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目的:探讨羧甲基壳聚糖抑制重组人白细胞介素-1β(rhIL-1β)刺激下的兔软骨细胞合成基质金属蛋白酶-1,3(MMP-1,3)的作用机制。方法:分离、培养兔软骨细胞,用rhIL-1β刺激,同时加入不同浓度的羧甲基壳聚糖(CM-chitosan)或L-精氨酸甲酯(L-NAME),培养24h后,检测上清液中的一氧化氮含量和软骨细胞内的诱导性一氧化氮合酶(iNOS)活力。提取细胞总RNA,采用逆转录聚合酶链方法(RT-PCR)检测iNOS,MMP-1,MMP-3的mRNA表达。通过ELISA方法检测上清液中MMP-1,MMP-3的含量。结果:IL-1β能够增加软骨细胞一氧化氮的释放及iNOS的酶活性,诱导细胞iNOS,MMP-1,3的mRNA表达,增加MMP-1,3的分泌。羧甲基壳聚糖对软骨细胞一氧化氮的释放,iNOS酶活力及iNOSmRNA表达均有抑制作用,不同浓度的抑制效果相近;它对软骨细胞的MMP-1,3mRNA的表达及MMP-1,3的分泌也有明显的抑制作用,且呈剂量依赖性。L-NAME对软骨细胞的MMP-1,3mRNA的表达及分泌也有抑制作用,但L-NAME与羧甲基壳聚糖对一氧化氮的影响相似时,羧甲基壳聚糖对MMP-1,3mRNA表达和分泌的抑制作用强于L-NAME。结论:羧甲基壳聚糖抑制软骨细胞合成MMP-1,MMP-3是部分通过降低iNOS表达,减少一氧化氮合成来实现的。
AIM: To investigate the mechanism of carboxymethyl chitosan inhibiting the synthesis of matrix metalloproteinase-1 (MMP-1,3) in rabbit chondrocytes stimulated with recombinant human interleukin-1β (rhIL-1β). Methods: Rabbit chondrocytes were isolated and cultured. The cells were stimulated with rhIL-1β and different concentrations of CM-chitosan or L-NAME were added. After culturing for 24 hours, Nitric oxide content in supernatant and inducible nitric oxide synthase (iNOS) activity in chondrocytes. The total RNA was extracted and the mRNA expressions of iNOS, MMP-1 and MMP-3 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The contents of MMP-1 and MMP-3 in the supernatant were detected by ELISA. Results: IL-1β could increase the release of nitric oxide and the activity of iNOS in chondrocytes, induce the mRNA expression of iNOS and MMP-1, and increase the secretion of MMP-1 andβ3. Carboxymethyl chitosan had inhibitory effects on the release of nitric oxide, iNOS activity and iNOS mRNA expression in chondrocytes, and the inhibitory effect at different concentrations was similar. The effects of carboxymethyl chitosan on the expression of MMP-1 and MMP-1, 3 secretion also had a significant inhibitory effect, and in a dose-dependent manner. L-NAME also inhibited the expression and secretion of MMP-1 and MMP-3 in chondrocytes. However, when L-NAME and carboxymethyl chitosan had similar effects on nitric oxide, , 3mRNA expression and secretion of inhibition stronger than L-NAME. CONCLUSION: Carboxymethyl chitosan inhibits the synthesis of MMP-1 and MMP-3 by chondrocytes in part through decreasing iNOS expression and decreasing nitric oxide synthesis.