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-中国药房

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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