Swertiamarin attenuates carbon tetrachloride (CCl4)-induced liver injury and inﬂammation in rats by regulating the TLR4 signaling pathway

The aim of the present study is to illustrate the effects of swertiamarin (STM), a natural iridoid from herbal medicines, on hepatic inflammation induced by carbon tetrachloride (CCl4) in rats. Male Sprague Dawley rats were exposed to CCl4 with or without STM co-administration for 8 weeks. Our results revealed that STM administration (100 and 200 mg/kg b.w.) significantly attenuated inflammation in livers of CCl4-treated rats. STM remarkably reduced the production of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-1a (MIP1α), and monocyte chemotactic protein-1 (MCP-1) in liver tissue of CCl4-treated rats. In addition, STM treatment downregulated connective tissue growth factor (CTGF) and ser307pIRS-1 expression, which was induced by CCl4 exposure. In the process of exploring the anti-inflammatory mechanisms of STM action, we demonstrated that STM significantly inhibited Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) p65 expression in the liver. In conclusion, these results suggested that the inhibition of CCl4-induced inflammation by STM was, at least in part, due to its regulation of the TLR4 /NF-κB signaling pathway.


INTRODUCTION
Swertiamarin (STM) is a bitter secoiridoid glycoside isolated from gentianaceae medicinal plants exerting notable hepatoprotective effects (Jaishree, Badami 2010;Zhang et al., 2015). In addition, we have previously demonstrated that STM could alleviate oxidative stress in CCl 4 -induced hepatotoxicity . However, the underlying molecular mechanisms for its hepatoprotective effects have not been fully elucidated.
There are many molecular events involved in the pathogenesis of the liver injury such as oxidative stress, inflammation, and immune reactions. Among them, tissue damage associated with hepatic inflammation can be mediated by the proinflammatory cytokines such as inducible nitric oxide synthase, interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) (Ma et al., 2014). These cytokines can stimulate the Kupffer cells to release various inflammatory mediators and free radicals (Decker 1990). The activation of inflammatory cells, including Kupffer cells, is a crucial step for activating hepatic stellate cells. Toll-like receptors (TLRs) have been reported to play an essential role in the activation of innate immunity (Pestka, Zhou, 2006). In response to stimulation of external risk factors, tissues engage the innate immune response and up-regulate TLR4 expression. Activated TLR4 will then stimulate further immune responses. Various signaling molecules have been shown to regulate the TLR pathway to modulate innate immune responses (Fang et al., 2016). The nuclear factor kappa B (NF-κB) is one of the most ubiquitous transcription factors modulating the immune response to infection or stimuli (Tornatore et al., 2012). Expression of the proinflammatory cytokines is modulated by NF-κB. Therefore, NF-κB and TLRs have attracted interest as targets for the treatment of inflammatory liver damage (Shin et al., 2013;Ma et al., 2015).
The present study aimed to explore the anti-inflammatory effects and mechanisms of STM on CCl 4induced liver injury.

Animals
This study was carried out in strict accordance with the guideline of the Council on Animal Care of Academia Sinica. The protocol was approved by the Ethical Committee on Animal Experimentation of Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. Adult male Sprague Dawley rats weighing 250-280 g were obtained from the Center of Experimental Animals of Hubei Province (Wuhan, China). All animals were kept under the same laboratory conditions of temperature (25 ± 2 °C) and lighting (12:12 h light: dark cycle) and were given free access to standard laboratory chow and tap water. All rats were allowed to acclimatize for 1 week before the experiment.

