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

目的研究丹参酮ⅡA对IL^(-1)β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(6.25,12.5,25,50μmol·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^(-1)β(10 ng·mL^(-1))组和IL^(-1)β+丹参酮ⅡA(6.25,12.5,25,50μmol·L^(-1))组共6组,IL^(-1)β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(12.5,25,50μmol·L^(-1))丹参酮ⅡA干预24 h可增加细胞增殖;对于IL^(-1)β诱导的炎性软骨细胞,与IL^(-1)β组相比,丹参酮ⅡA(25,50μmol·L^(-1))可增加细胞增殖(P<0.01)。IL^(-1)β+丹参酮ⅡA(12.5μmol·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^(-1)β+丹参酮ⅡA(25,50μmol·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^(-1)β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。

OBJECTIVE To study the protective effect of tanshinone ⅡA on IL^(-1)β induced inflammatory chondrocytes and the effect on PI3 K/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^(-1)β(10 ng·mL^(-1)) group, and IL^(-1)β+tanshinone ⅡA(6.25, 12.5, 25, 50 μmol·L–1) groups, IL^(-1)β 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 PI3 K/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^(-1)β induced inflammatory chondrocytes, compared with IL^(-1)β group, tanshinone ⅡA(25, 50 μmol·L–1) could increase cell proliferation(P<0.01). In IL^(-1)β+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^(-1)β+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-PI3 K, 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^(-1)β, and play a protective role in osteoarthritis by promoting chondrocyte proliferation, inhibiting inflammatory response and inhibiting PI3 K/AKT/NF-κB pathway.