青藤碱对脂多糖诱导的人成纤维滑膜细胞增殖的影响

Effect of sinomenine on the proliferation of human fibroblast-like synoviocyte induced by lipopolysaccharide

目的:探讨青藤碱(Sinomenine)对脂多糖诱导的人成纤维样滑膜细胞(Human fibroblast-like synoviocyte,HFLS)增殖的作用及其机制。方法:随机分为空白对照组、脂多糖模型组、青藤碱10-7mol/L、10-6mol/L、10-5mol/L组。利用脂多糖诱导HFLS的增殖,48 h后加入不同浓度的青藤碱,通过MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Western Blot检测凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、P53、细胞周期依赖激酶4(CDK4)、细胞周期蛋白D(CyclinD)的表达,检测核转录因子-κB(NF-κB)、核因子κB受体活化因子(RANK)、核因子κB受体活化因子配体(RANK-L)蛋白表达;ELISA法检测白介素10(IL-10)、白介素6(IL-6)、肿瘤坏死因子-α(TNF-α)与白介素1β(IL-1β)的含量。结果:MTT结果显示与空白对照组比较,模型组细胞增殖明显;与模型组比较,不同浓度的青藤碱均能抑制脂多糖诱导HFLS增殖,结果呈浓度依赖性;流式结果表明青藤碱可促进HFLS的凋亡;Western Blot结果提示与模型组相比,青藤碱组Bax、P53的蛋白表达明显升高,Cyclin D、CDK4、NF-κB、RANK、RANK-L蛋白的表达显著降低。结论:青藤碱可以通过抑制RANK及NF-κB的活化减轻脂多糖诱导的HFLS细胞的炎症反应,促进HFLS凋亡,抑制其增殖。

Objective: To investigate the effect and mechanism of Sinomenine on the proliferation of human fibroblast-like synoviocyte(HFLS) induced by lipopolysaccharide(LPS). Methods: HFLS were randomly divided into the control group, LPS model group, three doses of sinomenine groups(10-7 mol/L, 10-6 mol/L and 10-5 mol/L). Lipopolysaccharide was used to induce the proliferation of HFLS, and sinomenine was added after 48 hours. Cell proliferation was detected by MTT assay and the apoptosis was detected by flow cytometry. The expression levels of apoptosis-related proteins, such as B lymphocytoma-2(bcl-2), bcl-2-related X protein(Bax), P53, cyclin-dependent kinase 4(CDK4), Cyclin D, nuclear transcription factor κB(NF-κB), nuclear factor kappa B receptor activator(RANK) and nuclear factor kappa B receptor activator ligand(RANK-L) were determined by Western Blot. Contents of IL-10, IL-6, TNF-α and IL-1β in the cell supernatant were determined by ELISA. Results:Compared with the control group, MTT results showed that LPS significantly increased the proliferation of HFLS. Compared with the model group,different concentrations of sinomenine inhibited the proliferation of HFLS induced by LPS, in a dose-dependent manner. The flow cytometry result showed that sinomenine promoted the apoptosis of HFLS. Compared with the model group,Western Blot results showed that the protein expressions of Bax and P53 were significantly increased, the protein expressions of Cyclin D, CDK4, NF-κB, RANK and Rank-L protein were decreased. Conclusion: Sinomenine can alleviate the inflammatory response of LPS-induced HFLS by inhibiting the activation of RANK and NF-κB, promoting HFLS apoptosis and inhibiting its proliferation.