Mechanism Studies Of Zn²⁺ Mediates Resveratrolinduced Myocardial Protection

Objectives The purpose of this study was to investigate the detailed action mechanism of resveratrol to inhibit endoplasmic reticulum stress?ERS?and protect H9c2 cells.The role of zinc ion and mitochondrial permeability transition pores?m PTP?is mainly studied.Methods H9c2 cells?derived from rat embryonic heart tissue?were cultured in DMEM medium,and the ERS model was prepared using the ERS inducer 2-deoxy-D-glucose?2-DG?.Resveratrol and zinc chloride?ZnCl₂?required drug pretreatment,Zn²⁺ chelating agent N,N,N',N'-tetrakis ethylenediamine?TPEN?and protein kinase inhibitors acted before drug pretreatment.Transfection of si RNAs was performed in cell culture.Cell viability was detected by MTT cell proliferation assay kit.Cytotoxicity was detected by a lactate dehydrogenase cytotoxicity test kit.The expressions of the endoplasmic reticulum chaperone protein glucose regulated protein 78?GRP78?,glucose regulated protein?GRP94?and ERS-related apoptosis protein C/EBP homology protein?CHOP?,cysteinyl aspartate specific proteinase 12?Caspase 12?,jun N-terminal kinase?JNK?,and phosphorylations of extracellular regulated protein kinases?ERK?,phosphatidylinositol 3 kinase?PI3K?/protein kinase B?AKT?,glycogen synthase kinase-3??GSK-3??were detected by western blot.The expressions of GRP 94 and JNK were detected by immunocytochemical staining techniques.The mitochondrial membrane potential???m?was measured by laser confocal microscopy using TMRE red fluorescent dye to measure the openness of m PTP,and the concentration of Zn²⁺ in the cells was measured using newport green DCF fluorescent dye.Results 1 Zn²⁺ participated in the effect of resveratrol on increasing cell activity and reducing toxicity.The results of MTT and LDH experiments showed that compared with the control group,the activity of H9c2 cells was significantly reduced,and the toxicity was significantly enhanced in the 2-DG group.The resveratrol pretreatment inhibited the change induced by 2-DG,but was reversed by the Zn²⁺ chelating agent TPEN.2 Zn²⁺ participated in the effect of resveratrol to inhibit ERS and related apoptosis.Western blot and cell immunochemical staining results showed that compared with the control group,the expressions of GRP 78,GRP 94,CHOP,Caspase 12 and JNK increased significantly in 2-DG group.Resveratrol inhibited the protein expression induced by 2-DG,but was reversed by Zn²⁺ chelating agent TPEN.3 Zn²⁺ participated in the role of resveratrol in preventing the opening of m PTP.Confocal results showed that compared with the control group,the red fluorescence intensity of TMRE was significantly reduced in 2-DG group,that is,the mitochondrial membrane potential decreased,m PTP was open,and resveratrol inhibited the change induced by 2-DG,but was reversed by Zn²⁺ chelating agent TPEN.4 ERK/GSK-3? participated in resveratrol's role in inhibiting ERS and related apoptosis and preventing m PTP from opening.The experimental results showed that compared with the control group,the expressions of GRP 78,GRP 94,CHOP,Caspase 12 and JNK were significantly increased,the intensity of TMRE red fluorescence was significantly reduced in the 2-DG group.Resveratrol pretreatment inhibited the changes caused by 2-DG.While ERK inhibitor PD98059,GSK-3? inhibitor SB216763,ERK si RNA and GSK-3? si RNA inhibited the action of resveratrol,which reduced protein expressions and enhanced TMRE red fluorescence intensity.AKT inhibitor LY294002 and AKT si RNA were no significant change on the effect of resveratrol.In addition,resveratrol significantly increased the phosphorylations of ERK and GSK-3?,and had no significant effect on the phosphorylation of AKT.5 Resveratrol increased the concentration of Zn²⁺ in H9c2 cells.The results of the confocal experiment showed that compared with the control group,resveratrol could simulate the effect of ZnCl₂,which significantly enhanced the fluorescence intensity of Newport Green DCF.However,ERK inhibitor PD98059,GSK-3? inhibitor SB216763,ERK si RNA and GSK-3? si RNA reversed the effect of resveratrol,AKT inhibitor LY294002 and AKT si RNA had no obvious effect.Conclusions These datas suggest that resveratrol reduces endoplasmic reticulum stress and related apoptosisand and prevents m PTP from opening by increasing Zn²⁺ to protect H9c2 cells.The ERK/GSK-3? signaling pathway is involved in the role of resveratrol,and the AKT signal may not be involved in this process.Figure 19;Table 2;Reference 157...