中医手法对兔膝骨关节炎软骨退变及MAPK信号转导通路的影响
|
摘要
目的观察中医手法对实验性兔膝骨关节炎软骨退变的干预作用,并从细胞信号转导角度探讨其作用机制。方法 24只新西兰大白兔随机分为3组,即正常组、模型组和手法组,除正常组外,其余两组动物均采用手术方法建立膝骨关节炎模型。造模4周后对手法组给予髌骨按揉、关节被动屈伸等形式的手法治疗。干预8周后处死动物,暴露膝关节行形态学观察,并取股骨髁部做病理切片,行HE染色,镜下观察其组织病理学特征并进行Mankin’s评分,比较各组关节软骨退变情况。应用RT-PCR方法分别检测滑膜组织中IL-1β、i NOS、MMP-13和软骨组织中P38、ERK1/2的mRNA表达水平。应用Western blot方法检测软骨组织内MMP-13及磷酸化的ERK1/2(p-ERK1/2)的蛋白表达。比较各组实验数据并进行统计学分析。结果手法组关节软骨退变程度较模型组明显减轻,Mankin’s评分分值显著低于模型组(P<0.05)。手法组软骨P38、ERK1/2及滑膜组织IL-1β、i NOS、MMP-13的mRNA表达水平较模型组显著下调(P<0.05)。模型组和手法组的MMP-13、p-ERK1/2的蛋白表达较正常关节软骨明显增强,而手法组MMP-13、p-ERK1/2的蛋白活化程度与模型组比较明显受到抑制。结论中医手法能够有效抑制膝骨关节炎的软骨退变程度,影响炎症介质和细胞因子的表达,其机制可能是通过抑制软骨MAPK信号转导通路发挥作用。
Objective To observe the intervention effects of Chinese medical manipulation on the articular cartilage degeneration in rabbit model of knee osteoarthritis,and explore its mechanism from the perspective of cell signaling transduction.Methods 24 New Zealand white rabbits were randomly divided into the normal group,model group and manipulation group.Except the normal group,the model of knee osteoarthritis was established by operation in the other two groups.After modeling for 4 weeks,the manipulation group was treated with manipulation therapy such as patella massage and joint passive flexion and extension.After intervention for 8 weeks,the rabbits were sacrificed and the knee joints were exposed for morphological observation.The femoral condyle was collected and made into pathological sections for HE staining.The histopathological features were observed under microscope and evaluated by Mankin 's score,and the degeneration of articular cartilage in each group was compared.The mRNA expression levels of IL-1β、i NOS and MMP-13 in synovial tissues and P38 as well as ERK1/2 in cartilage tissue were detected by RT-PCR.The protein expression levels of MMP-13 and phosphorylated ERK1/2(p-ERK1/2) in cartilage tissue were detected by Western-blot.All the experimental data were compared and conducted the statistical analysis.Results The degree of articular cartilage degeneration in the manipulation group was obviously alleviated compared with the model group,and the Mankin's score in the manipulation group was significantly lower than that in the model group(P<0.05).Compared with the model group,the mRNA expression levels of P38 and ERK1/2 in cartilage and IL-1β,i NOS and MMP-13 in synovial tissues in the manipulation group were significantly down-regulated(P<0.05).Compared with the normal articular cartilage,the protein expressions of MMP-13 and p-ERK1/2 were significantly up-regulated in the model group and manipulation group.While the protein activation degree of MMP-13 and p-ERK1/2 in the manipulation group was obviously inhibited compared with that in the model group.Conclusion Chinese medical manipulation can effectively inhibit the cartilage degeneration degree of knee osteoarthritis,and affect the expressions of inflammatory mediators and cytokines.Its mechanism may be associated with inhibition of mitogen-activated protein kinase(MAPK) signal transduction pathway in articular cartilage.
