The Mechanism Of Indoleamine 2,3-dioxygenase 1 And Regulation Of Dahuang Zhechong Pill In Hepatic Fibrosis

BackgroundIndoleamine 2,3-dioxygenase 1(IDO1)is an intracellular rate-limiting enzyme in the metabolism of tryptophan along the kynurenine pathway,subsequently mediating the immune response;however,there is no intensive study of IDO1 in liver fibrosis and the role of it in this disease is still unclear.In the meanwhile,Chinese medicine in the regulation of immune and prevention of liver fibrosis has a unique advantage.Dahuang Zhechong Pill is clinically used to treat hepatic fibrosis and cirrhosis induced by various causes.Therefore,for the effective prevention and regulation of hepatic fibrosis,we explored the immune micro-environmental mechanism of Dahuang Zhechong Pill against liver fibrosis and regulatory mechanism of IDO1 as a target.Moreover,exploring a new way for development of new drugs against liver fibrosis is of great important.MethodsFor animals,carbon tetrachloride(CCl4)was used to establish liver fibrosis in wild-type and IDO1 knockout mice.Additionally,an IDO1 inhibitor,Dahuang Zhechong Pill and colchicine was administered to WT fibrosis mice.After modelling,the situation of liver lesions and apoptosis were observed by Western blot and TUNEL;the degree of hepatic fibrosis,expressions of IDO 1 related proteins and the changes of immune microenvironment were observed by H&E staining,Sirius red staining,Immunofluorescence staining,ELISA,Western blot and qPCR.Patients with hepatitis B virus-induced cirrhosis and healthy volunteers were enrolled and the correlations between IDO 1 serum levels and liver lesions and stiffness in the subjects were observed by ELISA.All data were analysed and performed using SPSS 22.0 and GraphPad Prism software respectively.All the data are presented as the means ± standard deviation.Values of P<0.05 were considered statistically significant.ResultsFirst,IDO1 knockout mice were protected from CCl4-induced liver fibrosis,as reflected by unchanged serum alanine transaminase and aspartate transaminase levels and lower collagen deposition,a-smooth muscle actin expression and apoptotic cell death rates in comparison with WT fibrosis mice(P<0.05).On the other hand,the expression of tryptophan 2,3-dioxygenase(TDO)and general control nonderepressible 2 kinase(GCN2)in liver tissues of IDO1 knockout mice was increased(P<0.05)and the levels of interleukin-17a(IL-17a)and downstream cytokines’ mRNA were reduced(P<0.05).Moreover,liver lesions were positively correlated with serum IDO1 levels in both the clinical subjects and hepatic fibrosis mice(P<0.05).A positive correlation between serum IDO1 levels and liver stiffness values was found in the cirrhosis patients(P<0.05).Further more,CCl4-induced hepatic fibrosis injury was resisted by high dose of Dahuang Zhechong Pill.Compared with WT fibrosis mice,the hepatic expression of TDO was increased(P<0.05)while the levels of IDO1 and hepatic IL-17 mRNA were reduced in mice administered by high dose of Dahuang Zhechong Pill(P<0.05).ConclusionsIn this present study,IDO1 deficiency attenuated the development of CC14-induced fibrosis in mice.As a result of IDO1 deficiency,TDO exhibited a compensatory increase,subsequently inducing the level of IL-17a.This appearance caused the reduction of release of hepatic cytokines and inhibited the activation of hepatic stellate cells,which attenuated the development of liver fibrosis.Moreover,the level of serum IDO1 was positively correlated with liver lesions both in mice and clinical subjects.Thus,the level of serum IDO1 might be regarded as a potential hallmark of liver lesions and the degree of liver fibrosis.At last,it was proved that Dahuang Zhechong Pill protected mice from CCl4-induced fibrosis mediated by IDO1 regulation.This application supports the feasibility of IDO1 as a potential target for screening drugs with therapeutic effect,or even as a therapeutic molecule for clinic treatment,against hepatic fibrosis.