青藤碱对FLS增殖及α7nAChR、TLR2和TLR4表达的影响  

Effect of Sinomenine on Proliferation of Fibroblast-like Synoviocytes and Expression of α7nAChR,TLR2 and TLR4

作　　者：彭冲[1] 易浪[1] 吕彦军[1] 白莎莎[1] 李景[1] 董燕[1] 王培训[1] 

机构地区：[1]广州中医药大学免疫研究室,广东广州510405

出　　处：《中药新药与临床药理》2016年第4期503-508,共6页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81573672);澳门科技大学中药质量研究国家重点实验室开放课题资助(MUST-SKL-2016-03)

摘　　要：目的探讨青藤碱(sinomenine,SIN)对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)刺激的大鼠成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)增殖及α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptors,α7n ACh R)、Toll样受体2(toll-like receptor 2,TLR2)和Toll样受体4(toll-like receptor 4,TLR4)表达的影响。方法组织块法分离培养原代FLS,流式细胞术检测FLS表面标志物CD90。FLS设对照组、TNF-α组、SIN组(100,200,400μmol·L^(-1)),SIN组给予SIN干预30 min后,TNF-α组、SIN组均给予20 ng·m L^(-1)TNF-α刺激24 h,采用MTT法检测各组FLS增殖,酶联免疫吸附(ELISA)法检测细胞上清白细胞介素-6(interleukin-6,IL-6)分泌量,RT-PCR法检测α7n ACh R、TLR2和TLR4 m RNA表达及Western bolt法检测α7n ACh R、TLR2和TLR4蛋白表达。结果 TNF-α刺激FLS后,细胞明显增殖,分泌IL-6明显增加,α7n ACh R、TLR2和TLR4 m RNA及蛋白表达增加,SIN(200,400μmol·L^(-1))能抑制FLS增殖,抑制IL-6分泌,并且下调α7n ACh R、TLR2和TLR4 m RNA及蛋白表达。结论 FLS增殖与α7n ACh R、TLR2和TLR4表达相关,且SIN能抑制FLS增殖,并下调α7n ACh R、TLR2和TLR4表达。Objective To study the effect of sinomenine（SIN）on the proliferation of rat fibroblast-like synoviocytes（FLS）stimulated by tumor necrosis factor-α（TNF-α）and on the expression of α7 nicotinic acetylcholine receptors（α7n ACh R）,toll-like receptor 2（TLR2）and TLR4 in rats. Methods Primary FLS were cultured using tissue explant method. And then CD90,the surface marker of FLS,was measured by flow cytometry. FLS were divided into blank control group,TNF-α group and SIN groups（the concentration being 100,200,400 μmol·L^（-1）,respectively）. SIN groups were pretreated with SIN for 30 min,and then TNF-α group and SIN groups were stimulated by 20 ng·m L^（-1）TNF-α for 24 h. The proliferation of FLS of each group was measured by MTT assay,the release of serum interleukin6（IL-6） was measured by enzyme-linked immunosorbent assay（ELISA）,and the m RNA expression of α7n ACh R,TLR2 and TLR4 was measured by RT-PCR and their protein expression was detected by Western blot method. Results After stimulating by TNF-α, the proliferation of FLS was significantly increased, and the release of IL-6 in the supernatant was also increased distinctly. Furthermore,the m RNA and protein expression of α7n ACh R,TLR2 and TLR4 were upregulated. However,the proliferation of FLS and the release of supernatants IL-6 were counteracted,and the expression of m RNA and protein of α7n ACh R,TLR2 and TLR4 were downregulated by SIN at 200,400μmol·L^（-1）. Conclusion The proliferation of FLS is related with the expression of α7n ACh R,TLR2 and TLR4. SIN can inhibit the proliferation of FLS,and downregulate the expression of α7n ACh R,TLR2 and TLR4.

关 键 词：青藤碱 成纤维样滑膜细胞 Α7烟碱型乙酰胆碱受体 Toll样受体2 Toll样受体4 增殖 

分 类 号：R285.5[医药卫生—中药学]