB activation in a dose-dependent manner (Fig. 4A). As is shown in Fig. 4B, C, D, and E that LPS/D-GalN-induced TLR4, phosphor-NF-κB, and phosphor-
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Fig. 3. Effects of morin on liver by the expression of Nrf2 and HO-1. Mice were given an intraperitoneal injection of morin 1 h prior to administration of LPS/D-GalN. After 3 h LPS/D-GalN challenge, we harvested liver tissues for the analysis of protein. Then the total expressed HO-1 protein and the nuclear expressed Nrf2 protein were analyzed by Western blot (A and B). The β-actin and Lamin B were acted as an internal control. All data were presented as means ± SEM of three independent experiments. ##p b 0.01 vs. the control group; ⁎⁎p b 0.01 vs. the LPS/D-GalN group.
IκB upswing were inhibited after pretreatment with morin in a dose-dependent manner. 3.9. Effects of morin on HepG2 cells toxicity In order to evaluate the cytotoxicity of the morin, HepG2 cells were treated with morin at concentrations of 0 to 128 μg/ml 12 h after the CCK8 test. These results showed that morin under concentrations from 0 to 128 μg/ml had no cell toxicity on HepG2 cells (Fig. 5A). So we selected concentrations (8, 16, and 32 μg/ml) for future research. 3.10. Effects of morin on Nrf2 and HO-1 expression in HepG2 cells To study the antioxidant mechanism of morin, we detected the levels of Nrf2 and HO-1 expression on HepG2 cells. We selected three non-toxic concentrations of morin (8, 16, and 32 μg/ml) to detect Nrf2 and HO-1 levels on HepG2 cells, the effects of morin on Nrf2 and HO-1 expression were detected by Western blotting. As is shown in Fig. 5B, C and D, we were surprised to find that the levels of Nrf2 and HO-1 were up-regulated after only morin treatment in a dose-dependent manner. The result indicated morin had a positive antioxidant effect. 4. Discussion Previous studies indicated that morin had anti-inflammatory, antioxidant and antineoplastic effects and could protect from carbon tetrachloride and paracetamol induced acute liver injury [9,10,11,17]. But the therapeutical effect of morin on LPS/D-GalN-induced liver damages has not been reported. We aimed to explore the function of morin in acute liver injury caused by LPS/D-GalN. In the recent study, we found that morin attenuated ALT and AST levels in LPS/D-GalN-induced acute liver injury. ALT and AST have been acknowledged as the marker of the liver cell damage [18], whose
levels represent the degree of liver injury. The experimental results showed that LPS/D-GalN caused significant increase of ALT and AST in mice. However, morin significantly reduced serum ALT and AST levels on LPS/D-GalN-induced acute liver injury. In addition, our histological analysis demonstrated that morin clearly repaired hemorrhage and cellular necrosis in mice. These results indicated that morin played a positive role in LPS/D-GalN-induced liver damage. TNF-α and IL-6 have been reported to play a critical role in the inflammatory pathogenesis of liver injury [19]. They could cause hepatocyte necrosis and organ failure and contribute to the severity of liver injury [20]. Meanwhile, the generation of TNF-α can promote the progression of other pathological mechanism [21]. Increased TNF-α level could cause renal injury [22]. Furthermore, previous reports also demonstrated that down-regulation of TNF-α and IL-6 production could alleviate liver injury [23]. We found that could reduce TNF-α and IL-6 levels in LPS/D-GalN-induced acute liver injury. MPO level was used to imply the amount of neutrophils, monocytes, and macrophages. The increase of MPO can cause severe oxidative stress and oxidative tissue damages. In our experiment morin could decrease MPO levels in this model. Thus, the results of this research showed that morin markedly inhibited TNF-α, IL-6 and MPO levels. The results suggested that morin could protect mice against LPS/D-GalN-induced acute liver injury by ameliorating inflammatory and oxidative response. Nuclear factor κB (NF-κB) is a nuclear transcription factor that regulates expression of various genes that are critical for inflammatory responses [24,25]. In its inactive form, NF-κB is normally sequestered in the cytoplasm by its inhibitory proteins IκBα [26]. In response to stimuli, the cytoplasmic NF-κB/IκB complex is activated by phosphorylation on conserved serine residues of IκBα. On dissociation from the inhibitor IκBα, NF-κB can translocate into the nucleus and activate the expression of pro-inflammatory mediator [27,28]. In this study, we examined the effects of morin on NF-κB activation and IκB changes. We found that morin inhibited phosphor-NF-κB and phosphor-IκB activation on LPS/ D-GalN induced acute liver injury in a dose-dependent manner.
