中医正骨2025，Vol.37Issue(5)：21-29,9.
基于核因子κB通路探讨壮骨健膝方对人膝骨关节炎滑膜炎成纤维样滑膜细胞炎症反应的影响及其作用机制
Study on effects and mechanism of Zhuanggu Jianxi Fang(壮骨健膝方)on inflammatory response of fibroblast-like synoviocytes in human knee osteoarthritis-associated synovitis based on the nuclear factor-κB pathway
摘要
Abstract
Objective:To observe the therapeutic effects of Zhuanggu Jianxi Fang(壮骨健膝方,ZGJXF)on inflammatory response of fibroblast-like synoviocytes(FLS)in human knee osteoarthritis(KOA)-associated synovitis,and to explore its underlying mechanism based on the nuclear factor-κB(NF-κB)pathway.Methods:The knee synovial tissues were harvested from patients with anterior cruciate ligament(ACL)injuries but not synovitis and the ones with KOA and synovitis,respectively,and the primary FLS were isolated from the knee synovi-al tissues and cultured.The third-generation normal FLS and KOA-associated synovitis-derived FLS were collected and identified by immu-nofluorescence staining for subsequent experiments.Additionally,the New Zealand white rabbits were obtained and intervened by intragastric administration with ZGJXF solution and normal saline,respectively,to acquire ZGJXF medicated serum and blank serum.The normal FLS were designated as the normal group,while the KOA-associated synovitis-derived FLS were randomized into model group,ZGJXF group,and NF-κB activator group.The FLS in the 4 groups were intervened by 10％blank serum,10％blank serum,10％ZGJXF medicated serum,and 10％ZGJXF medicated serum combined with 1 μmol/mL phorbol 12-myristate 13-acetate(PMA),respectively,for 24 hours.After the end of the intervention,the FLS and supernatants were collected from each group.After that,the proliferation activity of FLS in each group was detected by CCK-8 assay,the levels of NF-κB pathway downstream-related inflammatory mediators including interleukin-1β(IL-1 β),tumor necrosis factor-α(TNF-α),matrix metalloproteinase(MMP)-3,and MMP-13 in the supernatants of each group were measured by ELISA.Furthermore,the mRNA expression levels of key factors in the NF-κB pathway,including nuclear factor-κB subunit p50(p50),nu-clear factor-κB subunit p65(p65),and nuclear factor-κB inhibitor alpha(IκBα),in the FLS of each group were detected by real-time fluo-rescence quantitative PCR technology;and the protein expression levels of key factors in the NF-κB pathway,including p50,phospho-nucle-ar factor-κB subunit p50(p-p50),p65,phospho-nuclear factor-κB subunit p65(p-p65),IκBα,and phospho-nuclear factor-κB inhibitor al-pha(p-IκBα),in the FLS of each group were detected by Western Blot.Results:①The proliferation activity of FLS.The proliferation activi-ty of FLS increased in model group compared to normal group(P=0.000),decreased in ZGJXF group compared to model group(P=0.000),and increased in NF-κB activator group compared to ZGJXF group(P=0.000).②The NF-κB pathway downstream-related inflam-matory mediators.The levels of IL-1β,TNF-α,MMP-3,and MMP-13 increased in model group compared to normal group(P=0.000,P=0.000,P=0.000,P=0.000),decreased in ZGJXF group compared to model group(P=0.000,P=0.000,P=0.000,P=0.000),and increased in NF-κB activator group compared to ZGJXF group(P=0.000,P=0.000,P=0.000,P=0.000).③The mRNA expression levels of key factors in the NF-κB pathway.The mRNA expression levels of p50 and p65 were upregulated,while that of IκBα was downregu-lated in model group compared to normal group(P=0.000,P=0.000,P=0.000).The mRNA expression levels of p50 and p65 were downregulated,while that of IκBα was upregulated in ZGJXF group compared to model group(P=0.000,P=0.000,P=0.000).The mR-NA expression levels of p50 and p65 were upregulated,while that of IκBα was downregulated in NF-κB activator group compared to ZGJXF group(P=0.000,P=0.000,P=0.000).④The protein expression levels of key factors in the NF-κB pathway.The protein expression le-vels of p50,p-p50,p65 and p-p65 were upregulated,while those of IκBα and p-IκBα were downregulated in model group compared to nor-mal group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).The protein expression levels of p50,p-p50,p65 and p-p65 were downregulated,while those of IκBα and p-IκBα were upregulated in ZGJXF group compared to model group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).The protein expression levels of p50,p-p50,p65 and p-p65 were upregulated,while those of IκBα and p-IκBα were downregulated in NF-κB activator group compared to ZGJXF group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).Conclusion:ZGJXF can inhibit the proliferation of FLS and reduce the secretion of pro-inflammatory cyto-kines and MMP in human KOA-associated synovitis.It may work by upregulating the expression of IκBα and suppressing the expression of p50 and p65 in the NF-κB pathway,thereby inhibiting the activation of NF-κB pathway.关键词
骨关节炎,膝/滑膜炎/成纤维样滑膜细胞/NF-κB/信号传导/壮骨健膝方/体外试验Key words
osteoarthritis,knee/synovitis/fibroblast-like synoviocytes/NF-kappa B/signal transduction/Zhuanggu Jianxi Fang/in vitro test引用本文复制引用
詹洋,李炜明,吴立忠,刘俊,陈小华,郑萍,林自力,郭洁梅,翁绳健,吴星,陈鹏..基于核因子κB通路探讨壮骨健膝方对人膝骨关节炎滑膜炎成纤维样滑膜细胞炎症反应的影响及其作用机制[J].中医正骨,2025,37(5):21-29,9.基金项目
福建省自然科学基金项目(2021J011320) （2021J011320）
国家自然科学基金项目(82274349) （82274349）
福建省创伤骨科急救与康复临床医学研究中心项目(2020Y2014) （2020Y2014）
