二仙丸加减方含药血清抑制巨噬细胞焦亡的物质基础和作用机制  

Material basis and action mechanism of drug-containing serum of Modified Erxian Pill inhibiting macrophage pyroptosis

作　　者：李思源 王瑜茹 徐烨 郭地 南楠 刘杨 赵杰 郝慧琴 Li Siyuan;Wang Yuru;Xu Ye;Guo Di;Nan Nan;Liu Yang;Zhao Jie;Hao Huiqin(Third Clinical College of Shanxi University of Chinese Medicine,Jinzhong 030619,Shanxi Province,China;Basic Laboratory of Integrated Traditional Chinese and Western Medicine,Shanxi University of Chinese Medicine,Jinzhong 030619,Shanxi Province,China;Shanxi Provincial Key Laboratory of Classical Prescription Strengthening Yang,Taiyuan 030013,Shanxi Province,China)

机构地区：[1]山西中医药大学第三临床学院,山西省晋中市030619 [2]山西中医药大学中西医结合基础实验室,山西省晋中市030619 [3]经方扶阳山西省重点实验室,山西省太原市030013

出　　处：《中国组织工程研究》2025年第19期4029-4037,共9页Chinese Journal of Tissue Engineering Research

基　　金：中央引导地方科技发展资金项目(YDZJSX2021A040),项目负责人:郝慧琴;中西医结合防治风湿免疫病山西省科技创新人才重点团队(202204051002033),项目负责人:郝慧琴;山西中医药大学风湿免疫性疾病中西医结合基础学科建设项目(2023XKJS-03),项目负责人:郝慧琴;经方扶阳山西省重点实验室开放课题研究基金资助(202104010910011),项目负责人:赵杰;山西中医药大学2022年研究生教育创新项目(2022CX014),项目负责人:李思源。

摘　　要：背景:课题组前期研究发现二仙丸加减方可以缓解胶原诱导性关节炎大鼠炎症反应,但其作用机制尚需进一步验证。目的:分析二仙丸加减方入血成分,观察二仙丸加减方含药血清对J774A.1巨噬细胞焦亡的影响。方法:①二仙丸加减方入血成分分析:采用超高效液相色谱-高分辨质谱(UHPLC-HRMS)对二仙丸加减方及入血成分进行检测和鉴定。②二仙丸加减方含药血清对J774A.1巨噬细胞焦亡的影响:采用分子对接技术对二仙丸加减方入血成分倍半萜类化合物与NLRP3进行初步验证。将J774A.1巨噬细胞随机分为空白对照组、脂多糖+三磷酸腺苷组及脂多糖+三磷酸腺苷+二仙丸加减方含药血清低(2.5%)、中(5%)、高(10%)剂量组。根据试剂盒说明书检测各组细胞上清液中乳酸脱氢酶释放情况;ELISA检测各组细胞上清液中白细胞介素1β、白细胞介素18水平;Hoechst/PI染色法检测各组细胞膜受损情况;Western blot检测各组细胞中NLRP3、Caspase-1、GSDMD及GSDMD-N蛋白表达水平。结果与结论:①共鉴定出二仙丸加减方药效成分32个,入血成分21个,其中入血成分主要包括多种倍半萜类化合物;②分子对接结果显示3-O-Acetyl-13-deoxyphomenone、Incensol oxide、Atractylenolide Ⅲ、Rupestonic acid、3,7-Dihydroxy-9,11-eremophiladien-8-one与NLRP3之间结合活性较好;③与空白对照组相比,脂多糖+三磷酸腺苷组细胞上清中乳酸脱氢酶活性及白细胞介素1β、白细胞介素18水平均显著升高(P<0.001),Hoechst/PI染色可见PI阳性细胞数量显著增加。脂多糖+三磷酸腺苷+二仙丸加减方含药血清各组上述指标均显示不同程度降低;④与空白对照组相比,脂多糖+三磷酸腺苷组NLRP3、Caspase-1、GSDMD及GSDMD-N蛋白表达显著升高(P<0.05);与脂多糖+三磷酸腺苷组相比,脂多糖+三磷酸腺苷+二仙丸加减方含药血清各组细胞中NLRP3、Caspase-1、GSDMD及GSDMD-N蛋白表达均有不同程度降BACKGROUND:Our previous study found that Modified Erxian Pill could alleviate inflammation in collagen-induced arthritis rats,but its mechanism needs to be further verified.OBJECTIVE:To analyze the components absorbed in the blood of Modified Erxian Pill,and observe the effect of the drug-containing serum of Modified Erxian Pill on pyroptosis of J774A.1 macrophages.METHODS:(1)Analysis of components absorbed in the blood of Modified Erxian Pill:Ultra-high performance liquid chromatography-high resolution mass spectrometry was used to detect and identify Modified Erxian Pill and its components absorbed in the blood.(2)Effect of the drug-containing serum of Modified Erxian Pill on pyroptosis of J774A.1 macrophages:Molecular docking technology was used to initially verify the sesquiterpenoids and NLRP3 in components absorbed in the blood of Modified Erxian Pill.J774A.1 macrophages were randomly divided into blank control group,lipopolysaccharide+adenosine triphosphate group,and lipopolysaccharide+adenosine triphosphate+Modified Erxian Pill with low(2.5%),medium(5%),and high(10%)dose groups.The release of lactate dehydrogenase in the cell supernatant of each group was detected according to the kit instructions.The levels of interleukin-1β and interleukin-18 in cell supernatant were detected in each group by ELISA.The cell membrane damage was detected by Hoechst/PI staining.The expression levels of NLRP3,Caspase-1,GSDMD,and GSDMD-N protein in the cells of each group were detected by western blot assay.RESULTS AND CONCLUSION:(1)A total of 32 active components of Modified Erxian Pill were identified,and 21 components entered the blood.The main components into blood included a variety of sesquiterpenoids.(2)Molecular docking results showed that 3-O-Acetyl-13-deoxyphomenone,Incensol oxide,Atractylenolide Ⅲ,Rupestonic acid,and 3,7-Dihydroxy-9,11-eremophiladien-8-one had good binding activity with NLRP3.(3)Compared with the blank control group,lactate dehydrogenase activity and the expression levels of interleukin-1βand

关 键 词：二仙丸加减方 含药血清 细胞焦亡 J774A.1巨噬细胞 倍半萜类 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R255