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Fig. 4. Effects of morin treatment on LPS/D-GalN-activated TLR4 and NF-κB signaling pathways in acute liver injury mice. Mice were pre-treated with morin (25, 50, and 100 mg/kg) 1 h prior to exposure to LPS/D-GalN for 3 h. The protein levels were analyzed by Western blot. Quantification of relative expression of (A, C, and D) Quantification of p-IκBα and IκBα expression were normalized than that of β-actin. (A and B) Quantification of p-NF-κB (p-p65) expression was normalized than that of β-actin. (A and E) Quantification of TLR4 expression was normalized than that of β-actin. Similar data were repeated in three independent experiments, and one of three representative experiments is shown. ##p b 0.01 vs. the control group; ⁎⁎p b 0.01 vs. the LPS/D-GalN group.
Previous studies showed that LPS/D-GalN-induced acute liver injury could enhance MPO level, leading to the loss of antioxidant capacity. Meanwhile, Nrf2 can improve antioxidant defenses by regulating antioxidant enzymes expression [29,30]. Nrf2, a detoxification gene, has an important antioxidant capacity [31,32]. HO-1 has anti-inflammatory function against acute and chronic inflammatory diseases, including experimental colitis, bronchitis and hepatitis [33,34]. Our results showed that the increases of Nrf2 and HO-1 expression were augmented by morin on LPS/D-GalN-induced acute liver injury. These results suggested that morin could decrease oxidative stress induced by LPS/DGalN through activating oxidative defense system. Meanwhile, we found that morin only could augment Nrf2 expression in HepG2 cells in a dose-dependent manner, which illustrated that morin possessed activating Nrf2 function. Previous studies indicated that there was an interaction between Nrf2 and NF-κB. Activation of Nrf2 signal pathway could inhibit NF-κB signaling pathway [35]. Other studies also demonstrated that Nrf2-deficient mice showed augmented NF-κB activation in response to LPS stimulation [37]. Thus, the reason responsible for
the activation of Nrf2 may impact NF-κB signaling in this experiment. Morin could promote Nrf2 expression and suppress NF-κB activation. In addition, we found morin could suppress NF-κB activation. Because TLR and its upstream signaling can lead to the production of pro-IL-6 and the activation of TLR4 which cause the cracks of pro-IL-6 to mature IL-6. Previous study has reported that TLR4 agonists could augment NFκB expression [36]. Our western results showed that LPS/D-GalN induction increased TLR4 expression markedly. However, morin could downregulate TLR4 expression obviously in this model. These results indicated that morin inhibited LPS/D-GalN-induced inflammation by inhibiting TLR4 which affected NF-κB phosphorylation to protect liver. In conclusion, the results of this study indicate that morin plays a role in liver protection by inhibiting oxidative stress and inflammation and that these hepatoprotective effects are due to Nrf2 activation and NF-κB suppression. The results of this study demonstrated that morin possessed antioxidant effects through Nrf2 activation and anti-inflammatory effects by the inhibition of TLR4 leading to reduction of NF-κB expression against LPS/D-GalN-induced liver injury. In the future,
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Fig. 5. Effects of morin on HepG2 cell by the expression of Nrf2 and HO-1. (A) HepG2 cells were treated with increasing concentrations of morin (0, 2, 4, 8, 16, 32, and 64 μg/ml) for 12 h and subsequently cultured for an additional 3 h. Cell viability was analyzed by CCK8 assay. All of the data shown represent the average from three independent experiments. (B) Cells were pretreated with morin (0, 8, 16, and 32 μg/ml) 12 h. The protein levels were analyzed by Western blot. (C) Quantification of relative expression of Nrf2 expression was normalized than that of Lamin B. (D) Quantification of HO-1 expression was normalized than that of β-actin. Similar data were repeated in three independent experiments, and one of three representative experiments is shown. ⁎p b 0.05 vs. the morin (0 μg/ml) group; ⁎⁎p b 0.01 vs. the morin (0 μg/ml) group.
combination therapies with phosphor-NF-κB inhibition and Nrf2 activation may be developed as an effective strategy that reduces LPS/D-GalNinduced hepatotoxicity. Hence, further investigation into the roles of morin-like agents in modulating LPS/D-GalN toxicity is warranted.
[4]
[5]
Conflict of interest The authors declared that there is no conflict of interest.
