The Effect Of Strychnine On Apoptosis In Chondtocyte Of In-vitro Through Mitochondrial Pathway

Objective: To establish a model of cultivating the SD rat chondral cells outside the body and nitric oxide induced apoptosis in chondrocyte, observe the effect of Strychnine on the apoptosis on the level of cell and molecule and explore the mechanism of curing osteoarthritis by using Strychnine.Method: Separate the chondral cell of SD rat for observations of morphology, identify typeⅡcollangen of the primary chondral cells, establish the cultivating system of the SD rat chondral cells outside the body to supply applicable cells for the experiments.The SD rat chondral cells outside the body were divided into five groups : the blank group, SNP group, SNP+high dose Strychnine group(500mg/mL), SNP+middle dose Strychnine group(250mg/mL), SNP + low dose Strychnine group(125mg/mL),added separatedly the normal medium,S - nitroso -N-acetylpenicillamine(SNP) Which was apoptotic inducer 2mmol/L and different dose Strychnine.After 20 hours,Annexin-V/PI was used to detect the early and advanced apoptotic cell by flow cytometry,to analysis and compare the main stage on Strychnine effecting chondrote apoptosis.The Immune histocytochemistry method was detected expressions of cytochrome c (CytC),and western-blot analysis was used to determine expression of \apoptosis cell related proteins , prelimenary study on mitochondrial pathway of chondrote apoptosis effected by Strychnine.Result: The SD rat chondral cell cultivated outside the body have the expression of typeⅡcollangen,which means the cultivated cells are chondral cell;SNP induced chondrocyte apoptosis,the early apoptosis rate was 26.85±2.01,and the late apoptosis rate was 13.14±2.19;Stychnine can decrease chondrocyte apoptosis obviously ,the apoptosis rates were decreasing with Stychnine doses increasing,and compared the SNP group,the difference was significant because of P<0.05;Meanwhile the early apoptosis ratio of each group was obviously higer than the advanced and the difference was significant because of P<0.05;In the SNP group,Cytc were expressed in the most chondrocyte pulp, and the stained cell rate 2.6±0.55,moderately stained;The Cytc were expressed becoming less by the increasing doses of Stychnine.Compared the blank group, the difference was significant because of F=15.588,P<0.05;Western Blot identified that The expression of Smac activity was decreased as Stychnine dose increasing after SNP induced apoptosis.Conclusion: SNP induced SD rat chondrocyte apoptosis, and Stychnine inhibited chondrocyte apoptosis rat that was decreasing as Stychnine dose increasing, especially the early apoptosis ratio.CytC and Smac are not expressed on the normal chondrocyte pulp,but when it is apoptosis that are released from mitochondria,and Stychnine was inhibiting them released as dose increasing.Stychnine can decrease chondrocyte apoptosis of SD rat induced by SNP. The protection function for articular cartilage is likely realized by the mitochondrial pathway.