| Objective:The study was to investigate the the role and precise molecular mechanisms for explaining how gastrodin suppresses the autophagy flux and inflammatory in the serum deprivation/reperfusion induced H9C2 cardiomyocytes.Methods:H9C2 cardiomyocytes were cultured and treated differently in vitro and randomly divided into following groups,including the blank control group,the serum deprivation/reperfusion(2/4h)group,gastrodin(20μM)was added with serum deprivation/reperfusion(2/4h)treatment group,pretreatment with autophagy inhibitor 3-meth-yladennine(5mM,3-MA)for 1 h then added gastrodin(20μM)and serum deprivation/reperfusion(2/4h)treatment group and corresponding positive control group,pretreatment with autophagy inducer rapamycin(1 μM)for 1 h then added gastrodin(20μM)and serum deprivation/reperfusion(2/4h)group and corresponding positive control group,pretreatment with Beclin-1 overexpression(5μG)group,pretreatment with Beclin-1 overexpression(5μG)for 1 h then was added in serum deprivation/reperfusion(2/4h)group,pretreatment with Beclin-1 overexpression(5 pG)for 1 h then added gastrodin(20μM)and serum deprivation/reperfusion(2/4h)group and corresponding positive control group.The expression of autophagosome and autolysosome were used by mRFP-GFP-LC3 under confocal microscope.The variation of protein expression levels of autophagy related protein microtubule-associated protein 1 light chain 3(LC3),Beclinl and P62,LAMP2 and inflammatory related protein iNOS,COX-2,TNF-and IL-6 were measured by western blot.The inflammatory related genes iNOS,COX-2,TNF-and IL-6 were measured by QPCR.The expression of LAMP2 was measured by Lyso-trcacter under confocal microscope,and the expression of Beclin-1 was measured by double immunofluorescence labeling.Results:The serum deprivation/reperfusion(2/4h)was strongly upregulated the expression of Beclin-1、LC3Ⅱ、P62 and downregulated the LAMP2.Gastrodin(10、20、50μM)treatment strongly suppressed the upregulation of Beclin-1、LC3Ⅱ、P62 expression and the activation levels of LAMP2 by serum deprivation/reperfusion(2/4h)in H9C2 cardiomyocytes.Beclin-1 overexpression partially abolished the downregulation of gastrodin on the the protein expression of LC3Ⅱ、P62 and LAMP2.Also Beclin-1 overexpression partially abolished the downregulation of gastrodin on the the protein and mRNA expression of NOS、COX-2、TNF-α、IL-6.Conclusions:Gastrodin mediated the inflammatory response induced by ischemic reperfusion in H9C2 cardiomyocytes via down regulation of Beclin-1. |
