The Mechanic Effects On Retinoids Pathway In Activated Hepatic Stellate Cells

BackgroundAll the chronic liver could change to hepatic fibrosis and then become cirrhosis. Thefibrosis is reversible, but the cirrhosis is not. So we should recombine the mechanisms ofhepatic fibrosis to prevent the cirrhosis.Hepatic stellate cell is important in the development of hepatic fibrosis. HSC couldexcrete the ECM which is relevant with hepatic fibrosis. With the development of fibrosis,HSC which exist in the Disse clearance could bear more pressure and strech, and then tobe activated.In recent study,“lipid droplet”which formed by retinoids in HSCs was detected bytransmission electron microscope,and when hepatic fibrosis happened,“lipid droplet”waslost. So we think that the change of extracellular mechanical environment may regulateretinoids in HSCs.So the aim of our study is discover the role of mechanical in retinoids of HSC,elucidate the mechanism of mechanics in fibrosis and provide a new way to intervenefibrosis. The current study consists of three sections: isolate, identify and cultivate the HSC;load the pressure and stretch to the activation,and detect the correlational factor; explorethe underlying mechanisms of hepatic fibrosis.Methods1．Isolate, identification and cultivate HSCHSC were segregated from the hepatic of male SD rats by using pronase andcollagenase, and abstracted by using28.7%Nycodenz to centrifugate. Isolated HSC werecultured in the DMEM which include10%FBS.Identify the HSC by using Trypan blue staining、328nm ultraviolet light exposureand immunity fluorescence decoration.2．pressures in metabolism and activation of retinoids in HSC(1)Loading the pressureThe activated HSC were cultured on the plant and then put in to the pressure loadingequipment. Opened the gas valve, make the gass went to the equipment to make sure the (2)Detection the relevant factor of retinolAfter the pressure loading, total RNA and protein were drawn from HSC. We used todetect the mRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-α、type collagenⅠ and α-SMA by Real-time PCR and Western blot analysis.3．Mechanical stretch in metabolism and activation of retinoids in HSC(1)Mechanical stretch-loading modelThe stretch were mimiced by using Flexcell-4000T apparatus respectively. The cellwere planted on the Flexcell boards which were coating by muse tail collagen. Then weputted the boards on the BioFlex Cultur Plate which in the incubator house. Opened thevacuum air pump and the bridge networdking, started the Flexercell Strain central controlunit which was setting stretch parameter. The Flexcell-4000T apparatus was startedworking.(2)Detection the relevant factor of retinolAfter the stetch loading, total RNA and protein were drawn from HSC. We used todetect the mRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-α、type collagenⅠ and α-SMA by Real-time PCR and Western blot analysis.4．The mechanisms in pressure-induced activation of HSCThe Specific inhibitors of HX531and LE135were used to inhibit the RXR-αandRAR-βrespectively. We used to detect the mRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-α、type collagen Ⅰa nd α-SMA by Real-time PCR andWestern blot analysis.5．The mechanisms in stretch-induced activation of HSCThe specific inhibitors of HX531and LE135were used to inhibit the RXR-αandRAR-βrespectively. We used to detect the mRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-α、type collagen Ⅰa nd α-SMA by Real-time PCR andWestern blot analysis.6．Statistical analysisAll data were analysised by SPSS13.0. The P value≤0.05was considered statisticallysignificant.Results1．The identification of HSCMore than about95%HSC survived evaluated by the trypan blue staining. The autofluorescence and immunofluorescence coloretur make sure the purity of HSC wasnearly over95%.2．Effects of pressure on tmetabolism and activation of retinoids in HSCPressurization at10mmHg for one hour notably promoted the proliferation ofactivated HSC (P<0.01). Pressurization at10mmHg for one hour decreased mRNA andprotein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-α(P<0.05).3．Effects of stretch on metabolism and activation of retinoids in HSCStretch condition of10%elongation,0.5Hz frequency,24-hours duration promotedthe proliferation of activated HSC (P<0.01). this condition decreased mRNA and proteinexpressions of LRAT、CRBP-I、REH、RAR-β、RXR-α(P<0.05).4．Effects of inhibitors on pressure-induced HSC activation signaling pathway of retinoidsThe experimental HSC consist of4groups: Control group (C), pressure group (P),pressure+HX531group (PHX531), pressure+LE135group (PLE135).The mRNA andprotein expressions of α-SMA and collagen-I of P group is higer than C group,whilemRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-αis lower than Cgroup;The mRNA and protein expressions of α-SMA and collagen-I of P group is higerthan PHX531group and PLE135group,while mRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-αis lower than PHX531group and PLE135group.5．Effects of inhibitors on stretch-induced HSC activation signaling pathway in retinodsThe experimental HSC consist of4groups: Control group (C), Stretch group (S),stretch+HX531group (SHX531), stretch+LE135group (SLE135). The mRNA andprotein expressions of α-SMA and collagen-I of S group is higer than C group,whilemRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-αis lower than Cgroup;The mRNA and protein expressions of α-SMA and collagen-I of P group is higerthan SHX531group and SLE135group,while mRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-α is lower than SHX531group and SLE135group.Conclusions1．Pressure and stretch could increases the mRNA and protein expressions of α-SMA, Typecollagen Ⅰ, decreases the mRNA and protein expressions of LRAT、CRBP-I、REH、RAR-β、RXR-α,which could make the HSC activation to encourage the development ofhepatic fibrosis and portal hypertension,as well promotes the decomposition of retinoids.2．The retinol lipid droplet lost is involved into the mechanics promotes the HSC activation. We could slowdown the HSC activation by obstructed the retinol acceptor combined theligand.