连翘提取物通过调节巨噬细胞凋亡和极化抑制LPS诱导的炎症作用

Forsythia suspensa extract inhibits LPS-induced inflammation by regulating macrophage apoptosis and polarization

目的 研究连翘提取物(FSE)对脂多糖(LPS)诱导炎症的抑制作用,以及LPS诱导的RAW264.7细胞极化模型建立。方法 MTT法检测FSE和LPS对RAW264.7细胞的毒性作用;倒置显微镜检测不同浓度的FSE对LPS刺激后的细胞形态变化,酶标仪检测细胞上清液的一氧化氮(NO)生成量;荧光显微镜检测细胞内活性氧(ROS)和细胞凋亡的荧光强度;Real-time PCR法检测巨噬细胞中iNOS、TNF-α、IL-1β、IL-6、IL-10、Arginase-1和CD206 mRNA的表达水平。结果 MTT结果发现FSE在50μg/mL浓度时无细胞毒性,LPS作用36 h时,不同浓度FSE对LPS刺激的细胞形态变化和NO生成有抑制作用,同时FSE可以抑制LPS刺激后活性氧(ROS)的减少和细胞的凋亡,Real-time PCR结果显示,用不同浓度(100、250、500、750 ng/mL)LPS处理细胞24 h,或者用500 ng/mL浓度的LPS处理细胞不同时间后,RAW264.7细胞M1型标记基因(TNF-α、IL-1β、iNOS、IL-6)mRNA的表达水平明显升高,RAW264.7细胞M2型标记基因(IL-10、Arginase-1、CD206)mRNA的表达水平明显降低;FSE可降低LPS诱导的M1型标记基因mRNA的表达水平,升高M2型标记基因mRNA的表达水平。结论 FSE可通过抑制RAW264.7细胞凋亡和促进RAW264.7细胞的极化来抑制LPS诱导的炎症反应。

Objective We investigated the inhibitory effect of Forsythia suspensa extract(FSE) on lipopolysaccharide(LPS)-induced inflammation and establishment of an LPS-induced polarization model of RAW264.7 cells. Methods MTT assays were used to assess cytotoxic effects of FSE and LPS on RAW264.7 cells. Inverted microscopy was used to assess morphological changes of cells after stimulation with LPS and various concentrations of FSE. An enzyme marker was used to detect nitric oxide(NO) in culture supernatants. Fluorescence microscopy was used to detect intracellular reactive oxygen species(ROS) and apoptosis.;Real-time PCR was used to detect iNOS expression in macrophages. iNOS, TNF-α, IL-1β, IL-6, IL-10, Arginase-1 and CD206 mRNA expression was measured by Real-time PCR. Results FSE was not cytotoxic at 50 μg/mL. At 36 h of LPS stimulation, various concentrations of FSE inhibited LPS-induced morphological changes, NO production and FSE inhibited LPS-induced the reduction of reactive oxygen species(ROS) and apoptosis. Expression of M1-type marker genes(TNF-α, IL-1β, iNOS and IL-6) in RAW264.7 cells was significantly increased after treatment with various concentrations(100, 250, 500 and 750 ng/mL) of LPS for 24 h or with 500 ng/mL LPS for various times. The mRNA levels of M2 marker genes(IL-10, Arginase-1 and CD206) were significantly decreased in RAW264.7 cells. FSE decreased the expression of LPS-induced M1 marker genes and increased expression of M2 marker genes. Conclusions FSE inhibits LPS-induced inflammatory responses by inhibiting RAW264.7 cell apoptosis and promoting polarization.