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    丹参酮ⅡA抑制PI3K/AKT/NF-κB通路对炎性软骨细胞的保护作用

    Protective Effect of Tanshinone IIA on Inflammatory Chondrocytes by Inhibiting PI3K/AKT/NF-κB Pathway

      摘要
    • 摘要: 目的 研究丹参酮ⅡA对IL-1β诱导炎性软骨细胞的保护作用及PI3K/AKT/NF-κB通路的影响。方法 取SD大鼠10只，分离培养软骨细胞，分为正常组、丹参酮ⅡA（6.25，12.5，25，50 μmol·L-1）剂量组，培养24 h后CCK8法检测软骨细胞增殖情况。另取软骨细胞分为正常组、IL-1β（10 ng·mL-1）组和IL-1β+丹参酮ⅡA（6.25，12.5，25，50 μmol·L-1）组共6组，IL-1β与丹参酮ⅡA共培养24 h。CCK8检测细胞增殖情况，ELISA法检测软骨细胞TNF-α、IL-6、PGE2、NO的含量水平。qRT-PCR和Western blotting检测软骨细胞iNOS、COX-2、collagen-Ⅱ、aggrecan、MMP-13、ADAMTS-5的mRNA和蛋白表达水平及PI3K/AKT/NF-κB通路蛋白表达水平。结果 对于正常软骨细胞，给予（12.5，25，50 μmol·L-1）丹参酮ⅡA干预24 h可增加细胞增殖；对于IL-1β诱导的炎性软骨细胞，与IL-1β组相比，丹参酮ⅡA（25，50 μmol·L-1）可增加细胞增殖（P<0.01）。IL-1β+丹参酮ⅡA（12.5 μmol·L-1）组IL-6、PGE2、NO含量，COX-2、ADAMTS-5的mRNA表达及ADAMTS-5蛋白表达水平显著下降（P<0.05或P<0.01），aggrecan蛋白表达水平上升（P<0.05）；IL-1β+丹参酮ⅡA（25，50 μmol·L-1）组TNF-α、IL-6、PGE2、NO含量，iNOS、COX-2、MMP-13、ADAMTS-5的mRNA及蛋白表达，p-PI3K、p-AKT及p-P65蛋白表达水平显著下降（P<0.05或P<0.01），collagen-II、aggrecan蛋白表达水平显著上升（P<0.05或P<0.01）。结论 丹参酮ⅡA可有效保护IL-1β诱导的炎性软骨细胞，可通过促进软骨细胞增殖，抑制炎症反应，抑制PI3K/AKT/NF-κB通路，发挥保护作用。

       

      Abstract: OBJECTIVE To study the protective effect of tanshinone ⅡA on IL-1β induced inflammatory chondrocytes and the effect on PI3K/AKT/NF-κB pathway.METHODS Chondrocytes were isolated from 10 SD rats and divided into five groups: normal group, tanshinone ⅡA(6.25, 12.5, 25 and 50 μmol·L–1), after co-cultured with tanshinone ⅡA for 24 h, CCK8 assay was used to detect the proliferation effect on normal chondrocytes.At the same time, chondrocytes were divided into another 6 groups: normal group, IL-1β(10 ng·mL-1) group, and IL-1β+tanshinone ⅡA(6.25, 12.5, 25, 50 μmol·L–1) groups, IL-1β and tanshinone ⅡA were co-cultured for 24 h, and CCK8 assay was used to detect the cell proliferation.TNF-α, IL-6, PGE2 and NO levels in chondrocytes were detected by ELISA.The mRNA and protein expressions of iNOS, COX-2, collagen II, aggrecan, MMP-13, ADAMTS-5 were detected by qRT-PCR and Western blotting, and expression level of PI3K/AKT/NF-κB pathway protein was detected.RESULTS For normal chondrocytes, tanshinone ⅡA(12.5, 25, 50 μmol·L-1) co-incubation for 24 h could increase cell proliferation.For IL-1β induced inflammatory chondrocytes, compared with IL-1β group, tanshinone ⅡA(25, 50 μmol·L–1) could increase cell proliferation(P<0.01).In IL-1β+tanshinone ⅡA(12.5 μmol·L-1) group the contents of IL-6, PGE2, NO, the mRNA expression of COX-2, ADAMTS-5, the expression of ADAMTS-5 protein were decreased significantly(P<0.05 or P<0.01); the expression of aggrecan protein was increased(P<0.05).In IL-1β+tanshinone ⅡA(25, 50 μmol·L-1) group the contents of TNF-α, IL-6, PGE2, NO, the mRNA and proteins expression of iNOS, COX-2, MMP-13, ADAMTS-5, the proteins expression of p-PI3K, p-AKT and p-P65 were decreased significantly(P<0.05 or P<0.01); the proteins expression of collagen-II and aggrecan were increased significantly(P<0.05 or P<0.01).CONCLUSION Tanshinone ⅡA can effectively protect chondrocytes from inflammation induced by IL-1β, and play a protective role in osteoarthritis by promoting chondrocyte proliferation, inhibiting inflammatory response and inhibiting PI3K/AKT/NF-κB pathway.

       

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