左归降糖舒心方含药血浆对脂多糖诱导巨噬细胞损伤及焦亡的影响  

Effect of Zuogui Jiangtang Shuxin Formula-containing Plasma on LPS-induced Macrophage Damage and Pyroptosis

作　　者：杨金伟 周毅[2] 肖凡[1] 喻嵘 YANG Jinwei;ZHOU Yi;XIAO Fan;YU Rong(College of Integrative Medicine,Hunan University of Chinese Medicine,Changsha 410208 Hunan,China;The Second Affiliated Hospital of Integrative Medicine,Hunan University of Chinese Medicine,Liuyang 410300 Hunan,China;School of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208 Hunan,China)

机构地区：[1]湖南中医药大学中西医结合学院,湖南长沙410208 [2]湖南中医药大学附属第二中西医结合医院,湖南浏阳410300 [3]湖南中医药大学中医学院,湖南长沙410208

出　　处：《中药新药与临床药理》2025年第2期206-213,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(82205077);湖南省自然科学基金项目(2022JJ40315);湖南省教育厅资助科研项目(21B0390);湖南省卫生健康委科研计划项目(D202303067601);中药粉体与创新药物省部共建国家重点实验室培育基地开放基金项目(21PTKF1016)。

摘　　要：目的基于TLR4/NF-κB/NLRP3通路探讨左归降糖舒心方含药血浆对脂多糖(LPS)诱导小鼠J774A.1巨噬细胞损伤及焦亡的作用及机制。方法将分化完全的J774A.1巨噬细胞随机分为空白对照组(10%空白血浆)、模型组(10μg·mL^(-1)LPS+10%空白血浆)、5%中药含药血浆组(10μg·mL^(-1)LPS+5%左归降糖舒心方含药血浆)、10%中药含药血浆组(10μg·mL^(-1)LPS+10%左归降糖舒心方含药血浆)。除空白对照组外,其余各组以10μg·mL^(-1)LPS处理,诱导巨噬细胞炎性损伤及细胞焦亡。按照分组条件干预后,采用ELISA法检测细胞上清中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)和白细胞介素18(IL-18)含量;免疫荧光法检测细胞中NLRP3、ASC蛋白共表达情况;Western Blot法检测细胞TLR4/NF-κB/NLRP3通路及焦亡相关蛋白的表达水平。结果与空白对照组比较,模型组J774A.1细胞上清液中的TNF-α、IL-6、IL-1β、IL-18含量均显著升高(P<0.01);细胞的NLRP3、ASC荧光强度显著增强,蛋白共表达明显上调;TLR4、p-NF-κBp65、NLRP3、GSDMD-N、IL-1β蛋白表达显著上调(P<0.01)。与模型组比较,5%、10%中药含药血浆组细胞上清液中的TNF-α、IL-6、IL-1β、IL-18含量均显著降低(P<0.01);细胞的NLRP3、ASC荧光强度均明显减弱,蛋白共表达明显下调;TLR4、p-NF-κBp65、NLRP3、GSDMD-N、IL-1β蛋白表达显著下调(P<0.01)。结论左归降糖舒心方含药血浆能够抑制LPS诱导的J774A.1巨噬细胞损伤及炎症反应,其作用机制可能与调控TLR4/NF-κB/NLRP3炎症通路,抑制细胞焦亡及炎症因子表达有关。Objective To investigate the effect and mechanism of Zuogui Jiangtang Shuxin Formula-containing plasma on LPS-induced macrophage damage and pyroptosis based on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)pathway.Methods Fully differentiated J774A.1 cells were randomly divided into the blank control group(10%blank plasma),the model group(10μg·mL^(-1) LPS+10%blank plasma),5%(10μg·mL^(-1) LPS+5%Zuogui Jiangtang Shuxin Formula-containing plasma)and 10%(10μg·mL^(-1) LPS+10%Zuogui Jiangtang Shuxin Formula-containing plasma)Zuogui Jiangtang Shuxin Formula-containing plasma group.Except for the blank control group,other groups were treated with 10μg·mL^(-1) LPS to induce inflammatory injury and pyroptosis of macrophage.After the intervention,ELISA was used to detect concentration of tumor necrosis factor-alpha(TNF-α),interleukin 6(IL-6),interleukin 1 beta(IL-1β)and interleukin 18(IL-18)in macrophage supernatant.The co-expression of NOD-like receptor protein 3(NLRP3)and apoptotic spot-like protein(ASC)were detected by immunofluorescence assay.The expression of TLR4/NF-κB/NLRP3 signaling pathway and pyroptosis-related proteins were detected by Western Blot.Results Compared with the blank control group,the levels of TNF-α,IL-6,IL-1βand IL-18 in the supernatant of LPS-treated J774A.1 cells significantly increased(P<0.01).It was also found that the fluorescence intensity of NLRP3 and ASC in cells was significantly enhanced,and protein co-expression was significantly up-regulated.The protein co-expressions of TLR4,p-NF-κB p65,NLRP3,GSDMD-N and IL-1βwere significantly up-regulated(P<0.01).Compared with the model group,the levels of TNF-α,IL-6,IL-1βand IL-18 in the supernatant of Zuogui Jiangtang Shuxin Formulacontaining plasma-treated J774A.1 cells were obviously decreased(P<0.01).The fluorescence intensity of NLRP3 and ASC in cells was significantly reduced,and protein co-expression was significantly down-regulated.The protein coexpr

关 键 词：左归降糖舒心方含药血浆 J774A.1巨噬细胞 TLR4/NF-κB/NLRP3炎症通路 细胞焦亡 炎症因子 

分 类 号：R285.5[医药卫生—中药学]