Mechanism of Suxiao Jiuxin Pills on Hypoxia/Reoxygenation Injury in Cardiomyocytes Based on ALKBH5 Regulation of GSK3β/mTOR Pathway to Inhibit Excessive Autophagy
LI Cha
LI Yiping
WANG Keyan
WANG Dan
WANG Xiaolong
Abstract：Objective:Based on the level of autophagy in HL-1 cells and the expression of key proteins of related pathways,to explore the mechanism of Suxiao Jiuxin Pills on hypoxia/reoxygenation(H/R)injury through the Alk B homolog 5(ALKBH5)regulation of glycogen synthetase kinase 3beta(GSK3β)/mammalian target of rapamycin(mTOR)pathway to inhibit excessive autophagy.Methods:After construction of lentiviral interference carrier and screening of optimal interfering lentiviruses,the HL-1 cells were randomly divided into 3 groups and blank transfection,ALKBH5 transfection,and GSK3βtransfection.Each group was randomly divided into 4 groups;hypoxia/reoxygenation(model group),hypoxia/reoxygenation + Suxiao Jiuxin Pills treatment(Suxiao group),hypoxia/reoxygenation+ligustrazine treatment(ligustrazine group),hypoxia/reoxygenation +borneol treatment(borneol group).Autophagy flow was detected by double fluorescence staining of autophagy double-labeled adenovirus(mRFP-eGFP-LC3),cell proliferation was detected by cell counting(CCK8),apoptosis was detected by double-stained bivariate flow cytometric analysis(Annexin V/PI),the number and morphology of autophagic vesicles were observed by transmission electron microscopy,and GSK3β,mTOR content,and autophagy-related gene(autophagy-related gene,Atg5),benzyl chloride 1(Beclin1),autophagy-related protein microtubule-associated protein light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ),and autophagy marker p62 were detected by Western Blot.Results:ALKBH5 overexpression inhibited GSK3βexpression and enhanced mTOR expression(P<0.05).mRFP-eGFP-LC3 dual fluorescence staining for autophagy flow detection showed that compared with the blank transfected under the condition of H/R,ALKBH5 transfection promoted autophagy and GSK3βtransfection inhibited autophagy(P<0.05).The results of CCK8 assay showed that compared with the model group,cell proliferation was promoted in the Suxiao group,ligustrazine group,and borneol group.Compared with the blank transfection,cell proliferation was inhibited in the ALKBH5 group,and cell proliferation was promoted in the GSK3β group(P<0.05).Annexin V/PI double staining assay was used to detect apoptosis in cardiomyocytes,the result suggested that compared with the model group,in the Suxiao group,ligustrazine group,and borneol group apoptosis was inhibited,and ALKBH5 transfection and GSK3β transfection increased apoptosis compared with the blank group(P<0.05).Western Blot suggested that in the Suxiao group,ligustrazine group,and borneol group LC3Ⅱ/Ⅰ,Atg5,Atg7,and p62 expressions were inhibited and mTOR expression was enhanced(P<0.05).Compared with blank transfection,ALKBH5 transfection enhanced LC3Ⅱ/Ⅰ,Atg5,Atg7,p62 expression and inhibited mTOR expression;GSK3β transfection inhibited LC3Ⅱ/Ⅰ,Atg5,Atg7,p62 expression and enhanced mTOR expression(P<0.05).Conclusion:Suxiao Jiuxin Pills could inhibit excessive autophagy via the ALKBH5/GSK3β/mTOR signaling pathway,and alleviate the hypoxia/reoxygenation injury in cardiomyocytes.
Keywords：cardiomyocyteshypoxia/reoxygenation injurySuxiao Jiuxin PillsAlk B homolog 5ALKBH5glycogen synthetase kinase 3betaGSK3βmammalian target of rapamycinmTORautophagyexperimental study
Publication Date：2023-12-16
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：10( 4510-4519 )