Anti-inflammatory And Antioxidant Effects Of Dapagliflozin On LPS-induced Rat Chondrocytes And Its Mechanism

Objective:To explore the anti-inflammatory and antioxidant effects of dapagliflozin on LPS-induced rat chondrocytes and its mechanism,and to find a safer and more effective new drug for osteoarthritis prevention and treatment.Methods: Rat chondrocytes were isolated,cultured and identified.The effects of different concentrations of dapagliflozin on cell proliferation,cell viability and apoptosis were detected by CCK8 method,live/dead cell staining and flow cytometry,so as to screen out the optimal intervention concentration of dapagliflozin.chondrocytes were incubated with LPS 10ug/m L to construct in vitro OA model.Real-time quantitative PCR was used to detect the expression of IL-1β,COX-2 and MMP-13 in cells at different times,and the optimal induction time was selected.The effect of dapagliflozin on LPS-induced chondrocytes inflammation and oxidative stress and its mechanism were detected by Western Blot and fluorescent probe.Results: 1.Isolation,culture and identification of rat chondrocytes.The results of CCK8 method showed that compared with the blank group,the proliferation activity of rat chondrocytes decreased with the increase of the net concentration of dapagliflozin.When the concentration was 50μmol/L,the proliferation activity of rat chondrocytes decreased significantly,the difference was statistically significant.2.The results of live/dead cell staining showed that compared with the blank group,the green visual field gradually decreased with the increase of the net concentration of dapagliflozin,indicating that the cell vitality and the number of living cells decreased gradually.When the concentration was 500μmol/L,the red visual field was obvious,and the green visual field was almost invisible,indicating the cell vitality and the number of living cells were weak.3.Flow cytometry results showed that with the increase of dapagliflozin concentration,cell apoptosis gradually increased.When the concentration was,the proportion of cell apoptosis was significantly increased compared with the blank group,and the difference was statistically significant.4.Real-time quantitative PCR results showed that compared with blank group,the expressions of IL-1β,COX-2 and MMP-13 in chondrocytes induced by LPS gradually increased with the increase of time,and the secretion of inflammatory factors was significantly increased at 24 h,with statistical significance.5.The results of Western Blot and fluorescent probe showed that dapagliflozin could inhibit the expression of IL-1β,COX-2,MMP-13 and ROS in LPS-induced chondrocytes,and the inhibitory effect was more obvious when the concentration was 10μmol/L.6.Western Blot results showed that the expression of NF-κB in chondrocytes induced by LPS was increased,while the expression of NF-κB was significantly decreased after dapagliflozin intervention,with statistical significance.Conclusion:1.The proliferation activity of chondrocytes was inhibited by high concentration of dapagliflozin,but not by low concentration.2.High concentration of dapagliflozin could induce apoptosis of chondrocytes,while low concentration had little effect on apoptosis.3.Dapagliflozin inhibited LPS-induced inflammation and oxidative stress in chondrocytes,which may be related to the down-regulation of NF-κB protein expression in chondrocytes.