An Experimental Study On The Preventative Treatment Effects Of AKF-PD On Rat Liver Fibrosis Induced By Dimethyl Nitrosamine

Background:Liver fibrosis is a self-wound healing process following different chronic liver injuries,and a common pathological basis of liver cirrhosis developed from various chronic liver diseases. With a high incidence of liver diseases,China takes heavy burden in the treatment of liver fibrosis and cirrhosis.The active early prevention and treatment onchronic liver diseases,which aims to slowdown the occurrence and progression or even to reverse liver fibrosis,is of important and urgent significance.The anti-hepatic fibrosis effects of chemical drugs,natural medicines,biological agent,and gene therapy on the development and progression of liver fibrosis had been previously approached.The application of corticosteroid,silymarin,interferon, colchicines,and gene transfer mediated by vectors had been successively carried out for the treatment of liver fibrosis.The prevention and treatment effects of salvia,astragalus,peach kernel,861 mixtures, Fuzheng Huayu Capsules,and shell liver softening tablets on liver fibrosis had also been investigated in China.However,all the antifibrotic strategies listed above have gotten limited efficacy so far.Pirfenidone (PFD) is found to be a novel pyridone agent with broad spectrum high potency in anti-fibrosis.A series of in vitro and in vivo experiments have reported that PFD can attenuate liver fibrosis,pulmonary interstitial fibrosis,as well as renal fibrosis.With regard to the treatment of pulmonary fibrosis,PFD has been issued the permit for stage-Ⅲclinical trails by FDA in the United States.Meanwhile,PFD exerts its antifibrotic functions in the liver via inhibiting lipid peroxidation and some factors, such as tumor necrosis factor-α(TNF-α) and nuclear factor-κB(NF-κB), alleviating inflammation,regulating Various growth factors,as well as inhibiting the activation and proliferation of hepatic stellate cells(HSCs). Moreover,PFD can even reverse liver fibrosis by regulating the degradation of accumulated extracellular matrix(ECM).Fluorofenidone (AKF-PD) is a brand new pyridone agent synthesizedby Dr.Lijian Tao and Gaoyun Hu,Central South University,China.Receiving a national patent,AKF-PD has potential broad-spectrum antifibrotic characteristics due to the similar chemical structure to pirfenidone.The preliminary experiments in our GLP laboratory have demonstrated its low toxicity and antifibrotic functions in renal interstitial fibrosis.However,its preventive and therapeutic effects on liver fibrosis remain unclear. Constructing a rat model of liver fibrosis induced by dimethyl nitrosamine(DMN),the present study is to investigate the antifibrotic effects of AKF-PD on liver fibrosis and the related molecular mechanisms. Part 1 The preventive treatment effects of AKF-PD on rat liver fibrosis induced by DMNObjectives:To investigate the preventive treatment effects of AKF-PD on rat liver fibrosis induced by DMN.Methods:140male Wister rats were randomly divided into seven groups with 20 animals in each group as normal group(NG),normal rats treated with AKF-PD at the dose of 500mg/kg(AKF),model group of liver fibrosis induced by four-week intraperitoneal injection of 1%DMN at 1ml/kg body weight(DMN),DMN groups preventively treated with AKF-PD at three dosages(DMN+AKF,1000mg/kg,500mg/kg,and 125mg/kg),DMN group preventively treated with PFD at the dose of 500mg/kg(DMN+PFD),respectively.All the groups of drug treatment and drug excipient treatment as the control were administered by oral garage once daily from the beginning of rat model construction of liver fibrosis.All rats were sacrificed five days after the last DMN administration and drug treatment.The rats' serum were collected for further biochemical detection of serum alanine aminotransferase(ALT), aminotransferase(AST),total bilirubin(TBIL),and albumin(ALB).The liver tissue specimens were performed pathological examination and semi-quantitative scoring for liver inflammation and fibrosis through HE and Masson staining.The hydroxyproline content in liver tissue was measured by high perform liquid chromatography(HPLC).Results:All rats survived until the final processing.Compared with that in DMN group,serum AST and TBIL levels were significantly lower in NG(p＜0.01),AKF,DMN+AKF,and DMN+PFD groups(p＜0.05).The serum ALB level in NG,AKF,DMN+AKF,and DMN+PFD groups was significantly higher than that in DMN group(p＜0.01,p＜0.05,p＜0.05, respectively).Compared with that in NG,AKF,DMN+AKF,and DMN+PFD groups,the semi-quantitative