基于NLRP3炎症小体探讨葛根素对缺氧致肺动脉平滑肌细胞焦亡的影响  

Effects of Puerarin on Hypoxia Induced Pyroptosis of Pulmonary Artery Smooth Muscle Cells Based on NLRP3 Inflammasome

作　　者：张晓丹[1] 李文娣 张茹[1] 盛洁静[1] 张佳男 刘慧宇 李松林 ZHANG Xiaodan;LI Wendi;ZHANG Ru;SHENG Jiejing;ZHANG Jianan;LIU Huiyu;LI Songlin(School of Pharmacy,Harbin University of Commerce,Harbin 150076,China;School of Medical Laboratory Science and Technology,Daqing Campus of Harbin Medical University,Heilongjiang Daqing 163319,China)

机构地区：[1]哈尔滨商业大学药学院,哈尔滨150076 [2]哈尔滨医科大学大庆校区医学检验与技术学院,黑龙江大庆163319

出　　处：《中国药房》2021年第11期1337-1344,共8页China Pharmacy

基　　金：黑龙江省自然科学基金资助项目(No.ZD201416)。

摘　　要：目的:探讨葛根素(Pue)对缺氧诱导的肺动脉平滑肌细胞(PASMCs)焦亡的影响及其调控机制。方法:以大鼠PASMCs为对象,将其随机分为常氧组、缺氧组和缺氧+Pue组(0.2 mmol/L),除常氧组外,其余各组均于37℃、5%CO_(2)、3%O_(2)条件下培养24 h以建立缺氧模型。采用Western blot法检测细胞中焦亡相关蛋白[NOD样受体蛋白3(NLRP3)、胱天蛋白酶1、白细胞介素1β、凋亡相关斑点样蛋白]的表达水平;采用乳酸脱氢酶(LDH)释放实验检测细胞中LDH的释放量;采用Hoechst 33342/PI双染色法检测焦亡阳性细胞比例。另将PASMCs随机分为常氧+对照质粒组、缺氧+对照质粒组、缺氧+过表达质粒组和缺氧+过表达质粒+Pue组,除常氧+对照质粒组外,其余各组均同法建立缺氧模型;在分别转染对照质粒或NLRP3过表达质粒后,采用Western blot法、LDH释放实验和Hoechst 33342/PI双染色法检测Pue是否通过干扰NLRP3炎症小体来发挥对缺氧致PASMCs焦亡的影响。结果:与常氧组比较,缺氧组细胞中焦亡相关蛋白的表达水平、LDH释放量和焦亡阳性细胞比例均显著升高(P<0.05或P<0.01);而Pue可逆转上述指标(P<0.05或P<0.01)。当NLRP3炎症小体过表达时,细胞中焦亡相关蛋白的表达水平、LDH释放量和焦亡阳性细胞比例均显著升高(P<0.05或P<0.01);且Pue可通过调控NLRP3炎症小体而抑制上述现象(P<0.05或P<0.01)。结论:Pue可通过下调焦亡相关蛋白的表达、减少LDH的释放、降低焦亡阳性细胞比例,从而发挥对缺氧致PASMCs焦亡的抑制作用,其机制可能与抑制NLRP3炎症小体的活性有关。OBJECTIVE:To investigate the effects and mechanism of puerarin(Pue)on hypoxia-induced pyroptosis of pulmonary artery smooth muscle cells(PASMCs).METHODS:PASMCs of rats as research objects were randomly divided into normoxia group,hypoxia group and hypoxia+Pue group(0.2 mmol/L).Except for normoxia group,other groups were cultured with 5%CO_(2) and 3%O_(2) at 37℃for 24 hours to establish hypoxia model.Western blot assay was used to detect the expression of pyroptosis related proteins[NOD-like receptor protein-3(NLPR3),caspase-1,interleukin-1β(IL-1β),apoptosis-associated speck-like protein(ASC)].Lactate dehydrogenase(LDH)release assay was used to detect the release of LDH in cells;Hoechst 33342/PI double staining test was adopted to detect the proportion of pyroptosis positive cells.PASMCs was randomly divided into normoxia group+control plasmid group,hypoxia+control plasmid group,hypoxia+over-expression plasmid group and hypoxia+over-expression plasmid+Pue group.Except for the normoxia+control plasmid group,the other groups were established hypoxia model by the same method.After transfection of control plasmid or NLRP3 over-expression plasmid,Western blot,LDH release test and Hoechst 33342/PI double staining test were used to investigate whether Pue could inhibit hypoxia-induced PASMCs pyroptosis by interfering with the activity of NLRP3 inflammasomes.RESULTS:Compared with normoxia group,the expression of pyroptosis related proteins,the release of LDH and the proportion of pyroptosis positive cells were increased significantly in hypoxia group(P<0.05 or P<0.01).Pue had the effect of reversing the above indexes(P<0.05 or P<0.01).When the NLRP3 inflammasome was over-expressed,the expression of pyroptosis related proteins,the release of LDH and the proportion of pyroptosis positive cells were increased significantly(P<0.05 or P<0.01).Pue could inhibit the above phenomenon through regulating NLRP3 inflammasome(P<0.05 or P<0.01).CONCLUSIONS:Pue can significantly inhibit the hypoxia-induced pyroptosis of PASMCs by do

关 键 词：葛根素 肺动脉平滑肌细胞 焦亡 NOD样受体蛋白3炎症小体 

分 类 号：R285.5[医药卫生—中药学]