摘要
目的:探讨黄连乌梅提取物联合使用对脂多糖(Lipopolysaccharide,LPS)诱导小鼠单核巨噬细胞RAW264.7炎症反应的影响及作用机制.方法:采用LPS诱导RAW264.7细胞炎症模型,试验分为空白对照组、模型对照(LPS)组、二甲基亚砜(DM-SO)组、黄连乌梅提取物89、178、343 μg/mL组.采用CCK-8法检测细胞增殖率;流式细胞术检测细胞凋亡率、活性氧(Reactive oxy-gen species,ROS)水平和细胞表面分子CD80、CD83、CD86的水平;ELISA法检测其对细胞分泌炎症因子的影响;Western blot法检测细胞中高迁移率族蛋白B1(HMGB1)、P65、Toll样受体4(TLR4)蛋白的表达.结果:与空白对照组比较,LPS模型组细胞HMGB1、TLR4 蛋白表达明显上调(2.05±0.05、2.56±0.63,P＜0.05 或P＜0.01),细胞表面分子 CD80、CD83、CD86 显著增多(19.81±1.85、3.81±0.40、3.23±0.40,P＜0.01),细胞上清中 TNF-α、IL-6 和 IL-1β 含量显著升高(33.31±3.92、42.66±3.07、25.98±2.45,P＜0.01);与模型对照组比较,黄连乌梅提取物343 μg/mL组能下调HMGB1蛋白表达(1.50±0.08,P＜0.01),降低细胞表面分子CD80、CD83和 CD86 的水平(8.73±2.92、1.04±0.15、1.35±0.15,P＜0.01),明显减少细胞炎症因子 TNF-α、IL-6 和 IL-1β 的分泌(19.86±3.12、29.41±8.98、16.58±0.52,P＜0.05或P＜0.01).结论:黄连乌梅提取物联合使用对LPS所致的RAW264.7细胞炎症模型具有较好的抗炎作用,其作用机制可能与抑制ROS/HMGB1/TLR4炎症信号通路的激活有关.
Abstract
Objective:To investigate the effect and mechanism of combined use of COPTIDIS RHIZOMA and MUME FRUCTUS extracts on the li-popolysaccharide(LPS)-induced inflammatory response in mouse macrophage RAW264.7.Methods:LPS-induced inflammation model was established on RAW264.7 cells.In the experiment,the cells were divided into blank control group,model(LPS)group,dimethyl sulfoxide(DMSO)group and COPTIDIS RHIZOMA and MUME FRUCTUS extracts group.Cell proliferation was assessed using Cell Counting Kit-8(CCK-8).Flow cytometry was employed to measure cell apoptosis,reactive oxygen species(ROS)content,and the expressions of cell sur-face molecules CD80,CD83,and CD86.Enzyme-linked immunosorbent assay(ELISA)was used to determine the effect on inflammatory fac-tor secretion,and Western blot(WB)was used to analyze the protein expression levels of high mobility group protein B1(HMGB1),P65,and Toll-like receptor4(TLR4)in cells.Results:Compared with the blank control group,protein expressions of HMGB1 and TLR4 were up regulated in the model group(2.05±0.05,2.56±0.63,P＜0.05 or P＜0.01).The expression of cell surface molecules CD80,CD83,and CD86 were increased(19.81±1.85,3.81±0.40,and 3.23±0.40,P＜0.01),and contents of TNF-α,IL-6 and IL-1β in cell culture superna-tants were significantly increased(33.31±3.92,42.66±3.07,25.98±2.45,P＜0.01).Compared with the model group,the 343 μg/mL of COPTIDIS RHIZOMA and MUME FRUCTUS extracts down regulated the protein expression of HMGB1(1.50±0.08,P＜0.01),inhibited the expressions of cell surface molecules CD80,CD83,and CD86(8.73±2.92,1.04±0.15,1.35±0.15,P＜0.01),and significantly reduced the secretion of inflammatory factors TNF-α,IL-6 and IL-1β(19.86±3.12,29.41±8.98,16.58±0.52,P＜0.05 or P＜0.01).Conclusion:Combined use of COPTIDIS RHIZOMA and MUME FRUCTUS extracts demonstrates a potent anti-inflammatory effect in the LPS-induced in-flammation model of RAW264.7 cells.Its mechanism may be related to the inhibition of ROS/HMGB1/TLR4 signaling pathway.
基金项目
四川省中央引导地方科技发展专项(2022ZYD0106)
四川省科技计划(2022YFS0432)
国家科技期刊平台
NSTL
万方数据