Zhang Nian, Jiang Yi, Mu Fangxiang, Wu Hong, You Qingxia
Department of Traditional Chinese Medicine, Xinqiao Hospital, Chongqing 400037, China.
Department of Integrated TCM & Western Medicine Department, Southwest Hospital, Chongqing 400038, China.
Cell Mol Biol (Noisy-le-grand). 2019 Aug 5;65(6):85-90.
To investigate the effect of gentiopicrin on the expressions of inflammatory factors in human fibroblast-like synoviocytes (HFLS) and the underlying mechanism. Human fibroblast-like synoviocytes (HFLS) were cultured in vitro at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal bovine serum (FBS) in a humidified incubator containing 5 % CO2. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-tetrazolium bromide (MTT) assay, while real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expressions of interleukin 1β (IL-1β) and interleukin 6 (IL-6) mRNAs. The expressions of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) were determined using Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-1β and IL-6 in cell lysate. Treatment with 5-25 μM gentiopicrin did not significantly affect the number of viable cells, when compared with control group (p > 0.05). However, at 50 and 100 μM gentiopicrin, the number of viable cells were significantly increased, relative to control group (p < 0.05). Results of qRT-PCR showed that the expression levels of IL-1β and IL-6 mRNAs were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently decreased their expression levels compared with TNF-α group (p < 0.05). Western blotting results showed that the expressions of p-p38MAPK and NF-κB-p65 proteins were significantly upregulated in TNF-α group, when compared with control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently down-regulated the expression of these proteins compared with TNF-α group (p < 0.05). The levels of IL-1β and IL-6 in cell lysate were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin, and p38MAPK/NF-κB pathway inhibitors (SB203580 and BAY11-7082) significantly reduced the levels of these inflammatory factors compared with TNF-α group (p < 0.05). Gentiopicrin has therapeutic potential for Rheumatoid arthritis (RA ) through a mechanism involving the inhibition of p38MAPK/NF-κB pathway.
探讨龙胆苦苷对人成纤维样滑膜细胞（HFLS）中炎症因子表达的影响及其潜在机制。将人成纤维样滑膜细胞（HFLS）在37℃下于补充有5%胎牛血清（FBS）的杜氏改良 Eagle 培养基（DMEM）中体外培养，置于含5%CO₂的湿润培养箱中。使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法测定细胞活力，同时采用实时定量聚合酶链反应（qRT-PCR）测定白细胞介素1β（IL-1β）和白细胞介素6（IL-6）mRNA的表达。使用蛋白质印迹法测定p38丝裂原活化蛋白激酶（p38 MAPK）和活化B细胞核因子κB轻链增强子（NF-κB）的表达。采用酶联免疫吸附测定（ELISA）法测定细胞裂解液中IL-1β和IL-6的水平。与对照组相比，用5-25μM龙胆苦苷处理对活细胞数量无显著影响（p＞0.05）。然而，在50和100μM龙胆苦苷处理时，相对于对照组，活细胞数量显著增加（p＜0.05）。qRT-PCR结果显示，TNF-α组中IL-1β和IL-6 mRNA的表达水平显著高于对照组（p＜0.05）。然而，与TNF-α组相比，龙胆苦苷处理显著且剂量依赖性地降低了它们的表达水平（p＜0.05）。蛋白质印迹结果显示，与对照组相比，TNF-α组中p-p38MAPK和NF-κB-p65蛋白的表达显著上调（p＜0.05）。然而，与TNF-α组相比，龙胆苦苷处理显著且剂量依赖性地下调了这些蛋白的表达（p＜0.05）。TNF-α组细胞裂解液中IL-1β和IL-6的水平显著高于对照组（p＜0.05）。然而，与TNF-α组相比，龙胆苦苷以及p38MAPK/NF-κB通路抑制剂（SB203580和BAY11-7082）显著降低了这些炎症因子的水平（p＜0.05）。龙胆苦苷通过抑制p38MAPK/NF-κB通路对类风湿关节炎（RA）具有治疗潜力。
