Study On Effect Of Drug-Containing Serum Of Tougu Xiaotong Capsule To Chondrocyte Cycle And Apoptosis

ObjectiveTo discuss mechanism of effect of drug-containing serum of Tougu Xiaotong Capsule chondrocyte cycle and apoptosis.Methods1.60 New Zealand white rabbits, were randomly divided into 5 groups, blank group (0.9% normal saline gavage), control group (Bolus 5 mg/ml solution gavage), experimental group 1 (Tougu Xiaotong Capsule 2.5 mg/ml solution gavage), experimental group 2 (Tougu Xiaotong Capsule 5 mg/ml solution gavage), experimental group 3 (Tougu Xiaotong Capsule 10 mg/ml solution gavage),20 ml/time,2 times/day,3 consecutive days, abdominal aortic blood collected and prepared drug-containing serum.2.Cartilage was isolated from the knee joint of 32 New Zealand white rabbits and used to establish cultured primary chondrocytes. Chondrocytes were identified with toluidine blue staining and the mRNA expression of Collageâ…¡by RT-PCR. The 3rd generation of chondrocytes after synchronization, were randomly divided into blank group, control group, experimental group 1,experimental group 2, experimental group 3,which were added to each group containing 15% serum DMEM and cultured for 24 h,48 h,72 h to detect the activity of chondrocytes through MTT, cycle distribution of chondrocytes with flow cytometry to determine effective intervention time of drug-containing serum.The 3rd generation of chondrocytes after synchronization, were added to each group containing 15% serum DMEM and cultured for 72 h, chondrocytes morphology were observed under fluorescent inverted phase contrast microscope, optical microscopy and transmission electron microscopic, the mRNA expression of CyclinDl,CDK4 and p21 by RT-PCR.3.The 3rd generation chondrocytes cultured for 72 h, were randomly divided into apoptosis 1 group (SNP final concentration of 0 mmol/L), apoptosis 2 group (SNP final concentration of 0.5 mmol/L), apoptosis 3 group (SNP final concentration of 1 mmol/L), apoptosis 4 group (SNP final concentration of 2 mmol/L) and further cultured for 24 h. The induced apoptosis and morphological changes of chondrocytes were observed under fluorescent inverted phase contrast microscope, Chondrocytes apoptosis were identified with Hocehst 33342 staining, the activity of Chondrocytes through MTT to determine dose-effect relationship of the SNP-induced chondrocyte apoptosis.4.The 3rd generation chondrocytes cultured for 72 h, were randomly divided into blank group, control group, experimental group 1,experimental group 2, experimental group 3, which were added 15% each group serum DMEM containing SNP final concentration of 1 mmol/L and cultured for 12 h,24 h,36 h, the activity of Chondrocytes through MTT to determine effective intervention time of drug-containing serum. The 3rd generation chondrocytes cultured for 72 h, were added 15% each group serum DMEM containing SNP final concentration of 1 mmol/L and cultured for 24 h, the activity of Chondrocytes through MTT, chondrocytes apoptosis rate were detected using Hocehst 33342 staining, chondrocytes morphology were observed under fluorescent inverted phase contrast microscope and transmission electron microscopy, early apoptosis of chondrocytes Annexin V-FITC staining followed by flow cytometry, the mRNA expression of p38 and p53 by RT-PCR, the activity of Caspase-3 and Caspase-9 were measured using colorimeters.Results1.Identification of chondrocytes with toluidine blue staining, the cytoplasm were purple metachromatic granules in the original generation,2nd generation,3rd generation. Expression of Collage II mRNA gradually decreased from the original generation to 5th generation, and the original generation,2nd generation,3rd generation were significantly higher than 4th generation,5th generation (P<0.01).Tougu Xiaotong Capsule containing serum promote Chondrocytes proliferation in effective concentration of 15%, effective time of 72 h. After 72 h invention, the phenotypic stability of chondrocytes;chondrocytes OD values of control group, experimental group 2, experimental group 3 were significantly higher than blank group (P<0.05); expression of CyclinDl,CDK4 mRNA were significantly higher in chondrocytes of control group, experimental group 2, experimental group 3 than in blank group (P<0.05); expression of p21 mRNA were significantly lower in chondrocytes of control group, experimental group 2, experimental group 3 than in blank group (P<0.05).2.After 24 h invention with SNP, effective concentration induced chondrocytes apoptosis was 1 mmol/L. Effective intervention time of Tougu Xiaotong Capsule drug-containing serum inhibiting chondrocyte apoptosis was 24 h. After 24 h invention, chondrocytes OD values of control group, experimental group 2, experimental group 3 were significantly higher than blank group (P<0.05);chondrocytes apoptosis rate of control group, experimental group 2, experimental group 3 were significantly lower than blank group (P<0.05);expression of p53 mRNA were significantly lower in chondrocytes of control group, experimental group 2, experimental group 3 than in blank group (P<0.05); expression of Bcl-2 mRNA were significantly higher in chondrocytes of control group, experimental group 2, experimental group 3 than in blank group (P<0.05);activity of Caspase-3,Caspase-9 were significantly lower in chondrocytes of control group, experimental group 2, experimental group 3 than in blank group (P<0.05).Conclusions1.Tougu Xiaotong Capsule drug-containing serum may promote the expression of chondrocyte cycle positive regulators including CyclinDl and CDK4, inhibit negative regulator p21,which activate the key point of switch from G1 phase to S phase during chondrocyte cycle to advance the process of chondrocytes and maintain their stable phenotypes.2.Tougu Xiaotong Capsule drug-containing serum may down-regulate chondrocyte pro-apoptosis gene expression of p53,Caspase-9 and Caspase-3, up-regulate anti-apoptosis gene expression of Bcl-2, and inhibit chondrocyte mitochondrial signal transduction pathway of apoptosis.