Effect And Mechanism Of Erythropoietin Regulating Toll-like Receptor4of Cardiac Fibroblast

Objectives:The purpose of this study is to explore the effect and mechanism of EPOregulates the expression of toll-like receptor4in cardiac fibroblasts.Methods:We induced proliferation and inflammation of cardiac fibroblasts bypretreatment Ang Ⅱ before EPO intervention. Transfected cardiac fibroblasts withTLR4virus, which used to certified that EPO exert inhibit proliferation andinflammation induced by Ang Ⅱ via regulate TLR4expression levels.Therefore,cardiac fibroblasts were divided into the following seven groups, normal controlgroup:cardiac fibroblasts cultured with high glucose medium, AngⅡgroup: cardiacfibroblasts pretreatment with10-6mol AngⅡ,AngⅡ+EPO group: plus EPO on thebasis of AngⅡgroup, AngⅡ+TLR4virus group: basic AngⅡ+EPO group in plusTLR4lentiviral, AngⅡ+EPO+empty virus group: empty virus on the basis of AngⅡ+EPO group on,Ang Ⅱ+EPO+LY294002group:plus PI3K inhibitorLY294002(10-5mol/L) on the basis of AngⅡ+EPO group,AngⅡ+EPO+Aktinhibitor group: plus Akt inhibitor (10-5mol/L) on the basis of Ang Ⅱ+EPOgroup.After4days, we detect the growth of cardiac fibroblasts by cell count andCCK-8.To observation the migration capability of CFs pretreatment with PDGF-BBinstead of Ang Ⅱ by scratch test. To invest the MMP9levels of each group byenzyme-linked immunosorbent assay.Discovering the mechanism by EPO inhibitsproliferation and inflammation of cardiac fibroblasts via regulates the expression oftoll-like receptor4by detect the expression levels of TLR4in each group byqRT-PCR and flow cytometry.Western blot is used to discover the expression levelsof phosphorylated Akt.Results:Data of CCK8and cell count indicated EPO alley proliferation effect of cardiacfibroblasts induced by AngⅡ,but the effect of EPO was abated when pretreatmentcardiac fibroblasts with TLR4over-expression virus.The results of ELISA demonstrated that MMP9levels of Ang Ⅱ group were significantly increased (P<0.01).The MMP9levels of AngⅡ+EPO group were significantly lower than AngⅡgroup. The MMP9levels of increased when added the PI3K inhibitor (LY294002) orAkt inhibitor. Scratch test results certified that PDGF-BB induce migration of cardiacfibroblasts.EPO can abrogate the migration capability of cardiac fibroblasts inducedby PDGF-BB,but the migration capability of cardiac fibroblast increased when TLR4over-expression virus plus in. Flow cytometry and qRT-PCR results showed thattheprotein and mRNA expression levels of TLR4in Ang Ⅱ group was significantlyhigher than the control group(P <0.01).AngⅡ+EPO group was lower than AngⅡgroup (P <0.01).The protein and mRNA expression levels of TLR4of AngⅡ+EPO+LY294002group and AngⅡ+EPO+Akt inhibitor group were higher than AngⅡ+EPO group(P <0.01). Westernblot results showed no significant difference onexpression levels of t-Akt between each group(P>0.05).The expression levels ofp-Akt in AngⅡ+EPO group was significantly higher than AngⅡgroup (P<0.01).Compared with AngⅡ+EPO group,The expression levels of p-Akt in AngⅡ+EPO+TLR4virus group, Ang Ⅱ+EPO+LY294002group and AngⅡ+EPO+Akt decreased (P <0.01).Conclusions:EPO down-regulated the expression levels of TLR4by activated PI3K/Aktpathway in cardiac fibroblasts and result in anti-myocardial fibrosis.