黄芩苷对多杀性巴氏杆菌感染猪巨噬细胞后炎症因子表达及信号通路影响的研究
Effects of baicalin on expression of inflammatory cytokines and inflammatory signaling pathways in porcine macrophages infected with Pasteurella multocida
刘静 1周红蕾 1戈文 1徐一天 1张一多 1王丽华 1安东1
作者信息
- 1. 江苏农牧科技职业学院,江苏 泰州 225300
- 折叠
摘要
为研究不同浓度黄芩苷对多杀性巴氏杆菌(Pm)HN-13株感染的猪巨噬细胞(HD11)内炎症因子表达的影响及与炎症信号通路的相互作用关系,本研究采用western blot检测HN-13株感染(0、30 min、60 min、90 min)HD11细胞中促分裂原活化蛋白激酶(MAPK)信号通路3个关键蛋白(p-ERK/ERK、p-p38/p38、p-JNK/JNK)的表达水平;采用ELISA和荧光定量RT-PCR(RT-qPCR)分析检测低浓度剂量黄芩苷(L组,20 μmol/L)、中浓度剂量黄芩苷(M组,40 μmol/L)和高浓度剂量黄芩苷(H组,60 μmol/L)对Pm感染的猪巨噬细胞内肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和IL-1β分泌水平及其mRNA转录水平的影响,同时设置对照组(Control)和模型组(HN-13);利用抑制剂SCH772984阻断ERK通路,采用ELISA和RT-qPCR检测黄岑苷对Pm感染的HD11细胞内炎症因子TNF-α、IL-6和IL-1β表达及mRNA转录水平的影响,同时设置对照组(Control)、模型组(HN-13)、抑制组(HN-13+抑制剂).结果显示:与HN-13株处理0 min组相比,随着感染时间的延长,HN-13株感染HD11细胞中p-ERK/ERK的蛋白表达量逐渐升高(P<0.01),而p-p38/p38和p-JNK/JNK蛋白表达量均未发生显著变化(P>0.05),表明,HN-13株可使HD11细胞炎症相关ERK通路蛋白表达增强,且与作用时间呈正相关.与模型组(HN-13)相比,3个浓度剂量的黄芩苷均显著抑制TNF-α、IL-6和IL-1β的分泌水平及mRNA转录水平(P<0.05),且抑制作用随着黄芩苷浓度升高而增强.与抑制组相比,黄芩苷与抑制剂共处理组可以显著抑制HN-13感染的HD11细胞中TNF-α、IL-1β和IL-6的分泌及mRNA转录水平(P<0.05).综上所述,黄芩苷可以抑制Pm感染的HD11细胞中 ERK通路相关蛋白的表达和分泌,进而发挥抗炎作用.本研究为研制防治Pm引起的猪肺疫的新药提供了思路.
Abstract
In this study,we explored the effect of different concentrations of baicalin on the expression of inflammatory factors in porcine macrophages(HD11)infected with Pasteurella multocida(HN-13 strain)and their relationship with baicalin and inflammatory signaling pathway.Western blot was used to detect the expression levels of three key proteins(p-ERK/ERK、p-p38/p38,p-JNK/JNK)in the mitogen-activated protein kinase(MAPK)signaling pathway in the HN-13 infected HD11 cells(0,30min,60min,90min).Enzyme-related immunosorbent assay(ELISA)and real-time quantitative fluorescent PCR(RT-qPCR)were used to analyze the effects of different doses of baicalin:L group(20μmol/L),M group(40μmol/L),H group(60μmol/L)on the secretion and mRNA expression of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in porcine macrophages infected with P.multocida,control group(Control)and model group(HN-13)were set up simultaneously.The ERK pathway inhibitor SCH772984 was used to block this pathway,ELISA and PCR(RT-qPCR)were used to detect the secretion and mRNA expression of TNF-α,IL-1β and IL-6 of baicalin on HD11 cells infected with HN-13.Four groups were set up:control group(control group),model group(HN-13),inhibition group(HN-13 + ERK pathway inhibitor SCH772984),baicalin and inhibition group(HN-13+60μmol/L baicalin+ERK pathway inhibitor SCH772984).The results showed that the expression of p-ERK/ERK protein in HN-13 infected HD11 cells increased gradually with the extension of infection time(P<0.01),compared with the 0 min group(HN-13).There was no significant change in the expression of p-p38/p38 and p-JNK/JNK proteins(P>0.05).Different doses of baicalin significantly inhibited the secretion and mRNA expression levels of TNF-α,IL-1β,IL-6(P<0.05),compared with model group(HN-13),and the inhibitory effect in a concentration-dependent manner.Compared with inhibition group,baicalin and inhibition group significantly inhibited the secretion and mRNA expression levels of TNF-α,IL-1β,IL-6(P<0.05).In conclusion,baicalin can inhibit the expression and secretion of ERK pathway related proteins in porcine macrophages(HD11)infected with P.multocida(HN-13 strain),thereby exerting anti-inflammatory effects.This study provides ideas for the research of new drugs for the prevention and treatment of swine pulmonary disease caused by P.multocida.
关键词
黄芩苷/多杀性巴氏杆菌/猪巨噬细胞/炎症表达/MAPK信号通路Key words
Baicalin/Pasteurella multocida/porcine macrophage/inflammatory expression/MAPK signal path way引用本文复制引用
基金项目
江苏农牧科技职业学院学校科研立项项目(自然科学类)(NSF201903)
江苏省高等学校大学生创新创业训练计划(202012806091Y)
泰州市"311高层次人才培养工程"项目(第五期)()
出版年
2024
NETL
NSTL
维普
万方数据