Role Of β-arrestin2 In Hepatic Fibrosis And The Effect Of CP-25

Hepatic fibrosis is a pathologic process that leads to deposition of an excess of extracellular matrix（ECM）,and hepatic stellate cell（HSC）is a major fibrogenic cell type that contributes to collagen accumulation by producing ECM.β-arrestin2 is adaptor protein and signal transduction protein belong to arrestin family,which is well known for regulating G-protein-coupled receptor（GPCRs）signaling and participate in receptor desensitization and internalization.Recent studies have determined thatβ-arrestins not only desensitize GPCR transduction pathways,but also activate signaling cascades independent of G-protein activation and scaffold many intracellular signaling networks by diverse types of receptor,such as the type III TGF-β receptor（TβRIII）.Our previous studies have shown that collagen I expression was decreased in HSC after transfecting β-arrestin2 si RNA and the proliferation of HSC was inhibited.But the role of β-arrestin2 in hepatic fibrosis has not been determined in vivo.Total glucosides of peony（TGP）was extracted from Paeonia lactiflora pall root,it mainly consists of paeoniflorin（Pae）.In order to improve the bioavailability of Pae,a new Pae monomer derivative,paeoniflorin-6’-O-benzene sulfonate（CP-25）is derived from Pae structural modification.Our previous studies showed that both TGP and Pae exert the antifibroticeffects.The present study was designed to investigate whether CP-25 has protective effect on liver fibrosis and its possibile mechanism.To that end,firstly,human liver tissues from liver fibrotic patients in different stages were used to assess the expression of β-arrestin2 and TβRIII.In vivo studies,β-arrestin2-/-mice were used to investigate the role of β-arrestin2 in carbon tetrachloride（CCl₄）-induced liver fibrogenesis.In addition,liver fibrotic mice were intragastrically given CP-25 to investigate its effect.Furthermore,LX-2 cells overexpressed β-arrestin2 were used to explore the mechanism of CP-25.OBJECTIVE Clinical specimens from patients with hepatic fibrosis were used to observe the expression of β-arrestin2 and TβRIII.In vivo studies,β-arrestin2-/-mice were used to investigate the role of β-arrestin2 in carbon tetrachloride（CCl₄）-induced liver fibrogenesis.In addition,liver fibrotic mice were intragastrically given CP-25 to investigate its effect.In vitro,LX-2 cell overexpression β-arrestin2 were applied to explore the mechanism of CP-25.METHODS The expression of β-arrestin2 and TβRIII in liver fibrosis patients and intrahepatic biliary lithiasis patients were determined by immunohistochemistry.CCl₄-induced liver fibrosis was established in wide type（WT）and β-arrestin2-/-C57BL/6J mice.The transaminase activities were detected in serum,the oxidative and antioxidant indices,Hyp were detected in liver homogenate.The proliferation of T,B lymphocyte cells was observed by CCK-8 assay.Percentages of T cell subsets were quantified using flow cytometry.The expression of β-arrestin2,TβRIII,TGF-β1,collagen expression and related signaling pathways were detected by Western blot.Liver fibrosis was induced in male C57BL/6J mice by intraperitoneal injection of CCl₄.The mice were randomly divided into normal control group,liver fibrosis model group,CP-25（25,50,100 mg/kg）group,TGP（100 mg/kg）and colchicine（0.15 mg/kg）group.Indicators related to liver fibrosis were detected.Furthermore,effect of CP-25 on the expression of β-arrestin2,TβRIII,collagen and related signaling pathways in LX-2 cellsoverexpressed β-arrestin2 was detected in vitro.RESULTS1.The expression of β-arrestin2 and TβRIII in liver tissues from liver fibrotic patients The results of immunohistochemistry showed that the expression of β-arrestin2 in liver fibrosis patients was significantly higher than that in intrahepatic biliary lithiasis patients.The expression of β-arrestin2 increased along with the severity of liver fibrosis,while the TβRIII expression decreased in liver fibrotic patients.2.Effect