摘要
目的探讨丹参酮ⅡA通过核转录因子-κB(NF-κB)通路抑制脂多糖(LPS)诱导人脐静脉血管内皮细胞(HUVECs)炎性反应的作用机制。方法通过预实验筛选出脂多糖最佳的诱导浓度;采用最适浓度的脂多糖模拟HUVECs炎性损伤模型,通过CCK-8测定不同浓度的丹参酮ⅡA对损伤细胞凋亡率的影响;酶联免疫吸附法检测各组细胞培养液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)的含量变化;流式细胞仪检测各组细胞中血管细胞黏附分子-1(VCAM-1)的表达情况;免疫细胞化学染色法检测各组细胞中VCAM-1表达水平;蛋白质印迹法(Western Blot)检测各组细胞NF-κB信号通路相关蛋白TNF-α、NF-κB、NF-κB抑制蛋白α(IκBα)的表达情况。结果CCK-8检测脂多糖浓度为100μg/mL时,细胞抑制率为47.5%,此时细胞的损伤适中,确定本实验诱导浓度为100μg/mL(P<0.01);在0.1~5.0μg/mL内,不同浓度的丹参酮ⅡA能明显提高损伤后HUVECs细胞活性,但在10.0μg/mL时,HUVECs细胞的增殖明显减少(P<0.05),因此,后续实验丹参酮ⅡA的浓度选择为5.0μg/mL。与对照组比较,脂多糖模型组HUVECs培养液中TNF-α、IL-1β、IL-6含量均增加(P<0.01),IL-10含量减少(P<0.01);与脂多糖模型组比较,丹参酮ⅡA组HUVECs培养液中TNF-α、IL-1β、IL-6含量均减少(P<0.05或P<0.01),IL-10含量增加(P<0.05);与对照组比较,脂多糖刺激诱导HUVECs中VCAM-1的表达增加,也增加了平均荧光强度(P<0.001),丹参酮ⅡA组显示出对脂多糖刺激的抑制作用,即平均荧光强度降低(P<0.05);对照组细胞质中有弱VCAM-1蛋白荧光表达,脂多糖模型组细胞质中有强VCAM-1蛋白荧光表达,而丹参酮ⅡA组中VCAM-1荧光表达弱于脂多糖模型组;与对照组比较,脂多糖模型组HUVECs中TNF-α、NF-κB蛋白表达均上调(P<0.01),IκBα蛋白表达下调(P<0.01),与脂多糖模型组比较,丹参酮ⅡA组HUVECs中TNF-α、NF-κB p65蛋白表达均下调(P<0.05),IκBα蛋白表达上调。结论丹参酮ⅡA能够逆转脂多糖诱导的HUVECs损伤,其机制可能与抑制NF-κB通路激活有关。
Objective To investigate the mechanism of tanshinoneⅡA inhibition of lipidpolysaccharide(LPS)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs)through the nuclear transcription factor-κB(NF-κB)pathway.Methods The optimal induction concentration of LPS was screened by preliminary experiments.The optimal concentration of LPS was simulated the inflammatory injury model of HUVECs,and the different concentration of tanshinoneⅡA on the apoptosis rate of injured cells was determined by CCK-8.The levels of Tumor necrosis factor-α(TNF-α),interleukin 1β(IL-1β),interleukin-6(IL-6)and interleukin 10(IL-10)in the cell culture fluid were detected by enzyme-linked immunosorbent assay.The expression of vascular cell adhesion molecule-1(VCAM-1)was detected by flow cytometry and immunocytochemical staining.The expressions of NF-κB signaling pathway-related proteins(TNF-α,NF-κB,IκBα)were detected by Western Blotting.Results When the concentration of LPS was 100μg/mL by CCK8,the cell inhibition rate was 47.5%.At this time,the damage of the cells was moderate,and the induced concentration was determined to be 100μg/mL(P<0.01).The concentration of LPS was 0.1-5.0μg/mL,different concentrations of TanshinoneⅡA could significantly improve the cell viability of HUVECs after injury.The concentration of LPS was 10.0μg/mL,the proliferation of HUVECs cells was significantly reduced(P<0.05),and the concentration of TanshinoneⅡA was selected to be 5.0μg/mL.Compared with the control group,the expressions of TNF-α,IL-1βand IL-6 in the culture medium of HUVECs in the LPS model group increased(P<0.01),and the expression of IL-10 decreased(P<0.01).Compared with the LPS model group,the expressions of TNF-α,IL-1βand IL-6 in the HUVECs culture medium of TanshinoneⅡA group decreased(P<0.05)and IL-10 content increased(P<0.05).Compared with control group,the expression of VCAM-1 in HUVECs induced by LPS increased,and also increased the mean fluorescence intensity(P<0.001),TanshinoneⅡA group showed inhibition of LPS stimulation,the average fluorescence intensity decreased(P<0.05).The expression of weak VCAM-1 protein was observed in the cytoplasm of the control group,the expression of VCAM-1 protein in the cytoplasm of the LPS model group was strongly expressed,while the fluorescence expression of VCAM-1 in the tanshinoneⅡA group was weaker than that in the LPS model group.Compared with the control group,the expressions of TNF-αand NF-κB in HUVECs of LPS model group were up-regulated(P<0.01),and the expression of IκBαprotein down-regulated(P<0.01).Compared with the LPS model group,the expression of TNF-αand NF-κB p65 protein in HUVECs of tanshinoneⅡA group down-regulated(P<0.05),and the expression of IκBαprotein up-regulated.Conclusion TanshinoneⅡA could reverse injury of HUVECs induced by LPS,and its mechanism might be related to the inhibition of NF-κB pathway activation.
作者
何德全
陈友权
王世祥
HE Dequan;CHEN Youquan;WANG Shixiang(The Third Affiliated Hospital of Guangzhou Medical University,Guangzhou 510150,Guangdong,China)
出处
《中西医结合心脑血管病杂志》
2021年第3期428-432,共5页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
基金
广东省中医药局科研课题(No.20191211)。
关键词
丹参酮ⅡA
NF-ΚB通路
人脐静脉血管内皮细胞
炎性反应
TanshinoneⅡA
nuclear transcription factor-κB
human umbilical vein endothelial cells
inflammatory response
作者简介
通讯作者:陈友权,E-mail:lidd1981xj@163.com。