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Authors' contributions Y.T., H.H.F. contributed to the design. Y.T., Z.L., did the data collection. Y.T., H.H.F. did the analysis. Y.T. did the writing of the article. B.Y.S., H.H.F. did the revisions of the article. Acknowledgement
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effects of morin on lipopolysaccharide/ D-galactosamine-induced acute liver injury by inhibiting TLR4/NF-κB and activating Nrf2/HO-1 signaling pathways Ye Tian 1, Zheng Li 1, Bingyu Shen, Qiaoling Zhang, Haihua Feng ⁎ a
Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China
a r t i c l e
i n f o
Article history: Received 27 September 2016 Received in revised form 4 February 2017 Accepted 7 February 2017 Available online xxxx Keywords: LPS/D-GalN Morin Nrf2 NF-κB
a b s t r a c t Morin, a bioactive flavonoid extracted from the bark of Moraceae plants and many medicinal herbs, has anti-inflammatory and antioxidative effects. In this research, we explored the protective effects of morin against lipopolysaccharide (LPS) and D-galactosamine (D-GalN) induced acute liver injury in mice. Mice were given an intraperitoneal injection of morin before LPS and D-GalN treatment and the HepG2 cells were only given morin to investigate its effects. The results showed that morin markedly inhibited the production of serum alanine transaminase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), tumor necrosis factor (TNFα) and hepatic TNF-α, IL-6, and myeloperoxidase (MPO) induced by LPS/D-GalN. In order to evaluate morin effect in the future, we investigated the expression of nuclear factor E2 related factor 2 (Nrf2), nuclear factorkappaB (NF-κB), toll like receptor 4 (TLR4) on liver injury. Taken together, these results suggested that morin could exert the anti-inflammatory and anti-oxidative effects against LPS/D-GalN-induced acute liver injury by activating Nrf2 signal pathways and inhibiting NF-κB activation. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The occurrence of liver injury is often accompanied by endotoxemia and sepsis and can cause high mortality in animals and human beings due to the lack of effective drugs [1,2]. Therefore, finding an effective drug to treat acute liver injury is in desperate need. LPS and D-GalN-induced acute liver injury in mice model is widely used in human liver damage [3,4]. The mice model has been recognized as a potential treatment of drug research method [5]. D-GalN is a kind of hepatotoxic agent and can cause hepatotoxicity in liver mainly through inhibiting hepatocyte RNA and protein synthesis [6]. LPS is a kind of endotoxin that could activate inflammatory cytokines, which can bring about liver tissue injury [7,8]. Nuclear factor E2 related factor 2 is an important regulatory factor that regulates cellular defense against oxidative stress. It can protect against oxidative damage triggered by liver injury and inflammation by regulating the expression of antioxidant proteins. Nuclear factor κB, a nuclear transcription factor, plays a great role in the regulation of inflammation. The activation of NF-κB is considered to respond to the ⁎ Corresponding author. E-mail address: [email protected] (H. Feng). 1 The authors Ye Tian, Zheng Li, contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2017.02.010 1567-5769/© 2017 Elsevier B.V. All rights reserved.
oxidative stress. Recent study revealed that LPS/D-GalN was capable of activating NF-κB and Nrf2 signaling [9]. However, the molecular basis of morin mitigating the LPS/D-GalN-induced liver damage by induction of Nrf2 remains elusive. This led us to reason whether morin can activate Nrf2 and inhibit NF-κB signaling, which exert protective effect against LPS/D-GalN-mediated oxidant damage. If the above hypothesis is feasible, our results will show that morin may be a potential drug target to mitigate liver damage via inhibition of NF-κB and up-regulation of Nrf2 signal pathways. Morin (20, 3, 40, 5, 7-pentahydroxyflavone), a kind of flavonol, is extracted from herbal medicine which has anti-oxidant [10], anti-hyperglycemic [11] and liver-protective effects [12]. Previous studies showed that morin could inhibit the Fyn kinase, an enzyme linked to Nrf2 degradation [13,14] and influence LPS-BV2 cells by suppressing NF-κB activity and activating HO-1 induction [15]. In addition, morin was also found to inhibit LPS-induced acute lung injury in mice [16]. However, the effects of morin on LPS/D-GalN-induced liver injury remain unclear. In this study, we evaluated whether morin played a positive role in protecting liver tissue against LPS/D-GalN-induced acute liver injury. We demonstrated, for the first time, that morin could show functional and morphological protection against LPS/D-GalN-induced liver injury by alleviating inflammatory and oxidative stress.
