The Impatct Of Fuyuan Capsule To The Chondrocyte Hypertrophy Through Inhibiting DDR2

Objective:This research is to investigate the hypertrophy mechanism ofof chondrocyte after the induced by COLⅡ-DDR2,as well as the treatmenteffect of Fuyuan capsule to the hypertrophy of chondrocytes throughinhibiting COLⅡ-DDR2. To cultivate primary cells from costicartilage ofrat,and to establish plasmid vector for transfection, then to detect the targetgene and protein expression,to seek for the role of COLⅡ-DDR2duringthe progress of chondrocyte hypertrophy, as well as the intracellular signaltransduction pathway and downstream molecular mechanism. We wishedto find out the hypertrophy pathogenesis of OA and the role of Fuyuancapsule in it.Methods: Samples were from The First Affiliated Hospital ofChongqing Medical University, case inclusion criteria:fitting clinicaldiagnostic criteria of OA, After formalin fixation,the samples were soakedin15%ethylenediaminetetraacetic acid solution for decalcification. HEstainning and Immumohistochemical staining would be done for chondrocyte Hypertrophy markers alkaline phosphatase (ALP), matrixmetalloproteinase13(MMP13), Type Ⅹ collagen (COLⅩ); mice cartilagocostalis cells were cultured and identified, Plasmid expression vector beingstructured, purification, transform, recombination being transfected intochondrocyte, Real-time PCR for type Ⅱcollage,DDR2, MMP13,ALP,andcollagenΧ expression, hypertrophy markers detection by western-blotting;chondrocyte hypertrophy was detected after inhibitor of intracellular signaltransduction pathway of p38(SB203580)、MEK(PD98059)、Wnt（Dkk1）being jointed into cells respectively; drug serum of Fuyuan capsule,drugserum of Zhuangguguanjie pill, blank serum would be administrated3groups respectively, model group and negative control group with noadministration,and the hypertrophy markers would be checked.Results:1. Genetic expression of COLⅡand DDR2were fund in early stage ofOA, the classic alteration of chondrocyte hypertrophy was found by HEstaining, which exhibits4zones as “resting zone-proliferation zone-hypertrophy zone-subchondral zone”.2. recombination genes were establishmented and transformatedintoDH5α competent cell，then were transfected into chondrocytes afterobtaining target clone.3. Relative DNA expressing of DDR2was remarkable distinguishing，according with the situation of FD group> no transfection group> DS group> blank group,hypertrophy marker MMP13,ALP and collagenΧ weresimilar to the tendency of DDR2. variance analysis demonstrated thedifferences among groups had statistical significance.4. The inhibitor of intracellular signal transduction pathway of P38andMEK had little effect to the expressing of hypertrophy markers,and theinhibitor of intracellular signal transduction pathway of Wnt had aremarkable effect on the chondrocyte hypertrophy and the difference hasastatistical significance(p<0.05).5. Inhibitory effect of Fuyuan Capsule among5groups to the expressing ofhypertrophy markers induced by COLⅡ-DDR2showed that large dose andmiddle dose of Fuyuan capsule group had remarkable impact on expressionof target proteins,the small dose group had little effect on the expression ofhypertrophy proteins(p<0.05).Conclusion:DDR2expressing in cartilago articularis in early stage ofOA, COLⅡ-DDR2can promote chondrocyte hypertrophy differentiation,the inhibitor of intracellular signal transduction pathway of Wnt canweaken the effect,which demonstrates that COL Ⅱ-DDR2induceschondrocyte hypertrophy by activating Wnt pathway.Fuyuan capsule canrestrain hypertrophy of chondrocytes,perhaps the mechanism relates todown-regulate DDR2.