Hepatoprotective effect of Herpetospermum caudigerum Wall. on carbon tetrachloride‐induced hepatic fibrosis in rats

Abstract Tibetan medicine Herpetospermum caudigerum Wall. (HCW) has long been employed to treat hepatitis, inflammatory diseases and jaundice according to the records of “The Four Medical Tantras” in China. This study was investigated to explore the protective effects of HCW on hepatic fibrosis and the possible mechanism in a rat model. Hepatic fibrosis was established by intragastric administration of 3 ml/kg carbon tetrachloride (CCl4) twice a week for 6 weeks. CCl4‐treated rats were received HCW (1 and 3 g/kg/d) and silymarin (0.1 g/kg/d) from 3 to 6 weeks. The results showed that HCW could significantly decrease the levels of AST, ALT, HA, LN, PCIII, Col IV, TNF‐α, IL‐1β and IL‐6. Moreover, HCW could effectively inhibit collagen deposition and reduce the pathological damage. Analysis experiments finally exhibited that HCW was able to markedly inhibit hepatic fibrosis by modulating the expressions of NF‐κB p65, IκBα, Samd3 and TGF‐β1 proteins. Therefore, our results suggest that HCW has hepatoprotective activity against CCl4‐induced hepatic fibrosis in rats by regulating the inflammatory responses.


| INTRODUCTION
Hepatic fibrosis is a serious disease which affects human health around the world. 1 Evidences suggest that the pathogenesis of hepatic fibrosis is related to the inflammation in activated hepatic stellate cells. 2,3 During the activation of hepatic stellate cells, plenty of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-1b and tumour necrosis factor a (TNF-a) are produced and play important pathogenic roles. 4 Transforming growth factor-b1 (TGF-b1), nuclear factor-jB (NF-jB) and multiple growth factors can also promote the activation of hepatic stellate cells and accumulation of extra cellular matrix proteins which contributed to fibrosis. 5 To control the cirrhosis progression, hepatoprotective drugs are used properly at the stage of hepatic fibrosis. Recently, researchers identify the effective hepatoprotective drugs from herbal medicines due to their low toxicity and free from side effects.  The detector wavelength was set at 282 nm. The flow rate was 0.8 mL/min, and the injection volume was 2 lL. The mobile phase consists of water containing 50 mmol (w/v) KH 2 PO 4 (A) and methanol (B) (pH = 3.0). Four reference substances including dehydrodiconiferyl alcohol, herpetrione, herpetin and herpetotriol were used for the qualitative analysis. All rats were randomly divided into 5 groups (n = 12 per group):

| Animals and experimental model
normal control rats and CCl 4 -treated model rats were orally received 3 ml/kg olive oil and 3 ml/kg CCl 4 (mixed 1:1 in olive oil; Sigma-Aldrich, Co., MO, USA) twice a week for 6 weeks and normal saline daily from week 3 to week 6, respectively; rats in treatment groups were orally received 3 ml/kg CCl 4 twice a week for 6 weeks and silymarin (0.1 g/kg) or HCW (1 and 3 g/kg) daily from week 3 to week 6.
At the end of 6 weeks, rats were killed after overnight fast.
Serum samples and liver samples were collected. Some liver tissues immediately were stored at À80°C for analysis, and the others were used for histopathologic examination.

| Histopathological examination
Liver tissues were instantly fixed in 10% formalin and processed by routine histopathological procedures. The samples were stained with haematoxylin and eosin (HE) for photomicroscopic assessment.

| Immunohistochemical analysis
Liver tissues were fixed in 4% PFA and sectioned in 50 lm/piece.
The section was initially permeabilized with 0.4% Triton X-100 and 1% BSA for 2 hours and then incubated with anti-TGF-b1 and anti-F I G U R E 2 Effect of Herpetospermum caudigerum Wall. (HCW) on serum AST and ALT and TNF-a, IL-6 and IL-1b in serum and liver. Values are expressed as mean AE SD. # P < .05, ## P < .01 as compared with the normal control group; *P < .05, **P < .01 as compared with the CCl 4treated model group   Finally, the membranes were exposed to X-ray film for 1 minute.

