黄芪甲苷抑制NLRP3/IL-1β轴改善高糖诱导的血管平滑肌细胞炎症反应  

Astragaloside Ⅳ improves high glucose-induced inflammation in vascular smooth muscle cells by inhibiting NLRP3/IL-1β axis

作　　者：贾新菊[1] 康岩[1] 杜丽娜[2] 张洁[1] 周慧敏[1] 王丽娜[1] JIA Xinju;KANG Yan;DU Li'na;ZHANG Jie;ZHOU Huimin;WANG Li'na(Department of Endocrinology,First Hospital of Hebei Medical University,Shijiazhuang 050000,Hebei,China;Third Department of Neurology,First Hospital of Hebei Medical University,Shijiazhuang 050000,Hebei,China)

机构地区：[1]河北医科大学第一医院内分泌科,河北石家庄050000 [2]河北医科大学第一医院神经内三科,河北石家庄050000

出　　处：《上海中医药大学学报》2020年第3期62-69,共8页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：河北省卫计委2017年医学科学研究重点项目(20170505)。

摘　　要：目的:探讨黄芪甲苷(AS-Ⅳ)对高糖诱导的血管平滑肌细胞(VSMCs)炎症反应的作用及机制。方法:①采用不同浓度的葡萄糖(5.5、10、15、25 mmol/L)刺激VSMCs细胞24 h,或高浓度葡萄糖(25 mmol/L)处理VSMCs不同时间(0、6、12、24 h)。Western blot检测NOD样受体蛋白3(NLRP3)蛋白表达;ELISA检测白介素-1β(IL-1β)含量。②采用不同浓度(0、10、20、40μg/ml)AS-Ⅳ干预VSMCs细胞24 h后,CCK8法检测并计算细胞活力。③将VSMCs细胞分为对照组、高糖组、AS-Ⅳ组和AS-Ⅳ+高糖组。先给予AS-Ⅳ组和AS-Ⅳ+高糖组20μg/ml AS-Ⅳ预孵育2 h后,再向高糖组和AS-Ⅳ+高糖组加入25 mmol/L葡萄糖刺激24 h。Western blot检测NLRP3蛋白表达;ELISA检测IL-1β含量。④构建si-NLRP3 RNA转染的VSMCs细胞。将细胞分为对照组、高糖组、si-NLRP3组和si-NLRP3+高糖组。细胞转染6 h后,高糖组和si-NLRP3+高糖组细胞加入25 mmol/L葡萄糖刺激24 h。PCR检测NLRP3 mRNA表达;ELISA检测IL-1β含量。⑤应用转染技术构建NLRP3过表达的VSMCs细胞。将细胞分为对照组、AS-Ⅳ组、NLRP3过表达组和NLRP3过表达+AS-Ⅳ组。细胞转染6 h后,AS-Ⅳ组和NLRP3过表达+AS-Ⅳ组加入20μg/ml AS-Ⅳ干预24 h。PCR检测NLRP3 mRNA表达;ELISA检测IL-1β含量。结果:①高糖刺激能够上调VSMCs细胞NLRP3和IL-1β蛋白表达(P<0.01),且呈一定的浓度/时间依赖性,故选取25 mmol/L葡萄糖作用24 h进行后续研究。②10、20μg/ml AS-Ⅳ作用VSMCs细胞24 h后,细胞活力无显著变化(P>0.05),选择20μg/ml浓度进行后续实验。③与对照组相比,AS-Ⅳ组细胞NLRP3蛋白表达量显著下调(P<0.05),IL-1β含量显著降低(P<0.05);与高糖组相比,AS-Ⅳ+高糖组NLRP3蛋白表达量显著减少(P<0.01),IL-1β含量显著降低(P<0.01)。④与对照组相比,si-NLRP3组细胞NLRP3 mRNA表达量显著降低(P<0.01),IL-1β含量没有明显变化;与高糖组相比,si-NLRP3+高糖组NLRP3 mRNA表达量显著减少(P<0.01),IL-1β含量显著降�Objective: To study the effects and mechanisms of astragaloside Ⅳ(AS-Ⅳ) on high glucose-induced inflammation in vascular smooth muscle cells(VSMCs). Methods: ①The VSMCs were stimulated with glucose at different concentrations(5.5, 10, 15, 25 mmol/L) for 24 hours, or treated with glucose at high concentration(25 mmol/L) for different time(0, 6, 12, 24 hours).The protein expression of Nod-like receptor protein 3(NLRP3) was detected by Western blot. The content of interleukin-1β(IL-1β) was detected by ELISA. ②The VSMCs were treated with AS-Ⅳ at different concentrations(0, 10, 20, 40 μg/ml) for 24 hours, the cell vitality was detected and calculated by CCK8 assay. ③The VSMCs were divided into the control group, high glucose group, AS-Ⅳ group and AS-Ⅳ + high glucose group. The AS-Ⅳ group and AS-Ⅳ + high glucose group were treated with 20 μg/ml AS-Ⅳ for 2 hours, and then 25 mmol/L glucose was added to the high glucose group and AS-Ⅳ + high glucose group for 24 hours stimulation. The protein expression of NLRP3 was detected by Western blot. The content of IL-1β was detected by ELISA. ④The si-NLRP3 RNA transfected VSMCs were established. The cells were divided into the control group, high glucose group, si-NLRP3 group and si-NLRP3 + high glucose group. Six hours after transfection, 25 mmol/L glucose was added to the high glucose group and si-NLRP3 + high glucose group for 24 hours stimulation. The mRNA expression of NLRP3 was detected by PCR. The content of IL-1β was detected by ELISA. ⑤The NLRP3 overexpressed VSMCs were constructed by transfection technique. The cells were divided into the control group, AS-Ⅳ group, NLRP3 overexpression group and NLRP3 overexpression + AS-Ⅳ group. Six hours after transfection, 20 μg/ml AS-Ⅳ was added to the AS-Ⅳ group and NLRP3 overexpression + AS-Ⅳ group for 24 hours. The mRNA expression of NLRP3 was detected by PCR. The content of IL-1β was detected by ELISA. Results: ①High glucose stimulation could up-regulate the protein expressions of

关 键 词：黄芪甲苷 炎症 血管平滑肌细胞 NLRP3 IL-1Β 

分 类 号：R285[医药卫生—中药学]