AIM： To investigate the anti-inflammatory effects of asiatic acid（AA） on lipopolysaccharide（LPS）-induced inflammatory response in human corneal epithelial cells（HCECs）.METHODS： Cell viability was measured usin...AIM： To investigate the anti-inflammatory effects of asiatic acid（AA） on lipopolysaccharide（LPS）-induced inflammatory response in human corneal epithelial cells（HCECs）.METHODS： Cell viability was measured using a cell counting kit-8（CCK-8） assay.Quantitative real-time polymerase chain reaction（qR T-PCR） was used to determine the mR NA expression of interleukin-8（IL-8）,interleukin-6（IL-6）,interleukin-1β（IL-1β）,tumor necrosis factor-alpha（TNF-α）,and transforming growth factor-β（TGF-β） in HCECs.Intracellular reactive oxygen species（ROS） was measured using the ROS assay kit.Glutathione（GSH） concentration was measured using the total GSH assay kit.Akt1 and Akt phosphorylation（p-Akt1） levels were measured by Western blotting and immunofluorescence.RESULTS： AA induced toxicity at high concentrations and significantly stimulated the proliferation of HCECs at concentrations of 20 μmol/L for 1h.LPS at concentrations of 300 ng/mL for 1h significantly stimulated the mR NA expression of IL-8,IL-6,IL-1β,TNF-α,and TGF-β in HCECs,while the stimulation effects were significantly inhibited by AA（20 μmol/L）.In addition,AA was found to decrease the content of ROS,increase GSH generation,and also inhibit LPS-induced p-Akt in HCECs.CONCLUSION： AA decreases the generation of inflammatory factors IL-8,IL-6,IL-1β,TNF-α,and TGF-β in LPSstimulated HCECs.AA significantly inhibites the intracellular concentrations of ROS and increases GSH generation.AA also inhibites LPS-induced p-Akt in HCECs.These findings reveal that AA has anti-inflammation effects in LPS-stimulated HCECs.展开更多
本研究旨在探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对脂多糖(lipopolysaccharide,LPS)诱导人视网膜内皮细胞(human retinal endothelial cells,HRECs)炎症反应通路中调节活化正常T细胞表达与分泌趋化因子(regulat...本研究旨在探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对脂多糖(lipopolysaccharide,LPS)诱导人视网膜内皮细胞(human retinal endothelial cells,HRECs)炎症反应通路中调节活化正常T细胞表达与分泌趋化因子(regulated upon activation normal T cell expressed and secreted,RANTES)表达的影响及可能机制。将HRECs作为研究对象,分别用实时细胞计数法和非同位素细胞增殖法检测LPS(50～250 ng/mL)和EGCG(0～200μmol/L)对HRECs的毒性作用,确定合适的实验药物浓度。再将细胞随机分为正常对照组、LPS组和LPS+不同浓度EGCG(100、50、25、12.5、6.25μmol/L)共7组,用不同浓度EGCG预处理2 h,再加入LPS刺激24 h后,酶联免疫吸附法测定各组培养上清液中RANTES的表达水平,Western免疫印迹法检测蛋白激酶Akt及其磷酸化水平。结果显示,LPS可显著刺激诱导HRECs产生RANTES,EGCG抑制LPS诱导的RANTES表达,作用呈剂量依赖性。免疫印迹结果也显示,LPS对HRECs炎症过程中的Akt通路起重要作用,EGCG可显著抑制LPS诱导HRECs中Akt信号分子的蛋白磷酸化水平。以上结果提示,EGCG能有效抑制LPS诱导HRECs中RANTES的表达,其机制可能与抑制Akt信号通路有关。展开更多
基金Supported by Medical Program of Shandong Province(No.2014WS0441)Science and Technology Program of Shandong Province(No.2013YD21009)Innovative Team and Young Teachers Training Project of Qingdao Medical College(No.600201304)
文摘AIM： To investigate the anti-inflammatory effects of asiatic acid（AA） on lipopolysaccharide（LPS）-induced inflammatory response in human corneal epithelial cells（HCECs）.METHODS： Cell viability was measured using a cell counting kit-8（CCK-8） assay.Quantitative real-time polymerase chain reaction（qR T-PCR） was used to determine the mR NA expression of interleukin-8（IL-8）,interleukin-6（IL-6）,interleukin-1β（IL-1β）,tumor necrosis factor-alpha（TNF-α）,and transforming growth factor-β（TGF-β） in HCECs.Intracellular reactive oxygen species（ROS） was measured using the ROS assay kit.Glutathione（GSH） concentration was measured using the total GSH assay kit.Akt1 and Akt phosphorylation（p-Akt1） levels were measured by Western blotting and immunofluorescence.RESULTS： AA induced toxicity at high concentrations and significantly stimulated the proliferation of HCECs at concentrations of 20 μmol/L for 1h.LPS at concentrations of 300 ng/mL for 1h significantly stimulated the mR NA expression of IL-8,IL-6,IL-1β,TNF-α,and TGF-β in HCECs,while the stimulation effects were significantly inhibited by AA（20 μmol/L）.In addition,AA was found to decrease the content of ROS,increase GSH generation,and also inhibit LPS-induced p-Akt in HCECs.CONCLUSION： AA decreases the generation of inflammatory factors IL-8,IL-6,IL-1β,TNF-α,and TGF-β in LPSstimulated HCECs.AA significantly inhibites the intracellular concentrations of ROS and increases GSH generation.AA also inhibites LPS-induced p-Akt in HCECs.These findings reveal that AA has anti-inflammation effects in LPS-stimulated HCECs.
基金supported by the Public Technology Application Research Grant of Zhejiang ProvinceChina(No.2011C33029)+1 种基金the Natural Science Foundation of Zhejiang ProvinceChina(No.LY13B020002)
文摘本研究旨在探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对脂多糖(lipopolysaccharide,LPS)诱导人视网膜内皮细胞(human retinal endothelial cells,HRECs)炎症反应通路中调节活化正常T细胞表达与分泌趋化因子(regulated upon activation normal T cell expressed and secreted,RANTES)表达的影响及可能机制。将HRECs作为研究对象,分别用实时细胞计数法和非同位素细胞增殖法检测LPS(50～250 ng/mL)和EGCG(0～200μmol/L)对HRECs的毒性作用,确定合适的实验药物浓度。再将细胞随机分为正常对照组、LPS组和LPS+不同浓度EGCG(100、50、25、12.5、6.25μmol/L)共7组,用不同浓度EGCG预处理2 h,再加入LPS刺激24 h后,酶联免疫吸附法测定各组培养上清液中RANTES的表达水平,Western免疫印迹法检测蛋白激酶Akt及其磷酸化水平。结果显示,LPS可显著刺激诱导HRECs产生RANTES,EGCG抑制LPS诱导的RANTES表达,作用呈剂量依赖性。免疫印迹结果也显示,LPS对HRECs炎症过程中的Akt通路起重要作用,EGCG可显著抑制LPS诱导HRECs中Akt信号分子的蛋白磷酸化水平。以上结果提示,EGCG能有效抑制LPS诱导HRECs中RANTES的表达,其机制可能与抑制Akt信号通路有关。