Experimental design
The animals were randomly assigned to five experimental groups (n=12): (1) Control group, rats were given 1% Tween-20 saline by gavage once per day for 8 consecutive weeks with co-administration of vehicle (peanut oil, solvent of carbon tetrachloride, 0.3 mL/100 g, subcutaneously [s. c.] twice a week); (2) STM 200 mg/ kg group, rats were given STM dissolved in 1% Tween-20 saline (200 mg/kg body weight [b.w.]) by gavage once per day for 8 consecutive weeks with co-administration of vehicle (peanut oil, solvent of carbon tetrachloride, 0.3 mL/100 g, s. c. twice a week); (3) CCl 4 group, rats were given 1% Tween-20 saline by gavage once per day for 8 consecutive weeks with co-administration of 40% CCl 4 mixed peanut oil solution (0.3 mL/100 g, s. c. twice a week); (4) CCl 4 +STM 10 0 mg/kg group, rats were treated with STM dissolved in 1% Tween-20 saline (100 mg/kg b.w.) by gavage once per day for 8 consecutive weeks with co-administration of 40% CCl 4 mixed peanut oil solution (0.3 mL/100 g, s. c. twice a week); (5) CCl 4 +STM 200 mg/kg group, rats were treated with STM dissolved in 1% Tween-20 saline (200 mg/kg B.W.) by gavage once per day for 8 consecutive weeks with co-administration of 40% CCl 4 mixed peanut oil solution (0.3 mL/100 g, s. c. twice a week). The rats were sacrificed at the end of 8 weeks and 24 h after the last dose of CCl 4 . Blood and liver samples were collected for further analysis.

Histological examination
Liver samples were derived from the central part of the right large lobe of the rats. For hematoxylin and eosin staining, the liver tissues were fixed with 10% formalin for 24 h, and then washed with tap water, dehydrated, and embedded in paraffin. Each slide was subjected to histological assessment.

Measurement of serum aminotransferase activities
The activities of ALT and AST in serum were determined spectrophotometrically using commercial diagnostic kits (Jiancheng Institute of Biotechnology, Nanjing, China).

Western blot analysis
The whole liver lysate was prepared to evaluate the expression level of CTGF and TLR4. The nuclear extracts of liver tissue were obtained to evaluate the expression level of nucleus NF-κB p65 by using a nuclear/cytoplasmic isolation kit (Beyotime Institute of Biotechnology, Beijing, China). The protein concentration was determined using the bicinchoninic acid assay and samples were stored at -80 °C. An equal amount of protein per lane was separated by standard 10% SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane (Millipore). Then the membranes were blocked with Tris-buffered saline containing 5% nonfat milk at 4°C. After that, the membranes were respectively incubated overnight at 4°C in solution containing 0.1% Tween 20, 5% nonfat milk and the following primary antibodies: CTGF (1:500), TLR4 (1:500), NF-κB p65 (1:400); ser307pIRS-1 (1:400); GAPDH (1:20000); Histone H3 (1:1000). After three washes in Tris-buffered saline Tween 20, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies at room temperature. The protein bands were quantified using the Gel-Pro Analyzer software (Media Cybernetics, Bethesda, MD). GAPDH or Histone H3 was used as an internal control.

Statistical analysis
Data were expressed as mean ± S.D. The significant differences between groups were assessed with SPSS version 13.0. The differences between group means were calculated by one-way ANOVA with LSD post hoc analysis. Difference was considered statistically significant when p < 0.05, and extremely significant when p < 0.01.

STM alleviated CCl 4 -induced liver tissue damage
The hepatic samples of the control and STM 200mg/ kg groups presented normal architecture. However, liver tissues from rats intoxicated with CCl 4 showed extensive histopathological changes, characterized by severe hepatocytes degeneration/necrosis, fatty changes, and inflammatory cell infiltration and congestion. The incidence and severity of histopathological lesions in CCl 4 +STM groups were less than those in the CCl 4 group (Figure 1).

STM reduced CCl 4 -elevated serum ALT and AST activities
Repeated CCl 4 exposure showed elevated activities of serum ALT and AST. Treatment with STM obviously reduced serum ALT and AST activities as compared with the CCl 4 group (p < 0.01, Figure 2A-B), which indicated the restoration of liver function.

