The Role Of Orphan Nuclear Receptor NR4A2 In The Regulation Of Hepatic Stellate Cell Activation And Hepatic Fibrosis

Hepatic fibrosis is pathological repair secondary to kinds of insults.It is characterized with excessive deposition of extracellular matrix in liver.Myofibroblast is the center cell of producing collagen and plays an important role in liver fibrogenesis.And activated hepatic stellate cell is the main cell source of myofibroblast.Multiple cytokines and signal transduction pathways are involved in the activation of hepatic stellate cells.But the exact mechanism of hepatic stellate cell activation is not yet entirely clear,especially the details of signaling pathways downstream and the signaling molecule regulation on the gene transcription need to clarify.In recent years,scholars have focused on the correlation between nuclear transcription factors and hepatic stellate cell activation in the liver fibrogenesis and thought that AP-1,Smads,Foxf1,JunD,cAMP response element binding protein,c-Myb and many other transcription factors could activate hepatic stellate cells and promote collagen secretion as transcription factors.Our previous study showed that luteolin inhibited the activation of hepatic stellate cells through ERK5 signaling pathway and thus prevented liver fibrosis.In another study we found Hyperoside induced expression of NR4 A subfamily via ERK /CREB pathway in vascular smooth muscle cells which resulted in suppressed proliferation.So,we speculate NR4 A family may be involved in the regulation of hepatic stellate cell activation.The preliminary experiments showed:(1)In activated hepatic stellate cells NR4A2 expression was significantly reduced while no change occurred in NR4A1 and NR4A3 level.(2)NR4A2 expression decreased in fibrotic liver tissue.Based on above research and previous report we hypothesized: NR4A2 orphan nuclear receptors could regulate hepatic stellate cells and liver fibrosis development.Objective: Study the effect and mechanism of orphan nuclear receptor NR4A2 on activated hepatic stellate cells and liver fibrosisMethods:(1)NR4A2 expression was measured by Western blot and quantitative real-time PCR after HSC-T6 cells were stimulated by PDGF and TGF-? respectively.The expression of ?-SMA and NR4A2 was measured by real time quantitative PCR after rat primary hepatic stellate cells were isolated.(2)NR4A2 expression in fibrotic liver tissue and normal liver tissue was examined by immunohistochemistry,immunofluorescence and quantitative real-time PCR.(3)Small interfering NR4A2 siRNA and AdNR4A2 were used to transfect HSC-T6 cells or primary rat hepatic stellate cells,then the expression of NR4A2,? –SMA and Col1 was measured by quantitative PCR,cell cycle and apoptosis was examined by flow cytometric analysis and cell proliferation was examined by CCK8 assay,phosphorylated protein of P38,JNK and ERK1 / 2 was measured by Western blot,the NR4A2 translocation was measured by cell fluorescence assay and the migration ability was measured by Transwell assay.(4)SD rats were randomly divided into four groups and DMN solution and AdNR4A2 adenovirus were injected.Liver tissue paraffin sections were subjected to HE staning,Sirius Red and Masson staining analysis.Hydroxyproline content was measured by alkaline hydrolysis assay.Apoptosis in liver tissue was examined by TUNEL in situ apoptosis detection assay.Results:(1)In activated HSC-T6 cells NR4A2 expression significantly reduced,no change occurred in NR4A1 and NR4A3 expression.?-SMA expression increased while NR4A2 expression reduced significantly after primary rat hepatic stellate cells were isolated.(2)NR4A2 expression markedly decreased in both human liver cirrhosis tissue and rat fibrotic liver tissue compared with that in normal tissue.(3)With NR4A2 knockout in hepatic stellate cells ?-SMA and Col1 was up-regulated,cell apoptosis rate decreased,proliferative ability enhanced,the percentage of cells in G1 phase decreased and the percentage in S phase increased,the proteinphosphorylation of P38,JNK and ERK1 / 2 downregulated.(4)When NR4A2 was over-expressed in primary rat hepatic stellate cells,the mRNA level of ?-SMA decreased by more than 50%.While NR4A2 was over-expressed in HSC-T6 cells,the mRNA level of ?-SMA decreased by more than 70%,cell apoptosis rate increased,the percentage of cells in G1 phase increased and the percentage in G2 phase decreased,protein phosphorylation of P38 and ERK1 / 2 up-regulated,more NR4A2 distributed in cytoplasm and the ability of migration and invasion weakened.(5)Adenovirus AdNR4A2 treatment ameliorated liver fibrosis in rats.Hydroxyproline content in the model group was significantly higher than that in the normal group while the level of AdNR4A2 group was less than the level of model group or AdNC group.NR4A2 expression significantly reduced while ?-SMA increased in fibrotic rats compared with that in normal group.NR4A2 expression significantly increased while ?-SMA decreased in AdNR4A2 group than that in AdNC group.In normal liver tissue apoptotic cells were rarely observed and there were more apoptotic cells in AdNR4A2 group than AdNC group.Conclusion:(1)In activated hepatic stellate cells and fibrotic liver tissue NR4A2 expression reduces evidently.(2)The NR4A2 knockout in hepatic stellate cells could promote cell proliferation,reduce cell apoptosis,inhibit MAPK pathway.Overexpression of NR4A2 in hepatic stellate cells leads to cell cycle arrest,increased cell apoptosis rate,MAPK pathway activation,increased NR4A2 cytoplasmic translocation and weakened ability of migration and invasion.NR4A2over-expression inhibits liver fibrosis and induces cell apoptosis in vivo.This study will help to understand comprehensively the molecular mechanism of liver fibrosis,on one hand to elucidate the effect and mechanism of orphan nuclear receptor NR4A2 on liver fibrosis,on the other hand to provide new targets and ideas for anti-hepatic fibrosis.