Yang Junling, Zhao Feicui, Nie Jihong
College of Traditional Chinese Medicine, Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China.
Department of Pharmacy, Chinese Medicine Hospital Affiliated to Xinjiang Medical University, Urumqi, Xinjiang 830000, P.R. China.
Exp Ther Med. 2017 Jul;14(1):453-460. doi: 10.3892/etm.2017.4503. Epub 2017 May 23.
The aim of the present study was to investigate the effects of Worosch. crude drug, processed products and monomer components on human fibroblast-like synoviocyte rheumatoid arthritis (HFLS-RA) cells, and its associated mechanisms. Following drug treatment, cell proliferation was assessed using a Cell Counting Kit-8 assay. Cellular apoptosis and cell cycle were evaluated using flow cytometry. Levels of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF) and toll-like receptor 4 (TLR4) mRNA and protein were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. Levels of pro-inflammatory cytokines were evaluated using ELISA. Analysis of cell proliferation indicated that crude drug and processed products markedly inhibited the cell proliferation. Compared with the control group, the apoptosis rates were significantly elevated in all treatment groups (all P<0.05). Furthermore, the proportion of cells in G0/G1 phase was significantly decreased in all treatment groups compared with the control group (all P<0.05). RT-qPCR and western blotting indicated that, compared with the control group, mRNA and protein expression levels of HIF-1α, and TLR4 were significantly downregulated in all treatment groups (P<0.05). The mRNA and protein expression levels of VEGF in all treatment groups were decreased compared with those in the control group, but the difference was not significant. Results from ELISA demonstrated that the levels of interleukin (IL)-6, IL-1β and tumor necrosis factor-α in the cell culture supernatant were all significantly decreased following drug treatment in HFLS-RA cells (all P<0.05). Therefore, Worosch. crude drug, processed products and monomer components may exert anti-rheumatic effects on HFLS-RA cells, inhibiting cell proliferation and enhancing cellular apoptosis. These effects may be attributable to the downregulated expression of HIF-1α and TLR4, as well as decreased levels of pro-inflammatory cytokines.
本研究旨在探讨Worosch.生药、炮制品及单体成分对人成纤维样滑膜细胞类风湿关节炎（HFLS-RA）细胞的影响及其相关机制。药物处理后，使用细胞计数试剂盒-8法评估细胞增殖。采用流式细胞术评估细胞凋亡和细胞周期。分别使用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹分析评估缺氧诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF）和Toll样受体4（TLR4）的mRNA和蛋白质水平。使用酶联免疫吸附测定（ELISA）评估促炎细胞因子水平。细胞增殖分析表明，生药和炮制品显著抑制细胞增殖。与对照组相比，所有治疗组的凋亡率均显著升高（均P<0.05）。此外，与对照组相比，所有治疗组处于G0/G1期的细胞比例均显著降低（均P<0.05）。RT-qPCR和蛋白质免疫印迹表明，与对照组相比，所有治疗组中HIF-1α和TLR4的mRNA和蛋白质表达水平均显著下调（P<0.05）。所有治疗组中VEGF的mRNA和蛋白质表达水平均低于对照组，但差异不显著。ELISA结果表明，药物处理后HFLS-RA细胞培养上清液中白细胞介素（IL）-6、IL-1β和肿瘤坏死因子-α水平均显著降低（均P<0.05）。因此，Worosch.生药、炮制品及单体成分可能对HFLS-RA细胞发挥抗风湿作用，抑制细胞增殖并增强细胞凋亡。这些作用可能归因于HIF-1α和TLR4表达下调以及促炎细胞因子水平降低。
