目的:研究香豆雌酚(coumestrol)对膝骨关节炎患者软骨细胞OPG/RANKL及MMP-1、MMP-3表达的影响,初步探讨其对骨关节炎的治疗机制。方法:分离、培养因膝骨关节炎行膝关节置换患者的膝关节软骨细胞,运用Ⅱ型胶原免疫荧光染色进行软骨细胞鉴定。用不同浓度(10-9、10-8、10-7mol·L-1)香豆雌酚干预第3代细胞,并设0浓度为空白对照组,分别于培养后3、7 d用real-time PCR法测定细胞骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子配基(receptor activator of nuclear factor-κB ligand,RANKL)、基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)和基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)mRNA的表达。结果:干预3 d,10-8 mol·L-1组OPG及RANKL mRNA表达均增加,其OPG/RANKL相对表达是空白对照组的2.59倍(P<0.05);干预7 d,10-9和10-8 mol·L-1组OPG mRNA表达均增加,10-7 mol·L-1组RANKL mRNA表达增加,10-8 mol·L-1组OPG/RANKL相对表达是空白对照组的3.85倍(P<0.05)。干预3 d,10-8 mol·L-1组MMP-1 mRNA表达减少(P<0.05);干预7 d,10-8 mol·L-1组MMP-1和MMP-3 mRNA表达均减少(P<0.05)。结论:香豆雌酚能增加膝骨关节炎患者关节软骨细胞OPG表达及OPG/RANKL值,减少其MMP-1、MMP-3表达,具有一定的软骨保护作用。 |
Objective: to study effect of coumestrol on the expression of OPG, RANKL, MMP-1 and MMP-3 in the chondrocytes of patients with knee osteoarthritis(KOA).Methods: Chondrocytes from KOA patients who had taken a total knee arthroplasty(TKA) were isolated and cultured, and cells were identified with collagen typeⅡ immunofluorescence staining. The third generation cells were incubated in presence of various concentrations(10-9, 10-8, 10-7mol·L-1) of coumestrol, or 0 mol·L -1 for negtive control. real-time PCR assay was used to determine the OPG, RANKL, MMP-1 and MMP-3 mRNA expression level of the cells at the 3th and 7th day. Results: OPG/RANKL expression: At the 3th day, 10-8 mol·L-1 coumestrol significantly increased OPG and RANKL mRNA expression and the ratio of OPG/RANKL gene expression was 2.59 times as much as that of the negtive control(P<0.05). At the 7th day, 10-9 and 10-8 mol·L-1 coumestrol significantly increased OPG mRNA expression, while 10-7 mol·L-1 coumestrol significantly increased RANKL expression. And, in 10-8 mol·L-1 coumestrol group, the ratio of OPG/RANKL gene expression was 3.85 times as much as that of the negtive control(P<0.05). MMP-1, MMP-3 expression: At the 3th day, 10-8 mol·L-1 coumestrol significantly decreased MMP-1 mRNA expression(P<0.05). At the 7th day, 10-8 mol·L-1 coumestrol significantly decreased MMP-1 and MMP-3 mRNA expression(P<0.05). Conclusion: Coumestrol has its protective effects on cartilage by increasing the OPG mRNA expression and the ratio of OPG/RANKL gene expression and decreasing the MMP-1 and MMP-3 expression in the chondrocytes of patients with knee osteoarthritis. |
[1] PARAZZINI F,Progretto Menopausa Italia Study Group.Menopausal status,hormone replacement therapy use and risk of self reported physician-diagnosed osteoarthrit is in women attending menopause clinics in Italy[J].Maturitas,2003,46(3):207-212.
[2] RICHMOND R S,CARLSON C S,REGISTER T C,et al.Functional estrogen receptors in adult articular cartilage: estrogen replacement therapy increases chondrocyte synthesis of proteoglycans and insulin-like growth factor binding protein 2[J].Arthritis Rheum,2000,43(9):2081-2090.
[3] CLAASSEN H,HASSENPFLUG J,SCHVNKE M,et al.Immunohistochemical detection of estrogen receptor alpha in articular chondrocytes from cows,pigs and humans:in situ and in vitro results[J].Ann Anat,2001,183(3):223-227.
[4] WANG K,LIN Y F,QIN C H,et al.Bisphenol-A interferes with estradiol-mediated protection in osteoarthritic chondrocytes[J].Toxicology Lett,2012,198(2):127-133.
[5] HORST C,REINHARD S,JOACHIM H,et al.17β-estradiol reduces expression of MMP-1,-3,and-13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system[J].Cell Tissue Res,2010,342(2):283-293.
[6] SONDERGAARD B C,SCHUHZ N,MADSEN S H,et al.MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation-divergencein pathways leading to aggrecanase and MMP-mediated articular cartilage degradation[J].Osteoarthritis Cartilage,2010,18(3):279-288.
[7] YUNG C L,THANASEKARAN J,YEH F D.Chondmpmtective Role of Sesamol by Inhibiting MMPs Expression via Retaining NF-KB Signaling in Activated SWl353 Cells[J].J Agile Food Chem,2011,59(9):4969-4978.
[8] Da SILVA M A,YAMADA N,CLARKE N M,et al.Cellular and epigenetic features of a young healthy and a young osteoarthritic cartilage compared with aged control and OA cartilage[J].J Orthop Res,2009,27(5):593-601.
[9] AGARWAL S,LONG P,SEYEDAIN A,et al.A central role for the nuclear factor-kappaB pathway in anti-inflammatory and proinflammatory actions of mechanical strain[J].FASEB J,2003,17(8):899-901.
[10] CSAKI C,MOBASHERI A,SHAKIBAEI M.Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1beta-induced NF-kappaB-mediated inflammation and apoptosis[J].Arthritis Res Ther,2009,11(6):R165.
[11] WU X T,WEI J N,LI Y G,et al.Effects of coumestrol on expression of bone marker during primary osteoblastic cells differentiation[J].中国骨质疏松杂志,2009,15(7):490-495.
[12] WU X T,WANG B,WEI J N.Coumestrol promotes proliferation and osteoblastic differentiation in rat bone marrow stromal cells[J].J Biomed Mater Res B Appl Biomater,2009,90(2):621-628.
[13] KOMURO H,OLEE T,KVHN K,et al.The osteoprotegerin/receptor activator of nuclear factor kappaB/receptor activator of nuclear factor kappaB ligand system in cartilage[J].Arthritis Rheum,2001,44(12):2768-2776.
[14] 刘维嘉,黄有荣,潘能富,等.膝关节骨性关节炎与骨质疏松症的相关性研究[J].中国骨质疏松杂志,2008,14(3):187-188.
[15] NEVITT M C,FELSON D T,WILLIAMS E N,et al.The effect of estrogen plus progestin on knee symptoms and related disability in postmenopausal women[J].Arthritis Rheum,2001,44(4):811-818.
[16] 李贺,王宸,陈昌红,等.骨碎补总黄酮治疗兔膝骨关节炎的实验研究[J].现代医学,2010,38(3):208-211. |