Objective To observe the intervention effects of Chinese medical manipulation on the articular cartilage degeneration in rabbit model of knee osteoarthritis,and explore its mechanism from the perspective of cell signaling transduction.Methods 24 New Zealand white rabbits were randomly divided into the normal group,model group and manipulation group.Except the normal group,the model of knee osteoarthritis was established by operation in the other two groups.After modeling for 4 weeks,the manipulation group was treated with manipulation therapy such as patella massage and joint passive flexion and extension.After intervention for 8 weeks,the rabbits were sacrificed and the knee joints were exposed for morphological observation.The femoral condyle was collected and made into pathological sections for HE staining.The histopathological features were observed under microscope and evaluated by Mankin 's score,and the degeneration of articular cartilage in each group was compared.The mRNA expression levels of IL-1β、i NOS and MMP-13 in synovial tissues and P38 as well as ERK1/2 in cartilage tissue were detected by RT-PCR.The protein expression levels of MMP-13 and phosphorylated ERK1/2(p-ERK1/2) in cartilage tissue were detected by Western-blot.All the experimental data were compared and conducted the statistical analysis.Results The degree of articular cartilage degeneration in the manipulation group was obviously alleviated compared with the model group,and the Mankin's score in the manipulation group was significantly lower than that in the model group(P<0.05).Compared with the model group,the mRNA expression levels of P38 and ERK1/2 in cartilage and IL-1β,i NOS and MMP-13 in synovial tissues in the manipulation group were significantly down-regulated(P<0.05).Compared with the normal articular cartilage,the protein expressions of MMP-13 and p-ERK1/2 were significantly up-regulated in the model group and manipulation group.While the protein activation degree of MMP-13 and p-ERK1/2 in the manipulation group was obviously inhibited compared with that in the model group.Conclusion Chinese medical manipulation can effectively inhibit the cartilage degeneration degree of knee osteoarthritis,and affect the expressions of inflammatory mediators and cytokines.Its mechanism may be associated with inhibition of mitogen-activated protein kinase(MAPK) signal transduction pathway in articular cartilage.
引文
[1]BROOKS P M.Impact of osteoarthritis on individuals and society:how much disability?Social consequences and health economic implications[J].Curr Opin Rheumatol,2002,14(5):573-577.
[2]HULTH A,LINDBERG L,TELHAG H.Experimental osteoarthritis in rabbits.Preliminary report[J].Acta Orthop Scand,1970,41(5):522-530.
[3]王学宗,曹月龙,王翔,等.手法治疗膝骨关节炎的系统回顾[J].中国中医骨伤科杂志,2011,19(2):3-5.
[4]王琼,朱国苗.手法治疗膝骨关节炎疗效的系统评价[J].上海中医药杂志,2013,47(11):11-15.
[5]滕蔚然,杜宁,史炜镔.手法治疗膝骨关节病对膝关节功能改善的研究[J].中国中医骨伤科杂志,2000,8(5):46-48.
[6]王加谋,刘彤鸥.中医手法对膝关节退行性骨关节炎作用效应及临床观察[J].中国中医骨伤科杂志,2008,16(12):10-11.
[7]赵长伟,梁运海,闻辉,等.理筋手法治疗膝骨性关节炎的疗效评价[J].中国医药指南,2014,12(27):251.
[8]JEVSEVAR D S,BROWN G A,JONES D L,et al.The American Academy of Orthopaedic Surgeons evidence-based guideline on:treatment of osteoarthritis of the knee,2nd edition[J].J Bone Joint Surg Am,2013,95(20):1885-1886.
[9]GOLDRING M B,GOLDRING S R.Osteoarthritis[J].J Cell Physiol,2007,213(3):626-634.
[10]PELLETIER J P,MARTEL-PELLETIER J,RAYNAULD J P.Most recent developments in strategies to reduce the progression of structural changes in osteoarthritis:today and tomorrow[J].Arthritis Res Ther,2006,8(2):206.
[11]高世超,殷海波,刘宏潇,等.MAPK信号通路在骨关节炎发病机制中的研究进展[J].中国骨伤,2014,27(5):441-444.
