Effect Of Leptin On Inducible NO And MMP-13 In Rabbit Articular Chondrocytes In Vitro

Objective: To explore, in vitro, the effect of leptin(OB), alone or in combination with tumor necrosis factor-alpha (TNF-α) on inducible nitric oxide(NO)and on inducible Matrix metalloproteinase 13(MMP-13) in rabbit articular chondrocytes, and to explore the mechanism of leptin in the development of osteoarthritis(OA).Methods:To observe the introduction of NO secretion of chondrocytes intervented by leptin, we draw assistance from classic inflammatory cytokine TNF-α, with its stimulating chondrocytes, the concentration of NO in the chondrocytes cultured supernant fluid was detected. And with the concentration of NO as reference, to observe the difference of effect of leptin alone or in combination with TNF-αin introduction of NO. The sample were divided into 10 groups, include a blank control group, TNF-αgroup, deferent concentration of leptin alone or in combination with TNF-αgroups. The chondrocytes from the articular cartilage of 2-month-old rabbit were cultivated and identified, and the second filial generation chondrocytes were cocultured on plates with different concentration of leptin alone or in combination with TNF-αfor 48 hours after 12 hours starvation. The concentration of NO was detected by nitrate reductase kit in the chondrocytes culture supernant fluid. To observe the introduction of MMP-13 secretion of chondrocytes intervented by leptin, the sample were divided into leptin 10ng/ml, 50ng/ml and 100ng/ml groups. In the same, the second filial generation chondrocytes were cocultured on plates with different concentration of leptin for 48 hours and 96 hours after 12 hours starvation. and MMP-13 was measured by enzyme-linked immunosorbent assay(ELISA) in the chondrocytes culture supernant fluid. The result were analyzed by statistical method.Results : the average value of NO was 5.95±0.51μmol/L in the blank control group, there was no significant difference of the concentration of NO between the different concentration of leptin alone groups and the blank control group(P>0.05). The average value of NO was 10.71±0.89μmol/L in the TNF-α10ng/ml group, the concentration of NO was significantly higher than that of blank control group(P<0.05). In combination with the same concentration of TNF-α10ng/ml, leptin could dose-dependently increase the concentration of NO in the chondrocytes culture supernant fluid in vitro. And the optimum concentration of leptin was 10～15μg/ml. The average value of MMP-13 was dose-dependently increased by leptin in the chondrocytes culture supernant fluid. And which in 96 hours plate was significantly higher than which in 48 hours plate(P<0.05). While there was no MMP-13 exist in the blank control group.Conclusion: leptin can induce matrix metalloproteinase 13(MMP-13) and have synergistic induction effect of nitric oxide(NO) with tumor necrosis factor-alpha (TNF-α) in rabbit articular chondrocytes in vitro.