摘要
目的探讨头花蓼(PC)对幽门螺杆菌(H.pylori)刺激骨髓来源巨噬细胞(BMDMs)炎症反应的作用及机制。方法室温复苏H.pylori SS2000菌株,培养48 h并鉴定,将H.pylori与BMDMs共孵育,按照不同感染复数(MOI,细菌数∶细胞数)分为空白、25∶1、50∶1、100∶1、200∶1及400∶1组,并培养48 h,采用四甲基偶氮唑盐比色法(MTT)检测各组BMDMs细胞活力;并于3、6、12及24 h收集细胞及上清,采用酶联免疫吸附法(ELISA)检测不同MOI及刺激时间下白细胞介素-1β(IL-1β)IL-18的含量;取BMDMs分为空白组[不加H.pylori,加改良依格尔(DMEM)培养液]、模型组(加H.pylori,DMEM培养液)及PC治疗组(加H.pylori,DMEM培养液,0.08μg/L PC),采用Western blot检测各组BMDMs细胞质与细胞核中核因子κB单体p65(NF-κB/p65)的表达及细胞裂解液中NOD样受体蛋白3(NLRP3)、IL-1β前体(Pro-IL-1β)及细胞上清液中IL-1β蛋白的表达。结果MOI高于100∶1时,细胞抑制率增高,BMDMs的数量减少(P<0.05);模型组BMDMs多数变圆,表面不光滑,颗粒物质明显增多;MOI为100∶1且作用12 h时,BMDMs中IL-1β及IL-18含量达到最高;模型组BMDMs细胞质内NF-κB/p65蛋白表达较空白组减少、细胞核内NF-κB/p65蛋白表达较空白组升高(P<0.05),PC治疗组BMDMs胞内NF-κB/p65蛋白表达较模型组升高、核内NF-κB/p65蛋白表达较模型组减少(P<0.05);模型组BMDMs中NLRP3、IL-1β蛋白表达较空白组升高(P<0.05),PC治疗组BMDMs中NLRP3、IL-1β蛋白表达较模型组减少(P<0.05);各组BMDMs中Pro-IL-1β蛋白表达比较,差异均无统计学意义(P>0.05)。结论PC可改善H.pylori引起的BMDMs细胞炎症反应,其机制可能与抑制NF-κB/NLRP3/IL-1β信号通路的激活有关。
Objective To investigate the effect of Polygonum capitatum(PC)on the inflammatory response in Helicobacter pylori(H.pylori)-stimulated bone marrow-derived macrophage(BMDMs)and its mechanism.Methods H.pylori SS2000 strain was resuscitated at room temperature,cultured for 48 h and identified.According to different multiplicity of infection(MOI,number of bacteria:number of cells),BMDMs were and divided into the following groups:blank,25∶1,50∶1,100∶1,200∶1,400∶1.BMDMs were cocutured with H.pylori for 48 h.The cell viability of BMDMs in each group was determined by tetramethylazole salt colorimetric assay(MTT).The supernatants derived from BMDMs coculture with H.pylori were collected at 3,6,12,and 24 h during co-culture.The levels of interleukin-1β(IL-1β)and IL-18 were measured by enzyme-linked immunosorbent assay(ELISA)at different MOIs and stimulation time points.BMDMs were divided into blank group(without H.pylori,with Modified Eagle Medium(DMEM)),model group(with H.pylori,DMEM)and PC treatment group(with H.pylori,DMEM,0.08μg/L PC).Western blot was performed to detect the p65 expression of nuclear factor kappa B(NF-κB)in the cytoplasm and nuclei,NOD-like receptor protein 3(NLRP3),and IL-βprecursor(Pro-IL-1β)in total cell lysates,and the expression of IL-1βprotein in the supernatants.Results When MOI was higher than 100∶1,the cell inhibition rates were increased and the number of BMDMs were decreased(P<0.05).Most of BMDMs in model group were round,and their surface was not smooth,and the granular material were significantly increased in BMDMs.A t an MOI of 100∶1 and 12 h of co-culture,the levels of IL-1βand IL-18 in BMDMs reached the highest.NF-κB/p65 expression level was decreased in cytoplasm but increased in nuclei in model group relative to control group(P<0.05).NF-κB/p65 expression level in cytoplasm was decreased but increased in nuclei in PC group relative to model group(P<0.05).The expression levels of NLRP3 and IL-1βin model group were increased relative to blank group(P<0.05),and they were reduced in PC group relative to model group(P<0.05).There were no statistically significant differences in Pro-IL-1βprotein expression in all BMDM groups(P>0.05).Conclu-sion PC ameliorated inflammatory response of BMDMs to H.pylori by a mechanism that may be related to the inhibition of activation of the NF-κB/NLRP3/IL-1βsignaling pathway.
作者
袁蕴馨
简单
张姝
何芸
孙朝琴
黄健
莫非
YUAN Yunxin;JIAN Dan;ZHANG Shu;HE Yun;SUN Chaoqin;HUANG Jian;MO Fei(School of Clinical Laboratory Science,Guizhou Medical University,Guiyang 550004,Guizhou,China;Clinical Laboratory Center,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2022年第1期13-19,共7页
Journal of Guizhou Medical University
基金
国家自然科学基金(81860723,81960589)
贵州省卫生计生委科学技术基金(gzwjkj2018-1-07)。
关键词
螺杆菌
幽门
核因子ΚB受体活化因子
白细胞介素-1Β
头花蓼
骨髓来源巨噬细胞
NOD样受体蛋白3
核因子-κB单体p65
Helicobacter pylori(H.pylori)
receptor activator of nuclear factor-kappa(NF-κB)
interleukin-1beta(IL-1β)
polygonum capitatum
bone marrow-derived macrophages(BMDMs)
NOD-like receptor protein3(NLRP3)
nuclear factor kappa-B/p65(NF-κB/p65)