STM reduced the production of pro-inflammatory cytokines
The proinflammatory cytokines (IL-1β, IL-6, TNF-α, MIP-1α, and MCP-1) profile in liver shown in  Figure 3 showed a significant increase in proinflammatory cytokines in the CCl 4 -intoxicated rats as compared with the control rats. The IL-1β, IL-6, TNF-α, MIP-1α, and MCP-1 content in liver tissue of the STM-treated rats was significantly lower than that in the CCl 4 group, suggesting the alleviation of liver inflammation with STM treatment. Note: treatment with STM alone did not have an obvious influence on the cytokine profile.

STM downregulated CTGF protein expression
The effect of STM on CTGF protein level in liver was determined. Western blotting showed that CTGF protein expression in liver tissue was significantly increased in the CCl 4 group as compared with the control group (p < 0.05). STM treatment down-regulated CTGF protein level notably in liver tissue as compared with the CCl 4 group (Figure 4).

STM regulated protein expression in the TLR4 signaling pathway
The effect of STM on the TLR4 signaling pathway activation was examined. We assessed the activation of the signaling molecules, including TLR4 and NF-κB p65. Western blotting showed that cytoplasmic TLR4 and nuclear NF-κB p65 protein expression in liver tissue was significantly induced in the CCl 4 group as compared with the control group (p < 0.01). STM treatment markedly inhibited the CCl 4 -induced increase of TLR4 and NF-κB p65 protein expression in liver tissue. The results are shown in Figure 4.

