Regulation Of Oxidative Stress By β-arrestin2 In Hepatic Fibrosis Via Modulation Of NOX4 And The Effect Of CP-25

Liver fibrosis is a pathological process characterized by liver structural damage and dysfunction caused by long-term stimulation of pathogenic factors,excessive deposition of extracellular matrix（ECM）and abnormal proliferation of connective tissue.Hepatic stellate cells（HSC）are the main source producing ECM.The liver is vulnerable to reactive oxygen species（ROS）and free radical mediated disorders,releasing a large number of inflammatory factors,which directly stimulate HSC activation,ultimately leading to fibrosis.The phagocyte NADPH oxidase（NOX）is the first identified enzyme system that generates ROS.HSC mainly express NOX1,NOX2,NOX4,and NOX4 is a major source of ROS.β-arrestin2 is a adaptor protein and signal transduction protein that play an important role in signal regulation.Previously,several studies have suggested thatβ-arrestins are well known for negatively regulating G protein-coupled receptor（GPCR）signaling and participate in receptor desensitization and internalization.Recent studies have determined thatβ-arrestins not only desensitize GPCR transduction pathways,but also affect fibrotic diseases by regulating intracellular signaling networks,ECM deposition,inflammation,and oxidative stress.Our previous study showed that the expression ofβ-arrestin2 significantly increased in the patients with hepatic fibrosis;in vitro,after transfectingβ-arrestin2 siRNA in HSC inhibited proliferation and collagen production.However,the relationship betweenβ-arrestin2 and NOX4/ROS in liver fibrosis has not been determined.Total glucosides of peony（TGP）is the first anti-inflammatory immune regulatory drug.Paeoniflorin-6’-O-benzene sulfonate（CP-25）is a monomer derived from a structural modification of Pae,which is the active ingredient of TGP.Both TGP and Pae have shown more potent antiinflammatory and antioxidant activities.The present study was designed to investigate whether CP-25 has protective effect on liver fibrosis by anti-oxidant via modulation of NOX4.In vivo studies,β-arrestin2^-/-mice were used to investigate the role ofβ-arrestin2 in carbon tetrachloride（CCl₄）-induced liver fibrogenesis.In addition,liver fibrotic mice were intragastrically given CP-25 to investigate its effect.In vitro,the small interfering RNA（siRNA）of NOX4 andβ-arrestin2 was transfected in LX-2 cells respectively,and the collagen production,ROS level and downstream signaling were detected.Furthermore,the effects of CP-25 on the expression of ROS,NOX4 and collagen were also investigated.OBJECTIVE WT C57BL/6J mice andβ-arrestin2^-/-mice were used to establish liver fibrosis model to investigate the role ofβ-arrestin2 and NOX4 in vivo.In addition,CCl₄-induced WT liver fibrosis mice were given CP-25 to investigate its effect.In vitro,β-arrestin2 and NOX4 siRNA was transfected in LX-2 cells to detect the expression of ROS,NOX4,collagen Ⅰ and the related signaling pathways.Furthermore,CP-25 was added to detect its possible mechanism.METHODS CCl₄-induced liver fibrosis was established in WT andβ-arrestin2^-/-C57BL/6J mice.Dihydroethidium（DHE）staining was performed to measure ROS generation.The expression of NOX4,collagen,MMP-13,TIMP-1 was detected by Western blot.In addition,C57BL/6J mice were intraperitoneally injected CCl₄ to induce liver fibrosis model,and were divided into normal group,model group,CP-25（25,50,100 mg/kg）group and TGP group（100 mg/kg）.The oxidative stress related indicators were measured.In vitro,β-arrestin2 and NOX4 siRNA was transfected in LX-2 cells to explore the oxidative stress related indicators and related signaling pathways.Furthermore,CP-25 was incubated with LX-2 cells to detect oxidative stress by DCFH-DA fluorescent probe and flow cytometry.RESULTS1.Effect ofβ-arrestin2 deficiency on liver fibrosis mice Liver fibrostic model was established in WT andβ-arrestin2^-/-mice.Western blot results showed that the expression of NOX4,collagen Ⅰ and Ⅲ in the liver was significantly decreased,and the ratio of MMP-13/TIMP-1 was increased inβ-arrestin2^-/-mice compared with the WT model mice.2.Effect ofβ-arrestin2 deficiency on ROS production The fibrotic mice liver was dehydrated and frozen.Dihydroethidium（DHE）staining was performed to measure ROS level.The results showed that the ROS level inβ-arrestin2^-/-fibrotic mice was significantly lower than that in WT model mice.3.Effect of CP-25 on CCl4-induced liver fibrosis mice CCl₄-induced liver fibrosis was established in WT mice.Western blot results showed that CP-25 decreased the expression of NOX4,collagen Ⅲ andβ-arrestin2 in liver of fibrotic mice.Meanwhile,CP-25 increased MMP-13/TIMP-1 ratio.4.Effects of NOX4 siRNA andβ-arrestin2 siRNA on the expression of ROS,NOX4,collagen Ⅰ and related signaling in LX-2 cells To investigate the role of NOX4 in LX-2,NOX4 siRNA was used to silence the mRNA expression of NOX4.The result showed that silencing of NOX4 gene expression significantly inhibited ROS production and collagen Ⅰ expression in AngⅡ stimulated LX-2 cells.After transfectingβ-arrestin2 siRNA in LX-2 cells,the ROS level and expressions of NOX4,collagen Ⅰ,p-ERK and p-JNK were significantly inhibited.5.Effect of CP-25 on the expression of ROS,NOX4,collagen Ⅰ andβ-arrestin2 in LX-2 cells The results of DCFH-DA fluorescent probe showed that CP-25 inhibited the ROS production in LX-2 cells stimulated by AngⅡ.Western blot results showed that CP-25decreased the ROS production and expression of NOX4,collagen Ⅰ,β-arrestin2 in LX-2cells stimulated by AngⅡ.CONCLUSIONS 1.Inhibition ofβ-arrestin2 alleviated the degree of liver fibrosis in CCl₄-induced liver fibrosis mice.In addition,Western blot results showed that NOX4 and collagen expression was significantly decreased,but MMP-13/TIMP-1 ratio was increased inβ-arrestin2^-/-mice.2.β-arrestin2 deficiency significantly inhibited the production of ROS in fibrotic mice.The result suggested thatβ-arrestin2 may be through regulating ROS involved in the development of liver fibrosis.3.Silence of NOX4 expression by siRNA decreased ROS and collagen Ⅰ expression,andβ-arrestin2 siRNA downregulated ROS level and NOX4,collagen Ⅰ expression,and ERK,JNK activation in LX-2 cells.These results indicate that decreasedβ-arrestin2expression may be through inhibiting NOX4 and downstream ERK,JNK signals,thus inhibit ROS production and collagen synthesis in LX-2 cells.4.CP-25 maybe through inhibitingβ-arrestin2 to decrease NOX4 expression,finally inhibited ROS and collagen production,has protective effect on liver fibrosis.