Diazoxide prevents rat chondrocyte apoptosis via Inhibition of endoplasmic reticulum stress

Objective To research the mechanism of diazoxide on hydrogen peroxide induced chondrocyte apoptosis. Methods We obtain the carilage cells from the 3 SD rat articular cartilages. The cartilage cells were divided into six groups, namely the control group(A), the H₂O₂-injured group(B), H₂O₂ + 100 μmol/L DZ group(C), H₂O₂ + 200 μmol/L DZ group(D), H₂O₂ + 300 μmol/L DZ group(E), H₂O₂ + 400 μmol/L(F). Group A had no special treatment, group B incubated with 400 μmol/L H₂O₂ for 8 hours in 37℃ incubator, the C, D, E group were pre-incubated with 100, 200, 300, 400 μmol/L DZ in a 37℃ incubator incubated 30 minutes, then incubated with 400 μmol/L H₂O₂ solution in 37℃ incubator. The activity of chondrocytes in group A, B, C, D, E and F were detected by CCK8 method. The apoptosis of chondrocytes were detected by flow cytometry. The function of chondrocytes were detected by RT-PCR and immunofluorescence, Western blot were used to detect the expression of endoplasmic reticulum stress proteins. Results ①Using the CCK8 assay, the order of the rate of cells activity:group A > group E > group D > group F > group B. ②The apoptosis rates of cells were detected by Flow cytometer. The order of apoptosis rates of chondrocyte as follow:group B > group F > group D > group E > group A. ③The Coll-Ⅱ protein expression of chondrocyte were detected by RTPCR analysis, as follow:group A > group E > group D > group F > group B. However, the aggrecanase expression Contrary to Coll-Ⅱ. ④the expression of CHOP in each chondrocytes group detected by immunofluorescence, as follow:group B > group F > group A. ⑤The Caspase-3, Bax, CHOP protein expression of chondrocyte were detected by Westernblot analysis, as follow:group B > group F > group A. Conclusion Diazoxide prevents H₂O₂-induced rat chondrocyte apoptosis via inhibition of endoplasmic reticulum stress.