Page 63 - 中国药房2021年11期

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 Δ

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张晓丹 ，李文娣 ，张 茹 ，盛洁静 ，张佳男 ，刘慧宇 ，李松林（1.哈尔滨商业大学药学院，哈尔滨 150076；
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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）
中图分类号 R285.5 文献标志码 A 文章编号 1001-0408（2021）11-1337-08
DOI 10.6039/j.issn.1001-0408.2021.11.10
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％CO2、3％O2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。
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

Effects of Puerarin on Hypoxia Induced Pyroptosis of Pulmonary Artery Smooth Muscle Cells Based on
NLRP3 Inflammasome
ZHANG Xiaodan ，LI Wendi ，ZHANG Ru ，SHENG Jiejing ，ZHANG Jianan ，LIU Huiyu ，LI Songlin（1. School
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of Pharmacy，Harbin University of Commerce，Harbin 150076，China；2. School of Medical Laboratory
Science and Technology，Daqing Campus of Harbin Medical University，Heilongjiang Daqing 163319，China）
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 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
Δ 基金项目：黑龙江省自然科学基金资助项目（No.ZD201416）
＊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 NLRP3 inflammasome （P＜0.05 or P＜0.01）.

中国药房 2021年第32卷第11期 China Pharmacy 2021 Vol. 32 No. 11 ·1337 ·