[1]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):8-16.
　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(08):8-16.
点击复制

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()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期数:
2017年08期
页码:
8-16
栏目:
基础研究
出版日期:
2017-08-20

文章信息/Info

Title:
Study on mechanism of action of Tiaogu Pian(跳骨片)medicated serum in inhibiting inflammatory reaction induced by lipopolysaccharides in chondrocytes
作者:
郑文伟1; 何晓娟1; 贾良良1; 马玉环1; 陈后煌1; 邵翔1; 陈达1; 刘献祥1; 李西海1; 叶蕻芝2
1.福建中医药大学,福建 福州 350122; 2.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):
ZHENG Wenwei1; HE Xiaojuan1; JIA Liangliang1; MA Yuhuan1; CHEN Houhuang1; SHAO Xiang1; CHEN Da1; LIU Xianxiang2; LI Xihai2; YE Hongzhi1
1.Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 2.Fujian Key Laboratory of Integrated Medicine on Geriatrics,Fuzhou 350122,Fujian,China
关键词:
骨关节炎;  软骨细胞;  跳骨片;  软骨退变;  Wnt/β-catenin信号通路;  脂多糖类;  基质金属蛋白酶类;  炎症反应;  动物实验
Keywords:
Key words osteoarthritis;  chondrocytes;  Tiaogu Pian;  cartilage degeneration;  Wnt/β-catenin signaling pathway;  lipopolysaccharides;  matrix metalloproteinases;  inflammatory reaction;  animal experimentation
摘要:
目的:探讨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。方法:将10只8周龄雄性SD大鼠随机分为跳骨片组和空白组,跳骨片组以0.32 g·kg-1剂量的跳骨片灌胃,空白组给予等量生理盐水灌胃; 每天灌胃1次,连续7 d; 末次灌胃1 h后,经腹主动脉取血,分别制备跳骨片含药血清和空白血清,低温保存备用。从10只4周龄SD大鼠膝关节软骨中分离软骨细胞并培养,光学显微镜下观察软骨细胞形态,并用Ⅱ型胶原酶免疫组化鉴定。将培养好的第2代软骨细胞随机分为空白血清组、模型组、跳骨片含药血清组,其中空白血清组以含10%空白血清的培养基(dulbecco modified eagle medium,DMEM)培养; 模型组以浓度为10 ng·mL-1的脂多糖和含10%空白血清的DMEM培养; 跳骨片含药血清组以浓度为10 ng·mL-1的脂多糖和含10%跳骨片含药血清的DMEM培养; 3组均连续干预培养8 h,采用酶联免疫吸附法检测软骨细胞基质金属蛋白酶(matrix metalloproteinase,MMP)3、MMP9含量,采用荧光定量RT-PCR法检测软骨细胞中Wnt/β-catenin信号通路相关基因表达水平,采用Western blot法检测软骨细胞中β-链蛋白(β-catenin)、卷曲蛋白-2(Frizzled-2)的蛋白表达量,采用免疫荧光检测法检测软骨细胞中β-catenin、糖原合成酶激酶-3β(glycogen synthasc kinase-3β,GSK-3β)、蛋白多糖1(proteoglycans 1,PGS1)的蛋白表达量。结果:①软骨细胞免疫组化鉴定结果。第2代软骨细胞胞浆及细胞膜呈棕黄色阳性染色,具有典型的软骨细胞生物学特征。②软骨细胞MMP3、MMP9含量。脂多糖干预8 h后,空白血清组、模型组和跳骨片含药血清组的软骨细胞MMP3、MMP9含量比较,组间差异均有统计学意义[(34.019±1.036)ng·mL-1,(44.645±2.473)ng·mL-1,(32.941±1.792)ng·mL-1,F=36.060,P=0.000;(1.348±0.038)ng·mL-1,(1.562±0.112)ng·mL-1,(1.331±0.015)ng·mL-1,F=11.319,P=0.000]; 模型组软骨细胞MMP3、MMP9含量均高于空白血清组(LSD-t=-7.016,P=0.000; LSD-t=-3.768,P=0.003); 跳骨片含药血清组软骨细胞MMP3、MMP9含量均低于模型组(LSD-t=7.652,P=0.000; LSD-t=4.066,P=0.002); 空白血清组软骨细胞MMP3、MMP9含量与跳骨片含药血清组比较,差异均无统计意义(LSD-t=0.635,P=0.549; LSD-t=0.299,P=0.770)。③软骨细胞中Wnt/β-catenin信号通路相关基因的表达。脂多糖干预8 h后,空白血清组、模型组和跳骨片含药血清组软骨细胞中β-catenin、GSK-3β、Frizzled-2、Wnt-4、CKI-ε基因表达量比较,组间差异均有统计学意义(1.000±0.275,2.258±0.206,1.431±0.304,F=36.709,P=0.000; 1.000±0.133,0.417±0.104,0.842±0.094,F=29.259,P=0.000; 1.000±0.191,1.737±0.238,1.445±0.337,F=7.027,P=0.015; 1.000±0.341,3.801±0.579,1.876±0.388,F=71.903,P=0.000; 1.000±0.309,2.208±0.708,1.441±0.421,F=64.178,P=0.000); 模型组软骨细胞中β-catenin、Frizzled-2、Wnt-4、CKI-ε基因表达量均高于空白血清组(LSD-t=-8.431,P=0.000; LSD-t=-3.723,P=0.005; LSD-t=8.062,P=0.000; LSD-t=-11.235,P=0.000),GSK-3β基因表达量低于空白血清组(LSD-t=7.397,P=0.000); 跳骨片含药血清组软骨细胞中β-catenin、Frizzled-2、Wnt-4、CKI-ε基因表达量均低于模型组(LSD-t=5.541,P=0.000; LSD-t=1.477,P=0.017; LSD-t=8.062,P=0.000; LSD-t=6.882,P=0.000),GSK-3β基因表达量高于模型组(LSD-t=-5.387,P=0.000); 空白血清组软骨细胞中β-catenin、Wnt-4、CKI-ε基因表达量均低于跳骨片含药血清组(LSD-t=-2.289,P=0.018; LSD-t=-3.658,P=0.005; LSD-t=-4.352,P=0.002); 空白血清组软骨细胞中GSK-3β、Frizzled-2基因表达量与跳骨片含药血清组比较,差异均无统计学意义(LSD-t=2.009,P=0.075; LSD-t=-3.658,P=0.051)。