Effects Of Paeoniflorin On Hepatic Fibrosis Induced By Schistosoma Japonicum In Mice And Roles Of TNF-α In The Signal Pathways

Objective To approach for the effect of paeoniflorin(PAE) on periovular granuloma and hepatic fibrosis in mice infected with Schistosoma japonicum in different periods of infection and treatment with praziquantel (PZQ), simultaneously to detect the change of tumor necrosis factor-α(TNF-a) during infection, and to explore the role of TNF-a in signaling of liver fibrosis induced by S.japonicum.Methods The models of hepatic fibrosis caused by S.japonicum were established by exposing mice percutaneously through the tail to cercariae of S.japonicum. The mice were randomly divided into four groups:the groups of pre-treatment(groupⅠ) treatment simultaneously(groupⅡ),post-treatment (groupⅢ) and normal group. Then each group except normal group was assigned to following three sub-groups including model group, paeoniflorin treatment group and menstruum control group.All groups, except the normal group, were orally given PZQ (500 mg/(kg. d)×2d) in 42nd after infection. Mice in paeoniflorin treatment group and control group were seperately treated with PAE(100mg/(kg-d)×30d) and 0.5% sodium carboxymethycellulose (0.2ml/d×30d) which respectively started from 30d before PZQ,simultaneously with PZQ or 30d after PZQ treatment in groupⅠ,ⅡorⅢ. Mice in groupⅠ,ⅡandⅢwere sacrificed two month later after treatment with paeoniflorin or sodium carboxymethycellulose, that is to say, on the 102nd,132nd or 162nd day after infection. Collecting the blood and liver of mice, we assayed serum hyaluronic acid(HA), amino-terminal peptide of type III procollagen(PⅢP) and TNF-a by radioimmunoassay. Hydroxyproline(Hyp) was detected from liver. The liver histopathology was also examined by HE and Masson staining and selected liver originated from the left lobe liver tissue.Degree of hepatic fibrosis and area of egg granuloma were analyzed. The expression of collagen I,a-smooth muscle actin (α-SMA)and TNF-αwere examined by immunohistochemical method.We also assayed transcription and gene expression of liver TNF-a in different stages of infection and treatment by RT-PCR and Western blotting.Results 1.We found that each data increased in the model group infected with S.japonicum.And there is no difference between values of model group and menstruum control group, which showed that sodium carboxymethycellulose just play a role of menstruum.It had no effects on hepatic fibrosis.But after therapy with PAE, in the group before and after treatment with PZQ, those data in PAE groups have decreased very obviously than control groups.The serum lever of HA, PIIIP and HYP in liver had decreased. From HE and Masson staining of liver biopsy, we also found that the area of granuloma and degrees of hepatic fibrosis scores in the PAE-treated groups were significantly lower than those in the model and control groups, PAE can reduce the size of periovular granuloma and the degree of hepatic fibrosis.Calculating the value of MOD in immunohistochemistry slice, the data of collagenⅠand a-SMA in PAE-treated group were lower than the control group too(P<0.05 or P<0.01).But in groupⅡ,except the area of granuloma and positive a-SMA cells decreased, there are no difference between treatment group and control group in other datas(P>0.05).2.In measuring TNF-a in sera and liver, it was firstly discovered that TNF-a was correlated with the severity of hepatic fibrosis. The value of TNF-a in mice will advanced in all levels including serum, gene transcription and protein expression after infection with S.japonicum, likewise the same results appeared in the data of MOD(P<0.05).Secondly we discovered that TNF-a decreased in PAE-treated group, which happened with the reduction of hepatic fibrosis simultaneously, suggesting that PAE intervene in hepatic fibrosis rely on TNF-a cytokine. It is obviously to appear in the pre-treated group and post-treated group, TNF-a were all down than control group in the lever of serum, gene transcription and protein expression after treatment with PAE(P<0.05).But in groupⅡ,TNF-αwas reduced in the lever of serum and gene transcription. There is no difference in the level of gene expression(P>0.05).Conclusions 1.PAE can prevent and control the extent of hepatic fibrosis.PAE possesses the effects of reducing the size of periovular schistosomal granuloma and the synthesis of collagen I and a-SMA to resist the development of hepatic fibrosis caused by S.japonicum.Moreover, paeoniflorin can play a role in pre-/post-treatment with PZQ.2.TNF-αwas well correlated with the severity of hepatic fibrosis. Our results indicate that PAE may improve hepatic schistosomiasis through inhabiting the production of TNF-a.