[1]许丽梅,李慧,许云腾,等.基于NF-κB信号通路探讨独活寄生汤抑制脂多糖诱导的软骨细胞炎症反应的作用机制[J].中医正骨,2019,31(07):9-14.
　XU Limei,LI Hui,XU Yunteng,et al.An experimental study of mechanism of action of Duhuo Jisheng Tang(独活寄生汤)in inhibiting inflammatory reaction induced by lipopolysaccharide in chondrocytes based on NF-κB signaling pathway[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(07):9-14.
点击复制

基于NF-κB信号通路探讨独活寄生汤抑制脂多糖诱导的软骨细胞炎症反应的作用机制()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第31卷
期数:
2019年07期
页码:
9-14
栏目:
基础研究
出版日期:
2019-07-20

文章信息/Info

Title:
An experimental study of mechanism of action of Duhuo Jisheng Tang(独活寄生汤)in inhibiting inflammatory reaction induced by lipopolysaccharide in chondrocytes based on NF-κB signaling pathway
作者:
许丽梅1; 李慧2; 许云腾1; 王圣杰2; 曾建伟1; 王丽丽1; 叶蕻芝3; 李西海1
(1.福建中医药大学中西医结合研究院,福建 福州 350122; 2.福建中医药大学药学院,福建 福州 350122; 3.福建省中西医结合老年性疾病重点实验室,福建 福州 350122)
Author(s):
XU Limei1; LI Hui2; XU Yunteng1; WANG Shengjie2; ZENG Jianwei1; WANG Lili1; YE Hongzhi3; LI Xihai1
1.Academy of Integrated Medicine affiliated to Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 2.Pharmaceutical college of Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 3.Fujian Key Laboratory of Integrated Medicine on Geriatrics,Fuzhou 350122,Fujian,China
关键词:
骨关节炎;  软骨细胞;  独活寄生汤;  炎症反应;  脂多糖类;  NF-κB
Keywords:
osteoarthritis;  chondrocytes;  Duhuo Jisheng Tang;  inflammatory response;  lipopolysaccharides;  NF-kappa B
摘要:
目的:基于NF-κB信号通路探讨独活寄生汤抑制脂多糖诱导的软骨细胞炎症反应的作用机制。方法:取4周龄SPF级SD大鼠双侧膝关节软骨,分离软骨细胞进行体外培养。取培养的第2代软骨细胞,随机分为5组。空白组中加入含10%FBS的DMEM培养基,模型组加入含10 ng·mL-1脂多糖的10%FBS DMEM培养基,独活寄生汤低、中、高浓度组加入含脂多糖(10 ng·mL-1)和独活寄生汤(浓度分别为100 μg·mL-1、200 μg·mL-1、300 μg·mL-1)的10%FBS DMEM培养基。干预8 h后,采用ELISA法测定各组细胞培养液上清液中的基质金属蛋白酶(matrix metalloproteinase,MMP)-9含量,确定独活寄生汤最佳干预浓度。随后取培养的第2代软骨细胞,随机分为4组。空白组不进行干预,模型组加入10 ng·mL-1脂多糖,独活寄生汤组加入10 ng·mL-1脂多糖和最佳浓度的独活寄生汤,抑制剂组加入10 ng·mL-1脂多糖和10 μmol吡咯烷二硫代甲酸铵盐。干预8 h后,采用荧光定量PCR法检测软骨细胞中NF-κB基因含量、用免疫荧光检测法观察软骨细胞中NF-κB蛋白核转位情况。结果:①独活寄生汤最佳干预浓度测定结果。空白组、模型组、独活寄生汤低浓度组、独活寄生汤中浓度组、独活寄生汤高浓度组的MMP-9含量比较,差异有统计学意义[(0.125±0.012)ng·mL-1,(0.154±0.008)ng·mL-1,(0.148±0.012)ng·mL-1,(0.148±0.010)ng·mL-1,(0.134±0.002)ng·mL-1,χ2=10.034,P=0.040]。模型组的MMP-9含量高于空白组和独活寄生汤高浓度组(P=0.002,P=0.006); 空白组与独活寄生汤高浓度组比较,差异无统计学意义(P=0.573); 独活寄生汤低、中浓度组MMP-9含量与模型组比较,差异均无统计学意义(P=0.483,P=0.356)。上述测定结果显示独活寄生汤最佳干预浓度为300 μg·mL-1。②软骨细胞中NF-κB基因含量。空白组、模型组、独活寄生汤组、抑制剂组的NF-κB基因含量比较,差异有统计学意义[1.013±0.015,1.848±0.216,1.454±0.250,1.289±0.332,F=8.859,P=0.002]。模型组的NF-κB基因含量高于空白组、独活寄生汤组和抑制剂组(P=0.000,P=0.035,P=0.006); 独活寄生汤组的NF-κB基因含量高于空白组(P=0.021); 独活寄生汤组的NF-κB基因含量与抑制剂组比较,差异无统计学意义(P=0.341)。③软骨细胞中NF-κB蛋白核转位情况。各组软骨细胞核均呈蓝色; 软骨细胞NF-κB蛋白染色明显,呈绿色; 模型组NF-κB蛋白核转位最为明显,空白组、抑制剂组NF-κB蛋白核转位相对较少,独活寄生汤组NF-κB蛋白核转位情况介于空白组与模型组之间。结论:独活寄生汤可通过抑制NF-κB蛋白核转位,减轻脂多糖诱导的软骨细胞炎症反应,从而延缓软骨退变。
Abstract:
Objective:To explore the mechanism of action of Duhuo Jisheng Tang(独活寄生汤,DHJST)in inhibiting inflammatory reaction induced by lipopolysaccharide(LPS)in chondrocytes based on NF-κB signaling pathway.Methods:The 4-week-old SPF-grade SD rats were executed and their articular cartilages were fetched out from both knees,and the chondrocytes were extracted from the knee cartilage tissues and were cultured in vitro.The second-generation chondrocytes were divided into 5 groups,and were cultured in DMEM added with 10% FBS(blank group),10% FBS and LPS(10 ng/mL)(model group),10% FBS,LPS(10 ng/mL)and DHJST(100 μg/mL)(DHJST low-concentration group),10% FBS,LPS(10 ng/mL)and DHJST(200 μg/mL)(DHJST middle-concentration group)and 10% FBS,LPS(10 ng/mL)and DHJST(300 μg/mL)(DHJST high-concentration group)respectively.The content of matrix metalloproteinase(MMP)-9 in supernatant of cell culture fluid of each group were measured by using ELISA method after 8-hour intervention to determine the optimal intervention concentration of DHJST.Then the second-generation chondrocytes were randomly divided into blank group,model group,DHJST group and inhibitor group.The chondrocytes in blank group were not intervened and the chondrocytes in the other three groups were cultured in DMEM added with LPS(10 ng/mL),DMEM added with LPS(10 ng/mL)and DHJST with optimal concentration and DMEM added with LPS(10 ng/mL)and PDTC(10 μmol)respectively.After 8-hour intervention,the gene content of NF-κB in chondrocytes were detected by using fluorescent quantitative PCR method,and nuclear translocation of NF-κB protein in chondrocytes were detected by using immunofluorescence assay.Results:There was statistical difference in content of MMP-9 between blank group,model group,DHJST low-,middle- and high-concentration group(0.125+/-0.012,0.154+/-0.008,0.148+/-0.012,0.148+/-0.010,0.134+/-0.002 ng/mL,χ2=10.034,P=0.040).The content of MMP-9 was higher in model group compared to blank group and DHJST high-concentration group(P=0.002,P=0.006),and there was no statistical difference in content of MMP-9 between blank group and DHJST high-concentration group(P=0.573).There was no statistical difference in content of MMP-9 between DHJST low-concentration group and model group and between DHJST middle-concentration group and model group(P=0.483,P=0.356).Above results showed that the optimal intervention concentration of DHJST was 300 μg/mL.There was statistical difference in gene content of NF-κB between blank group,model group,DHJST group and inhibitor group(1.013+/-0.015,1.848+/-0.216,1.454+/-0.250,1.289+/-0.332,F=8.859,P=0.002).The gene content of NF-κB was higher in model group compared to blank group,DHJST group and inhibitor group,and was higher in DHJST group compared to blank group(P=0.000,P=0.035,P=0.006; P=0.021).There was no statistical difference in gene content of NF-κB between DHJST group and inhibitor group(P=0.341).The cytoblast of chondrocytes presented with blue staining in each group.The chondrocytes presented with obvious green staining of NF-κB protein.The most obvious nuclear translocations of NF-κB protein were found in model group,and relatively less nuclear translocations of NF-κB protein were found in blank group and inhibitor group,and the nuclear translocation of NF-κB protein in DHJST group was between blank group and model group.Conclusion:DHJST can alleviate inflammatory reaction induced by LPS in chondrocytes through inhibiting nuclear translocation of NF-κB protein,which may be the mechanisms of action for delaying articular cartilage degeneration.

参考文献/References:


[1] CORR E M,CUNNINGHAM C C,HELBERT L,et al.Osteoarthritis-associated basic Calcium phosphate crystals activate membrane proximal kinases in human innate immune cells[J].Arthritis Res Ther,2017,19(1):23.
[2] SCANZELLO C R.Role of low-grade inflammation in osteoarthritis[J].Curr Opin Rheumatol,2017,29(1):79-85.
[3] SHEN J,ABU-AMER Y,O'KEEFE R J,et al.Inflammation and epigenetic regulation in osteoarthritis[J].Connect Tissue Res,2017,58(1):49-63.
[4] 周江涛,赵依娜,王庆来.独活寄生汤口服联合玻璃酸钠关节腔注射治疗早中期膝骨关节炎[J].中医正骨,2019,31(2):17-21.
[5] 何晓娟,林平冬,马玉环,等.独活寄生汤含药血清抑制白细胞介素1β诱导的软骨细胞炎症反应的作用机制研究[J].中医正骨,2017,29(8):1-7.
[6] 郑文伟,翁霞萍,黄绥心,等.独活寄生汤对骨关节炎软骨退变的影响及其作用机制[J].中医正骨,2017,29(7):5-11.
[7] OLIVOTTO E,OTERO M,MARCU K B,et al.Pathophysiology of osteoarthritis:canonical NF-kappaB/IKKbeta-dependent and kinase-independent effects of IKKalpha in cartilage degradation and chondrocyte differentiation[J].RMD Open,2015,1(Suppl 1):e 000061.
[8] ZHONG J H,LI J,LIU C F,et al.Effects of microRNA-146a on the proliferation and apoptosis of human osteoarthritis chondrocytes by targeting TRAF6 through the NF-kappa B signalling pathway[J].Biosci Rep,2017,37(2)[2019-04-01].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408711.
[9] 陈后煌,邵翔,叶蕻芝,等.独活寄生汤对脂多糖诱导的大鼠软骨细胞炎症模型IL-1β、TNF-α表达的影响[J].中华老年骨科与康复电子杂志,2017,3(2):77-84.
[10] 李西海,刘献祥.骨关节炎的核心病机——本痿标痹[J].中医杂志,2014,55(14):1248-1249.
[11] 李西海,梁文娜,叶蕻芝,等.独活寄生汤调控风寒湿痹型骨关节炎软骨下骨重建失衡的作用机制探讨[J].风湿病与关节炎,2014,3(8):62-64.
[12] HWANG H S,KIM H A.Chondrocyte apoptosis in the pathogenesis of osteoarthritis[J].Int J Mol Sci,2015,16(11):26035-26054.
[13] RAHMATI M,NALESSO G,MOBASHERI A,et al.Aging and osteoarthritis:Central role of the extracellular matrix[J].Ageing Res Rev,2017,40:20-30.
[14] CUI N,HU M,KHALIL R A.Biochemical and biological attributes of matrix metalloproteinases[J].Prog Mol Biol Transl Sci,2017,147:1-73.
[15] ZENG G Q,CHEN A B,LI W,et al.High MMP-1,MMP-2,and MMP-9 protein levels in osteoarthritis[J].Genet Mol Res,2015,14(4):14811-14822.
[16] SESTITO S E,FACCHINI F A,MORBIOLI I A,et al.Amphiphilic guanidinocalixarenes inhibit lipopolysaccharide(LPS)- and Lectin-Stimulated toll-like receptor 4(TLR4)signaling[J].J Med Chem,2017,60(12):4882-4892.
[17] LU Y C,YEH W C,OHASHI P S.LPS/TLR4 signal transduction pathway[J].Cytokine,2008,42(2):145-151.
[18] ROY A,SRIVASTAVA M,SAQIB U,et al.Potential therapeutic targets for inflammation in toll-like receptor 4(TLR4)-mediated signaling pathways[J].Int Immunopharmacol,2016,40:79-89.
[19] LEPETSOS P,PAPAVASSILIOU K A,PAPAVASSILIOU A G.Redox and NF-kappa B signaling in osteoarthritis[J].Free Radic Biol Med,2019,132:90-100.
[20] SAITO T,TANAKA S.Molecular mechanisms underlying osteoarthritis development:Notch and NF-kB[J].Arthritis Res Ther,2017,19(1):94.
[21] MITCHELL J P, CARMODY R J.NF-κB and the transcriptional control of inflammation[J].Int Rev Cell Mol Biol,2018,335:41-84.
[22] LIN T H,PAJARINEN J,LU L,et al.NF-κB as a therapeutic target in inflammatory-associated bone diseases[J].Adv Protein Chem Struct Biol,2017,107:117-154.
[23] HAYDEN M S,GHOSH S.Regulation of NF-kappa B by TNF family cytokines[J].Semin Immunol,2014,26(3):253-266.

相似文献/References:

[1]孟维娜,明立功,王新德,等.关节镜下清理联合腓骨近1/3段截骨治疗膝骨关节炎[J].中医正骨,2015,27(11):40.
[2]明立功,孟维娜,王新德,等.腓骨近端截骨治疗内侧间室膝骨关节炎的近期疗效观察[J].中医正骨,2015,27(10):25.
[3]张杰,王人彦,张玉柱.膝骨关节炎的治疗进展[J].中医正骨,2015,27(10):68.
[4]梁朝,蔡静怡,闫立,等.针刀疗法改善膝骨关节炎早期疼痛症状的疗效评价[J].中医正骨,2015,27(09):9.
　LIANG Zhao,CAI Jingyi,YAN Li,et al.Evaluation of the curative effect of needle-knife therapy for relieving knee pain in patients with early knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(07):9.
[5]王建武,党建军,李强,等.四联疗法治疗膝骨关节炎[J].中医正骨,2015,27(08):44.
[6]刘红娟,郭会利,郭树农.云克联合中药治疗膝骨关节炎的护理[J].中医正骨,2015,27(08):75.
[7]陈卫衡.探索建立系统的膝骨关节炎中医临床科研范式 和理论体系[J].中医正骨,2015,27(07):1.
[8]郑春松,叶蕻芝,李西海,等.透骨消痛胶囊中补肾柔肝药和活血祛风药治疗 骨关节炎作用方式的计算机模拟比较[J].中医正骨,2015,27(07):6.
　ZHENG Chunsong,YE Hongzhi,LI Xihai,et al.Comparison of the mode of action of Bushen Rougan(补肾柔肝)drugs versus Huoxue Qufeng(活血祛风)drugs contained in Tougu Xiaotong Jiaonang(透骨消痛胶囊)for the treatment of osteoarthritis:A computer simulation study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(07):6.
[9]帅波,沈霖,杨艳萍,等.加味青娥丸治疗膝骨关节炎的作用机制研究[J].中医正骨,2015,27(07):15.
　SHUAI Bo,SHEN Lin,YANG Yanping,et al.Study on the mechanism of action of Jiawei Qing'e Wan(加味青娥丸)for the treatment of knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(07):15.
[10]梅其杰,袁长深,段戡,等.壮药骨痹方烫熨联合运动疗法治疗膝骨关节炎的临床研究[J].中医正骨,2015,27(07):27.
　MEI Qijie,YUAN Changshen,DUAN Kan,et al.Clinical study of the curative effect of hot compressing and rubbing with packet of Gubi Fang(骨痹方)combined with exercise therapy in the treatment of knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(07):27.
[11]郑春松,叶蕻芝,李西海,等.独活寄生汤含药血清对白细胞介素1β诱导的 退变关节软骨细胞中基质金属蛋白酶 和环氧化酶2表达的影响[J].中医正骨,2015,27(12):1.
　ZHENG Chunsong,YE Hongzhi,LI Xihai,et al.Impact of Duhuo Jisheng Tang(独活寄生汤)medicated serum on expression of matrix metalloproteinase and cyclooxygenase 2 in degenerative articular chondrocytes induced by interleukin-1 beta[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(07):1.
[12]林木南,林艳红,刘献祥,等.温热疏密波对膝骨关节炎模型大鼠软骨细胞 RAS/丝裂原活化蛋白激酶信号通路的影响[J].中医正骨,2016,28(02):1.