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2. Materials and methods 2.1. Materials Morin (purity ≥98%) was purchased from Chengdu Reference Products (Chengdu, China). Dimethylsulfoxide (DMSO), LPS (Escherichia coli lipopolysaccharide, 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CCK8 was purchased from Beyotime Biotechnology (Beijing. China). Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM), penicillin and streptomycin were purchased from Invitrogen-Gibco (Grand Island, NY). We purchased D-galactosamine hydrochloride from Aladdin Industrial Corporation (Shanghai, China). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits were provided by Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Mouse TNF-α, and IL-6 enzymelinked immunosorbent assay (ELISA) kits were provided by BioLegend (CA, USA), and Rabbit Ab Phosphor-NF-κB, Rabbit Ab phosphor-IᴋB and IᴋB were purchased from Abcam (USA). Anti-HO-1 and anti-Nrf2 monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-Lamin B, anti-TLR4 monoclonal antibodies were purchased from Proteintech Group Inc. (Boston, MA, USA), and HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). All other chemicals were of reagent grade. 2.2. Animals C57/BL6 mice (male, 6–8 weeks old, weighing approximately 18 to 22 g each) were purchased from Liaoning Changsheng Biotechnology (Liaoning, China). These mice were given adequate food and water ad libitum and housed in clean cages for 2–3 d. The laboratory temperature was 24 ± 1 °C and relative humidity was 40%–80%. All studies were performed in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health.
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injection (i.p.) of different concentrations of morin. Then PBS was given in the control group. One hour later, the mice were given LPS/DGalN treatment. In addition, others mice were given morin (25, 50, and 100 mg/kg) treatment. Mice blood and liver tissues were collected subsequently for future research.
2.4.2. MPO (myeloperoxidase) assay MPO is a marker of neutrophil activation, oxidative stress and oxidative tissue damage. MPO levels were detected 3 h after LPS/D-GalN injection. Liver tissues were homogenized for further research through using MPO ELISA kit (Jiancheng Company, Nanjing, China). MPO levels were determined by measuring the absorbance under 450 nm using a spectrophotometer.
2.4.3. Cytokine assays The C57/BL6 mice were randomly divided into six groups and were treated as described before. In the end, the blood and liver tissues were collected for measuring TNF-α and IL-6 by using ELISA kits (BioLegend) according to the production directions.
2.4.4. Histopathology evaluation of the liver tissues Livers were collected 3 h after LPS/D-GalN treatment and tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sliced and stained with hematoxylin and eosin (H&E). The pathological changes of liver tissues were observed with a light microscope.
2.4.5. Analysis of liver enzymes Blood were collected from all mice to detect plasma enzyme of ALT and AST changes. The ALT and AST levels marked the degree of Hepatocyte damage. Serum was collected to detect ALT and AST levels with test kits purchased from Jiancheng Bioengineering Institute of Nanjing according to the instructions.
2.3. In vitro study 2.5. Western blot 2.3.1. Cell culture and treatment The HepG2 human hepatoma cell line was purchased from the China Cell Line Bank (Beijing, China). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated Fetal bovine serum, 3 mM Glutamine, antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37 °C and 5% CO2 conduction. All cell experiments were incubated in the presence or absence of various concentrations of morin for 12 h followed by natural culture. 2.3.2. CCK8 assay for cell viability HepG2 cells were plated at a density of 4 × 105 cells/ml onto 96-well plates and incubated in 37 °C and 5% CO2 incubator for 12 h (100 μl/ well) after trypsin treated. Then the cell culture medium was discarded, and the cells were treated with 100 μl morin of distinctive concentrations (0–128 μg/ml). After 12 h, 10 μl CCK8 was added to each well, and the cells were incubated for an extra 3 h at 37 °C with 5% CO2. The optical density was measured at 450 nm on a microplate reader (TECAN, Austria). 2.4. In vivo study 2.4.1. LPS/D-GalN-induced acute liver injury mode The C57/BL6 mice were randomly divided into six groups: the control group, LPS/D-GalN-stimulated group (intraperitoneal injection with 600 mg/kg D-GalN and 10 μg/kg LPS) and morin (25, 50, and 100 mg/kg) groups which were treated with morin 1 h before LPS/DGalN stimulation. The negative control was only given morin (100 mg/kg) treatment. Firstly, mice were given intraperitoneal
HepG2 cells were trypsinized and plated onto 6-well plate. The density of cells was 4 × 105 cells/ml. The cells were incubated in 37 °C and 5% CO2 for 12 h. afterwards, morin in different concentrations (8, 16, and 32 μg/ml) were added into every plate. After 8 h, the cellular proteins were collected. Liver tissues were collected 3 h after LPS/D-GalN injection. Nuclear and cytoplasmic proteins of the liver tissues and cells were extracted from livers by using Nuclear and cytoplasmic protein Extraction Kit (Beyotime). The protein concentrations were assayed by using BCA protein assay kit (Thermo, USA) following the manufacturer's instructions. 30 μg of sample proteins were regularly collected, followed by being fractionated on 12% polyacrylamide-SDS gel and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked in 5% skim milk on the table for 2 h at room temperature and incubated with primary antibody (1:1000) at 4 °C overnight. The membranes were washed by TBST four times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h (1:5000). Then the membranes were washed by TBST four times and the immunoreactive proteins were detected by chemiluminescence (ECL) Western blotting detection kit (Thermo, USA).
