雷公藤红素对高糖诱导的H9C2细胞损伤的保护作用

The protective effect of Celastrol on H9C2 cell injury induced by high glucose

目的:通过高糖条件培养大鼠心肌H9C2细胞模拟糖尿病心肌病(DCM)体外细胞模型,并探讨雷公藤红素对H9C2细胞的保护作用及机制。方法:实验设立低糖组(5.6 mmol/L低糖培养)、等渗组(甘露醇等渗培养)、高糖组(30 mmol/L高糖培养)和雷公藤红素组(高糖培养并加入10 ng/mL和100 ng/mL雷公藤红素干预)。采用MTT法分析高糖组和雷公藤红素组H9C2细胞的活力;应用活性氧簇荧光探针染色法和流式细胞术分析低糖组、等渗组、高糖组和100 ng/m L雷公藤红素组H9C2细胞中ROS产生水平;RT-PCR和Western blot实验技术分析低糖组、等渗组、高糖组和雷公藤红素组H9C2细胞中NF-κB、TGF-1β和IL-1β的mRNA和蛋白表达水平。结果:高糖组中高糖培养条件对H9C2细胞活力有抑制作用,与低糖组和等渗组比差异有统计学意义(P<0.05),雷公藤红素干预可以降低高糖培养对H9C2细胞的生长抑制作用;高糖组细胞内的ROS水平明显高于低糖组和等渗组(P<0.05),100 ng/m L雷公藤红素可抑制细胞内ROS产生;RT-PCR和Western blot结果显示,与低糖组和等渗组相比,高糖组细胞中NF-κB、TGF-1β和IL-1βm RNA和蛋白表达上调,差异有统计学意义(P<0.05);与高糖组相比,10 ng/mL和100 ng/mL雷公藤红素组细胞中的NF-κB、TGF-1β和IL-1β基因mRNA和蛋白表达水平均出现下调,差异有统计学意义(P<0.05)。结论:雷公藤红素可以明显抑制H9C2细胞在高糖培养条件下ROS的产生水平并降低高糖培养对细胞的生长抑制作用,发挥对心肌细胞的保护作用,其机制可能与抑制炎症因子相关基因的表达有关。

Objective:To simulate in vitro the cellular model of DCM by high glucose culture medium of rat cardiomyocytes H9 C2 and to study the protective effect and mechanism of Celastrol on H9 C2 cells.Methods:In the experiment,low glucose group(5.6 mmol/L),isoosmotic group(mannitol isoosmotic culture),high glucose treatment group(30 mmol/L)and Celastrol group(high glucose culture and intervention with 10 ng/mL and 100 ng/mL Celastrol)were established.MTT assays were used to analyze the viability of H9 C2 cells in high glucose treatment group and Celastrol group.Reactive oxygen species cluster fluorescence probe staining and flow cytometry were performed to analyze the production of ROS in H9 C2 cells induced by high glucose culture and intervened with Celastrol.The mRNA and protein expression level of NF-κB,TGF-1βand IL-1βwere detected by RT-PCR and Western blot.Results:Significant inhibitory effects on the viability of H9 C2 cells were observed in high glucose treatment group,with statistically significant difference compared with the low glucose group(P<0.05).The proliferation inhibitory effect was attenuated when intervened with different concentrations of Celastrol.The ROS levels in high glucose treatment group was significantly higher than the low glucose group(P<0.05),and the ROS levels were decreased after intervention of 100 ng/mL Celastrol.RT-PCR,Western blot results showed that the mRNA and protein expression levels of NF-κB,TGF-1βand IL-1βgenes were upregulated in high glucose treatment group,compared with low glucose and the isotonic group,the differences were statistically significant(P<0.05).The mRNA and protein expression levels of NF-κB,TGF-1βand IL-1βin 10 ng/mL and 100 ng/mL Celastrol group were down-regulated compared with high glucose treatment group,the differences being significant(P<0.05).Conclusion:Celastrol could inhibit production of ROS in H9 C2 cells in high glucose culture and reduce the growth inhibition effect on cells,thus producting protective effects on cardiac myocytes.The mechanism may be related to the inhibited expression of inflammatory factor-related genes.