摘要
目的:探究通管方含药血清对LPS诱导的RAW264.7细胞炎症的影响,从而明确通管方治疗输卵管炎性不孕的细胞机制。方法:选用脂多糖(LPS)刺激RAW264.7巨噬细胞建立体外模型,检测细胞氧化应激ROS水平、细胞凋亡水平和PI3K/Akt信号通路蛋白表达。CCK-8检测通管方含药血清对细胞炎症的影响。将细胞分为对照组、LPS炎症细胞模型组和不同浓度的通管方含药血清处理LPS炎症细胞模型组。qPCR检测各组细胞中IL-1β、IL-6和TNF-α的mRNA表达水平。免疫荧光和Western blot检测PI3K/Akt信号通路的磷酸化水平,同时检测细胞凋亡基因Bcl-2、Bax和caspase-3蛋白水平。利用Hoechst 33342/PI染色和流式细胞术检测通管方含药血清对细胞凋亡的影响。利用DCFH-DA探针检测通管方含药血清对细胞ROS含量的影响。结果:①通管方含药血清减轻LPS对细胞的损伤,具有剂量依赖性;②通管方含药血清降低LPS诱导的促炎细胞因子IL-6、IL-1β和TNF-α的产生;③通管方含药血清减少了LPS诱导的细胞内ROS产生;④通管方含药血清预处理激活了PI3K/Akt信号通路;⑤通管方含药血清促进了细胞凋亡。结论:通管方含药血清处理可以减轻细胞损伤和炎症反应,并通过激活PI3K/Akt信号通路和促进细胞凋亡来保护细胞免受LPS的损伤,进一步降低细胞炎症水平。
Objective:To investigate the effect of Tongguan Fang drug-containing serum on LPS-induced inflammation of RAW264.7 cells,so as to clarify the cellular mechanism of the Tongguan Fang in treating tubal inflammatory infertility.Methods:Lipo-polysaccharide(LPS)was used to stimulate the RAW264.7 macrophage cell line as an in vitro model,Detected levels of cellular oxida-tive stress(ROS),apoptosis and expressions of PI3K/Akt signaling pathway proteins.CCK-8 was used to detect effect of Tongguan Fang drug-containing serum on cellular inflammation.Effects of Tongguan Fang-containing serum on cell inflammation were assessed using CCK-8 assay.Cells were divided into control group,LPS-induced inflammation model group,and Tongguan Fang-containing serum-treated LPS-induced inflammation model groups of different concentrations.qPCR was performed to detect mRNA expression levels of IL-1β,IL-6 and TNF-αin different groups.Immunofluorescence and Western blot were used to evaluate phosphorylation levels of the PI3K/Akt signaling pathway and protein levels of Bcl-2,Bax and caspase-3 of apoptotic.Hoechst 33342/PI staining and flow cytometry were employed to assess the impact of Tongguan Fang-containing serum on cell apoptosis.DCFH-DA probe was used to measure cellular ROS levels affected by Tongguan Fang-containing serum.Results:①Tongguan Fang-containing serum reduced LPS-induced cellular injury in a dose-dependent manner;②Tongguan Fang-containing serum decreased production of pro-inflammatory cytokines IL-6,IL-1βand TNF-αinduced by LPS;③Tongguan Fang-containing serum reduced intracellular ROS generation induced by LPS;④Pre-treatment with Tongguan Fang-containing serum activated PI3K/Akt signaling pathway;⑤Tongguan Fang-containing serum promoted cell apoptosis.Conclusion:Treatment with Tongguan Fang-containing serum can reduce cellular injury and inflam-mation,protect cells from LPS-induced cellular injury by activating the PI3K/Akt signaling pathway and promoting cell apoptosis,and further alleviate cellular inflammation levels.
作者
李萍
徐佳
黄紫纯
王淑婷
匡继林
LI Ping;XU Jia;HUANG Zichun;WANG Shuting;KUANG Jilin(Hunan Provincial Hospital of Traditional Chinese Medicine,Changsha 410005,China;Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中国免疫学杂志》
北大核心
2025年第8期1840-1846,1852,共8页
Chinese Journal of Immunology
基金
湖南省中医药科研计划项目(2020011)
湖南省中医药管理局中医药科研计划一般项目(E2022016)子课题(D2022112)。
