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
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篇名: 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
TITLE: Effects of Puerarin on Hypoxia Induced Pyroptosis of Pulmonary Artery Smooth Muscle Cells Based on NLRP 3 Inflammasome
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(0.2mmol/L),除常氧组外,其余各组均于37℃、5%CO2、3%O2条件下培养24h以建立缺氧模型。采用Westernblot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Hoechst33342/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Westernblot法、LDH释放实验和Hoechst33342/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。
ABSTRACT: 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% CO2 and 3% O2 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-1 β (IL-1 β),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 NLRP 3 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 NLRP 3 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 NLRP 3 inflammasome was over-expressed ,the expression of pyroptosis related proteins ,the release of LDH and the proportion of Δ 基金项目:黑龙江省自然科学基金资助项目(No.ZD201416) pyroptosis positive cells were increased significantly (P<0.05 *教授,博士生导师 ,博士。研究方向 :心血管药理学 。电话: or P<0.01). Pue could inhibit the above phenomenon through 0451-58853046。E-mail:zhangxd85@163.com regulating NLRP 3 inflammasome (P<0.05 or P<0.01). 中国药房 2021年第32卷第11期 China Pharmacy 2021Vol. 32 No. 11 ·1337· CONCLUSIONS:Pue can significantly inhibit the hypoxia-induced pyroptosis of PASMCs by down-regulating the expression of pyroptosis related proteins ,reducing the release of LDH and proportion of pyroptosis positive cells. The mechanism is related to the activity inhibition of NLRP 3 inflammasome.
期刊: 2021年第32卷第11期
作者: 张晓丹,李文娣,张茹,盛洁静,张佳男,刘慧宇,李松林
AUTHORS: ZHANG Xiaodan ,LI Wendi ,ZHANG Ru,SHENG Jiejing ,ZHANG Jianan,LIU Huiyu,LI Songlin
关键字: 葛根素;肺动脉平滑肌细胞;焦亡;NOD样受体蛋白3炎症小体
KEYWORDS: Puerarin;Pulmonary artery smooth muscle cells ;Pyroptosis;NLRP3 inflammasome
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