| Background:Osteoarthritis is a chronic progressive disease characterized by joint pain, stiffness and limited activity. At present, the treatment of OA mostly concentrated in relieve symptoms. To Repair articular cartilage and to regulate chondrocytes’ function can inhibit the progress of OA. It has been demonstrated that curcumin has multi-factors and multi-targets in protecting cartilage tissue, and was clinical effective in the treatment of OA. Neverthless, the intervention mechanism in proliferation and apoptosis of chondrocytes has not been clearfied.Objective:To observe the effect of curcumin on survival and apoptosis of chondrocytes, and explore the possible mechanism of curcumin to anti chondrocytes apoptosis and protect the cartilage tissue.Methods:Isolated, cultured and indentificated rabbit knee articular chondrocytes in vitro, observe the characteristic of growth and morphology. CCK-8 method was used to observe the effect of different concentrations of curcumin on cell survival rate. Established apoptosis model of chondrocytes exposed to sodium nitroprusside(SNP) in vitro, measured the nitric oxide(NO) level in the culture culture media of different groups by nitratase reduction method and observed the effect of curcumin on chondrocytes apoptosis rate by flow cytometry. Western blot was used to analyzed the expression of Caspase-3, GSK-3β and β-catenin.Results:(1) 80μmol·L-1 and 160μmol·L-1 curcumin showed obvious cytotoxic effect on the chondrocytes compared with the control group(P<0.01) after 12 hours.40 μmol·L-1 curcumin had weaker cytotoxicity than higher doses, but the cell survival rate was lower than that of control group(P<0.05). The cytotoxicity increased with time prolonged at 40μmol·L-1 of curcumin.2.5,5,10,20μmol·L-1 curcumin had no obvious cytotoxic effect on chondrocytes compared with the control group(P>0.05) within 48 hours, and there was no significant difference between each groups.(2) NO level decreased significantly in the curcumin mid-dose group compared with the model group(P<0.05). NO level had the tendency of reduction in other groups of curcumin, but not significant(P>0.05). High dose curcumin could decrease the chondrocytes apoptosis rate obviously compared with that in lower dose(P<0.01), and low dose curcumin showed no effect in preventing cell apoptosis (P>0.05).(3) Western blot results showed:The expression of Caspase-3, GSK-3β and P-catenin enhanced obviously in the model group than that in the normal group(P<0.01). The expression of Caspase-3, GSK-3P and β-catenin decreased separately in the cucumin high dose group compared with the model group(P<0.05 or P<0.01). The were differences between the curcumin mid-dose group and the model group in the expression of Caspase-3 and β-catenin(P<0.05). It had no significant differences of the expression of the proteins between the curcumin low-dose group and the model group(P>0.05).Conclusion:(1) Curcumin revealed no promoting effect in chondrocytes proliferation in all concentrations at different time points. In the safe concentration range from 0 μmol·L-1 to 20 μmol·L-1, curcumin showed no cytotoxicity on the rabbit knee articular chondrocytes in vitro. There were obvious cytotoxic effect on the chondrocytes exposed to the curcumin of 40 μmol·L-1 above, and the cell viabilitiy decreased with time prolonged.(2) Large amount of NO was released to the culture media in the process of chondrocytes apoptosis in vitro. Curcumin could downregulate NO content in the culture media and inhibit chondrocytes apoptosis. Middle dose curcumin is superior to other doses in the ability of eliminating NO. Nevertheless, high dose curcumin had better effect in downregulating the cell apoptosis rate of chondrocytes campared with other intervention groups.(3) Wnt/β-catenin signaling pathway may mediate the prosess of chondrocytes apopotsis. Curcumin could protect rabbit articular chondrocytes against SNP induced apoptosis in vitro, perhaps through the inhibition of GSK-3β, P-catenin and Caspase-3 via Wnt/β-catenin signaling pathway. |
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