Experimental Study Of A Novel Pan-PPAR Agonist MBT1805 On The Treatment Of Cholestatic Liver Injury

Background and aims:Cholestatic liver injury is a disease in which bile acids accumulate in the liver resulting in hepatic cells damage due to a variety of causes,such as impaired bile acids production or transport,or mechanical obstruction of intrahepatic/extrahepatic biliary ducts.Primary biliary cholangitis(PBC),drug induced liver injury(DILI),and primary sclerosing cholangitis(PSC)are included.Chronic cholestasis may gradually aggravate liver damage,leading to cirrhosis and end-stage liver disease eventually.At present,there is still a clinical need for the treatment of cholestatic liver injury.Ursodeoxycholic acid(UDCA)was the only recommended treatment for PBC until 2016 when obeticholic acid(OCA)was approved for the treatment of PBC in combination with UDCA in adults with an inadequate response to UDCA or as monotherapy in adults unable to tolerate UDCA.However,up to 30-40%PBC patients have inadequate response to standard UDCA treatment,and OCA has the side effect of aggravating itching.Therefore,novel therapeutic targets need to be further explored.In recent years,clinical trials targeting peroxisome proliferator-activated receptor(PPAR)for the treatment of PBC have achieved encouraging results.Treatment combination with UDCA and PPAR agonists(bezafibrate,fenofibrate)significantly reduced alkaline phosphatase(ALP)levels and improved the response rates of PBC patients who did not respond adequately to UDCA monotherapy.In addition,relieved pruritus was observed in some PBC patients in combination therapy group.In the latest Asian pacific association for the study of the liver(APASL)clinical guidelines on the management of PBC,fibrates(PPAR agonists)are recommended as combination therapy for PBC.Peroxisome proliferator-activated receptors belong to the nuclear receptor superfamily,including PPAR?,PPAR?/?and PPAR?,which control gene expression of energy metabolism,cell development and differentiation.The important biological roles determine their important significance in medical research and drug discovery.Due to differences in tissue distribution,ligand sensitivity,and target genes,the three PPAR isoforms have different but complementary physiological function.In clinical practice,fibrates as selective PPAR?agonists are often used in the treatment of hyperlipidemia.Equally,thiazolidinediones(TZDs)as selective PPAR?agonist including pioglitazone and rosiglitazone are used for the management of type 2 diabetes mellitus.The clinical success of fibrates and TZDs has led to the development of a variety of single receptor agonists as well as novel PPAR agonists,including dual/pan PPAR agonists,with a view to synthesizing the advantages of activation of single receptor and avoiding the side effects of single receptor overactivation.MBT1805 in this study is a novel pan-PPAR agonist.Previous studies have demonstrated that MBT1805 could activate PPAR?,PPAR?/?and PPAR?equally(EC₅₀:8.46?M,11.15?M,11.94?M respectively).Considering the prospect of PPAR agonist on the treatment of PBC and the pharmacological properties of MBT1805,we investigated the anti-cholestatic effects of MBT1805 in vitro and in vivo,and evaluated whether MBT1805 could be a potential treatment for cholestatic liver injury.Materials and methods:We constructed a cell model of in vitro cholestatic injury in Hepa RG cell lines with bile acids mixtures.Cell viability,release of lactic dehydrogenase(LDH),and the contents of intracellular total bile acid(TBA)were detected to evaluate the protective effect of MBT1805 on Hepa RG cell.Quantitative real-time PCR(q RT-PCR)was used to detect the effects of MBT1805 on the expression of bile acid synthase and transporters.In addition,PPAR?was knocked down by small interfering RNA(si RNA),further verifying the PPAR?dependence of MBT1805.Inflammation is an important factor in the progression of cholestatic liver injury.Phorbol 12-myristate 13-acetate(PMA)was used to induce the differentiation of human THP-1 cell into M0 macrophage.Lipopolysaccharide(LPS)was used to induce the formation of inflammation.Enzyme linked immunosorbent assay(ELISA),flow cytometry(FCM)and western blot were used respectively to detect the effects of MBT1805 on the proinflammatory factors expression including interleukin-1?