摘要
目的观察芪黄煎剂对脂多糖(lipopolysaccharide,LPS)刺激的巨噬细胞M1型极化及p38丝裂原活化蛋白激酶(p38 mitogen activated protein kinases,p38MAPK)/核因子-κB(nuclear factor-kappa B,NF-κB)通路的影响。方法对SD大鼠灌服芪黄煎剂制备含药血清。将RAW264.7巨噬细胞分为对照组、LPS组、LPS+芪黄煎剂组、芪黄煎剂+LPS组、LPS+NF-κB抑制剂组、LPS+p38MAPK抑制剂组、LPS+NF-κB抑制剂+芪黄煎剂组及LPS+p38MAPK抑制剂+芪黄煎剂组。除了对照组,其余各组均使用LPS诱导RAW264.7巨噬细胞24 h。采用流式细胞术检测巨噬细胞极化方向;ELISA法测定细胞培养上清液中白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)水平。RT-qPCR法检测细胞内IL-6、IL-1β、TNF-α、NF-κB p65、p38MAPK、核因子-κB抑制蛋白α(inhibitor of nuclear factor-kappa B alpha,IκBα)、Toll样受体4(Toll-like receptor 4,TLR4)、肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)mRNA表达水平,Western blot法检测NF-κB p65、p38MAPK、IκBα、TLR4、TRAF6蛋白表达水平。结果与对照组比较,LPS组细胞表面CD86表达水平升高,CD206表达水平降低(P<0.05);与LPS组比较,LPS+芪黄煎剂组细胞表面CD86表达水平降低,CD206表达水平升高(P<0.05);与LPS+NF-κB抑制剂组比较,LPS+NF-κB抑制剂+芪黄煎剂组细胞表面CD86、CD206表达水平降低(P<0.05)。芪黄煎剂、NF-κB抑制剂显著降低LPS诱导的RAW264.7巨噬细胞上清液中IL-6、IL-1β、TNF-α水平(P<0.05),芪黄煎剂加强了NF-κB抑制剂的作用(P<0.05)。芪黄煎剂、NF-κB抑制剂显著降低LPS诱导的RAW264.7巨噬细胞中IL-6、IL-1β、TNF-α、NF-κB p65、p38MAPK、IκBαmRNA及其蛋白表达水平(P<0.05),芪黄煎剂加强了NF-κB抑制剂的作用(P<0.05);NF-κB抑制剂不能显著降低LPS诱导的RAW264.7巨噬细胞中TLR4、TRAF6 mRNA及其蛋白表达水平(P>0.05),而芪黄煎剂显著加强了NF-κB抑制剂的作用(P<0.05)。结论芪黄煎剂含药血清可抑制LPS诱导的巨噬细胞炎症反应,其机制可能与调控p38MAPK/NF-κB信号通路抑制巨噬细胞向M1型极化有关。
Objective To investigate the effect of Qihuang Decoction on lipopolysaccharide(LPS)-induced M1 macrophage polarization and the p38 mitogen-activated protein kinase(p38MAPK)/nuclear factor-kappa B(NF-κB)signaling pathway.Methods Sprague-Dawley rats were given Qihuang Decoction by gavage to prepare drug-containing serum.RAW264.7 macrophages were divided into control group,LPS group,LPS+Qihuang Decoction group,Qihuang Decoction+LPS group,LPS+NF-κB inhibitor group,LPS+p38MAPK inhibitor group,LPS+NF-κB inhibitor+Qihuang Decoction group,and LPS+p38MAPK inhibitor+Qihuang Decoction group.All groups except the control group were given LPS to induce RAW264.7 macrophages for 24 hours.Flow cytometry was used to detect the polarization of macrophages;ELISA was used to measure the levels of interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α)in supernatant;RT-qPCR was used to measure the mRNA expression levels of IL-6,IL-1β,TNF-α,NF-κB p65,p38MAPK,inhibitor of nuclear factor-kappa B alpha(IκBα),Toll-like receptor 4(TLR4),and TNF receptor-associated factor 6(TRAF6),and Western blot was used to measure the protein expression levels of NF-κB p65,p38MAPK,IκBα,TLR4,and TRAF6.Results Compared with the control group,the LPS group had a significant increase in the expression level of CD86 and a significant reduction in the expression level of CD206 on cell surface(P<0.05);compared with the LPS group,the LPS+Qihuang Decoction group had a significant reduction in the expression level of CD86 and a significant increase in the expression level of CD206(P<0.05);compared with the LPS+NF-κB inhibitor group,the LPS+NF-κB inhibitor+Qihuang Decoction group had significant reductions in the expression levels of CD86 and CD206(P<0.05).Qihuang Decoction and NF-κB inhibitor significantly reduced the levels of IL-6,IL-1β,and TNF-αin the supernatant of RAW264.7 macrophages induced by LPS(P<0.05),and Qihuang Decoction enhanced the effect of NF-κB inhibitor(P<0.05).Qihuang Decoction and NF-κB inhibitor significantly reduced the mRNA and protein expression levels of IL-6,IL-1β,TNF-α,NF-κB p65,p38MAPK,and IκBαin RAW264.7 macrophages induced by LPS(P<0.05),and Qihuang Decoction enhanced the effect of NF-κB inhibitor(P<0.05);NF-κB inhibitor could not significantly reduce the mRNA and protein expression levels of TLR4 and TRAF6 in RAW264.7 macrophages induced by LPS(P>0.05),while Qihuang Decoction significantly enhanced the effect of NF-κB inhibitor(P<0.05).Conclusion The drug serum containing Qihuang Decoction can inhibit inflammatory response in macrophages induced by LPS,possibly by regulating the p38MAPK/NF-κB signaling pathway to inhibit M1 macrophage polarization.
作者
刘海韦
张琦
于庆生
刘举达
舒亚倩
刘旭
齐伟
黄龙
彭辉
LIU Haiwei;ZHANG Qi;YU Qingsheng;LIU Juda;SHU Yaqian;LIU Xu;QI Wei;HUANG Long;PENG Hui(The First Affiliated Hospital of Anhui University of Chinese Medicine,Anhui Hefei 230031,China;Institute of Chinese Traditional Surgery,Anhui Academy of Chinese Medicine,Anhui Hefei 230031,China)
出处
《安徽中医药大学学报》
2025年第3期75-81,共7页
Journal of Anhui University of Chinese Medicine
基金
安徽高校自然科学研究重点项目(2023AH050751)
安徽省卫生健康科研项目(AHWJ2023A20362)
安徽省临床医学研究转化专项(202427b10020025)。
关键词
胃切除术
巨噬细胞极化
p38MAPK/NF-κB信号通路
芪黄煎剂
Gastrectomy
Macrophage polarization
p38 Mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway
Qihuang Decoction
作者简介
刘海韦(1996-),女,硕士研究生;通信作者:张琦(1980-),男,博士,主任医师,博士研究生导师,zhangqi0551zq@163.com。