| Western blot analysis
The relative expression of various proteins was quantified by densitometric scanning using image analysis system.

| Statistical analysis
All of the data were presented as mean AE SD. Divergence between groups was compared by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. P < .05 was considered as significant.

| Identification of HCW
Four compounds (dehydrodiconiferyl alcohol, herpetrione, herpetin and herpetotriol) were identified by comparing the individual peak retention times with those of the authentic reference standards ( Figure 1).

| Effect of HCW on biochemical parameters
As illustrated in Figure 2A and Table 1, the serum levels of AST, ALT, HA, LN, PCIII and Col IV in CCl 4 -treated rats were significantly higher than those of the normal control rats, while treatment with HCW (1 and 3 g/kg) and silymarin (0.1 g/kg) effectively decreased these levels (P < .05).
In addition, there were significant increased levels of TNF-a, IL-6 and IL-1b in CCl 4 -treated rats both in serum and in liver tissues. To the respective, however, the production of TNF-a, IL-6 and IL-1b was effectively inhibited by HCW (1 and 3 g/kg) or silymarin (0.1 g/kg) ( Figure 2B and C).

| Immunohistochemical analysis
The expression of TGF-b1 and NF-jB p65 proteins in liver tissues was determined by immunohistochemical staining. The expression of TGF-b1 and NF-jB p65 proteins was significantly up-regulated in CCl 4treated rats, while HCW (1 and 3 g/kg) or silymarin (0.1 g/kg) obviously ameliorated these situations (as shown in Figure 3B and C).

| Effects of HCW on protein expressions in live tissues of CCl 4 -treated rats
The expressions of NF-jB p65, p-NF-jB p65, IjBa, p-IjBa, TGF-b1, p-Samd3 and Samd3 proteins in liver tissues were determined by

| DISCUSSION
Hepatic fibrosis is associated with most chronic liver diseases which results in significant mortality rate around the world. Attentions have been focused on the inflammation-related pathways as promising therapies for hepatic fibrosis. In the present study, we demonstrated that HCW administration prevented the development of hepatic fibrosis in CCl 4 -treated rats.
CCl 4 is a hepatotoxin and has been widely adopted for experimental induction of hepatic fibrosis. 9 In hepatic fibrosis, the TGF-b1 is an important mediator during the activation of hepatic stellate cells. It is ubiquitously distributed and has been recognized as one of most powerful pro-fibrogenic mediators. 11 Upon activation, TGF-b may bind to type I and type II serine/threonine kinase receptors and then activate receptor-regulated Smads including Smad3. 12 Previous studies have shown that TGF-b/Smad signalling pathway inactivation may relieve the hepatic fibrosis. 13,14 The present study manifested that the higher expression of TGF-b1 and ratio of p-Smad3/Samd3 were observed in CCl 4treated rats. HCW remarkably inhibited phosphorylation of Smad3 and activation of TGF-b1, suggesting that the inhibitory effects of HCW on hepatic fibrosis might be related to its action on hepatic stellate cells deactivation by controlling the TGF-b1/Smad3 signalling pathway.
However, which is the exert compound that contributes to the effect of HCW on hepatic fibrosis? As reported, dehydrodiconiferyl alcohol inhibits the activation of NF-jB protein in LPS-treated macrophage and suggests as an potential agent for the treatment of inflammatory diseases. 15,16 Herpetrione and herpetin have hepatoprotective effect on CCl4-treated mice. 17,18 Pinoresinol downregulated the inflammatory mediators by inhibiting the expression of NF-jB and AP-1 proteins in CCl4-induced liver injury. 19 On the basis of these above-mentioned researches, we suggest that the comprehensive effect of these compounds on HCW contributes to the hepatoprotective effect of HCW. Whether other compounds have the hepatoprotective effect or not needs to be addressed in our future research.
In conclusion, the present study demonstrates that HCW has effectively therapeutic activity in the CCl 4 -induced hepatic fibrosis in rats. The preliminary exploration of the underlying mechanisms exerts the hepatic protection via the modulations of NF-jB and TGF-b1/Smad3 signalling pathways. However, further researches are needed for more details in future. Therefore, based on our present study, HCW may be considered as a promising drug for the prevention of hepatic fibrosis.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.