(IL-1?),interleukin-6(IL-6)and tumor necrosis factor?(TNF?),M1/M2 macrophage polarization and NF-?B pathway.PPAR?/?was knocked down by si RNA as control as well as selective PPAR?/?agonist GW501516.Alpha-naphthylisothiocyanate(ANIT)induced cholestatic liver injury model and3,5-diethoxycarbonyl 1,4-dihydrocollidine(DDC)induced sclerosing cholangitis model were used to evaluate whether MBT1805 had an anticholestatic effect in vivo.Based on the results of in vivo studies,untargeted metabolomics and bile acid targeted metabolomics were used to investigate the changes of hepatic metabolism in ANIT induced cholestatic liver injury model.Possible mechanism of MBT1805 was further explored and confirmed by q RT-PCR and western blot.Results:MBT1805 had protective effects on Hepa RG cell lines treated with bile acids mixtures,including increased cell viability,decreased LDH release,and decreased intracellular TBA content.Protective effects of MBT1805 were reduced by si RNA knockout of PPAR?.q RT-PCR results indicated that MBT1805 significantly inhibited cholesterol 7?-hydroxylase(CYP7A1)and cholesterol 27?-hydroxylase(CYP27A1),which inhibited bile acid synthesis.Moreover,the expression of multidrug resistant associated protein 2(MRP2)and bile salt export pump(BSEP)was increased,indicating bile acids excretion was increased,thereby reducing the intracellular bile acid concentration and protecting cells from the toxic effects of bile acids.In THP-1 induced macrophage,LPS stimulation activated the proinflammatory expression of macrophages and increased the secretion of inflammatory cytokines.Flow cytometry results showed that MBT1805 promoted the differentiation of macrophages into M2 macrophages and had anti-inflammatory effects.ELISA results indicated that MBT1805 reduced the release of proinflammatory effects.Western blot results showed that MBT1805 inhibited NF-?B pathway.The above results about MBT1805 on THP-1 induced macrophage were similar to that of PPAR?/?agonist GW501516,but were opposite with si-PPAR?/?group.Above results indicate that MBT1805 has anti-cholestatic and anti-inflammatory effects in vitro.MBT1805 significantly reduces serum ALT,AST,ALP and Tbil levels of mice in ANIT induced cholestasis,and improved necrosis and inflammatory infiltration of liver tissues.While,in DDC induced sclerosing cholangitis model,the same results were not observed in MBT1805 treatment group.However,masson staining of liver tissues showed that MBT1805 effectively inhibits liver fibrosis compared with DDC model group.793 metabolites were identified in untargeted metabolomics about ANIT induced cholestasis.Principle component analysis(PCA)and partial least squares-discriminant analysis(PLS-DA)as well as analysis of variance were performed on all the metabolites,and 27 metabolites were identified as differential metabolites.Further pathway analysis about 27 metabolites indicated that MBT1805 affected the primary bile acid biosynthesis pathway.Bile acid targeted metabolomics quantifies more than20 bile acid,and MBT1805 significantly reduced the contents of abnormally elevated bile acids caused by ANIT.The expression of bile acid synthase and transporters of live tissues detected by western blot and q RT-PCR indicated that MBT1805 could inhibit bile acid synthesis,increase the phase?metabolic reaction of bile acid in hepatocytes,which reduce the toxicity of hydrophobic bile acid,thus playing an anti-cholestasis role.Conclusion:1.In vitro studies have shown that MBT1805 alleviates the cytotoxicity of Hepa RG cells caused by the accumulation of bile acids mixtures in a PPAR?dependent manner.2,MBT1805 promotes macrophages polarization into M2 macrophage,and has anti-inflammatory effects,inhibiting the expression of NF-?B pathway.3.MBT1805 alleviates ANIT induced cholestasis and inhibits liver fibrosis in DDC induced sclerosing cholangitis in mice.4.As a novel pan-PPAR agonist,MBT1805 is a potential treatment for cholestasis liver injury.