摘要
目的:采用四氢小檗碱(tetrahydroberberine,THB)作用于模拟脑缺血再灌注损伤的糖氧剥夺模型(oxygen glucose deprivation,OGD)和脂多糖(lipopolysaccharides,LPS)诱导血管内皮细胞损伤模型,探讨其对血管内皮细胞损伤的干预作用。方法:采用MTT比色法检测THB终浓度分别为0、2.5、5、10、20、40μg/mL对EA.hy926细胞存活率的影响。实验第I小组分组:正常对照组、OGD组、OGD+THB-10μg/mL组、OGD+THB-20μg/mL组、OGD+THB-40μg/mL组;实验第Ⅱ小组分组:正常对照组、LPS组、OGD+LPS组以及OGD+LPS+THB-20μg/mL组。通过荧光定量PCR检测实验I、Ⅱ处理小组的炎症相关因子如Caspase-1、ASC、IL-1β、IL-6等基因的相对表达量,同时Western Blot分析检测IL-1β、HO-1蛋白表达量。结果:MTT法结果表明四氢小檗碱对EA.hy926细胞存活无影响即不存在细胞毒性。实验I、Ⅱ处理小组的qPCR结果显示,OGD模型能明显通过下调Caspase-1、ASC、IL-1β与IL-6基因的表达发挥抗炎的应激保护作用;THB对于OGD模型的抗炎效果一般,但通过OGD+LPS组与OGD+LPS+THB-20μg/mL组对比发现THB也是通过下调炎症基因表达发挥抗炎效果的。Western Blot分析检测IL-1β蛋白表达量与qPCR结果一致,HO-1蛋白作为保护因子表达量与炎症因子荧光定量结果相反。结论:OGD模型可以提高细胞应激保护作用,四氢小檗碱通过下调Caspase-1、ASC、IL-1β与IL-6基因的表达,上调HO-1蛋白表达,抑制NLRP3炎症小体的活化发挥抗炎的作用。
Objective:To investigate the effects and mechanisms of tetrahydroberberine(THB)on vascular endothelial cell injury induced by oxygen glucose deprivation(OGD)and LPS-induced cell injury model.Methods: MTT assay was used to detect the effects of THB final concentration on the survival rate of EA.hy926 cells,which were 0μg/mL,2.5μg/mL,5μg/mL,10μg/mL,20μg/mL and 40μg/mL,respectively.GroupⅠof the experiment the normal control group,the OGD group,the OGD+THB-10μg/mL group,the OGD+THB-20μg/mL group,the OGD+THB-40μg/mL group;Experimental group II group the normal Control group,the LPS group,the OGD+LPS group and the OGD+LPS+THB-20μg/mL group.The relative expression of inflammation-related factors such as Caspase-1,ASC,IL-1βand IL-6 was detected by fluorescence quantitative PCR,and the expression of IL-1βand HO-1 was detected by Western Blot analysis.Results: The results of MTT assay showed that THB had no effect on the survival of EA.hy926 cells.The qPCR results of the experimental groupⅠandⅡshowed that the OGD model could play an anti-inflammatory protective role by down-regulating the expression of Caspase-1,ASC,IL-1βand IL-6 genes.The anti-inflammatory effect of THB on the OGD model was general,but it was found that THB also exerted an anti-inflammatory effect by down-regulating the expression of inflammatory genes by the OGD+LPS group compared with the OGD+LPS+THB-20μg/mL group.Western Blot analysis showed that the expression of IL-1βprotein was consistent with the results of qPCR.The expression of HO-1 protein as a protective factor was contrary to that of inflammatory factor fluorescence quantitative analysis.Conclusion: OGD model can improve the protective effect of cell stress.THB plays an anti-inflammatory role by down-regulating the expression of Caspase-1,ASC,IL-1βand IL-6 genes,up-regulating the expression of HO-1 protein and inhibiting the activation of NLRP3 inflammatory bodies.
作者
方星悦
刘启兵
杨潇
易国辉
樊好飞
黄静
FANG Xing-yue;LIU Qi-bing;YANG Xiao;YI Guo-hui;FAN Hao-fei;HUANG Jing(School of Basic and Life Science, Hainan Medical University, Haikou, 571199, China;The first Affiliated Hospital of Hainan Medical University, Haikou, 571199, China;Science Experiment Center of Hainan Medical College University, Haikou, 571199, China)
出处
《海南医学院学报》
CAS
2020年第15期1146-1151,1158,共7页
Journal of Hainan Medical University
基金
海南省自然科学基金青年基金项目(818QN247)
国家自然科学基金项目(81460550)
海南省重点科技计划项目(ZDYF2018154)。