of β-arrestin2 deficiency on liver fibrosis mice CCl₄-induced liver fibrosis was established in WT and β-arrestin2-/-mice.The serum transaminase activities were significantly decreased,and the activity of superoxide dismutase（SOD）,glutathione（GSH）were improved in β-arrestin2-/-mice compared with WT mice.In addition,deficiency of β-arrestin2 reduced the content of malonaldehyde（MDA）and hydroxyproline（Hyp）in liver homogenate.Moreover,the proliferation of T,B cells was inhibited in β-arrestin2-/-mice assayed by CCK-8.Flow cytometry results showed that the percentage of CD4+CD69+ T and Th17/Treg cell ratio were decreased in spleen from β-arrestin2-/-mice compared with WT mice,while the percentage of CD4+CD62L+ T cell was increased.Furthermore,the results of Western blot showed that collagen I and TGF-β1 expression was decreased,but TβRIII expression was increased in β-arrestin2-/-mice,and the activation of Smad2,Smad3,Akt signaling pathway were also inhibited.3.Effect of CP-25 on liver fibrosis mice CCl₄-induced liver fibrosis was established in WT mice.The transaminase activities in serum and the content of MDA,Hyp in liver homogenate were decreased after treatment with CP-25 compared with fibrotic mice.CP-25 also restored the decrease of SOD and GSH activities.In addition,administration of CP-25 significantly inhibited the proliferation of T,B cells.Flow cytometry results showed that the percentage ofCD4+CD69+ T and Th17/Treg cell ratio were decreased treatment with CP-25,while the percentage of CD4+CD62L+ T cell was increased.Moreover,Western blot results showed that CP-25 decreased the expression of β-arrestin2 and collagen I in liver of fibrotic mice.4.Effect of CP-25 on the expression of collagen I,β-arrestin2 and TβRIII in LX-2cell overexpression of β-arrestin2 Western blot results showed that CP-25 10-8¹0-5 mol/L inhibited the expression of collagen I in LX-2 cell stimulated by TGF-β1.In addition,treatment with CP-25 decreased the expression of β-arrestin2 and collagen I,and increased TβRIII expression in LX-2 cells overexpression of β-arrestin2.5.Effect of CP-25 on the expression of Smad2,Smad3,Akt signaling pathway in LX-2 cell overexpression of β-arrestin2 The results of Western blot showed that CP-25 significantly decreased the expression of p-Smad2,p-Smad3 and p-Akt.Treatment with CP-25 significantly inhibited the activation of Smad2,Smad3 and Akt signaling pathway in LX-2 cells overexpression of β-arrestin2.CONCLUSIONS1.β-arrestin2 expression was increased in liver tissue from liver fibrotic patients along with the severity of liver fibrosis.The result suggested that β-arrestin2 plays an important role in the progression of liver fibrosis.2.Deficiency of β-arrestin2 significantly reduced liver damage and symptoms of liver fibrosis in CCl₄-induced liver fibrotic mice.In addition,the percentage of CD4+CD69+T and Th17/Treg cell ratio were decreased,while the percentage of CD4+CD62L+ T cell was increased in β-arrestin2-/-mice.Furthermore,TβRIII expression was increased,the expression of p-Smad2,p-Smad3,p-Akt was decreased following β-arrestin2 deficiency.These results indicated that β-arrestin2 deficiency might alleviate liver fibrosis through regulating percentages of T cell subsets and suppressing Smad 2/3,Akt activation.3.The serum transaminase activities,the content of Hyp and the levels of collagen I,β-arrestin2 were decreased after treatment with CP-25 compared with fibrotic mice.In addition,the percentage of CD4+CD69+ T and Th17/Treg cell ratio were decreased treatment with CP-25,while the percentage of CD4+CD62L+ T cell increased.These results indicated that CP-25 could alleviate liver fibrosis.4.CP-25 inhibited the expression of collagen I,but promoted TβRIII expression in LX-2 cell overexpressed β-arrestin2.It also inhibited Smad 2/3 and Akt activation.These results indicated that CP-25 might alleviate liver fibrosis through downregulatingβ-arrestin2 and strengthen the effect of TβRIII on inhibition Smad 2/3 and Akt activation.