Effects Of Drug-containing Serum Of Tougu Xiaotong Capsule To Chondrocyte Activity And Apoptotic Pathway Of Mitochondria And Extracellular Matrix

ObjectiveTo discuss mechanism of effects of drug-containing serum of Tougu Xiaotong Capsule to chondrocyte activity and apoptotic pathway of mitochondria and extracellular matrix.Methods1. The 50 SD rats aged 4 weeks were randomly divided into 5 groups, i.e. blank group, control group, experimental group 1, experimental group 2, experimental group 3,10 rats for every group. All rats were enforced intragastric administration of equivalent dose for 3 days, abdominal aortic blood collected and to prepare drug-containing serum of Tougu Xiaotong Capsule.2. Cartilage was isolated from the knee joint of 20 SD rats aged 4 weeks, and used to establish a cultivation system of chondrocytes in vitro. Chondrocytes were identified with toluidine blue staining and immunohistochemistry staining of Collagenâ…¡.The growth curve of chondrocytes was described by MTT assay. The object was to obtain ideal seeding cells. The 3rd generation of chondrocytes after synchronization were added to each group (blank group, experimental group 2) containing 10% serum DMEM and cultivated for 24h,48h,72h,96h to detect the activity of chondrocytes through MTT assay, to determine effective intervention time of drug-containing serum. The 3rd generation of chondrocytes after synchronization were added to each group (blank group, experimental group 2) containing 5%,10%,15%,20% serum DMEM and cultivated for 72h to detect the activity of chondrocytes through MTT assay, to determine effective intervention concentration of drug-containing serum. 3. The 3rd generation of chondrocytes cultivated for 72h, were randomly divided into apoptosis 1 group, apoptosis 2 group, apoptosis 3 group, apoptosis 4 group, and further cultivated for 24h. The morphology was observed under fluorescent inverted phase contrast microscope. Chondrocyte apoptosis rate was detected with Hocehst 33342 staining to determine dose-effect relationship of the SNP-induced chondrocyte apoptosis. The 3rd generation of chondrocytes cultivated for 72h, were added to each group (blank group, control group, experimental group 1, experimental group 2, experimental group 3) containing 10% serum DMEM and SNP final concentration of 1 mmol/L, and cultivated for 24h to detect the activity of chondrocytes through MTT assay, the mRNA expression of Bax, Bcl-2, Caspase-9, Caspase-3 by Real time RT-PCR methods.4. The 3rd generation of chondrocytes after synchronization were added to each group (blank group, control group, experimental group 1, experimental group 2, experimental group 3) containing 10% serum DMEM and cultivated for 72h. The proliferation of chondrocytes was observed by MTT assay, the expression of Collagenâ…¡by immunohistochemistry staining, the mRNA expression of Collagenâ…¡,PG,Aggreca,Cathepsin K from the chondrocyte extracellular matrix by Real time RT-PCR methods.Results1. The original generation,2nd generation,3rd generation of chondrocytes grew well in vitro. Identification of chondrocytes with toluidine blue staining, in the kytoplasm were purple metachromatic granules; with immunohistochemistry staining of Collagenâ…¡, the kytoplasm appeared brown and no coloration in the nucleus. The 4th generation of chondrocytes gradually became fusiform and started to lose its special phaenotype. Observed from the proliferation curve, the 2nd generation and 3rd generation of chondrocytes grew vigorous, and their activity was higher than the 4th generation and 5th generation, especially for the 3rd generation.2. After intervention with drug-containing serum of Tougu Xiaotong Capsule, chondrocyte OD values of 72h intervention were significantly higher than 24h,48h,96h (P<0.05); chondrocyte OD values of 10% concentration of drug-containing serum were significantly higher than 5%,20%(P<0.01). So, we determined that 72h was the effective intervention time, and 10% was the effective intervention concentration.3. After 24h intervention with SNP, the effective concentration induced chondrocyte apoptosis was 1 mmol/L. After 24h intervention with drug-containing serum, chondrocytes OD values of control group, experimental group 2, experimental group 3 were significantly higher than blank group(P<0.05); the mRNA expression of Bcl-2 were significantly higher in chondrocytes of control group, experimental groups than in blank group(P<0.05); the mRNA expression of Bax, Caspase-9 and Caspase-3 were significantly lower in chondrocytes of control group, experimental groups than in blank group (P<0.01)4. After 72h intervention with 10% concentration of drug-containing serum, chondrocyte OD values of experimental groups of Tougu Xiaotong Capsule were significantly higher than blank group (P<0.01). The effect of proliferation appeared the most obvious in experimental group 2 (P<0.05). The expression of Collagenâ…¡by immunohistochemistry staining was higher in experimental groups of Tougu Xiaotong Capsule than in blank group and control group. The mRNA expression of Collagenâ…¡,PG,Aggrecan increased significantly in experimental groups of Tougu Xiaotong Capsule (P<0.01), with the most significantly in experimental group 2 (P<0.01). The mRNA expression of Cathepsin K decreased significantly in experimental groups of Tougu Xiaotong Capsule (P<0.01), with the most significantly in experimental group 2 (P<0.01).Conclusions1. The effective intervention time in which the drug-containing serum of Tougu Xiaotong Capsule may promote the activity of chondrocyte is 72h, and the effective intervention concentration is 10%.2. The drug-containing serum of Tougu Xiaotong Capsule may promote the proliferation of chondrocyte, retain the activity of chondrocyte, and the proliferation is concerned with the drug concentration, and the effect of proliferation appeared the most obvious in the midst-dose drug concentration (Containing Tougu Xiaotong Capsule 29mg/d).3. Drug-containing serum of Tougu Xiaotong Capsule may down-regulate the expression of chondrocyte pro-apoptosis gene of Bax, Caspase-9 and Caspase-3, up-regulate the expression of anti-apoptosis gene of Bcl-2, and inhibit signal transduction of apoptotic pathway of chondrocyte mitochondria.4. The drug-containing serum of Tougu Xiaotong Capsule may up-regulate the mRNA expression of Collagenâ…¡,PG,Aggrecan from the chondrocyte extracellular matrix, down-regulate the mRNA expression of Cathepsin K, and promote the synthesis of the extracellular matrix, regulate the dynamic balance between extracellular matrix and chondrocyte. And the effect of regulation appeared the most obvious in the midst-dose drug concentration (Containing Tougu Xiaotong Capsule 29mg/d).