Research On Effect And Mechanism Of Clematis Chinensis Osbeck Mediated By Simulated Microgravity On Chondrocyte Phenotype Maintenance

OBJECTIVE:to study the effect and mechanism of the extract of Clematis chinensis Osbeck(CCO)on the phenotypic maintenance of rabbit chondrocytes in simulated microgravity culture environment.METHODS:Extract The rabbit knee joint cartilage cells,chondrocytes(P3 generations)were divided into four groups:Control,CCO,microgravity group(RCCS),RCCS-CCO;RCCS and RCCS-CCO group were cultured with Rotary Cell Culture system(RCCS)to simulate microgravity environment.The rest of the two groups were cultured with two-dimensional plate for 7 days;Morphological changes of chondrocytes were observed;Energy metabolism was assessed by detecting concentration of intracellular glucose sugar,ATP and lactate;Chondrocytes apoptotic rate were detected using Tunel;The mRNA expression of Collagen type II,Aggrecan,TGF-?,SOX9 and MMP13,RUNX2;collagen type X collagen type ? and IHH were detected by RT-qPCR;The protein expression of type ? collagen and Aggrecan,TGF-?and MMP13 were detected by Western blotting.RESULTS:The morphology of chondrocytes was good in microgravity cultureenviroment,and spindle cells increased in plate culture.Compared with the Control group,the concentration of intracellular glucose and ATP in the other three groups increased,while the content of lactate decreased,and the CCO-RCCS group was more significant than the other two(P<0.05).The results of PCR and WB reveal that compared with the blank group,the other three groups'expression of Aggrecan,collagen type ?,TGF-beta,SOX9 are raised,and MMP13,RUNX2,collagen type X,IHH expression are downregulated,CCO-RCCS group is significantly higher than the other two groups(P<0.05);Tunel test results showed that the apoptosis rate of the CCO,RCCS and the CCO-RCCS group were all decreased compared with the blank group,and the CCO-RCCS group had the most significant effects(P<0.05).CONCLUSION:The extraction of Clematis chinensis Osbeck and the microgravity culture environment can improve the energy metabolism level of chondrocytes and promote the maintenance of chondrocytes phenotype,and the combined application of the two can play a synergistic action,more significant effects.