2.6. Statistical analysis All values were expressed as mean ± SEM. Differences between mean values of normally distributed data were assessed by two-tailed Student's t-test. Statistical significance was accepted at P b 0.05 or P b 0.01.
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Fig. 1. Effects of morin on MPO activities and mortality in the liver of acute liver injury. (A) The chemical structure of Morin. (B) Mice were administered morin (25, 50, and 100 mg/kg) i.p. for 1 h, followed by exposure to a lethal dose of LPS (10 μg/kg) and D-GalN (600 mg/kg). The data are expressed as the percentage of surviving mice at each time point. The values presented are the means ± SEM (n = 10 in each group) of three independent experiments. (C) Effects of morin on MPO activity in liver homogenates. The values presented are the means ± SEM (n = 9 in each group). ##P b 0.01 vs. the control group; ⁎P b 0.05 vs. the LPS/D-GalN group; ⁎⁎P b 0.01 vs. the LPS/D-GalN group.
3. Results 3.1. Effects of morin on mortality in LPS/D-GalN-induced mice As is shown in Fig. 1B, the mortality rate of the LPS (10 μg/kg) and DGalN (600 mg/kg)-treated mice was much higher than that of the LPS/ D-GalN plus morin (25, 50, and 100 mg/kg)-treated mice. In addition, we found that the morin (25, 50, and 100 mg/kg) plus LPS (10 μg/kg)/ D-GalN (600 mg/kg) groups could improve mice survival rate in a dose-dependent manner. The only morin (100 mg/kg)-treated mice group was not found toxic. These results suggested that morin effectively protected against LPS/D-GalN induced hepatotoxicity 3.2. Effects of morin on MPO activity in LPS/D-GalN-induced acute liver injury mice To research the effect of morin on neutrophil accumulation within the LPS/D-GalN-induced hepatic tissue, MPO activity was measured by MPO ELISA kit. According to the protocol of the manufacturer, The MPO activity in LPS/D-GalN group was higher than that in the control group (##P b 0.01). Nevertheless, morin could reduce MPO production after LPS/D-GalN stimulation efficiently (Fig. 1C). 3.3. Effects of morin on the secretion of cytokines The levels of TNF-α and IL-6 in serum and hepatic samples were detected by ELISA. As is shown in Fig. 2A, B, C, and D, the TNF-α and IL-6 levels in serum and hepatic samples increased significantly after LPS (10 μg/kg)/D-GalN (600 mg/kg) treatment. These results showed that LPS/D-GalN could improve inflammatory cytokines release. But the levels of TNF-α and IL-6 showed significant attenuation after morin
(25, 50, and 100 mg/kg) treatment in a dose-dependent manner. Our ELISA results indicated that morin could inhibit the production of LPS/ D-GalN-induced inflammatory cytokines. 3.4. Effects of Morin protects against LPS/D-GalN-induced hepatic damage We histopathologically examined the extent of liver injury to verify the protective effects of morin. As is shown in Fig. 2E (a), the livers of the control group showed normal liver architecture. As is indicated by the arrows in Fig. 2E (b), treatment of LPS/D-GalN led to severe damage to the liver, such as extensive hemorrhage, necrosis and neutrophil infiltration. As is indicated by the arrows in Fig. 2E (c, d, and e), treatment of morin markedly modify liver injury induced by LPS/D-GalN. 3.5. Effects of morin on ALT and AST levels induced by LPS/D-GalN We next assessed the effects of pretreatment with morin on LPS/DGalN-induced hepatotoxicity and found that LPS (10 μg/kg)/D-GalN (600 mg/kg) dramatically increased the serum AST and ALT levels, whereas morin (25, 50, and 100 mg/kg) reduced these increases (Fig. 2F and G). Serum ALT and AST levels were detected 3 h after LPS/DGalN injection. It was found that AST and ALT levels increased significantly in LPS/D-GalN stimulation group. The only morin (100 mg/kg)treated mice group was not found change. These increases were attenuated by morin administration. These results suggested that morin effectively protected against LPS/D-GalN-induced hepatotoxicity. 3.6. Effects of morin on ALT and AST levels in the serum There were no changes in serum levels of ALT and AST among groups treated with morin under different concentrations (0–100 mg/kg)