④软骨细胞中β-catenin、Frizzled-2的蛋白表达。脂多糖干预8 h后,空白血清组、模型组和跳骨片含药血清组软骨细胞中β-catenin、Frizzled-2蛋白表达量比较,组间差异均有统计学意义(0.449±0.063,0.746±0.156,0.549±0.056,F=5.323,P=0.026; 1.348±0.038,1.562±0.112,1.331±0.015,F=6.291,P=0.034); 模型组软骨细胞中β-catenin、Frizzled-2蛋白表达量高于空白血清组(LSD-t=-11.235,P=0.005; LSD-t=-3.104,P=0.021); 跳骨片含药血清组软骨细胞中β-catenin、Frizzled-2蛋白表达量低于模型组(LSD-t=6.883,P=0.037; LSD-t=3.039,P=0.023); 空白----------------------------------------------- 血清组软骨细胞中β-catenin蛋白表达量低于跳骨片含药血清组(LSD-t=-4.352,P=0.002); 空白血清组软骨细胞中Frizzled-2蛋白表达量与跳骨片含药血清组比较,差异无统计学意义(LSD-t=-0.065,P=0.950)。⑤软骨细胞中β-catenin、GSK-3β、PGS1的蛋白表达。脂多糖干预8 h后,软骨细胞中β-catenin、GSK-3β、PGS1蛋白染色明显,呈绿色; 空白血清组、模型组和跳骨片含药血清组软骨细胞中β-catenin、GSK-3β、PGS1蛋白表达量比较,组间差异均有统计学意义(0.014±0.002,0.029±0.006,0.018±0.002,F=9.910,P=0.013; 0.380±0.011,0.237±0.015,0.287±0.002,F=56.639,P=0.000; 0.034±0.003,0.022±0.002,0.029±0.003,F=27.232,P=0.001); 模型组软骨细胞β-catenin蛋白表达量高于空白血清组(LSD-t=-4.103,P=0.006),GSK-3β、PGS1蛋白表达量低于空白血清组(LSD-t=1.048,P=0.000; t=7.365,P=0.000); 跳骨片含药血清组软骨细胞β-catenin蛋白表达量低于模型组(LSD-t=-3.548,P=0.012),GSK-3β、PGS1蛋白表达量高于模型组(LSD-t=-3.657,P=0.011; LSD-t=-3.273,P=0.017); 空白血清组软骨细胞中β-catenin蛋白表达量与跳骨片含药血清组比较,差异无统计学意义(LSD-t=-0.554,P=0.599); 空白血清组软骨细胞中GSK-3β、PGS1蛋白表达量高于跳骨片含药血清组(LSD-t=6.827,P=0.000; LSD-t=4.092,P=0.010)。结论:跳骨片含药血清可以抑制脂多糖诱导的软骨细胞炎症反应,延缓关节软骨退变。其作用机制可能与Wnt/β-catenin信号通路的调控有关,其中β-catenin、Frizzled-2、GSK-3β、Wnt-4、CKI-ε基因可能是该信号通路的重要靶点。但是由于引起OA的因素众多且跳骨片含药血清成分多且复杂,有待于进一步研究证实。
Abstract:
ABSTRACT Objective:To explore the mechanism of action of Tiaogu Pian(跳骨片,TGP)medicated serum in inhibiting inflammatory reaction induced by lipopolysaccharides in chondrocytes.Methods:Ten 8-week-old male SD rats were randomly divided into TGP group and blank group.The rats in TGP group were intragastric administrated with TGP in dosage of 0.32 g/kg,while the others in blank group were intragastric administrated with the same dose of normal saline,once per day for 7 consecutive days.At 1 hour after the last intragastric administration,their blood were fetched out from abdominal aorta and were made into TGP medicated serum and blank serum respectively and the serum were reserved at low temperature for future use.The chondrocytes of ten 4-week-old SD rats were isolated from knee articular cartilage and were cultured.The chondrocytes morphology were observed under optical microscope,and immunohistochemical identification were carried out by using typeⅡcollagenase.The second-generation chondrocytes were randomly divided into blank serum group,model group and TGP medicated serum group.The chondrocytes in blank serum group were cultured in dulbecco modified eagle medium(DMEM)supplemented with 10% blank serum.The