[1]詹洋,刘俊,陈小华,等.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(05):21-29.
　ZHAN Yang,LIU Jun,CHEN Xiaohua,et al.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[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(05):21-29.
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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()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年05期
页码:
21-29
栏目:
基础研究
出版日期:
2025-05-20

文章信息/Info

Title:
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
作者:
詹洋1; 刘俊2; 陈小华3; 郑萍1; 林自力1; 郭洁梅4; 翁绳健1; 吴星1; 陈鹏2; 李炜明1; 吴立忠1
1.福州市第二总医院,福建 福州 350007; 2.福建中医药大学中医学院,福建 福州 350122; 3.福建中医药大学第一临床医学院,福建 福州 350004; 4.中医骨伤及运动康复教育部重点实验室,福建 福州 350122
Author(s):
ZHAN Yang1; LIU Jun2; CHEN Xiaohua3; ZHENG Ping1; LIN Zili1; GUO Jiemei4; WENG Shengjian1; WU Xing1; CHEN Peng2; LI Weiming1; WU Lizhong1
1.Fuzhou Second General Hospital,Fuzhou 350007,Fujian,China 2.College of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 3.The First Clinical Medical College,Fujian University of Traditional Chinese Medicine,Fuzhou 350004,Fujian,China 4.Key Laboratory of Orthopedics&Traumatology and Rehabilitation of Traditional Chinese Medicine of Ministry of Education,Fuzhou 350122,Fujian,China
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
Keywords:
osteoarthritis; knee;  synovitis;  fibroblast-like synoviocytes;  NF-kappa B;  signal transduction;  Zhuanggu Jianxi Fang;  in vitro test
摘要:
目的:基于核因子κB(nuclear factor-κB,NF-κB)通路探讨壮骨健膝方对人膝骨关节炎(knee osteoarthritis,KOA)滑膜炎成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)炎症反应的影响及其作用机制。方法:收集前交叉韧带损伤且未被诊断为滑膜炎的患者及KOA合并滑膜炎患者的膝关节滑膜组织,分离培养获得第3代正常FLS和KOA滑膜炎FLS用于后续实验。分别以壮骨健膝方药液和生理盐水灌胃干预新西兰白兔,获取壮骨健膝方含药血清和空白血清。将正常FLS设为正常组,将KOA滑膜炎FLS随机分为模型组、壮骨健膝方组、NF-κB激活剂组,分别用10%空白血清、10%空白血清、10%壮骨健膝方含药血清、10%壮骨健膝方含药血清+1 μmol·mL-1佛波醇12-十四酸酯13-乙酸酯干预,24 h后收集细胞及上清液。采用CCK-8法检测各组FLS增殖活性,采用ELISA法检测各组上清液中白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、基质金属蛋白酶(matrix metalloproteinase,MMP)-3、MMP-13等NF-κB通路下游相关因子含量,采用实时荧光定量PCR技术检测各组细胞中核因子κB亚基p50(nuclear factor-κB subunit p50,p50)、核因子κB亚基p65(nuclear factor-κB subunit p65,p65)、核因子κB抑制因子α(nuclear factor-κB inhibitor alpha,IκBα)等NF-κB通路关键因子的mRNA表达水平,采用Western Blot法检测p50、磷酸化核因子κB亚基p50(phospho-nuclear factor-κB subunit p50,p-p50)、p65、磷酸化核因子κB亚基p65(phospho-nuclear factor-κB subunit p65,p-p65)、IκBα、磷酸化核因子κB抑制因子α(phospho-nuclear factor-κB inhibitor alpha,p-IκBα)等NF-κB通路关键因子的蛋白表达水平。结果:①FLS增殖活性检测结果。模型组FLS增殖活性较正常组升高(P=0.000); 壮骨健膝方组FLS增殖活性较模型组降低(P=0.000); NF-κB激活剂组FLS增殖活性较壮骨健膝方组升高(P=0.000)。②NF-κB通路下游相关因子含量检测结果。模型组IL-1β、TNF-α、MMP-3、MMP-13含量均较正常组升高(P=0.000,P=0.000,P=0.000,P=0.000); 壮骨健膝方组IL-1β、TNF-α、MMP-3、MMP-13含量均较模型组降低(P=0.000,P=0.000,P=0.000,P=0.000); NF-κB激活剂组IL-1β、TNF-α、MMP-3、MMP-13含量均较壮骨健膝方组升高(P=0.000,P=0.000,P=0.000,P=0.000)。③NF-κB通路关键因子的mRNA表达水平检测结果。与正常组相比,模型组p50、p65的mRNA表达水平均升高(P=0.000,P=0.000),IκBα的mRNA表达水平降低(P=0.000); 与模型组相比,壮骨健膝方组p50、p65的mRNA表达水平均降低(P=0.000,P=0.000),IκBα的mRNA表达水平升高(P=0.000); 与壮骨健膝方组相比,NF-κB激活剂组p50、p65的mRNA表达水平均升高(P=0.000,P=0.000),IκBα的mRNA表达水平降低(P=0.000)。④NF-κB通路关键因子的蛋白表达水平检测结果。与正常组相比,模型组p50、p-p50、p65、p-p65的蛋白表达水平均升高(P=0.000,P=0.000,P=0.000,P=0.000),IκBα、p-IκBα的蛋白表达水平均降低(P=0.000,P=0.000); 与模型组相比,壮骨健膝方组p50、p-p50、p65、p-p65的蛋白表达水平均降低(P=0.000,P=0.000,P=0.000,P=0.000),IκBα、p-IκBα的蛋白表达水平均升高(P=0.000,P=0.000); 与壮骨健膝方组相比,NF-κB激活剂组p50、p-p50、p65、p-p65的蛋白表达水平均升高(P=0.000,P=0.000,P=0.000,P=0.000),IκBα、p-IκBα的蛋白表达水平均降低(P=0.000,P=0.000)。结论:壮骨健膝方能够抑制人KOA滑膜炎FLS增殖,并减少促炎性细胞因子及MMP的分泌,其机制可能与促进NF-κB通路中IκBα表达、抑制p50和p65表达,从而抑制NF-κB通路的活化有关。
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 synovial tissues and cultured.The third-generation normal FLS and KOA-associated synovitis-derived FLS were collected and identified by immunofluorescence 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-1β),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),nuclear factor-κB subunit p65(p65),and nuclear factor-κB inhibitor alpha(IκBα),in the FLS of each group were detected by real-time fluorescence quantitative PCR technology; and the protein expression levels of key factors in the NF-κB pathway,including p50,phospho-nuclear factor-κB subunit p50(p-p50),p65,phospho-nuclear factor-κB subunit p65(p-p65),IκBα,and phospho-nuclear factor-κB inhibitor alpha(p-IκBα),in the FLS of each group were detected by Western Blot.Results:①The proliferation activity of FLS.The proliferation activity 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 inflammatory 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 downregulated 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 mRNA 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 normal 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 cytokines 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.

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备注/Memo

备注/Memo:
基金项目:福建省自然科学基金项目(2021J011320); 国家自然科学基金项目(82274349); 福建省创伤骨科急救与康复临床医学研究中心项目(2020Y2014)
通讯作者:翁绳健 E-mail:13609591369@163.com
更新日期/Last Update: 1900-01-01