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Fig. 2. Effects of morin on LPS/D-GalN-induced cytokines and liver injury. (A, B, C, and D) Mice were given an intraperitoneal injection of morin (25, 50, and 100 mg/kg) 1 h prior to administration of LPS/D-GalN. The negative control was only given morin (100 mg/kg) treatment. After 3 h LPS/D-GalN challenge, we harvested serum and hepatic for the analysis of cytokines levels. The values presented are the means ± SEM (n = 9 in each group). (E) Mice were given an intraperitoneal injection of morin (25, 50, and 100 mg/kg) 1 h prior to LPS/D-GalN administration. After 3 h LPS/D-GalN challenge, we processed each group for histological evaluation. (a) Control group: Normal control mouse showing clear liver structure. (b) LPS (10 μg/kg)/D-GalN (600 mg/kg) group: LPS/D-GalN administered mouse showing collapsed liver structure with wide focal area of hemorrhage, inflammatory cell infiltration and necrosis. (c) LPS (10 μg/kg)/D-GalN (600 mg/kg) + morin (25 mg/kg) group, (d) LPS (10 μg/kg)/D-GalN (600 mg/kg) + morin (50 mg/kg) group, (e) LPS (10 μg/kg)/ D-GalN (600 mg/kg) + morin (100 mg/kg) group, (c, d, and e): morin treated mouse showing more obvious liver structure with mild area of focal hemorrhage. (Hematoxylin and eosin staining, magnification 200×). (F, G) At 1 h after the last dose of morin, LPS (10 μg/kg)/D-GalN (600 mg/kg) was administered for 3 h. Then, the serum levels of ALT and AST were measured. The date of ALT and AST are the mean ± SEM (n = 9 in each group) average from three independent experiments. (H) Mice only were given an intraperitoneal injection of morin (0, 25, 50, and 100 mg/kg). After 3 h, we harvested serum for the analysis of ALT and AST levels. Date is presented as mean ± SEM (n = 9 in each group) average from three independent experiments. ##P b 0.01 indicates significant differences from the control group; ⁎⁎P b 0.01 vs. the LPS/D-GalN group; ⁎P b 0.05 vs. the LPS/D-GalN group.
without LPS/D-GalN injection (Fig. 2H). The results confirmed that morin had no toxicity on the liver, and it had no effect on the experiment results.
simulation group, suggesting that morin could activate expression of Nrf2 and HO-1. 3.8. Effects of morin on TLR4 expression and NF-κB activation
3.7. Effects of morin on Nrf2 and HO-1 expression in liver issue We further examined the effects of morin on the activities of Nrf2 and HO-1, which may be responsible for its hepatoprotective effects, and the HO-1 in liver tissues. The effects of morin on LPS/D-GalN-induced Nrf2 and HO-1 expression were detected by Western blotting. Our western results showed that the expression of Nrf2 and HO-1 were up-regulated after morin treatment. As is shown in Fig. 3A and B, morin (25, 50, and 100 mg/kg) administration increased the HO-1 expression and nuclear translocation of Nrf2 better than LPS/D-GalN
To investigate the anti-inflammatory mechanisms by which morin inhibits LPS/D-GalN-induced cytokine production, we observed TLR4 and NF-κB signaling pathways by Western blotting analysis using different phosphor-special antibodies. In order to study the anti-inflammatory mechanism of morin, we detected the expression of NF-κB and TLR4. The results showed that LPS/D-GalN markedly enhanced the expression of TLR4 and the activation of NF-κB. But we found that morin treatment could attenuate LPS/D-GalN-induced TLR4 expression and NF-κB activation in a dose-dependent manner (Fig. 4A). As is shown in Fig. 4B, C, D, and E that LPS/D-GalN-induced TLR4, phosphor-NF-κB, and phosphor-
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Fig. 3. Effects of morin on liver by the expression of Nrf2 and HO-1. Mice were given an intraperitoneal injection of morin 1 h prior to administration of LPS/D-GalN. After 3 h LPS/D-GalN challenge, we harvested liver tissues for the analysis of protein. Then the total expressed HO-1 protein and the nuclear expressed Nrf2 protein were analyzed by Western blot (A and B). The β-actin and Lamin B were acted as an internal control. All data were presented as means ± SEM of three independent experiments. ##p b 0.01 vs. the control group; ⁎⁎p b 0.01 vs. the LPS/D-GalN group.