chondrocytes in model group were cultured in DMEM supplemented with lipopolysaccharide(LPS)with concentration of 10 ng/ml and 10% blank serum.The chondrocytes in TGP medicated serum group were cultured in DMEM supplemented with LPS with concentration of 10 ng/ml and 10% TGP medicated serum.The chondrocytes in the 3 groups were intervened and cultured for continuous 8 hours.The content of matrix metalloproteinase(MMP)3 and MMP9 in chondrocytes were detected by using enzyme-linked immunoadsordent assay(ELISA).The expression levels of gene related to Wnt/β-catenin signaling pathway in chondrocytes were detected by using fluorescence quantitative RT-PCR method.The protein expressions of β-catenin and Frizzled-2 in chondrocytes were detected by using Western blot method.The protein expressions of β-catenin,glycogen synthasc kinase-3β(GSK-3β)and proteoglycans 1(PGS1)in chondrocytes were detected by using immunofluorescence assay(IFA).Results:The second-generation chondrocytes had typical biological characteristics of chondrocytes and their endochylemas and cytomembranes presented with brown-yellow positive staining.After 8-hour intervention by LPS,there was statistical difference in the content of MMP3 and MMP9 in chondrocytes between blank serum group,model group and TGP medicated serum group(34.019+/-1.036,44.645+/-2.473,32.941+/-1.792 ng/ml,F=36.060,P=0.000; 1.348+/-0.038,1.562+/-0.112,1.331+/-0.015 ng/ml,F=11.319,P=0.000).The content of MMP3 and MMP9 in chondrocytes were higher in model group compared to blank serum group(LSD-t=-7.016,P=0.000; LSD-t=-3.768,P=0.003)and were lower in TGP medicated serum group compared to model group(LSD-t=7.652,P=0.000; LSD-t=4.066,P=0.002).There was no statistical difference in the content of MMP3 and MMP9 in chondrocytes between blank serum group and TGP medicated serum group(LSD-t=0.635,P=0.549; LSD-t=0.299,P=0.770).After 8-hour intervention by LPS,there was statistical difference in gene expression levels of β-catenin,GSK-3β,Frizzled-2,Wnt-4 and CKI-ε between blank serum group,model group and TGP medicated serum group(1.000+/-0.275,2.258+/-0.206,1.431+/-0.304,F=36.709,P=0.000; 1.000+/-0.133,0.417+/-0.104,0.842+/-0.094,F=29.259,P=0.000; 1.000+/-0.191,1.737+/-0.238,1.445+/-0.337,F=7.027,P=0.015; 1.000+/-0.341,3.801+/-0.579,1.876+/-0.388,F=71.903,P=0.000; 1.000+/-0.309,2.208+/-0.708,1.441+/-0.421,F=64.178,P=0.000).The gene expression levels of β-catenin,Frizzled-2,Wnt-4 and CKI-εin chondrocytes were higher and the gene expression levels of GSK-3βwere lower in model group compared to blank serum group(LSD-t=-8.431,P=0.000; LSD-t=-3.723,P=0.005; LSD-t=8.062,P=0.000; LSD-t=-11.235,P=0.000,LSD-t=7.397,P=0.000).The gene expression levels of β-catenin,Frizzled-2,Wnt-4 and CKI-ε in chondrocytes were lower and the gene expression levels of GSK-3β were higher in TGP medicated serum group compared to model group(LSD-t=5.541,P=0.000; LSD-t=1.477,P=0.017; LSD-t=8.062,P=0.000; LSD-t=6.882,P=0.000; LSD-t=-5.387,P=0.000).The gene expression levels of