摘要
目的:探讨盐酸小檗碱对脂多糖诱导的强直性脊柱炎成纤维样滑膜(FLS)细胞增殖、迁移及侵袭的影响及分子机制。方法:从强直性脊柱炎患者滑膜组织活检中分离出成纤维样滑膜细胞进行培养。待成纤维样滑膜细胞传至第3代后,用脂多糖提前预处理细胞24 h,再用含有不同浓度盐酸小檗碱的培养基干预细胞24 h。随后,采用倒置显微镜观察盐酸小檗碱对成纤维样滑膜细胞的形态学变化,CCK-8检测盐酸小檗碱对脂多糖刺激下成纤维样滑膜细胞增殖的影响,平板克隆形成实验和钙黄绿素/碘化丙啶(Calcein/PI)染色实验检测盐酸小檗碱对脂多糖刺激下成纤维样滑膜细胞增殖的影响,Hoechst33342染色实验和Annexin-V/PI流式细胞仪检测盐酸小檗碱对脂多糖刺激下成纤维样滑膜细胞凋亡的影响,ELISA检测盐酸小檗碱对脂多糖刺激下成纤维样滑膜细胞上清液中IL-6、TNF-α、IL-17、IL-23表达水平的影响,Transwell小室实验检测盐酸小檗碱对成纤维样滑膜细胞侵袭能力的影响,划痕实验检测盐酸小檗碱对成纤维样滑膜细胞迁移能力的影响,用蛋白质印迹技术检测盐酸小檗碱对脂多糖刺激下成纤维样滑膜细胞中相关凋亡、迁移及通路蛋白表达水平的影响。结果:与空白组相比,盐酸小檗碱处理组(观察组)对脂多糖刺激下成纤维样滑膜细胞的增殖、侵袭、迁移力及炎性因子均具有一定的抑制作用,且诱导脂多糖刺激下成纤维样滑膜细胞的凋亡。盐酸小檗碱可以上调Bax、Cleaved-casepase-3、E-cadherin的相对表达(P<0.01),且下调Bcl-2、IL-17、IL-23、c-Jun NH2末端激酶和细胞外信号调节蛋白激酶、N-cadherin的表达水平(P<0.01)。结论:盐酸小檗碱抑制成纤维样滑膜细胞的增殖、侵袭及迁移能力并诱导其凋亡,该机制可能与抑制p-JNK、p-ERK1/2通路有关。
Objective:To investigate effect and molecular mechanisms of berberine hydrochloride on the proliferation,migration,and invasion of fibroblast-like synovial(FLS)cells in lipopolysaccharide induced ankylosing spondylitis.Methods:FLS cells were isolated from synovial tissue biopsy of patients with ankylosing spondylitis and cultured.After the FLS cells were transferred to the third generation,the cells were pre-treated with lipopolysaccharide for 24 h,and then dried with culture media containing different concentrations of berberine hydrochloride for 24 h.Subsequently,an inverted microscope was used to observe the morphological changes of berberine hydrochloride on FLS cells.CCK-8 was used to detect the effect of berberine hydrochloride on the proliferation of FLS cells under lipopolysaccharide stimulation.Plate clone formation experiments and Calcein/PI staining experiments were used to examine the effect of berberine hydrochloride on the proliferation of FLS cells under lipopolysaccharide stimulation;Hoechst33342 staining experiment and Annexin-V/PI flow cytometry were used to detect the effect of berberine hydrochloride on apoptosis of FLS cells stimulated by lipopolysaccharide.The effects of berberine hydrochloride on the expression levels of IL-6,TNF-α,IL-17 and IL-23 in the supernatant of FLS by lipopolysaccharide were detected by ELISA.Transwell chamber experiment was used to detect the effect of berberine hydrochloride on the invasive ability of FLS cells;scratch experiment was used to detect the effect of berberine hydrochloride on the migration ability of FLS cells,and protein blotting technology was used to detect the effect of berberine hydrochloride on apoptosis,migration,and pathway protein expression levels in FLS cells under lipopolysaccharide stimulation.Results:Compared with the blank group,the berberine hydrochloride treatment group had a certain inhibitory effect on the proliferation,invasion,migration,and inflammatory factors of FLS cells under lipopolysaccharide stimulation,and induced apoptosis of FLS cells under lipopolysaccharide stimulation.Berberine hydrochloride can up-regulate the relative expression of Bax,cleared casease-3,and E-cadherin(P<0.01),and down-regulated the expression levels of Bcl-2,IL-17,IL-23,c-Jun NH2 terminal kinase,extracellular signal-regulated protein kinase,and N-cadherin(P<0.01).Conclusion:BBR inhibits the proliferation,invasion,and migration ability of AS FLS cells and induces their apoptosis,which may be related to the inhibition of the p-JNK and p-ERK1/2 pathways.
作者
刘跃振
张邦能
汪丽娟
孟祥云
张磊
李红专
张效收
LIU Yuezhen;ZHANG Bangneng;WANG Lijuan;MENG Xiangyun;ZHANG Lei;LI Hongzhuan;ZHANG Xiaoshou(Gansu University of Chinese Medicine,Lanzhou 730000,China;Gansu Provincial Hospital of Traditional Chinese Medicine,Lanzhou 730050,China)
出处
《中国中医骨伤科杂志》
CAS
2024年第7期1-10,17,共11页
Chinese Journal of Traditional Medical Traumatology & Orthopedics
基金
甘肃省科技重点研发项目(21YF5FA025)。
关键词
盐酸小檗碱
强直性脊柱炎
成纤维样滑膜细胞
增殖
侵袭
迁移
berberine hydrochloride
ankylosing-like spondylitis
proliferation
fibroblastic-like synovial cells proliferation
inva-sion
migration
作者简介
通信作者:张邦能,E-mail:zbn81@163.com。
