脂联素对高糖环境心肌细胞氧化应激及凋亡的影响

Effects of Adiponectin on Cell Oxidative Stress and Apoptosis in Human Cardiac Myocytes Cultured with High Glucose

目的通过高糖环境培养人心肌细胞(human cardiac myocytes,HCM)建立心肌细胞氧化应激模型,观察脂联素(ad-iponectin,ADPN)对心肌细胞各氧化应激指标及凋亡的影响,从而揭示脂联素对心肌细胞可能的保护机制。方法将体外培养的人心肌细胞分为3组:对照组、高糖组、高糖+脂联素组,分别干预24、48、72h。在倒置显微镜下观察心肌细胞形态变化,采用黄嘌呤氧化酶法检测超氧化物歧化酶(superoxide dismutase,SOD)含量。用代巴比妥酸法测定丙二醛(malondialdehyde,MDA)含量,用荧光定量RT-PCR法测定衔接蛋白(adaptinP66Shc)、血红蛋白加氧酶-1(heme oxygenase-1,Ho-1)在各个时间点上的表达,通过流式细胞术检测心肌细胞的凋亡率。结果与对照组相比,高糖组SOD含量显著下降,MDA含量显著上升(P<0.05),与高糖组相比,脂联素组SOD含量显著上升,MDA含量显著下降(P<0.05),并且呈时间依赖性。与对照组相比,高糖组Ho-1 mRNA表达增高;脂联素组较高糖组表达亦增高(P<0.05)。与对照组相比,高糖组P66Shc mRNA表达增高;脂联素组较高糖组表达降低(P<0.05),呈时间依赖性。高糖组细胞凋亡率较对照组明显增加,而脂联素组细胞凋亡率较高糖组显著下降。结论脂联素可通过减少P66Shc表达、增加Ho-1表达,增加抗氧化能力,减少氧化应激反应,减少细胞凋亡,可能对糖尿病心肌细胞起保护作用。

Objective To establish a myocardial cell oxidative stress model by cultivating human cardiac myocytes(HCM) in a high glucose environment,and to observe if there is a protective role of adiponectin(ADPN) on the oxidative stress and apoptosis of myocardial cells,and reveals the protection mechanism of the adiponectin.Methods The in vitro cultured HCM were divided into three groups: the control group,high glucose group,high glucose + adiponectin group,and these cells were cultured 24,48,72hours respectively.Then we observed the morphological change of the HCM under the inverted microscope and evaluated the level of superoxide dismutase(SOD) by the xanthineoxidase method.The level of malondialdehyde(MDA) was detected by benzodiazepines acid method.The expression of adaptin(P66Shc) and heme oxygenase-1(Ho-1) in three time points was detected by real-time PCR,and the apoptosis rate of the HCM was tested by the flow cytometry.Results Our findings showed significant increase of MDA levels(P < 0.05) and decrease of SOD activity(P < 0.05) in the high glucose group compared with the control group.However,in the adiponectin group,MDA levels decreased(P < 0.05) and SOD activity increased significantly(P < 0.05) compared with those in the high-glucose group and they were time dependent.Compared with the control group,the expression of Ho-1 mRNA and P66Shc mRNA increased in the high glucose group.The adiponectin group expressed a higher level of Ho-1 mRNA but a lower level of P66Shc mRNA compared with the glucose group(P < 0.05) and they were time dependent.The apoptosis rate of the HCM in the high-glucose group was higher than the control group;while the apoptotic rate in the adiponectin group declined remarkably compared with the high glucose group.Conclusion The adiponectin has a protective effect on diabetes myocardial cells by upregulating the expression of Ho-1 mRNA and downregulating the expression of P66Shc mRNA,which can increase the antioxidant capacity,reduce oxidative stress and reduce high glucose-induced apoptosis of HCM.