GSK-3β磷酸化与高糖因素取消七氟烷后处理心肌细胞保护作用的关系

Relationship between phosphorylation of GSK-3βand high glucose-caused abolition of cardioprotection induced by sevoflurane postconditioning

目的评价糖原合酶激酶3β(GSK-3β)磷酸化与高糖因素取消七氟烷后处理心肌细胞保护作用的关系。方法取H9c2大鼠心肌细胞分别培养于正常浓度葡萄糖(5.56 mmol/L)或高浓度葡萄糖(33 mmol/L)的DMEM培养基中。采用随机数字表法分为8组(n=24):正常对照组(NC组)、正常糖浓度缺氧复氧组(NH/R组)、正常糖浓度七氟烷后处理组(NS组)、正常糖浓度GSK-3β抑制剂SB216763组(NSB组)、高糖培养组(HC组)、高糖缺氧复氧组(HH/R组)、高糖七氟烷后处理组(HS组)和高糖GSK-3β抑制剂SB216763组(HSB组)。采用缺氧3 h复氧的方法制备心肌细胞缺氧复氧模型。NS组和HS组复氧即刻将心肌细胞暴露于2.4%七氟烷30 min,NSB组和HSB组复氧开始前加入GSK-3β抑制剂SB216763,终浓度10μmol/L。于复氧3 h时采用Anexin V/PI双染流式细胞术检测细胞凋亡率,Western blot法检测GSK-3β和磷酸化GSK-3β(p-GSK-3β)的表达,黄嘌呤氧化酶法测定SOD活性,比色法测定LDH活性和MDA含量。结果与NC组比较,NH/R组和HC组细胞凋亡率、LDH活性及MDA含量升高,SOD活性降低,NH/R组GSK-3β表达上调,p-GSK-3β表达下调,NS组p-GSK-3β表达上调,HC组p-GSK-3β表达下调(P<0.05);与NH/R组比较,NS组和NSB组细胞凋亡率、LDH活性及MDA含量降低,SOD活性升高,NS组GSK-3β表达下调,p-GSK-3β表达上调(P<0.05);与HC组比较,HH/R组细胞凋亡率、LDH活性及MDA含量升高,SOD活性降低,GSK-3β表达上调,p-GSK-3β表达下调(P<0.05);与HH/R组比较,HSB组细胞凋亡率、LDH活性及MDA含量降低,SOD活性升高(P<0.05)。结论高糖因素取消七氟烷后处理心肌细胞保护作用的机制与抑制GSK-3β磷酸化有关。

Objective To evaluate the relationship between phosphorylation of glycogen synthase kinase-3β(GSK-3β)and high glucose-caused abolition of cardioprotection induced by sevoflurane postconditioning.Methods H9c2 cells were incubated in normal glucose(5.56 mmol/L)DMEM culture medium or high glucose(33 mmol/L)DMEM culture medium.The cells were divided into 8 groups(n=24 each)using a random number table method:normal control group(group NC),normal glucose-cultured hypoxia/reoxygenation(H/R)group(group NH/R),normal glucose-cultured sevoflurane postconditioning group(group NS),normal glucose-cultured GSK-3βinhibitor SB216763 group(group NSB),high glucose-cultured group(group HC),high glucose-cultured H/R group(group HH/R),high glucose-cultured sevoflurane postconditioning group(group HS)and high glucose-cultured GSK-3βinhibitor SB216763 group(group HSB).The model of cardiomyocyte H/R was established by subjecting cardiomyocytes to 3 h of hypoxia followed by reoxygenation.Immediately after onset of reoxygenation,cardiomyocytes were exposed to 2.4%sevoflurane for 30 min in Ns and HS groups.Before the beginning of reoxygenation,GSK-3βinhibitor SB216763 was added to the culture medium with the final concentration of 10μmol/L in NSB and HSB groups.At 3 h of reoxygenation,the apoptosis rate was determined by Anexin V-PI flow cytometry,the expression of GSK-3βand phosphorylated GSK-3β(p-GSK-3β)was detected by Western blot,superoxide dismutase(SOD)activity was measured using xanthineoxidase method,and lactic dehydrogenase(LDH)activity and malondialdehyde(MDA)content were determined by colorimetric assay.Results Compared with group NC,apoptosis rate,LDH activity and MDA content were significantly increased,and SOD activity was decreased in group NH/R and group HC,expression of GSK-3βwas up-regulated,and expression of p-GSK-3βwas down-regulated in group NH/R,expression of p-GSK-3βwas up-regulated in group NS,and expression of p-GSK-3βwas down-regulated in group HC(P<0.05).Compared with group NH/R,apoptosis rate,LDH activity and MDA content were significantly decreased,and SOD activity was increased in group NS and NSB groups,and expression of GSK-3βwas down-regulated,and expression of p-GSK-3βwas up-regulated in group NS(P<0.05).Compared with group HC,apoptosis rate,LDH activity and MDA content were significantly increased,SOD activity was decreased,expression of GSK-3βwas up-regulated,and expression of p-GSK-3βwas down-regulated in group HH/R(P<0.05).Compared with group HH/R,apoptosis rate,LDH activity and MDA content were significantly decreased,and SOD activity was increased in group HSB(P<0.05).Conclusion The mechanism by which high glucose abolishes cardioprotection induced by sevoflurane postconditioning is related to inhibiting phosphorylation of GSK-3β.