α7烟碱型乙酰胆碱受体激动剂对缺氧复氧大鼠心肌细胞糖原合成酶激酶3β活性的影响  

Effect of α7 nicotinic acetylcholine receptor agonist on activity of glycogen synthase kinase-3β in rat cardiomyocytes subjected to anoxia/reoxygenation

作　　者：崔昕龙[1] 程怡[1] 薛富善[1] 王世玉[1] 李瑞萍[1] 刘高谱 杨桂珍[1] 廖旭[1] 刘建华[1] 

机构地区：[1]中国医学科学院北京协和医学院整形外科医院麻醉科,北京市100144

出　　处：《中华麻醉学杂志》2014年第11期1375-1378,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金（81170128）

摘　　要：目的 评价α7烟碱型乙酰胆碱（α7nACh）受体激动剂对缺氧复氧大鼠心肌细胞糖原合成酶激酶3β（GSK-3β）活性的影响.方法 大鼠心肌细胞培养72 h后,按照随机数字表法分为3组（n=18）：对照组（C组）、缺氧复氧组（AR组）和α7nACh受体激动剂组（PNU282987组）.C组心肌细胞正常培养;AR组心肌细胞缺氧6h后复氧;PNU282987组心肌细胞缺氧6h后复氧,在复氧培养基中加入用DMSO溶解的PNU282987,终浓度30 μmol/L,C组和AR组加入等浓度DMSO.于复氧6h时采用比色法检测心肌细胞乳酸脱氢酶（LDH）漏出率,Annexin V/PI双染流式细胞术检测凋亡情况,Westernblot法检测GSK-3β与磷酸化GSK-3β（p-GSK-3β Ser9）、NF-κcB p65与磷酸化NF-κB p65 （p-NF-κB p65Ser536）蛋白的表达,ELISA法检测心肌细胞培养上清液IL-6与TNF-α的浓度.结果 与C组比较,AR组和PNU282987组心肌细胞LDH漏出率升高,正常细胞百分比降低,早期凋亡细胞百分比和晚期凋亡细胞百分比均升高,心肌细胞p-GSK-3β Ser9、NF-κB p65和p-NF-κB p65 Ser536蛋白表达上调,心肌细胞培养上清液IL-6和TNF-α浓度升高（P＜0.01）,心肌细胞GSK-3β蛋白表达差异无统计学意义（P＞0.05）;与AR组比较,PNU282987组LDH漏出率降低,正常细胞百分比升高,早期凋亡细胞百分比降低（P＜O.01）,晚期凋亡细胞百分比差异无统计学意义（P＞0.05）,心肌细胞p-GSK-3β Ser9蛋白表达上调,心肌细胞p-NF-κB p65 Ser536蛋白表达下调,心肌细胞培养上清液IL-6和TNF-α浓度均降低（P＜0.01）,NF-κB p65蛋白表达差异无统计学意义（P＞0.05）.结论 α7nACh受体激动剂可通过降低GSK-3β活性抑制炎性反应,从而减轻大鼠心肌细胞缺氧复氧损伤.Objective To evaluate the effect of α7 nicotinic acetylcholine （α7nACh） receptor agonist on the activity of glycogen synthase kinase-3β （GSK-β） in rat cardiomyocytes subjected to anoxia/reoxygenation （A/R）.Methods After being cultured for 72 h,the cardiomyocytes were randomly divided into 3 groups （n =18 each） using a random number table：control group （group C）,A/R group and α7nACh receptor agonist PNU282987 group （PNU282987 group）.A/R and PNU282987 groups were exposed to 6 h anoxia followed by 6 h reoxygenation.In addition,PNU282987 with the final concentration of 30 μmol/L （in dimethyl sulfoxide） was added to the culture media for reoxygenation in PNU282987 group,while the equal concentration of dimethyl sulfoxide was added to the culture media for reoxygenation in C and AR groups.At 6 h of reoxygenation,the rate of lactate dehydrogenase （LDH） released was detected using colorimetric method,apoptosis in cardiomyocytes was detected u sing annexin V/PI double-staining assay,the expression of GSK-3β,phosphorylated GSK-3β Ser9 （pGSK-3β Ser9）,NF-κB p65 and phosphorylated NF-κB p65 Ser536 （p-NF-κB p65 Ser536） was detected by Western blot,and the concentrations of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in the supernatant were measured using ELISA.Results Compared with C group,the rate of LDH released was significantly increased,the percentage of normal cells was decreased,the percentage of apoptotic cells in the early and late stages was increased,the expression of p-GSK-3β Ser9,NF-κB p65 and p-NF-κB p65 Ser536 was upregulated,the concentrations of TNF-α and IL-6 were increased,and no significant change was found in the expression of GSK-3β in AR and PNU282987 groups.Compared with AR group,the rate of LDH released was significantly decreased,the percentage of normal cells was increased,the percentage of apoptotic cells in the early stage was decreased,no significant change was found in the percentage of apoptotic cells in the late s

关 键 词：胆碱能激动剂 缺氧 氧 肌细胞 心脏 糖原合成酶激酶 炎症 

分 类 号：R542.2[医药卫生—心血管疾病]