补阳还五汤调控TLR4-NFκB-NLRP3炎症小体信号通路及线粒体自噬缓解LPS诱导巨噬细胞炎症的机制OA
Mechanism of Buyang Huanwu Decoction(补阳还五汤)on LPS-induced macrophage inflam-mation via modulating the TLR4-NF-κB-NLRP3 inflammasome pathway and mitophagy
目的 探究补阳还五汤通过调控TLR4-NFκB-NLRP3炎症小体信号通路及线粒体自噬缓解 LPS 诱导巨噬细胞炎症的机制研究.方法 将RAW264.7巨噬细胞分为正常组、模型组、模型对照组、补阳还五汤低、中、高剂量组、NLRP3抑制剂组、线粒体自噬抑制剂组、线粒体自噬诱导剂组、ROS抑制剂组;CCK-8实验检测细胞活力值筛选合适的LPS造模浓度和NLRP3抑制剂(MCC950)、线粒体自噬抑制剂(cyclosporine)、ROS抑制剂(NAC)、线粒体自噬诱导剂(valinomycin)给药浓度;流式细胞仪检测细胞氧化应激及ROS;JC-1荧光染色观察线粒体结构功能变化;溶酶体荧光探针LysoTracker和线粒体荧光探针MitoTracker共定位观察溶酶体与线粒体的融合情况;mcherry-GFP-LC3双荧光体系等融合蛋白来示踪自噬体形成以及自噬流的顺畅程度;免疫荧光检测细胞中NLRP3定位及活化;Western blot检测细胞LC3B、NLRP3、ASC、pro-Caspase-1、TLR4-Myd88-IRAK4-TRAF6-NFκB通路、P-NFκB、P-IκB、NFκB、IκB、NIX、BNIP3、FUNDC1、P-PINK1、P-Parkin、PINK1、Parkin蛋白的表达;qPCR检测细胞中NIX、BNIP3、FUNDC1、PINK1、Parkin mRNA的变化.结果 补阳还五汤含药血清联合抑制剂组可显著降低ROS的表达水平(P<0.05,P<0.01),显著升高JC-1聚集体/单体荧光强度比值(Red/Green Ratio)、红/黄斑点数比值与皮尔逊相关系数(P<0.05,P<0.01),线粒体自噬NIX、BNIP3、FUNDC1、PINK1、Parkin mRNA基因及其蛋白LC3B、Beclin1、P62、NIX、BNIP3、FUNDC1、P-PINK/PINK、P-Parkin/Parkin表达水平升高(P<0.05,P<0.01),TLR4、Myd88、IRAK4、TRAF-6、P-NFκB、NFκB、IκB、P-IκB、NLRP3、pro-caspase-1、ASC表达水平降低(P<0.05,P<0.01).结论 补阳还五汤可能通过TLR4-MyD88-IRAK4-TRAF6-NFκB通路能够减轻巨噬细胞氧化应激及改善受损线粒体功能进而减少炎症反应.
Objective To investigate the mechanism of Buyang Huanwu Decoction(补阳还五汤,BYHWD)in alleviating lipopoly-saccharide(LPS)-induced macrophage inflammation by modulating the TLR4-NF-κB-NLRP3 inflammasome signaling pathway and mitophagy.Methods RAW264.7 macrophages were divided into the following groups:normal,model,model control,low/medium/high-dose BYHWD,NLRP3 inhibitor(MCC950),mitophagy inhibitor(cyclosporin A),mitophagy inducer(valinomycin),and reactive oxygen species(ROS)inhibitor(N-acetylcysteine,NAC).Cell Counting Kit-8(CCK-8)assay was performed to determine cell viability and to screen optimal concentrations for LPS modeling and pharmacological interventions.Flow cytometry(FCM)was used to assess oxidative stress and ROS levels.Mitochondrial membrane potential was evaluated by JC-1 fluorescence staining.Co-localization of LysoTracker and MitoTracker probes was analyzed to examine lysosome-mitochondria fusion.The mCherry-GFP-LC3 reporter system was employed to monitor autophagosome formation and autophagic flux.NLRP3 localization and activation were detected by immunofluorescence(IF).Protein expression levels of LC3B,NLRP3,ASC,pro-caspase-1,components of the TLR4-MyD88-IRAK4-TRAF6-NF-κB pathway(including phospho-/total NF-κB and IκB),and key mitophagy regulators(NIX,BNIP3,FUNDC1,phospho-/total PINK1 and Parkin)were measured by Western blot(WB).mRNA expression of NIX,BNIP3,FUNDC1,PINK1,and Parkin was quantified by quantitative polymerase chain reaction(qPCR).Results Treatment with BYHWD-containing serum,particularly in combination with specific inhibitors,significantly reduced ROS levels(P<0.05,P<0.01)and increased the JC-1 aggregate/monomer fluorescence ratio(red/green),the ratio of red-to-yellow puncta,and the Pearson's correlation coefficient(P<0.05,P<0.01).It also upregulated the mRNA and protein expression of mitophagy-related markers(NIX,BNIP3,FUNDC1,PINK1,Parkin,LC3B,Beclin1,P62)and the ratios of phosphorylated to total PINK1 and Parkin(P<0.05,P<0.01).Conversely,the expression of TLR4,MyD88,IRAK4,TRAF6,phospho-/total NF-κB,phospho-/total IκB,NLRP3,pro-caspase-1,and ASC was downregulated(P<0.05,P<0.01).Conclusion BYHWD may alleviate LPS-induced macrophage inflammation by attenuating oxidative stress and improving mitochondrial function via suppression of the TLR4-MyD88-IRAK4-TRAF6-NF-κB pathway and modulation of mitophagy.
贺智贤;常诗瑶;俞虹;游宇;刘玉晖
江西中医药大学,江西 南昌 330004江西中医药大学,江西 南昌 330004江西中医药大学,江西 南昌 330004南昌大学第一附属医院,江西 南昌 330006江西中医药大学,江西 南昌 330004
医药卫生
补阳还五汤氧化应激巨噬细胞动脉粥样硬化
Buyang Huanwu Decoction(补阳还五汤)Oxidative stressMacrophagesAtherosclerosis
《时珍国医国药》 2026 (9)
1645-1656,12
国家自然科学基金(82360785),江西省自然科学基金重点项目(20232ACB206058),江西省研究生创新专项资金项目(YC2025-S208)
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