　LIN Munan,LIN Yanhong,LIU Xianxiang,et al.Effect of warm sparse-dense wave on RAS/mitogen-activated protein kinase signaling pathway in chondrocytes of knee osteoarthritis rat models[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(07):1.
[13]原晓强,金王东,周云婧,等.纯化血小板对大鼠软骨细胞增殖及膝骨关节炎大鼠软骨修复的作用研究[J].中医正骨,2016,28(12):6.
　YUAN Xiaoqiang,JIN Wangdong,ZHOU Yunjing,et al.Effect of purified platelet rich plasma on chondrocyte proliferation in rats and cartilage repair in rats with knee osteoarthritis:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(07):6.
[14]刘启明,罗统富,董黎强,等.植物雌激素类中药延缓关节软骨细胞退变的研究进展[J].中医正骨,2017,29(05):27.
[15]叶锦霞,付长龙,林洁,等.透骨消痛胶囊对毒胡萝卜素诱导的内质网应激PEKR信号通路介导的大鼠体外培养关节软骨细胞凋亡的影响[J].中医正骨,2017,29(06):1.
　YE Jinxia,FU Changlong,LIN Jie,et al.Effect of Tougu Xiaotong Jiaonang(透骨消痛胶囊)on apoptosis mediated by endoplasmic reticulum stress(PEKR signaling pathway)and induced by thapsigargin in rat's articular chondrocytes cultured in vitro[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(07):1.
[16]何晓娟,林平冬,马玉环,等.独活寄生汤含药血清抑制白细胞介素1β诱导的软骨细胞炎症反应的作用机制研究[J].中医正骨,2017,29(08):1.
　HE Xiaojuan,LIN Pingdong,MA Yuhuan,et al.Study on mechanism of action of Duhuo Jisheng Tang(独活寄生汤)medicated serum in inhibiting inflammatory reaction induced by interleukin-1 beta in chondrocytes[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(07):1.
[17]郑文伟,何晓娟,贾良良,等.跳骨片含药血清抑制脂多糖诱导的软骨细胞炎症反应的作用机制研究[J].中医正骨,2017,29(08):8.
　ZHENG Wenwei,HE Xiaojuan,JIA Liangliang,et al.Study on mechanism of action of Tiaogu Pian(跳骨片)medicated serum in inhibiting inflammatory reaction induced by lipopolysaccharides in chondrocytes[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(07):8.
[18]陈世宣,刘益杰,胡笑燊,等.基于瞬时受体电位香草素亚家族4信号通路探讨牛蒡子苷元对体外培养软骨细胞增殖及Ⅱ型胶原蛋白和软骨蛋白聚糖表达的影响[J].中医正骨,2017,29(10):13.
　CHEN Shixuan,LIU Yijie,HU Xiaoshen,et al.An experimental study of effect of arctigenin on proliferation of chondrocyte cultured in vitro and expression of typeⅡcollagen protein and aggrecan based on the transient receptor potential vanilloid 4 signaling pathway[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(07):13.
[19]陈俊,吴广文,许惠凤,等.独活寄生汤含药血清对大鼠退变软骨细胞蛋白激酶 R样内质网激酶/免疫球蛋白结合蛋白信号通路的影响[J].中医正骨,2018,30(08):1.
　CHEN Jun,WU Guangwen,XU Huifeng,et al.Effect of Duhuo Jisheng Tang(独活寄生汤)medicated serum on protein kinase R-like endoplasmic reticulum kinase/immunoglobulin-binding protein signaling pathway in degenerated chondrocytes of rats[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(07):1.
[20]王会含,王永堂,苗建华,等.川芎嗪对人骨关节炎软骨细胞的影响及作用机制研究[J].中医正骨,2021,33(07):4.
　WANG Huihan,WANG Yongtang,MIAO Jianhua,et al.A study of the effects of tetramethylpyrazine on human osteoarthritic chondrocytes and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2021,33(07):4.

备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(81573998); 中国博士后科学基金第60批面上资助项目(2016M600625); 中国博士后科学基金第10批特别资助项目(2017T100591) 通讯作者:李西海 E-mail:lixihai79dahai@163.com(收稿日期:2019-06-05 本文编辑:李晓乐)
更新日期/Last Update: 2019-07-20