β-catenin,Wnt-4 and CKI-ε in chondrocytes were lower in blank serum group compared to TGP medicated serum group(LSD-t=-2.289,P=0.018; LSD-t=-3.658,P=0.005; LSD-t=-4.352,P=0.002).There was no statistical difference in gene expression levels of GSK-3β and Frizzled-2 between blank serum group and TGP medicated serum group(LSD-t=2.009,P=0.075; LSD-t=-3.658,P=0.051).After 8-hour intervention by LPS,there was statistical difference in protein expressions of β-catenin and Frizzled-2 between blank serum group,model group and TGP medicated serum group(0.449+/-0.063,0.746+/-0.156,0.549+/-0.056,F=5.323,P=0.026; 1.348+/-0.038; 1.562+/-0.112; 1.331+/-0.015,F=6.291,P=0.034).The protein expressions of β-catenin and Frizzled-2 in chondrocytes were higher in model group compared to blank serum group(LSD-t=-11.235,P=0.005; LSD-t=-3.104,P=0.021).The protein expressions of β-catenin and Frizzled-2 in chondrocytes were lower in TGP medicated serum group compared to model group(LSD-t=6.883,P=0.037; LSD-t=3.039,P=0.023).The protein expressions of β-catenin in chondrocytes were lower in blank serum group compared to TGP medicated serum group(LSD-t=-4.352,P=0.002).There was no statistical difference in protein expressions of Frizzled-2 between blank serum group and TGP medicated serum group(LSD-t=-0.065,P=0.950).After 8-hour intervention by LPS,the chondrocytes presented with obvious green staining of β-catenin,GSK-3β and PGS1 protein.There was statistical difference in protein expressions of β-catenin,GSK-3β and PGS1 between blank serum group,model group and TGP medicated serum group(0.014+/-0.002,0.029+/-0.006,0.018+/-0.002,F=9.910,P=0.013; 0.380+/-0.011,0.237+/-0.015,0.287+/-0.002,F=56.639,P=0.000; 0.034+/-0.003,0.022+/-0.002,0.029+/-0.003,F=27.232,P=0.001).The protein expressions of β-catenin were higher and the protein expressions of GSK-3β and PGS1 were lower in model group compared to blank serum group(LSD-t=-4.103,P=0.006; LSD-t=1.048,P=0.000; t=7.365,P=0.000).The protein expressions of β-catenin were lower and the protein expressions of GSK-3β and PGS1 were higher in TGP medicated serum group compared to model group(LSD-t=-3.548,P=0.012; LSD-t=-3.657,P=0.011; LSD-t=-3.273,P=0.017).There was no statistical difference in protein expressions of β-catenin between blank serum group and TGP medicated serum group(LSD-t=-0.554,P=0.599).The protein expressions of GSK-3βand PGS1 were higher in blank serum group compared to TGP medicated serum group(LSD-t=6.827,P=0.000; LSD-t=4.092,P=0.010).Conclusion:TGP medicated serum can inhibit inflammatory reaction induced by LPS in chondrocytes and delay the articular cartilage degeneration.The mechanisms of action may be related to the regulation of Wnt/β-catenin signaling pathway,in which β-catenin gene,Frizzled-2 gene,GSK-3β gene,Wnt-4 gene and CKI-ε gene may be the important action targets.However,many factors can cause OA and there are many complicated ingredients in TGP medicated serum,further studies are needed to confirm the specific action targets.

