自噬在右美托咪定减轻大鼠高糖离体心肌细胞缺氧复氧损伤中的作用

Role of autophagy in reduction of high glucose and hypoxia-reoxygenation injury to isolated cardiomyocytes by dexmedetomidine in rats

目的评价自噬在右美托咪定减轻大鼠高糖离体心肌细胞缺氧复氧损伤中的作用。方法正常培养的对数期大鼠H9c2心肌细胞,以1×10^(6)个/ml密度接种于6孔板,采用随机数字表法分为4组(n=15):对照组(C组)、高糖缺氧复氧组(HG+H/R组)、右美托咪定组(DEX组)和右美托咪定+自噬抑制剂3-甲基腺嘌呤组(3-MA组)。细胞密度达到50%时,使用含1%胎牛血清+1%双抗的培养基孵育24 h。采用高糖培养基(糖浓度33 mmol/L)培养24 h,37℃下培养箱(95%N2+5%CO2)培养4 h,复氧(90%O2+10%CO2)2 h,制备高糖心肌细胞缺氧复氧损伤模型,DEX组和3-MA组于缺氧前1 h时加入右美托咪定,终浓度5μmol/L,3-MA组右美托咪定孵育1 h时加入3-MA,终浓度5μmol/L。于复氧后2 h时采用CCK-8法测定细胞活力,采用试剂盒检测上清液LDH活性,采用Western blot法检测自噬相关蛋白LC3、P62和Beclin-1的表达,计算LC3Ⅱ/LC3Ⅰ比值,采用RT-PCR法检测P62和Beclin-1 mRNA的表达。结果与C组比较,HG+H/R组、DEX组和3-MA组细胞活力降低,HG+H/R组和3-MA组上清液LDH活性升高,细胞P62表达下调,3-MA组LC3Ⅱ/LC3Ⅰ比值降低,HG+H/R组Beclin-1表达下调(P<0.05);与HG+H/R组比较,DEX组上清液LDH活性降低,Beclin-1和P62及其mRNA表达上调,LC3Ⅱ/LC3Ⅰ比值升高,3-MA组上清液LDH活性升高(P<0.05);与DEX组比较,3-MA组细胞活力降低,上清液LDH活性升高,细胞Beclin-1和P62及其mRNA表达下调,LC3Ⅱ/LC3Ⅰ比值降低(P<0.05)。结论自噬参与了右美托咪定减轻大鼠高糖离体心肌细胞缺氧复氧损伤的过程。

Objective To evaluate the role of autophagy in reduction of high glucose and hypoxia-reoxygenation(HG+H/R)injury to isolated cardiomyocytes by dexmedetomidine in rats.Methods The normally cultured rat H9c2 cardiomyocytes at the logarithmic growth phase were seeded in 6-well plates at a density of 1×10^(6)cells/ml and divided into 4 groups(n=15 each)using a random number table method:control group(group C),group HG+H/R,dexmedetomidine group(group DEX)and dexmedetomidine+autophagy inhibitor 3-methylpurine group(group 3-MA).The cells were incubated in culture medium with 1%fetal bovine serum+1%double antibody for 24 h when the cell density reached 50%.To establish HG+H/R injury model,the cardiomyocytes were cultured in high-glucose culture medium(glucose concentration of 33 mmol/L)for 24 h,and then incubated in a 37℃incubator(95%N2+5%CO2)for 4 h followed by reoxygenation(90%O2+10%CO2)for 2 h.Dexmedetomidine was added until the final concentration reached 5μmol/L at 1 h before hypoxia in DEX and 3-MA groups,and 3-MA was added until the final concentration reached 5μmol/L at 1 h of incubation with dexmedetomidine.At 2 h after reoxygenation,the cell viability was recorded by the cell counting kit-8 assay,lactate dehydrogenase(LDH)activity was detected by LDH kit,the expression of autophagy-related protein LC3,P62 and Beclin-1 was detected by Western Blot,the ratio of LC3Ⅱ/LC3Ⅰwas calculated,and the expression of P62 and Beclin-1 mRNA was detected by real-time polymerase chain reaction.Results Compared with group C,the cell viability was significantly decreased in HG+H/R,DEX and 3-MA groups,LDH activity in the supernatant was increased and expression of P62 was decreased in HG+H/R and 3-MA groups,ratio of LC3Ⅱ/LC3Ⅰwas decreased in group 3-MA,and the expression of Beclin-1 was down-regulated in group HG+H/R(P<0.05).Compared with HG+H/R group,LDH activity in the supernatant was significantly decreased,expression of Beclin-1,P62 and its mRNA was up-regulated,and ratio of LC3Ⅱ/LC3Ⅰwas increased in DEX group,and LDH activity in the supernatant was increased in group 3-MA(P<0.05).Compared with DEX group,cell viability were decreased,LDH activity in the supernatant was increased,Beclin-1,P62 and its mRNA was down-regulated,and ratio of LC3Ⅱ/LC3Ⅰwas decreased in group 3-MA(P<0.05).Conclusion Autophagy is involved in the reduction of HG+H/R injury to isolated cardiomyocytes by dexmedetomidine in rats.