[12]LIN M,LIN Y,LI X,et al.Warm sparse-dense wave inhibits cartilage degradation in papain-induced osteoarthritis through the mitogenactivated protein kinase signaling pathway[J].Exp Ther Med,2017,14(4):3674-3680.
[13]张林,陆军,李永刚.骨关节炎中介导软骨细胞代谢失衡的相关信号通路研究进展[J].东南大学学报(医学版),2013,32(4):465-472.
[14]KIMURA H,YUKITAKE H,SUZUKI H,et al.The chondroprotective agent ITZ-1 inhibits interleukin-1beta-induced matrix metalloproteinase-13 production and suppresses nitric oxide-induced chondrocyte death[J].J Pharmacol Sci,2009,110(2):201-211.
[15]KIM S J,JU J W,OH C D,et al.ERK-1/2 and p38 kinase oppositely regulate nitric oxide-induced apoptosis of chondrocytes in association with p53,caspase-3,and differentiation status[J].Biol Chem,2002,277(2):1332-1339.
[16]WEI L,SUN X J,WANG Z,et al.CD95-induced osteoarthritic chondrocyte apoptosis and necrosis:dependency on p38 mitogenactivated protein kinase[J].Arthritis Res Ther,2006,8(2):R37.
[17]KüHN K,SHIKHMAN A R,LOTZ M.Role of nitric oxide,reactive oxygen species,and p38 MAP kinase in the regulation of human chondrocyte apoptosis[J].J Cell Physiol,2003,197(3):379-387.
[18]MENGSHOL J A,VINCENTI M P,COON C I,et al.Interleukin-1induction of collagenase 3(matrix metalloproteinase 13)gene expression in chondrocytes requires p38,c-Jun N-terminal kinase,and nuclear factor kappa B:differential regulation of collagenase 1 and collagenase 3[J].Arthritis Rheum,2000,43(4):801-811.
[19]WADA Y,SHIMADA K,SUGIMOTO K,et al.Novel p38 mitogenactivated protein kinase inhibitor R-130823 protects cartilage by downregulating matrix metalloproteinase-1,-13 and prostaglandin E2production in human chondrocytes[J].Int Immunopharmacol,2006,6(2):144-155.
[20]SUN H Y,HU K Z,YIN Z S.Inhibition of the p38-MAPK signaling pathway suppresses the apoptosis and expression of proinflammatory cytokines in human osteoarthritis chondrocytes[J].Cytokine,2017(90):135-143.
[21]SéGUIN C A,BERNIER S M.TNFalpha suppresses link protein and type II collagen expression in chondrocytes:Role of MEK1/2 and NFkappa B signaling pathways[J].J Cell Physiol,2003,197(3):356-369.
[22]SCOTTO D'ABUSCO A,CALAMIA V,CICIONE C,et al.Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes[J].Arthritis Res Ther,2007,9(5):R104.
[23]夏军,俞永林,杨丰建,等.丝裂酶原活化蛋白激酶激酶1/2抑制对家兔关节软骨保护作用的研究[J].中华骨科杂志,2008,28(1):63-67.
[2]HULTH A,LINDBERG L,TELHAG H.Experimental osteoarthritis in rabbits.Preliminary report[J].Acta Orthop Scand,1970,41(5):522-530.
[3]王学宗,曹月龙,王翔,等.手法治疗膝骨关节炎的系统回顾[J].中国中医骨伤科杂志,2011,19(2):3-5.
[4]王琼,朱国苗.手法治疗膝骨关节炎疗效的系统评价[J].上海中医药杂志,2013,47(11):11-15.
[5]滕蔚然,杜宁,史炜镔.手法治疗膝骨关节病对膝关节功能改善的研究[J].中国中医骨伤科杂志,2000,8(5):46-48.
[6]王加谋,刘彤鸥.中医手法对膝关节退行性骨关节炎作用效应及临床观察[J].中国中医骨伤科杂志,2008,16(12):10-11.
[7]赵长伟,梁运海,闻辉,等.理筋手法治疗膝骨性关节炎的疗效评价[J].中国医药指南,2014,12(27):251.