参考文献/References:

[1] MALAISE O,RELIC B,QUESADA-CALVO F,et al.Selective glucocorticoid receptor modulator compound A,in contrast to prednisolone, does not induce leptin or the leptin receptor in human osteoarthritis synovial fibroblasts[J].Rheumatology(Oxford),2015,54(6):1087-1092.
[2] ROCKEL JS,YU C,WHETSTONE H,et al.Hedgehog inhibits β-catenin activity in synovial joint development and osteoarthritis[J].J Clin Invest,2016,126(5):1649-1663.
[3] BOUAZIZ W,SIGAUX J,MODROWSKI D,et al.Interaction of HIF1α and β-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice[J].Proc Natl Acad Sci U S A,2016,113(19):5453-5458.
[4] 郑文伟,马玉环,林平冬,等.跳骨片水提物对体外大鼠关节软骨细胞活性的影响[J].风湿病与关节炎,2017,6(1):5-8.
[5] 黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[6] CHAN CM,MACDONALD CD,LITHERLAND GJ,et al.Cytokine-induced MMP13 Expression in Human Chondrocytes Is Dependent on Activating Transcription Factor 3(ATF3)Regulation[J].J Biol Chem,2017,292(5):1625-1636.
[7] KANEKO M,TOMITA T,NAKASE T,et al.Expression of proteinases and inflammatory cytokines in subchondral bone regions in the destructive joint of rheumatoid arthritis[J].Rheumatology(Oxford),2001,40(3):247-255.
[8] SUN S,BAYJENSEN AC,KARSDAL MA,et al.The active form of MMP-3 is a marker of synovial inflammation and cartilage turnover in inflammatory joint diseases[J].BMC Musculoskelet Disord,2014,15(1):93.
[9] ÖZLER K,AKTA �瘙 塁 E,ATAY Ç, et al.Serum and knee synovial fluid matrixmetalloproteinase-13 and tumor necrosis factor-alpha levels in patients with late stage osteoarthritis[J].Acta Orthop Traum Turc,2016,50(3):356.
[10] USAMI Y,GUNAWARDENA AT,IWAMOTO M,et al.Wnt signaling in cartilage development and diseases: lessons from animal studies[J].Lab Invest,2016,96(2):186-196.
[11] GAO ZH,SEELING JM,HILL V,et al.Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex[J].Proc Natl Acad Sci U S A,2002,99(3):1182-1187.
[12] UMAR S,WANG Y,MORRIS AP,et al.Dual alterations in casein kinase I-epsilon and GSK-3beta modulate beta-catenin stability in hyperproliferating colonic epithelia[J].Am J Physiol Gastrointest Liver Physiol,2007,292(2):G599-G607.
[13] DU G,SONG Y,WEI L,et al.Osthole inhibits proliferation and induces catabolism in rat chondrocytes and cartilage tissue[J].Cell Physiol Biochem,2015,36(6):2480-2493.
[14] 赵晋,闫振宇,张立智.骨性关节炎软骨和软骨下骨之间信号通路[J].中华骨质疏松和骨矿盐疾病杂志,2016,9(2):193-198.

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(08):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(08):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(08):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(08):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(08):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(08):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(08):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(08):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(08):1.
[17]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(08):13.
[18]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(08):1.
[19]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.
　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(08):9.
[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(08):4.

备注/Memo

备注/Memo:
基金项目:福建省科学技术厅重点资助项目(2014Y0064) 通讯作者:叶蕻芝 E-mail:yelin0930@163.com
更新日期/Last Update: 2017-12-29