Role Of Epac1 On Phenotypic Transformation Of Hepatic Stellate Cells And The Effect Of CP-25

Hepatic fibrosis is a pathological process of abnormal connective tissue hyperplasia caused by excessive deposition of extracellular matrix（ECM）after long-term stimulation by injury factors.Hepatic stellate cell（HSC）is the main source of ECM.Normally,HSC as a store of vitamin A remains stationary in the perinusional space,but in the case of injury,HSC transforms into myofibroblasts,releasing cytokines that promote ECM production,a process known as phenotypic transformation.The exchange protein activated by c AMP（Epac）is involved in many important signaling pathways,including fibrosis.When Epac1 is activated,Ras-related protein-1（Rap1）is replaced by active Rap1-GTP from inactive Rap1-GDP,thus activating the downstream signaling pathway to play its role.However,the role of Epac1 in phenotypic transformation of HSC remains unclear.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.Our previous studies showed that CP-25 had protective effect on CCl₄-induced hepatic fibrosis mice,and that CP-25 at 100 mg/kg had stronger protective activity than TGP at the same dose.In our present study,liver samples from patients with liver fibrosis were collected to observe the expression ofα-SMA and Epac1.CCl₄ was used to induce C57BL/6J mice to establish hepatic fibrosis model.At the same time,CCl₄-induced hepatic fibrosis mice were intragastrically given CP-25 to observe the expression of Epac1 and the effect of CP-25 on fibrosis.In vitro,LX-2 cells were treated with Epac1 agonist or antagonist,the activation and downstream signals in LX-2 cells were observed.In addition,transforming growth factor beta 1（TGF-β1）was used to stimulate LX-2 cells in vitro.Small interfering RNA（si RNA）targeting Epac1was transfected to detect changes of cell activation and related downstream signaling pathways.Different concentrations of CP-25 were given to investigate the expression ofα-SMA,collagen,p-ERK,p-AKT and Rap1-GTP.OBJECTIVESpecimens from patients with hepatic fibrosis were collected to detect the expression ofα-SMA and Epac1.CCl₄-induced hepatic fibrosis model was established and the mice were intragastrically administered CP-25 to observe the expression of Epac1.In vitro,the activation of HSC and the related signals were detected in LX-2 cells after activating or inhibiting Epac1.CP-25 was administered to observe its effect on phenotypic transformation of HSC.Thus tocertificate the possible role of Epac1 in HSC phenotypic transformation,and to clarify the partial mechanism of CP-25.METHODSDouble immunofluorescence staining was performed to detect the co-localization ofα-SMA and Epac1 in clinic specimens from hepatic fibrosis patients and fibrotic mice induced by CCl₄.The expression of Epac1 was detected by Western blot in fibrotic mice.In addition,mice were randomly divided into normal group,liver fibrosis model group,CP-25 group（25,50,100 mg/kg）,TGP group（100 mg/kg）.The expression ofα-SMA,type III collagen,Epac1,p-AKT,p-ERK in liver tissues was detected by Western blot and immunohistochemical staining.In vitro,after selective stimulation or inhibition of Epac1,CCK-8 and wound-healing assay were used to observe the proliferation and migration of LX-2 cells.Western blot was used to examine the expression ofα-SMA,type III collagen,type I collagen and the activation of AKT,ERK pathways.The level of Rap1-GTP was detected by Pull-down assay.After transfection with Epac1 si RNA,the expression ofα-SMA,type III collagen,downstream signaling pathway and Rap1-GTP level in HSC were further determined.Furthermore,the effect of CP-25 on LX-2 cells were determined by Western blot and pull-down assay.RESULTS1. Expression of Epac1 in activated HSC of patients with hepatic fibrosisThe results of immunofluorescence double staining showed that the expression ofα-SMA（the maker of activated HSC）in liver of hepatic fibrosis patients was higher than that of patients with intrahepatic biliary lithiasis,and the expression of Epac1 in hepatic fibrosis patients was also increased.2. Expression ofα-SMA and Epac1 in liver of fibrotic miceThe