DCA调控NF-κB/NLRP3信号通路在急性乳腺炎发生过程中的作用机制研究  

The Mechanism of DCA Regulation of NF-κB/NLRP3 Signaling Pathway in the Occurrence of Acute Mastitis

作　　者：赵婷 赵楠 ZHAO Ting;ZHAO Nan(Obstetrics Department,Xi’an Cunji Maternity Hospital,Xi’an 710000,China)

机构地区：[1]西安存济妇产医院产科,西安710000

出　　处：《中国细胞生物学学报》2024年第12期2064-2072,共9页Chinese Journal of Cell Biology

摘　　要：该研究旨在探究脱氧胆酸(deoxycholic acid,DCA)介导NF-κB/NLRP3信号通路在急性乳腺炎发生过程中的调控机制,为急性乳腺炎的预防和治疗提供理论依据。通过脂多糖(lipopolysaccharide,LPS)刺激人乳腺上皮细胞株DU4475建立炎症模型,实验分为对照组、模型组、DCA低浓度组(DCA-L,50μmol/L)、DCA中浓度组(DCA-M,100μmol/L)和DCA高浓度组(DCA-H,200μmol/L)。通过细胞计数试剂盒-8(cell counting kit-8,CCΚ-8)筛选LPS刺激DU4475细胞产生炎症反应的最佳浓度和DCA符合预期的浓度,并采用CCK-8法检测各组细胞活力;荧光染色法检测各组细胞内活性氧(reactive oxygen species,ROS)的产生水平;Western blot检测NF-κB/NLRP3通路相关蛋白及焦亡通路相关蛋白的表达情况;酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测各组细胞肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-1β(interleukin-1β,IL-1β)、IL6(interleukin-6,IL-6)和IL-18(interleukin-18,IL-18)炎症因子分泌水平。研究发现,相较于对照组,模型组细胞活力明显下降,TNF-α、IL-1β、IL-6和IL-18炎症因子水平明显提高,说明DU4575细胞在50μg/mL LPS处理下炎症模型构建成功。各DCA处理组细胞活力较模型组有明显上升趋势,ROS产生量明显下降,IκB-α、NF-κB p65蛋白磷酸化水平下降,NLRP3、GSDMD、Caspase-1、NLRC4等焦亡通路蛋白的表达量也有明显下降趋势。另外各组细胞TNF-α、IL-1β、IL-6和IL-18等炎症因子水平也显著下降。其中DCA-M组和DCA-H组改善细胞活力、抗氧化应激以及降低炎症水平效果最为明显。DCA可通过抑制NF-κB信号通路的活化,降低下游ROS产生水平,以及TNF-α、IL1β、IL-6和IL-18等炎症因子的表达量,起到抗炎作用。此外,NF-κB通过抑制NLRP3炎症小体的活性,降低炎症因子的表达水平。因此,DCA对急性乳腺炎具有潜在的治疗作用。The aim of this study was to investigate the regulatory mechanism of DCA-mediated NF-κB/NLRP3 signaling pathway in the development of acute mastitis,and to provide theoretical basis for the prevention and treatment of acute mastitis.Inflammation model was established by LPS stimulation of human mammary epithelial cell line DU4475,and the experiment was divided into a control group,a model group,a DCA low-concentration group(DCA-L,50μmol/L),a DCA-medium-concentration group(DCA-M,100μmol/L),and a DCA-high-concentration group(DCA-H,200μmol/L).The optimal concentration of LPS to stimulate DU4475 cells to produce inflammatory response and the concentration of DCA that met the expectation were screened by CCΚ-8(cell counting kit8),and the viability of each group was determined by CCK-8 assay;the level of intracellular ROS(reactive oxygen species)production in cells was detected by fluorescence staining in each group;and Western blot was performed to detect the NF-κB/NLRP3 pathway-related proteins and apoptosis pathway-related proteins;ELISA(enzyme-linked immunosorbent assay)was used to detect TNF-α(tumor necrosis factor-α),IL-1β(interleukin-1β),IL-6(interleukin-6),and IL-18(interleukin-18)levels in the cells of each group.It was found that compared with the control group,the cell viability of the model group was significantly decreased,and the levels of TNF-α,IL-1β,IL-6 and IL-18 were significantly increased,indicating that the inflammation model of DU4575 cells was successfully constructed under the treatment of 50μg/mL LPS.The cell viability of each DCA-treated group showed a significant upward trend compared with that of the model group,with a decrease in ROS production and a decrease in the phosphorylation levels of IκB-αand NF-κB p65 proteins,and a significant downward trend in the expression of pyroptosis pathway proteins,such as NLRP3,GSDMD,Caspase-1,and NLRC4,was also observed.In addition,the levels of inflammatory factors such as TNF-α,IL-1β,IL-6,and IL-18 in each group also significantly dec

关 键 词：脱氧胆酸 NF-κB/NLRP3信号通路 TLRP3炎症小体 炎症反应 急性乳腺炎 

分 类 号：R655.8[医药卫生—外科学]