甘草甜素调节HMGB1/RAGE信号通路对高糖诱导的心肌细胞凋亡和自噬的影响

Effects of Glycyrrhizin Modulating HMGB1/RAGE Signaling Pathway on Apoptosis and Autophagy of Cardiomyocytes Induced by High Glucose

目的:甘草甜素(Gly)调节高迁移率族蛋白(HMGB1)/晚期糖基化终产物受体(RAGE)信号通路对高糖诱导的心肌细胞凋亡和自噬的影响。方法:体外培养大鼠心肌细胞H9c2,将其分为7组:对照组、高糖组、高糖+Gly组、高糖+pcDNA3.1空载体组、高糖+pcDNA3.1-HMGB1组、高糖+Gly+pcDNA3.1空载体组、高糖+Gly+pcDNA3.1-HMGB1组。细胞计数试剂盒-8(CCK-8)法检测细胞活力;V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙啶(PI)法检测细胞凋亡率;透射电镜观察细胞自噬;蛋白免疫印迹法(Western Blot)检测B细胞淋巴瘤/白血病基因-2相关X蛋白基因(Bax)、微管相关蛋白1轻链3(LC3)和Beclin-1凋亡、自噬相关蛋白表达;酶联免疫吸附试验(ELISA)检测白介素-1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平;2,7-二氯荧光素二乙酸酯(DCFH-DA)荧光探针法检测活性氧(ROS)水平;实时荧光定量聚合酶链式反应(qRT-PCR)检测HMGB1、RAGE mRNA表达水平。结果:与对照组比较,高糖组H9c2细胞活力下降,细胞凋亡率、自噬小体数量及Bax、LC3、Beclin-1蛋白表达增加,IL-1β、TNF-α、ROS水平及HMGB1、RAGE mRNA表达增加,差异均有统计学意义(P<0.05);与高糖组比较,高糖+Gly组H9c2细胞活力升高,细胞凋亡率、自噬小体数量及Bax、LC3、Beclin-1蛋白表达减少,IL-1β、TNF-α、ROS水平及HMGB1、RAGE mRNA表达减少,差异均有统计学意义(P<0.05);与高糖+Gly组比较,高糖+Gly+pcDNA3.1-HMGB1组H9c2细胞活力下降,细胞凋亡率、自噬小体数量及Bax、LC3、Beclin-1蛋白表达增加,IL-1β、TNF-α、ROS水平及HMGB1、RAGE mRNA表达增加,差异均有统计学意义(P<0.05)。结论:Gly能够减轻高糖诱导的H9c2细胞炎症和氧化应激反应,抑制其凋亡和自噬,可能与抑制HMGB1/RAGE信号通路有关。

Objective:To investigate the effect of glycyrrhizin(Gly)on apoptosis and autophagy induced by high glucose in cardiomyocytes by regulating the high mobility group box protein(HMGB1)/receptor for advanced glycation end products(RAGE)signaling pathway.Methods:Rat cardiomyocytes H9c2 were cultured in vitro and divided into control(Ctrl)group,high glucose group,high glucose+Gly group,high glucose+pcDNA3.1 empty vector group,high-glucose+pcDNA3.1-HMGB1 group,high glucose+Gly+pcDNA3.1 empty vector group,and high glucose+Gly+pcDNA3.1-HMGB1 group.Cell viability was detected by cell counfing kit-8(CCK-8)assay.Apoptosis rate was detected by Annexin V-FITC/propyl iodide(PI)method.Autophagy was observed by transmission electron microscopy.The expression of B-cell lymphoma/leukemia gene-2-related X protein gene(Bax),microtubule-related protein 1 light chain 3(LC3)and Beclin-1 apoptosis and autophagy-related protein were detected by Western Blot.The levels of interleukin-1β(IL-1β)and tumor necrosis factorα(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).2,7-dichlorofluorescein diacetate(DCFH-DA)fluorescence probe method was performed to detect reactive oxygen species(ROS).Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was performed to detect the mRNA expression levels of HMGB1 and RAGE.Results:Compared with Ctrl group,H9c2 cell viability inhigh glucose group decreased,apoptosis rate,autophagosome number and protein expression of Bax,LC3 and Beclin-1 increased,IL-1β,TNF-α,ROS levels and mRNA expression of HMGB1 and RAGE increased,with statistical significance(P<0.05).Compared with the high glucose group,the H9c2 cell viability was increased in the high glucose+Gly group,the apoptosis rate,the number of autophagosomes and the expression of Bax,LC3 and Beclin-1 protein decreased,and the levels of IL-1β,TNF-αand ROS and the mRNA expressions of HMGB1 and RAGE decreased,with statistical significance(P<0.05).Compared with the high glucose+Gly group,the viability of H9c2 cells in the high glucose+Gly+pcDNA3.1-HMGB1 group decreased,the apoptosis rate,the number of autophagosomes and the expression of Bax,LC3 and Beclin-1 protein increased,the levels of IL-1β,TNF-αand ROS and the expression of HMGB1 and RAGE mRNA increased.The differences were statistically significant(P<0.05).Conclusion:Gly can reduce the inflammation and oxidative stress response of H9c2 cells induced by high glucose,inhibit their apoptosis and autophagy,which may be related to the inhibition of HMGB1/RAGE signaling pathway.