The Mechanism Of AS-IV Attenuates I/R Injury-induced Myocardial Fibrosis And CSPGs Expression By Targeting MTORC1

Background:Chondroitin sulfate proteoglycans(CSPGs)are highly expressed after cardiac remodeling and ischemia reperfusion(I/R)injury.Chondroitin-4-sulfate(C4S)is the predominant chondroitin sulfate component in the heart.However,the mechanisms that induce CSPGs expression in fibroblasts following myocardial infarction(MI)are not entirely clear.Objective:The purpose of this study was to elucidate the mechanism and intervention measures of CSPGs expression induced by I/R injury through in vitro and in vivo experiments.Methods:(1)By constructing a rat heart I/R injury model,the cardiac tissue was subjected to Masson's trichrome staining and Alcian blue staining to verify the expression of myocardial fibrosis and CSPGs in the model group,and WB was used to verify the expression of type I collagen in the model group..(2)q PCR and WB used to verified oxygen-glucose deprivation(OGD)+TGF?1induced fibroblast transdifferentiation into myofibroblastsd.q PCR and WB were used to verify the expression of CSPGs in the OGD+TGF?1-induced group.The activation of Smad3 signaling pathway and PI3K/Akt/m TOR signaling pathway in fibroblasts in OGD+TGF?1 induced group was observed by WB.By using small molecule inhibitors ZSTK474,MK2206,AZD8055 against PI3K/Akt/m TOR pathway and using small interfering RNA to knock down Smad3,Raptor,and Rictor,respectively,the cells were intervened,and the induction of fibroblasts by OGD+TGF?1 was verified by WB and cell immunofluorescence.(3)The proliferation of fibroblasts after OGD,OGD+TGF?1,OGD+TGF?1+AS-IV intervention was verified by MTT experiment.Cell cycle was detected by flow cytometry,and PCNA was detected by WB to verify the proliferation of fibroblasts after intervention.Scratch experiments were used to verify the effects of TGF?1 and AS-IV on fibroblast migration ability.Using WB,cell immunofluorescence detection of ?-SMA to verify the effect of AS-IV intervention on fibroblast transdifferentiation.Collagen gel contraction experiments were used to verify the effects of OGD+TGF?1,OGD+TGF?1+AS-IV intervention on the contractile properties of myofibroblasts.The activation of PI3K/Akt/m TOR signaling pathway in fibroblasts after AS-IV intervention was observed by WB.The rat model of cardiac I/R injury was treated by intragastric administration of AS-IV,and the effect of AS-IV on I/R injury-induced myocardial fibrosis and the expression of CSPGs was verified by Masson's trichrome and alcian blue staining of cardiac tissue.The effect of AS-IV intervention on the expression of type I collagen induced by I/R injury was verified by WB detection.Results:(1)Myocardial I/R injury induces myocardial fibrosis,and CSPGs are highly expressed and co-localized with the myocardial fibrosis area 7 days after myocardial I/R injury,and continue to be highly expressed 14 days and 28 days after modeling,myocardial I/R injury induces Type I collagen expression is increased.(2)This study found that OGD+TGF?1 stimulation induced myofibroblast transformation and C4 S synthesis in vitro.Increased expression of phosphorylated proteins of Smad3,Akt,PRAS40 and p70S6 K.Using the PI3 K inhibitor ZSTK474,the Akt inhibitor MK2206,or the m TOR inhibitor AZD8055,we observed that OGD+TGF?1 stimulation induced myofibroblast transformation and that C4 S synthesis was m TOR-dependent,whereas the upstream canonical PI3K/Akt axis was dispensable.si RNA knockdown of Smad3,Rptor,or Rictor,indicated that m TORC1 was critical for promoting OGD+TGF?1-induced myofibroblast transformation and C4 S synthesis.This response may be mediated via cooperation between canonical Smad3 and m TORC1 signaling.(3)After intervention with 30 ?M AS-IV in vitro,the proliferation of fibroblasts was inhibited,the cell cycle of fibroblasts was arrested in G1 phase,inhibited migration and transdifferentiation.Contractile properties of fibroblasts.AS-IV works by inhibiting phosphorylation of m TORC1,not PI3K/Akt.In vivo 20mg/kg AS-IV gavage in the I/R model suppressed myocardial fibrosis and CSPGs expression.Conclusion:mTORC1 is essential for OGD+TGF?1-induced myofibroblast transdifferentiation and C4 S secretion,whereas the upstream canonical PI3K/Akt axis is not functional.This may be mediated through cooperation between Smad3 and m TORC1 signaling.AS-IV inhibited fibroblast proliferation,migration,transdifferentiation and shrinkage.I/R injury induced myocardial fibrosis,and AS-IV gavage inhibited myocardial fibrosis and the expression of CSPGs.