results of immunofluorescence double staining showed thatα-SMA and Epac1expression in liver of mice after 8 weeks of CCl₄injection was increased compared with that of normal mice.Western blot assays indicated that the expression of Epac1 protein in the cell cytoplasm was significantly decreased,while the expression of Epac1 in the cell membrane was significantly increased at 4 weeks after CCl₄ injection.3. Effect of selective activation or inhibition Epac1 on LX-2 cell phenotypic transformation and downstream signalingCCK-8,wound-healing assay results showed that 8-p CPT（non-selective agonist of Epac）or selective activation of Epac2（ESI-05）significantly promoted the proliferation and migration of LX-2 cells.Compared with the 8-p CPT group,selective inhibition of Epac1（CE3F4）significantly inhibited the proliferation and migration of LX-2 cells.Western blot results showed that compared with control group,8-p CPT or ESI-05increased the expression ofα-SMA,type III collagen,type I collagen,p-ERK,p-AKT in LX-2 cells.Compared with the 8-p CPT group,CE3F4 reduced the expression ofα-SMA,type III collagen,type I collagen,p-ERK,and p-AKT.Pull-down results showed that 8-p CPT or ESI-05 increased Rap1-GTP levle in LX-2 cells.Compared with the 8-p CPT group,CE3F4 decreased Rap1-GTP level.4. Effect of Epac1 si RNA on the expression ofα-SMA,type III collagen and related signaling in LX-2 cells stimulated by TGF-β1Western blot showed that TGF-β1 significantly promoted the expression ofα-SMA,type III collagen,p-ERK,and p-AKT in LX-2 cells.After transfection of Epac1 si RNA,the expression ofα-SMA,type III collagen,p-ERK,and p-AKT in LX-2 cells significantly decreased compared with TGF-β1 stimulated cells.Pull-down results showed that TGF-β1 significantly increased the Rap1-GTP level in LX-2 cells,and Rap1-GTP level was significantly reduced after LX-2 cells transfected Epac1 si RNA.5. Effect of CP-25 onliver fibrosis miceThe results of HE and Masson staining showed that CP-25 could reduce collagen deposition and reduce the degree of liver fibrosis.Western blot results showed that the expression of collagen III andα-SMA in mice liver was decreased after CP-25 treatment.Furthermore,CP-25 could reduce Epac1 expression confirmed by immunohistochemical assay and Western blot.6. Effect of CP-25 on LX-2 cells stimulated by TGF-β1The results of Western blot indicated that CP-25 10^-6～10^-5 mol/L could inhibit the expression ofα-SMA,type III collagen,p-ERK,and p-AKT in LX-2 cells.Pull-down results showed that CP-25 10^-5 mol/L significantly inhibited the level of Rap1-GTP in LX-2 cells compared with the TGF-β1 stimulated cells.CONCLUSIONS1.The expression ofα-SMA and Epac1 was increased in liver fibrosis patients and CCl₄-induced liver fibrotic mice.In the progression of liver fibrosis,the distribution of Epac1 has been changed,suggesting that HSC activation and high expression of Epac1may be involved in the development of liver fibrosis.2.Selective activation of Epac1 can promote the proliferation and migration of LX-2cells,increase the expression ofα-SMA and type III collagen,elevate the level of Rap1-GTP,and promote the activation of ERK and AKT.The results suggested that selective activation of Epac1 may promote the activation of Rap1 and downstream ERK and AKT,thus to participate in the phenotype transformation of HSC.3.In vitro,silence of Epac1 by si RNA reduced the expression ofα-SMA and type III collagen,Rap1-GTP level and the expression of p-AKT,p-ERK in LX-2 cells.The results indicated that decreased Epac1 expression may inhibit the activation of AKT and ERK pathway and inhibit the expression ofα-SMA and collagen III in HSC by inhibiting the activation of Rap1.4.CP-25 alleviated the degree of liver fibrosis and decreased the expression of Epac1 in vivo.In vitro,CP-25 inhibited the expression ofα-SMA and type III collagen,the level of Rap1-GTP,and the expression of p-AKT,p-ERK in LX-2 cells.These provide evidence that CP-25 may play a protective effect on liver fibrosis through regulating the activity of Epac1,thereby inhibiting the activation of Rap1,and then inhibiting the activation of AKT and ERK signals.