基于TLR4/MyD88/NF-κB信号通路介导的巨噬细胞焦亡探讨黄芩黄连药对治疗动脉粥样硬化的作用机制  

Mechanism of Scutellariae Radix-Coptidis Rhizoma in the treatment of atherosclerosis based on TLR4/MyD88/NF-κB signaling pathway mediated pyrodeath of macrophages

作　　者：纪凌云 陈维达[2] 葛春蕾 吴俏兰 陈泽涛[2] 宋婷[2] JI Lingyun;CHEN Weida;GE Chunlei;WU Qiaolan;CHEN Zetao;SONG Ting(Shandong University of Traditional Chinese Medicine,Jinan 250355,China;Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250011,China;Linyi Hospital of Traditional Chinese Medicine,Linyi 276600,China)

机构地区：[1]山东中医药大学,济南250355 [2]山东中医药大学附属医院,济南250011 [3]临沂市中医医院,临沂276600

出　　处：《中华中医药杂志》2024年第5期2266-2272,共7页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：山东省自然科学基金项目(No.ZR2020MH354);济南市科技计划项目(No.202019038);齐鲁医派中医学术流派传承项目(No.202019151)。

摘　　要：目的:基于Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子κB(NF-κB)信号通路介导的巨噬细胞焦亡探讨黄芩黄连药对(芩连药对)治疗动脉粥样硬化(AS)的作用机制。方法:体外培养J774A.1细胞,SD大鼠灌胃芩连药对后制备含药血清,MTT法筛选芩连药对含药血清最佳干预浓度。采用不同浓度的含药血清预处理J774A.1细胞后,给予脂多糖(LPS)+三磷酸腺苷(ATP)诱导焦亡。光学显微镜下观察细胞形态变化,MTT法检测细胞活力,乳酸脱氢酶(LDH)释放法及Hoechst 33342/碘化丙锭(PI)染色用于检测细胞的损伤情况,酶联免疫吸附测定(ELISA)法测定细胞上清液中白细胞介素(IL)-1β、IL-18的水平。Western Blot法检测通路及焦亡相关蛋白TLR4、MyD88、NF-κB p65、p-NF-κB p65、NLRP3、ASC、Caspase-1、Cleave-Caspase-1、GSDMD、GSDMD-NT的表达。TLR4基因过表达后,检测通路相关蛋白表达水平及细胞焦亡情况。结果:芩连药对含药血清可改善造模后J774A.1的形态;提高造模后J774A.1细胞的活力、降低LDH释放率及PI细胞阳性率(P<0.05);降低细胞上清液中IL-1β及IL-18的水平(P<0.05);显著下调TLR4/MyD88/NF-κB信号通路及焦亡相关蛋白的表达(P<0.05);同时过表达TLR4可逆转芩连药对对J774A.1细胞焦亡的保护作用(P<0.05)。结论:芩连药对可通过抑制TLR4/MyD88/NF-κB信号通路降低巨噬细胞焦亡水平,从而发挥抗AS的作用。Objective:To investigate the mechanism of Scutellariae Radix-Coptidis Rhizoma drug(QLYD)in the treatment of atherosclerosis(AS)based on TLR4/MyD88/NF-κB signaling pathway mediated pyrodeath of macrophages.Methods:J774A.1 cell was cultured in vitro,and the drug-containing serum was prepared after inadministration of QLYD in SD rats.The optimal intervention concentration of the drug-containing serum was screened by MTT method.J774A.1 cells were pretreated with different concentrations of drug-containing serum,and lipopolysaccharide(LPS)+adenosine triphosphate(ATP)was given to induce pyrodeath.Morphological changes of cells were observed under optical microscope,cell viability was detected by MTT assay,and cell damage was detected by LDH release assay and Hoechst 33342/propyl iodide(PI)staining.The levels of interleukin(IL)-1βand IL-18 in cell supernatant were determined by enzyme-linked immunosorbent assay(ELISA).Western Blot assay was used to detect the expression of pathway and related proteins TLR4,MyD88,NF-κB p65,p-NF-κB p65,NLRP3,ASC,Caspase-1,Cleave-Caspase-1,GSDMD and GSDMD-NT.After TLR4 gene overexpression,the expression level of pathway-related proteins and the scorch death of cells were detected.Results:QLYD serum could improve the morphology of J774A.1 after molding.The activity of J774A.1 cells was increased,the LDH release rate and the positive rate of PI cells were decreased(P<0.05).The levels of IL-1βand IL-18 in cell supernatant were decreased(P<0.05).The expression of TLR4/MyD88/NF-κB signaling pathway and pyroptosis related protein were significantly down-regulated(P<0.05).Meanwhile,overexpression of TLR4 could reverse the protective effect of QLYD on J774A.1 cells(P<0.05).Conclusion:QLYD can reduce the pyroptosis level of macrophages by inhibiting TLR4/MyD88/NF-κB signaling pathway,so AS to play an anti-AS role.

关 键 词：黄芩黄连药对 J774A.1细胞 焦亡 含药血清 TLR4/MyD88/NF-κB信号通路 动脉粥样硬化 

分 类 号：R285.5[医药卫生—中药学]