IκB upswing were inhibited after pretreatment with morin in a dose-dependent manner. 3.9. Effects of morin on HepG2 cells toxicity In order to evaluate the cytotoxicity of the morin, HepG2 cells were treated with morin at concentrations of 0 to 128 μg/ml 12 h after the CCK8 test. These results showed that morin under concentrations from 0 to 128 μg/ml had no cell toxicity on HepG2 cells (Fig. 5A). So we selected concentrations (8, 16, and 32 μg/ml) for future research. 3.10. Effects of morin on Nrf2 and HO-1 expression in HepG2 cells To study the antioxidant mechanism of morin, we detected the levels of Nrf2 and HO-1 expression on HepG2 cells. We selected three non-toxic concentrations of morin (8, 16, and 32 μg/ml) to detect Nrf2 and HO-1 levels on HepG2 cells, the effects of morin on Nrf2 and HO-1 expression were detected by Western blotting. As is shown in Fig. 5B, C and D, we were surprised to find that the levels of Nrf2 and HO-1 were up-regulated after only morin treatment in a dose-dependent manner. The result indicated morin had a positive antioxidant effect. 4. Discussion Previous studies indicated that morin had anti-inflammatory, antioxidant and antineoplastic effects and could protect from carbon tetrachloride and paracetamol induced acute liver injury [9,10,11,17]. But the therapeutical effect of morin on LPS/D-GalN-induced liver damages has not been reported. We aimed to explore the function of morin in acute liver injury caused by LPS/D-GalN. In the recent study, we found that morin attenuated ALT and AST levels in LPS/D-GalN-induced acute liver injury. ALT and AST have been acknowledged as the marker of the liver cell damage [18], whose
levels represent the degree of liver injury. The experimental results showed that LPS/D-GalN caused significant increase of ALT and AST in mice. However, morin significantly reduced serum ALT and AST levels on LPS/D-GalN-induced acute liver injury. In addition, our histological analysis demonstrated that morin clearly repaired hemorrhage and cellular necrosis in mice. These results indicated that morin played a positive role in LPS/D-GalN-induced liver damage. TNF-α and IL-6 have been reported to play a critical role in the inflammatory pathogenesis of liver injury [19]. They could cause hepatocyte necrosis and organ failure and contribute to the severity of liver injury [20]. Meanwhile, the generation of TNF-α can promote the progression of other pathological mechanism [21]. Increased TNF-α level could cause renal injury [22]. Furthermore, previous reports also demonstrated that down-regulation of TNF-α and IL-6 production could alleviate liver injury [23]. We found that could reduce TNF-α and IL-6 levels in LPS/D-GalN-induced acute liver injury. MPO level was used to imply the amount of neutrophils, monocytes, and macrophages. The increase of MPO can cause severe oxidative stress and oxidative tissue damages. In our experiment morin could decrease MPO levels in this model. Thus, the results of this research showed that morin markedly inhibited TNF-α, IL-6 and MPO levels. The results suggested that morin could protect mice against LPS/D-GalN-induced acute liver injury by ameliorating inflammatory and oxidative response. Nuclear factor κB (NF-κB) is a nuclear transcription factor that regulates expression of various genes that are critical for inflammatory responses [24,25]. In its inactive form, NF-κB is normally sequestered in the cytoplasm by its inhibitory proteins IκBα [26]. In response to stimuli, the cytoplasmic NF-κB/IκB complex is activated by phosphorylation on conserved serine residues of IκBα. On dissociation from the inhibitor IκBα, NF-κB can translocate into the nucleus and activate the expression of pro-inflammatory mediator [27,28]. In this study, we examined the effects of morin on NF-κB activation and IκB changes. We found that morin inhibited phosphor-NF-κB and phosphor-IκB activation on LPS/ D-GalN induced acute liver injury in a dose-dependent manner.
