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 H
2O
2-injured group(B), H
2O
2 + 100 μmol/L DZ group(C), H
2O
2 + 200 μmol/L DZ group(D), H
2O
2 + 300 μmol/L DZ group(E), H
2O
2 + 400 μmol/L(F). Group A had no special treatment, group B incubated with 400 μmol/L H
2O
2 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 H
2O
2 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 H
2O
2-induced rat chondrocyte apoptosis via inhibition of endoplasmic reticulum stress.