丹参酮ⅡA磺酸钠对尿毒症毒素作用下人脐静脉内皮细胞功能的影响

Effect of tanshinoneⅡA on function of human umbilical vein endothelial cells after treated with uremic toxin

目的:探讨丹参酮ⅡA磺酸钠(STS)对尿毒症毒素作用下人脐静脉内皮细胞(hUVECs)功能的影响,并阐明其作用机制。方法:将hUVECs进行传代培养并分为空白对照组、尿毒症毒素刺激组、尿毒症毒素+STS组和尿毒症毒素+STS+细胞外信号调节激酶(ERK)抑制剂组,其中后2组中STS的浓度为10 mg·L^(-1);先给予各组剪切力刺激,剪切力大小为12 dyn·cm^(-2);采用CCK-8法测定各组细胞增殖活性,Western blotting法检测各组细胞中ERK、核因子κB(NF-κB)和Ⅰ型胶原蛋白表达水平,实时荧光定量PCR(RT-qPCR)法检测细胞中ERK、NF-κB和Ⅰ型胶原mRNA表达情况;原位末端转移酶标记技术(TUNEL)法检测各组细胞凋亡率。结果:CCK-8法检测,在剪切力作用后,尿毒症毒素刺激组和尿毒症毒素+STS+ERK抑制剂组细胞增殖活性低于尿毒症毒素+STS组(P<0.01)。Western blotting法检测,与尿毒症毒素组比较,尿毒症毒素+STS组细胞中ERK、NF-κB和Ⅰ型胶原蛋白表达水平升高(P<0.01);抑制ERK信号通路后,与空白对照组、尿毒症毒素组和尿毒症毒素+STS组比较,尿毒症毒素+STS+ERK抑制剂组细胞中ERK、NF-κB和Ⅰ型胶原蛋白表达水平明显降低(P<0.01)。RT-qPCR法检测,与尿毒症毒素组比较,尿毒症毒素+STS组细胞中ERK、NF-κB和Ⅰ型胶原mRNA表达水平升高(P<0.01);抑制ERK通路后,与空白对照组、尿毒症毒素组和尿毒症毒素+STS组比较,尿毒症毒素+STS+ERK抑制剂组细胞中ERK、NF-κB和Ⅰ型胶原mRNA表达水平明显降低(P<0.01)。TUNEL法检测,尿毒症毒素+STS组的细胞凋亡率小于尿毒症毒素刺激组和尿毒症毒素+STS+ERK抑制剂组(P<0.05)。结论:一定浓度STS能通过ERK信号通路调节NF-κB和Ⅰ型胶原mRNA及蛋白表达来改善尿毒症毒素作用下的内皮细胞增殖,减少细胞凋亡。

Objective:To discuss the effect of sodium tanshinoneⅡAsulfonate(STS)on the function of human umbilical vein endothelial cells(hUVECs)after treated with uremic toxin,and to clarify its mechanism.Methods:The hUVECs were passaged and divided into blank control group,uremic toxin-stimulation group,uremic toxin+STS group,and uremic toxin+STS+extracellular signal-regulated kinase(ERK)inhibitor group.The concentration of STS used in the last two groups was 10 mg·L^(-1).The shear stress stimulation at 12 dyn·cm^(-2) was applied to the cells in various groups.The proliferation activities of the cells in various groups were detected by CCK-8 assay;the expression levels of ERK,nuclear factor kappa B(NF-κB),and typeⅠcollagen proteins in the cells in various groups were detected by Western blotting method;the expression levels of ERK,NF-κB,and typeⅠcollagen mRNA in the cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method;the apoptotic rates the cells in various groups were detected by TUNEL method.Results:The CCK-8 assay results showed that after treated with shear stress,the probiferation activitres of the cells in uremic toxin-stimulation group and uremic toxin+STS+ERK inhibitor group were lower than that in uremic toxin+STS group(P<0.01).The Western blotting results showed that compared with uremic toxin group,the expression levels of ERK,NF-κB,and typeⅠcollagen proteins in the cells in uremic toxin+STS group were increased(P<0.01).After inhibiting the ERK pathway,compared with blank control group,uremic toxin group,and uremic toxin+STS group,the expression levels of ERK,NF-κB,and typeⅠcollagen proteins in the cells in uremic toxin+STS+ERK inhibitor group were significantly decreased(P<0.01).The RT-qPCR results showed that compared with uremic toxin group,the expression levels of ERK,NF-κB,and typeⅠcollagen mRNA in the cells in uremic toxin+STS group were increased(P<0.01).After inhibiting the ERK signaling pathway,compared with blank control group,uremic toxin group,and uremic toxin+STS group,the expression levels of ERK,NF-κB,and typeⅠcollagen mRNA in the cells in uremic toxin+STS+ERK inhibitor group were significantly decreased(P<0.01).The TUNEL method detection results showed that the apoptotic rate in the cells in uremic toxin+STS group was lower than those in uremic toxin-stimulation group and uremic toxin+STS+ERK inhibitor group(P<0.05).Conclusion:A certain concentration of STS can improve the proliferation of the endothelial cells and reduce the apoptosis of the cells after treated with uremic toxins by modulating the expressions of NF-κB and typeⅠcollagen mRNA and proteins through the ERK signaling pathway.