[8]JEVSEVAR D S,BROWN G A,JONES D L,et al.The American Academy of Orthopaedic Surgeons evidence-based guideline on:treatment of osteoarthritis of the knee,2nd edition[J].J Bone Joint Surg Am,2013,95(20):1885-1886.
[9]GOLDRING M B,GOLDRING S R.Osteoarthritis[J].J Cell Physiol,2007,213(3):626-634.
[10]PELLETIER J P,MARTEL-PELLETIER J,RAYNAULD J P.Most recent developments in strategies to reduce the progression of structural changes in osteoarthritis:today and tomorrow[J].Arthritis Res Ther,2006,8(2):206.
[11]高世超,殷海波,刘宏潇,等.MAPK信号通路在骨关节炎发病机制中的研究进展[J].中国骨伤,2014,27(5):441-444.
[12]LIN M,LIN Y,LI X,et al.Warm sparse-dense wave inhibits cartilage degradation in papain-induced osteoarthritis through the mitogenactivated protein kinase signaling pathway[J].Exp Ther Med,2017,14(4):3674-3680.
[13]张林,陆军,李永刚.骨关节炎中介导软骨细胞代谢失衡的相关信号通路研究进展[J].东南大学学报(医学版),2013,32(4):465-472.
[14]KIMURA H,YUKITAKE H,SUZUKI H,et al.The chondroprotective agent ITZ-1 inhibits interleukin-1beta-induced matrix metalloproteinase-13 production and suppresses nitric oxide-induced chondrocyte death[J].J Pharmacol Sci,2009,110(2):201-211.
[15]KIM S J,JU J W,OH C D,et al.ERK-1/2 and p38 kinase oppositely regulate nitric oxide-induced apoptosis of chondrocytes in association with p53,caspase-3,and differentiation status[J].Biol Chem,2002,277(2):1332-1339.
[16]WEI L,SUN X J,WANG Z,et al.CD95-induced osteoarthritic chondrocyte apoptosis and necrosis:dependency on p38 mitogenactivated protein kinase[J].Arthritis Res Ther,2006,8(2):R37.
[17]KüHN K,SHIKHMAN A R,LOTZ M.Role of nitric oxide,reactive oxygen species,and p38 MAP kinase in the regulation of human chondrocyte apoptosis[J].J Cell Physiol,2003,197(3):379-387.
[18]MENGSHOL J A,VINCENTI M P,COON C I,et al.Interleukin-1induction of collagenase 3(matrix metalloproteinase 13)gene expression in chondrocytes requires p38,c-Jun N-terminal kinase,and nuclear factor kappa B:differential regulation of collagenase 1 and collagenase 3[J].Arthritis Rheum,2000,43(4):801-811.
[19]WADA Y,SHIMADA K,SUGIMOTO K,et al.Novel p38 mitogenactivated protein kinase inhibitor R-130823 protects cartilage by downregulating matrix metalloproteinase-1,-13 and prostaglandin E2production in human chondrocytes[J].Int Immunopharmacol,2006,6(2):144-155.
[20]SUN H Y,HU K Z,YIN Z S.Inhibition of the p38-MAPK signaling pathway suppresses the apoptosis and expression of proinflammatory cytokines in human osteoarthritis chondrocytes[J].Cytokine,2017(90):135-143.
[21]SéGUIN C A,BERNIER S M.TNFalpha suppresses link protein and type II collagen expression in chondrocytes:Role of MEK1/2 and NFkappa B signaling pathways[J].J Cell Physiol,2003,197(3):356-369.
[22]SCOTTO D'ABUSCO A,CALAMIA V,CICIONE C,et al.Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes[J].Arthritis Res Ther,2007,9(5):R104.
[23]夏军,俞永林,杨丰建,等.丝裂酶原活化蛋白激酶激酶1/2抑制对家兔关节软骨保护作用的研究[J].中华骨科杂志,2008,28(1):63-67.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |