Mechanism Of Duhuo Jisheng Decoction On The Inhibition Of Chondrocyte Apoptosis

ObjectiveThe molecular mechanisms behind the effect of Duhuo Jisheng decoction (DHJSD) on chondrocyte apoptosis were explored.Methods1. Chondrocytes were obtained from the knee articular cartilage of 4-week Sprague Dawley rats by mechanical-enzyme digestion method and cultured in vitro. The images of chondrocytic morphology were captured under phase-contrast microscopes. Chondrocytes were identified by type II collagen immunohistochemistry and Toluidine blue staining. The growth curve of Passage 2 (P2) chondrocytes was described by MTT assay.2. The P2 chondrocytes were treated by different concentration of tunicamycin (TM) for 24 h, to determine a better concentration by MTT assay. The chondrocytes were observed under a phase-contrast microscope and established an endoplasmic reticulum (ER) stress-mediated chondrocyte apoptosis model. The viability of chondrocytes treated with DHJSD was evaluated by MTT assay, screening out the optimum concentration and time. Sodium 4-phenylbutyrate,an ERS inhibitor, was used as the positive control and the morphology of chondrocytes were observed under a phase-contrast microscope. The apoptosis of TM-induced chondrocytes was showed by DAPI. The related mRNA expressions and protein levels of Bip. Atf4. Chop. Xbpl. Xbp1s. Bax. Bcl-2, caspase-9 and caspase-3 were detected by Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The expression of miR-34a was tested using the TaqMan MicroRNA Assays.3. The P2 chondrocytes were treated by different concentration of sodium nitroprussiate (SNP) for 24 h, to determine a better concentration by MTT assay. The chondrocytes were observed under a phase-contrast microscope and established a mitochondrial-mediated chondrocyte apoptosis model. The viability of chondrocytes treated with DHJSD was evaluated by MTT assay, screening out the low, medium and high optimum concentration and time. The morphology of chondrocytes was observed under a phase-contrast microscope. The apoptosis of SNP-induced chondrocytes were showed by DAPI and Annexin V/PI. The mitochondrial membrane potential (ATm) of SNP-induced chondrocytes was detected by JC-1 staining. The related mRNA expressions and protein levels of Bax, Bcl-2, caspase-9 and caspase-3 were detected by RT-PCR and Westernblot, respectively.Results1. From the morphology of chondrocytes observed in the process of cultivation, we found that the P2 and P3 chondrocytes had strong ability to secrete matrix and grew much more rapidly with typical characteristics of chondrocytes. The P4 and P5 chondrocytes become large and fiber with poor state. Results showed cytoplasm stained brown represent positive expression of type Ⅱ collagen, while the negative control was failed to stain brown by immunohistochemical staining, and red/purple particles in the cytoplasm represent proteoglycans by toluidine blue staining. The growth of chondrocytes was "S" shape curve.2. The optimum concentration of TM on ERS-mediated chondrocyte apoptosis was 2 μg/ml. The viability of TM-induced chondrocyte treated with DHJSD 200 μg/ml for 24h was better. The mRNA and protein expressions of Xbpl, Xbpls and Bcl-2 were increased, while the mRNA and protein expressions of Bip, Atf4, Chop, Bax, caspase-9 and caspase-3 were decreased in TM-induced chondrocytes treated with DHJSD or PBA compared to that in Tm-induced chondrocytes. The expression of miR-34a was down-regulated in the TM-induced chondrocytes treated with DHJSD or PBA compared to that in TM-induced chondrocytes.3. The optimum concentration of SNP on mitochondrial-mediated chondrocyte apoptosis was 1 mM. The viability of SNP-induced chondrocyte treated with DHJSD 300,400 and 500 μg/ml for 24 h was better. The percentage of apoptotic cells (including the early and late apoptotic cells, and dead cells) in SNP-induced chondrocytes treated by DHJSD were significantly lower than that of the SNP-induced chondrocytes, while the ΔΨm in SNP-induced chondrocytes treated by DHJSD were higher compared to the SNP-induced chondrocytes in a dose-dependent manner. The Bcl-2 expression was extremely increased, whereas the Bax, caspase-9 and caspase-3 expressions were decreased in the SNP-induced chondrocytes treated by DHJSD, compared to that of the SNP-induced chondrocytes in a dose-dependent manner.Conclusions1. DHJSD inhibits TM-induced chondrocyte apoptosis by upregurating the expressions of Xbpl, Xbpls and Bcl-2 and downregurating that of Bip, Atf4, Chop, Bax. caspase-9 and caspase-3. DHJSD inhibits ER stress TM-induced chondrocyte apoptosis by down-regulating miR-34a.2. DHJSD inhibits SNP-induced chondrocyte apoptosis by upregurating the expressions of Bcl-2 and downregurating that of Bax, caspase-9 and caspase-3.