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Fig. 4. Effects of morin treatment on LPS/D-GalN-activated TLR4 and NF-κB signaling pathways in acute liver injury mice. Mice were pre-treated with morin (25, 50, and 100 mg/kg) 1 h prior to exposure to LPS/D-GalN for 3 h. The protein levels were analyzed by Western blot. Quantification of relative expression of (A, C, and D) Quantification of p-IκBα and IκBα expression were normalized than that of β-actin. (A and B) Quantification of p-NF-κB (p-p65) expression was normalized than that of β-actin. (A and E) Quantification of TLR4 expression was normalized than that of β-actin. Similar data were repeated in three independent experiments, and one of three representative experiments is shown. ##p b 0.01 vs. the control group; ⁎⁎p b 0.01 vs. the LPS/D-GalN group.
Previous studies showed that LPS/D-GalN-induced acute liver injury could enhance MPO level, leading to the loss of antioxidant capacity. Meanwhile, Nrf2 can improve antioxidant defenses by regulating antioxidant enzymes expression [29,30]. Nrf2, a detoxification gene, has an important antioxidant capacity [31,32]. HO-1 has anti-inflammatory function against acute and chronic inflammatory diseases, including experimental colitis, bronchitis and hepatitis [33,34]. Our results showed that the increases of Nrf2 and HO-1 expression were augmented by morin on LPS/D-GalN-induced acute liver injury. These results suggested that morin could decrease oxidative stress induced by LPS/DGalN through activating oxidative defense system. Meanwhile, we found that morin only could augment Nrf2 expression in HepG2 cells in a dose-dependent manner, which illustrated that morin possessed activating Nrf2 function. Previous studies indicated that there was an interaction between Nrf2 and NF-κB. Activation of Nrf2 signal pathway could inhibit NF-κB signaling pathway [35]. Other studies also demonstrated that Nrf2-deficient mice showed augmented NF-κB activation in response to LPS stimulation [37]. Thus, the reason responsible for
the activation of Nrf2 may impact NF-κB signaling in this experiment. Morin could promote Nrf2 expression and suppress NF-κB activation. In addition, we found morin could suppress NF-κB activation. Because TLR and its upstream signaling can lead to the production of pro-IL-6 and the activation of TLR4 which cause the cracks of pro-IL-6 to mature IL-6. Previous study has reported that TLR4 agonists could augment NFκB expression [36]. Our western results showed that LPS/D-GalN induction increased TLR4 expression markedly. However, morin could downregulate TLR4 expression obviously in this model. These results indicated that morin inhibited LPS/D-GalN-induced inflammation by inhibiting TLR4 which affected NF-κB phosphorylation to protect liver. In conclusion, the results of this study indicate that morin plays a role in liver protection by inhibiting oxidative stress and inflammation and that these hepatoprotective effects are due to Nrf2 activation and NF-κB suppression. The results of this study demonstrated that morin possessed antioxidant effects through Nrf2 activation and anti-inflammatory effects by the inhibition of TLR4 leading to reduction of NF-κB expression against LPS/D-GalN-induced liver injury. In the future,
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Fig. 5. Effects of morin on HepG2 cell by the expression of Nrf2 and HO-1. (A) HepG2 cells were treated with increasing concentrations of morin (0, 2, 4, 8, 16, 32, and 64 μg/ml) for 12 h and subsequently cultured for an additional 3 h. Cell viability was analyzed by CCK8 assay. All of the data shown represent the average from three independent experiments. (B) Cells were pretreated with morin (0, 8, 16, and 32 μg/ml) 12 h. The protein levels were analyzed by Western blot. (C) Quantification of relative expression of Nrf2 expression was normalized than that of Lamin B. (D) Quantification of HO-1 expression was normalized than that of β-actin. Similar data were repeated in three independent experiments, and one of three representative experiments is shown. ⁎p b 0.05 vs. the morin (0 μg/ml) group; ⁎⁎p b 0.01 vs. the morin (0 μg/ml) group.
combination therapies with phosphor-NF-κB inhibition and Nrf2 activation may be developed as an effective strategy that reduces LPS/D-GalNinduced hepatotoxicity. Hence, further investigation into the roles of morin-like agents in modulating LPS/D-GalN toxicity is warranted.
[4]
[5]
Conflict of interest The authors declared that there is no conflict of interest.
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Authors' contributions Y.T., H.H.F. contributed to the design. Y.T., Z.L., did the data collection. Y.T., H.H.F. did the analysis. Y.T. did the writing of the article. B.Y.S., H.H.F. did the revisions of the article. Acknowledgement
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[9]
[10] [11]
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