scores of liver inflammation and liver fibrosis,as well as the hydroxyproline content in liver tissue in DMN group increased significantly(p＜0.01,respectively).Conclusions:AKF-PD has a significant biological function of hepatocyte protection indicated by decreasing serum AST and TBIL levels,increasing serum ALB level,attenuating the semi-quantitative scores of liver inflammation via inhibiting inflammatory cell infiltration, as well as the degeneration and necrosis of rat liver tissue induced by DMN.Moreover,AKF-PD has a significant preventative treatment effect on rat liver fibrosis induced by DMN via reducing the hydroxyproline content and the deposition of fibrous matrix in liver tissue. Part 2 A study on the molecular mechanisms of the preventive treatment of AKF-PD on rat liver fibrosis induced by DMNObjectives:To explore the molecular mechanisms of the antifibrotic effects of AKF-PD in rat liver fibrosis model induced by DMN through the investigation of TypeⅠ,Ⅲcollagens,smooth muscleα-actin(α-SMA), transforming growth factor-β1(TGF-β1),connective tissue growth factor (CTGF),and tissue inhibitor of metalloproteinase 1(TIMP-1) in rat liver tissue.Methods:The liver tissue specimens were randomly collected from five rats in each group for immunohistochemical staining to detect the protein expression of TypeⅠ,Ⅲcollagens,α-SMA,TGF-β1,and CTGF. Meanwhile,mRNA expression levels of TypeⅠ,Ⅲcollagens,α-SMA, TGF-β1,and CTGF in rat liver tissue were approached by Real-time PCR. Western blot was employed to measure the protein expression of TIMP-1 in liver tissue.Results:The immunohistochemical staining demonstrated that the protein expression levels of TypeⅠ,Ⅲcollagens,α-SMA,TGF-β1,and CTGF were significantly higher in DMN group than that in NG,AKF, DMN+AKF,and DMN+PFD groups(p＜0.01,respectively).The mRNA expression detection results by Real-time PCR were consistent with that of protein expression levels measured by immunohistochemical staining (p＜0.01 or p＜0.05,respectively).However,the mRNA expression difference of TypeⅢcollagen among DMN and treatment groups was not statistically significant(p＞0.05).The results of Western blot indicated that TIMP-1 protein expression of liver tissue in DMN group was significantly higher than that in NG,AKF,DMN+AKF,and DMN+PFD groups(p＜0.01,or p＜0.05,respectively).Conclusions:AKF-PD attenuate rat liver fibrosis via down-regulating mRNA and protein expression of TypeⅠ,Ⅲcollagens,α-SMA,TGF-β1,as well as CTGF in liver tissue to inhibit the HSCs activation.Meanwhile,AKF-PD regulates the degradation of ECM through inhibiting the expression of TIMP-1.SummaryAKF-PD is a novel pyridone agent being issued a national invention patent in Sept.2005(Patent No.:ZL02114190.8).Our early experiments have demonstrated its antifibrotic functions in renal interstitial fibrosis. Moreover,AKF-PD exerted its potential biological effects on the prevention and reversal of liver fibrosis.Constructing a rat model of liver fibrosis induced by dimethyl nitrosamine(DMN),AKF-PD was applied for the first time to preventively treat rat liver fibrosis in the present study.The preventive treatment effects of AKF-PD on rat liver fibrosis were assessed by both pathological and serological assays,such as HE tissue staining,Masson collagen staining,and HPLC for hydroxyproline detection in liver tissue. The antifibrotic mechanisms of AKF-PD were explored respectively at histology,mRNA,and protein level by the methods of immunohistochemical staining,Real-time PCR,and Western blot.The results demonstrated that AKF-PD significantly decreased rat serum AST and TBIL,elevated serum ALB level,attenuated inflammatory cell infiltration,and inhibited the degeneration and necrosis of rat liver tissue induced by DMN,so as to relieve the inflammation of rat liver induced by DMN.Meanwhile,AKF-PD significantly decreased the hydroxyproline content and the deposition of fibrous matrix in liver tissue. Moreover,AKF-PD attenuated rat liver fibrosis via down-regulating mRNA and protein expression of TypeⅠ,Ⅲcollagens,α-SMA,TGF-β1, as well as CTGF in liver tissue.Furthermore,AKF-PD significantly down-regulated the degradation of ECM via its inhibition on TIMP-1 expression in rat liver tissue.The present study demonstrates the preventative treatment effects of AKF-PD on rat liver fibrosis induced by DMN.The molecular mechanisms are supposed to be associated with the attenuation of liver inflammation,the inhibition on the exPression of TypeⅠandⅢcollages, α-SMA,CTGF,TGF-β1,and TIMP1,as well as the regulation of ECM synthesis and degradation in rat liver tissue.