摘要
目的:探讨蛭龙活血通瘀胶囊对U937巨噬细胞焦亡及NLRP3炎症小体活化的影响,验证其抑制细胞焦亡的机制。方法:利用佛波酯(PMA)将U937单核细胞诱导为巨噬细胞,LPS+ATP激活NLRP3炎症小体建立细胞焦亡模型;瑞氏染色观察细胞分化情况;CCK-8法筛选超滤后蛭龙活血通瘀胶囊成分对U937巨噬细胞活力值影响最适宜的用药浓度;将实验分为正常对照组、模型对照组及蛭龙活血通瘀胶囊3μg/mL、10μg/mL、30μg/mL组,处理细胞后免疫印迹法测定各组细胞裂解物和培养上清液中核苷酸结合寡聚结构域样受体蛋白3(NLRP3)、Gsdmd-N、活化天冬氨酸蛋白酶-1(Caspase-1)、白介素-1β(IL-1β)蛋白的表达水平;高质粒转染NLRP3蛋白在细胞中的表达,流式细胞术及免疫印迹法检测蛭龙活血通瘀胶囊成分对转染后U937巨噬细胞抑制NLRP3蛋白表达的水平。结果:免疫印迹实验提示,与正常对照组相比,模型对照组NLRP3、Gsdmd-N、Caspase-1、IL-1β蛋白表达水平显著上调(P<0.01);经过蛭龙活血通瘀胶囊3μg/mL、10μg/mL、30μg/mL干预后,NLRP3、Gsdmd-N和Caspase-1蛋白表达水平下调P<0.05或P<0.01);IL-1β表达水平显著下调(P<0.01)。经高质粒转染NLRP3后,与模型对照组相比,NLRP3高质粒转染组可以增强细胞焦亡,上调NLRP3蛋白表达水平(P<0.01);与NLRP3高质粒转染+蛭龙活血通瘀胶囊30μg/mL组相比,空质粒转染+蛭龙活血通瘀胶囊组可以减少细胞焦亡,降低NLRP3蛋白表达水平(P<0.01)。蛭龙活血通瘀胶囊成分无法逆转高质粒转染NLRP3蛋白表达。结论:蛭龙活血通瘀胶囊能够通过NLRP3信号途径抑制U937巨噬细胞焦亡。
Objective:To investigate the effect of Zhilong Huoxue Tongyu capsule on pyroptosis of U937 macrophages and the activation of NLRP3 Inflammosome.Methods:U937 monocytes were induced into macrophages by PMA,the NLRP3 inflammasome was activated by LPS+ATP to establish a pyroptosis model.The cell differentiation was observed by Wright staining.CCK 8 assay was used to screen the most optimal concentration of Zhilong Huoxue Tongyu capsule.The cells were divided into the control group,the model group and Zhilong Huoxue Tongyu(ZL)capsule groups(3μg/mL,10μg/mL,30μg/mL).After treatment,the expression levels of NLRP3,Gsdmd-N,Caspase-1 and IL-1βin cell lysates and culture supernatant were determined by western blot.The expression of NLRP3 protein in cells was transfected with high plasmid and the protein expression of NLRP3 inhibited by U937 macrophages after transfected was detected by flow cytometry and western blot.Results:Western blotting result showed,compared with the control group,the expression levels of NLRP3,Gsdmd-N,Caspase-1 and IL-1βwere increased in the model group(P<0.01).After treated with ZL(3μg/mL,10μg/mL,30μg/mL),the protein expression levels of NLRP3,Gsdmd-N and Caspase-1 were down regulated(P<0.05,P<0.01),and IL-1βexpression level was down regulated(P<0.01).After transfected with high plasmid,compared with the model group,pIRES2,-EGFP,-NLRP3 enhanced pyroptosis and up-regulated the expression level of NLRP3 protein(P<0.01).Compared with pIRES2-EGFP-NLRP3+30μg/mL ZL group,the pyroptosis was reduced and the expression level of NLRP3 protein was decreased in empty plasmid transfected NLRP3+ZL group(P<0.01).Zhilong Huoxue Tongyu Capsule didn’t reverse the expression of NLRP3 protein when high plasmid transfection was performed.Conclusion:Zhilong Huoxue Tongyu capsule can inhibit U937 macrophage pyroptosis by NLRP3 signaling pathway.
作者
刘孟楠
任维
张伟
杨廷富
李亚琴
罗钢
周华
杨思进
Liu Mengnan;Ren Wei;Zhang Wei;Yang Tingfu;Li Yaqin;Luo Gang;Zhou Hua;Yang Sijin(Department of Cardiovascular Medicine,The TCM Affiliated Hospital of Southwest Medical University,Luzhou 646000;Faculty of Chinese Medicine,Macao University of Science and Technology,Macao,China 999078)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2020年第6期156-161,共6页
Pharmacology and Clinics of Chinese Materia Medica
基金
2020年国家自然科学基金(编号:82074378)
四川省科技厅项目(编号:2018SZ0060)
西南医科大学—西南医科大学附属中医医院联合项目(编号:2018XYLH-O05)
泸州市科技局项目(编号:2019-RCM-95)。
