乌头汤对骨关节炎软骨细胞中基质金属蛋白酶13和肿瘤坏死因子-α表达的影响机制研究

Mechanism Study of Wu-tou Decoction on the Expression of Matrix Metalloproteinase-13 and Tumor Necrosis Factor-αin Osteoarthritis Chondrocytes

目的:探讨乌头汤对骨关节炎(OA)软骨细胞中基质金属蛋白酶13(MMP-13)和肿瘤坏死因子-α(TNF-α)表达的影响,以及其对沉默信息转录调控因子1(Silent Information Regulator 1,SIRT1)/转录因子叉头框蛋白O1(Forkhead Box Transcription Factor O1,FOXO1)信号的调控机制。方法:以脂多糖模拟炎性微环境构建骨关节炎软骨细胞模型;以SIRT1信号特异性抑制剂EX527做功能挽救实验,细胞实验分为正常对照组、脂多糖组、乌头汤低剂量组、乌头汤高剂量组、抑制剂组;钙黄绿素-AM(Calcein-AM)/碘化丙啶(Propidium Iodide,PI)染色检测各组细胞的生长活性;流式细胞仪检测各组细胞的凋亡率,免疫荧光检测各组细胞中TNF-α和MMP-13的表达;免疫组化检测各组细胞中蛋白聚糖(Aggrecan)和Ⅱ型胶原(CollagenⅡ)的表达;Western Blot法检测细胞和软骨组织中SIRT1、乙酰化叉头转录因子1(Ac-FOXO1)、B淋巴细胞瘤-2(B Cell Lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl2-associated X Protein,Bax)的表达。结果:在骨关节炎细胞模型中,细胞的生长活性明显降低,凋亡率明显升高,TNF-α和MMP-13以及Ac-FOXO1和Bax的表达明显升高,Aggrecan和CollagenⅡ、SIRT1和Bcl-2的表达明显降低,乌头汤低、高剂量能明显阻滞这些参数的恶化,发挥保护软骨细胞的作用,而EX527可部分逆转乌头汤的保护作用,差异均有统计学意义(P<0.05)。结论:乌头汤能明显下调TNF-α和MMP-13的表达,抑制骨关节炎软骨细胞的凋亡,这可能与激活SIRT1信号、抑制FOXO1的乙酰化有关。

Objective:To investigate the effect of Wu-tou decoction on the expressions of matrix metalloproteinase 13(MMP-13)and tumor necrosis factor-α(TNF-α)in osteoarthritis(OA)chondrocytes cell,and its regulatory mechanism on silent information regulator 1(SIRT1)/forkhead box transcription factor O1(FOXO1)signaling pathway.Methods:Lipopolysaccharide was used to simulate inflammatory microenvironment to construct osteoarthritis chondrocyte model.EX527,SIRT1signal specific inhibitor,was used to perform functional rescue experiment.Cell experiments were divided into normal control group,lipopolysaccharide group,low dose group of Wu-tou decoction,high dose group of Wu-tou decoction and inhibitor group.Calcein AM/propidium iodide(PI)staining was used to detect the growth activity of cells in each group.Flow cytometry was used to detect the apoptosis rate.Immunofluorescence was used to detect the expression of TNF-αand MMP-13.Immunohistochemistry was used to detect the expression of Aggrecan and CollagenⅡ.Western Blot was used to detect the expression of SIRT1,acetylated FOXO1(Ac-FOXO1),B cell lymphoma-2(Bcl-2)and Bcl-212 Chinese J Trad Med Traum&Orthop,Feb.2024,Vol.32,No.2 associated X protein(Bax)in cells.Results:In osteoarthritis cells,the growth activity of cells decreased significantly,the apoptosis rate increased significantly,the expressions of TNF-αand MMP-13,Ac-FOXO1and Bax were increased significantly,and the expressions of Aggrecan and CollagenⅡ,SIRT1and Bcl-2were decreased significantly.Low and high dose of Wu-tou decoction could significantly block the deterioration of these parameters and exert a protective effect of chondrocytes,while EX527could partially reverse the protective effect of Wu-tou decoction.The differences were statistically significant(P<0.05).Conclusion:Wu-tou decoction could significantly down-regulate the expression of TNF-αand MMP-13 and inhibit the apoptosis of chondrocytes in osteoarthritis,which may be related to the activation of SIRT1signal and inhibition of FOXO1acetylation.