STM downregulated pIRS-1
The expression level of phosphorylated ser-307 IRS-1 in a liver sample was determined with western blotting. The ser307pIRS-1 expression in liver tissue was significantly elevated in the CCl 4 group compared with the control group (p < 0.05). STM treatment (200 mg/kg b.w.) downregulated the ser307pIRS-1 level notably in liver in comparison with the CCl 4 group ( Figure 5).
In this article we showed that STM, a natural iridoid compound, was instrumental in attenuating liver injury in rats induced by a repeated CCl 4 intoxication. The doses of STM (100 and 200 mg/kg b.w.) were based on the previously published articles on experimental liver injury (Jaishree, Badami 2010;Zhang et al., 2015;Chen et al., 2017). In the present study, a repeated CCl 4 exposure resulted in a significant increase in serum ALT and AST levels, biomarkers of liver injury. In addition, severe liver lesion and inflammatory response were also triggered by CCl 4 . All of the aforementioned pathological changes were significantly improved with STM treatment. Additionally, cytoplasmic TLR4 and nuclear accumulation of the NF-κB p65 in the livers of CCl 4 -intoxicated rats were significantly decreased by the administration of STM, suggesting a possible mechanism of action.
to activate quiescent HSCs that are localized in the perisinusoidal space, resulting in an abnormal quantity and composition of extracellular matrix, which in turn leads to hepatitis, liver fibrosis, and cirrhosis (Wu, Zern, 2000). Thus, it is important to suppress hepatic inflammation in the early stages of liver fibrosis.
Cytokines/chemokines may promote inflammatory development via a wide variety of cascade events. IL-6 release can relate to acute phase of liver lesions associated with inflammation and may induce the release of other proinflammatory cytokines (Liang et al., 2014). Considerable evidence suggests that IL-1β and TNF-α contribute to the pathogenesis of liver inflammatory diseases by activating the NF-κB signaling pathway (Muriel, 2009). In addition, it has been reported that acute hepatotoxicity with CCl 4 leads to NF-κB activation and very high expression of IL-1β, IL-6, and TNF-α (Reyes-Gordillo et al., 2007). In addition, chronic CCl 4 treatment could also induce a severe degree of liver inflammation and necrosis evidenced by an increase in the mRNA levels of various proinflammatory cytokines and chemokines such as TNF-α, IL-1β, MIP-1α, MIP-2, and MCP-1 (Mukhopadhyay et al., 2014). These cytokines are also valuable indicators of hepatic inflammation. In our study, STM treatment significantly inhibited these cytokines (IL-1β, IL-6, TNF-α, MIP-1α, and MCP-1) induced by CCl 4 that were consistent with relieved liver injury.
Hepatic fibrogenesis depends on preexistent and continuous liver injury, in part due to inflammatory cell infiltration (Gao, Jeong, Tian, 2008). CTGF is a matricellular protein that participates in critical processes including differentiation, development, tumor growth, and FIGURE 4 -STM repressed the protein expression of CTGF, TLR4 and NF-κB p65 in the liver. The CTGF protein level of rats in CCl 4 +STM 100 mg/kg and CCl 4 +STM 200 mg/kg groups were downregulated significantly compared with the CCl4 group. The protein expression of TLR4 and NF-κB p65 of rats in CCl 4 +STM 100 mg/kg and CCl 4 +STM 200 mg/kg groups were significantly lower than that in the CCl 4 group. Data are represented as means ± S.D for 3-4 animals per group. * p < 0.05 versus control, ** p < 0.01 versus control; # p < 0.05 versus CCl 4 , ## p < 0.01 versus CCl 4 by one-way ANOVA and LSD post hoc test. wound healing (Brigstock, 1999). CTGF has been shown to be a pivotal mediator of hepatic fibrogenesis (Tong et al., 2009). The expression of CTGF is obviously elevated in livers from rodents subjected to bile duct ligation or exposure to CCl 4 (Williams et al., 2000;Sedlaczek et al., 2001). Our results showed that STM significantly downregulated CTGF expression, which was induced by CCl 4 , contributing to the restrictions on fibrogenesis and inflammatory reaction in the liver.
C C l 4 i n t o x i c a t i o n c a n a c t i v a t e n u m e r o u s intracellular signaling pathways (such as MAPK, TGF-β1), which may converge on NF-κB (Ma et al., Page 7 / 9 2011; Wang et al., 2013). Activation of NF-κB in liver cells results in the recruitment of inflammatory cytokines/mediators, thus inducing fibrosis (Luedde, Schwabe, 2011). In addition, many studies from mouse and human had indicated that TLRs are central mediators of the inflammatory response and crucial links between inflammation and fibrosis in chronic liver diseases (Schwabe, Seki, Brenner, 2006;Berzsenyi et al., 2011). TLR4 exhibits a fundamental role in managing innate immunity activation and regulates the recruitment of cytokines that are necessary for the progression of inflammation (Wei et al., 2015). TLR4 is also strongly associated with the progression of liver fibrosis (Aoyama, Paik, Seki, 2010). Specifically, modulation of TGF-β signaling by a TLR4-MyD88-NF-κB axis provides a novel link between proinflammatory and profibrogenic signals (Seki et al., 2007). The present findings revealed that high levels of NF-κB and TLR4 in liver cells of CCl 4 -treated rats reflected liver impairments that are associated with inflammation. Interestingly, these two proinflammatory genes were downregulated by administration of STM. As a result, we postulated that the molecular mechanism against CCl 4triggered hepatotoxication is linked to STM-mediated inactivation of the TLR4/NF-κB pathway, in which the benefit contributes to synergistic roles of attenuating immunotoxicity and inflammatory response in CCl 4lesioned liver tissue, further improving liver functions.
CCl 4 intoxication could induce diffuse hepatosteatosis in rats (Cetinkaya et al., 2013). Excess free fatty acids are activators of various protein kinases that lead to Ser 307 phosphorylation of IRS-1, a hallmark of insulin resistance (Patel et al., 2016). The phosphorylation of IRS-1 has been reported to play a role in an in vitro model of obesity-induced inflammation (Lopez-Vazquez et al., 2017). Here we tested the influence of swertiamarin on the ser307pIRS-1 expression in CCl 4 -induced hepatotoxicity. The increased expression of the ser307pIRS-1 in CCl 4 -intoxicated rats was notably down-regulated by swertiamarin treatment (200 mg/kg b.w.), suggesting a novel mechanism.
In summary, our findings demonstrate that STM may be an alternative detoxifying medication due to its potential hepatoprotective and anti-inflammatory effects. Although underlying mechanisms have been discussed (Figure 6), further studies need to be explored.