Wu Rui, Long Li, Chen Qiqi, Wu Xiaodan, Zhu Jing, Zhou Bin, Cheng Jia
Department of Rheumatology and Immunology, Sichuan Academy of Medical Sciences, Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, P.R. China.
Exp Ther Med. 2017 Sep;14(3):2721-2727. doi: 10.3892/etm.2017.4819. Epub 2017 Jul 19.
The objective of the present study was to investigate the effects of Tim-3 silencing on cell viability and lipopolysaccharide (LPS)-induced inflammatory reactions in fibroblast-like synoviocytes (FLS). T-cell immunoglobulin mucin domain molecule (Tim)-3 expression in FLS obtained from patients with rheumatoid arthritis (RA) and normal controls were detected by western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). Small interfering (si)RNA was transfected using Lipofectamine 2000 to decrease Tim-3 expression. Following transfection, FLS were stimulated by LPS. An MTT assay, RT-PCR and western blot analysis were performed to measure cell viability, Toll-like receptor 4 (TLR4) signaling pathway-related protein expression and inflammatory cytokine release, respectively. The results of the present study indicated that Tim-3 expression was increased in FLS from patients with RA compared with FLS from healthy controls. Transfection of Tim-3 siRNA significantly decreased Tim-3 expression in FLS from patients with RA. Notably, Tim-3 silencing decreased FLS cell viability. Following stimulation with LPS, cell viability and the expression of TLR4, myeloid differentiation protein gene 88 (MyD88) and nuclear factor-κB (NF-κB) p65 were enhanced in FLS. By contrast, Tim-3 silencing attenuated LPS-induced cell proliferation and the expression of TLR4, MyD88 and NF-κB p65. In addition, LPS significantly increased levels of cytokines in the supernatant, including tumor necrosis factor-α, interferon-γ and interleukin-6 (P<0.01). By contrast, Tim-3 silencing significantly decreased LPS-induced cytokine release (P<0.01). However, Tim-3 silencing did not affect TLR4, MyD88 and NF-κB p65 expression and the release of cytokines in cells that did not undergo treatment with LPS. Therefore, the results of the present study indicate that Tim-3 silencing decreases the viability of FLS in RA and attenuates the LPS-induced inflammatory reaction.
本研究的目的是探讨沉默Tim-3对类风湿关节炎（RA）患者成纤维样滑膜细胞（FLS）活力及脂多糖（LPS）诱导的炎症反应的影响。通过蛋白质免疫印迹分析和逆转录-聚合酶链反应（RT-PCR）检测RA患者和正常对照者的FLS中T细胞免疫球蛋白黏蛋白结构域分子（Tim）-3的表达。使用Lipofectamine 2000转染小干扰（si）RNA以降低Tim-3表达。转染后，用LPS刺激FLS。分别进行MTT试验、RT-PCR和蛋白质免疫印迹分析以检测细胞活力、Toll样受体4（TLR4）信号通路相关蛋白表达及炎性细胞因子释放。本研究结果表明，与健康对照者的FLS相比，RA患者的FLS中Tim-3表达增加。转染Tim-3 siRNA可显著降低RA患者FLS中Tim-3的表达。值得注意的是，沉默Tim-3可降低FLS细胞活力。LPS刺激后，FLS的细胞活力以及TLR4、髓样分化蛋白基因88（MyD88）和核因子-κB（NF-κB）p65的表达增强。相比之下，沉默Tim-3可减弱LPS诱导的细胞增殖以及TLR4、MyD88和NF-κB p65的表达。此外，LPS显著增加上清液中细胞因子水平，包括肿瘤坏死因子-α、干扰素-γ和白细胞介素-6（P<0.01）。相比之下，沉默Tim-3可显著降低LPS诱导的细胞因子释放（P<0.01）。然而，沉默Tim-3不影响未用LPS处理的细胞中TLR4、MyD88和NF-κB p65的表达以及细胞因子的释放。因此，本研究结果表明，沉默Tim-3可降低RA患者FLS的活力，并减弱LPS诱导的炎症反应。
