摘要
目的:研究青藤碱(SIN)对人类风湿关节炎(RA)成纤维细胞样滑膜细胞(FLS)凋亡的影响及分子机制。方法:分离培养人RA FLS,用100 mg/L的脂多糖(LPS)处理记为LPS组;3.2 mmol/L SIN和100 mg/L LPS共同处理细胞,记为LPS+SIN组;不作任何处理的细胞作为空白(blank)组。将miR-con、miR-23b-3p、si-con和si-成纤维细胞生长因子9(FGF9)转染至RA FLS中再用100 mg/L LPS处理,分别记为LPS+miR-con组、LPS+miR-23b-3p组、LPS+si-con组和LPS+si-FGF9组;将anti-miR-con、anti-miR-23b-3p、pcDNA和pcDNA-FGF9转染至RA FLS中,再用3.2 mmol/L SIN和100 mg/L LPS共同处理,分别记为LPS+SIN+anti-miR-con组、LPS+SIN+anti-miR-23b-3p组、LPS+SIN+pcDNA组和LPS+SIN+pcDNA-FGF9组。CCK-8法检测细胞活力;集落形成实验检测细胞的集落形成数;West⁃ern blot法检测FGF9、cleaved caspase-3、Bax和Bcl-2蛋白水平;流式细胞术检测细胞凋亡;RT-qPCR检测miR-23b-3p和FGF9 mRNA的表达水平;双萤光素酶报告基因实验验证miR-23b-3p和FGF9的靶向关系。结果:SIN促进LPS诱导的RA FLS凋亡,抑制细胞增殖,上调miR-23b-3p表达,下调FGF9表达。miR-23b-3p靶向调控FGF9,过表达miR-23b-3p或沉默FGF9抑制LPS诱导的RA FLS增殖并促进细胞凋亡。干扰miR-23b-3p或过表达FGF9逆转了SIN对LPS诱导的RA FLS增殖和凋亡的影响。结论:青藤碱可通过miR-23b-3p/FGF9轴诱导RA FLS凋亡,抑制细胞增殖。
AIM:To investigate the effect of sinomenine(SIN)on the apoptosis of human rheumatoid arthritis(RA)fibroblast-like synoviocytes(FLS)and its molecular mechanism.METHODS:Human RA FLS were isolated and cultured.The cells treated with lipopolysaccharide(LPS)at 100 mg/L was recorded as LPS group.The cells treated with SIN at 3.2 mmol/L and LPS at 100 mg/L were recorded as LPS+SIN group.The cells without any treatment served as blank group.The cells transfected with miR-con,miR-23b-3p,si-con and si-fibroblast growth factor 9(FGF9)and treated with 100 mg/L LPS were recorded as LPS+miR-con group,LPS+miR-23b-3p group,LPS+si-con group and LPS+si-FGF9 group,respectively.The anti-miR-con,anti-miR-23b-3p,pcDNA and pcDNA-FGF9 were also transfected into RA FLS,and then the cells were treated with SIN at 3.2 mmol/L and LPS at 100μg/mL.These cells were recorded as LPS+SIN+anti-miR-con group,LPS+SIN+anti-miR-23b-3p group,LPS+SIN+pcDNA group,LPS+SIN+pcDNA-FGF9 group,respectively.The cell viability was measured by CCK-8 assay.The number of colonies was accessed by colony formation experiment.The protein levels of FGF9,cleaved caspase-3,Bax and Bcl-2 were determined by Western blot.The apoptosis was analyzed by flow cytometry.The expression of miR-23b-3p and FGF9 mRNA was detected by RT-qPCR.Dual-luciferase reporter assay was used to verify the targeting relationship between miR-23b-3p and FGF9.RESULTS:Treatment with SIN promoted LPS-induced apoptosis of RA FLS,inhibited cell proliferation,up-regulated miR-23b-3p expression,and down-regulated FGF9 expression.miR-23b-3p targeted FGF9.Over-expression of miR-23b-3p or silencing of FGF9 inhib ited LPS-induced proliferation and enhanced apoptosis of the RA FLS.Interfering with miR-23b-3p or over-expression of FGF9 reversed the effects of SIN on the proliferation and apoptosis of LPS-induced RA FLS.CONCLUSION:Sinome nine induces RA FLS apoptosis and inhibits cell proliferation through miR-23b-3p/FGF9 signaling.
作者
李嘉
付婷婷
张光峰
刘向前
陈民
LI Jia;FU Ting-ting;ZHANG Guang-feng;LIU Xiang-qian;CHEN Min(Department of Orthopedics,Guangdong Provincial People's Hospital,Guangzhou 510080,China;Department of Traditional Chinese Medicine,Guangdong Provincial People's Hospital,Guangzhou 510080,China;Department of Rheumatism,Guangdong Provincial People's Hospital,Guangzhou 510080,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2020年第9期1653-1660,共8页
Chinese Journal of Pathophysiology
基金
广东省中医药局科研项目(No.20171003)。