桑叶多糖对猪肺泡巨噬细胞的免疫调节作用  

Immunomodulatory Effects of Mulberry Leaf Polysaccharide on Porcine Alveolar Macrophages

作　　者：韩辰淼 王靖萱 杨海峰[1] 陈晓兰[1] 卜仕金[2,3] HAN Chenmiao;WANG Jingxuan;YANG Haifeng;CHEN Xiaolan;BU Shijin(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]江苏农牧科技职业学院,泰州225300 [2]扬州大学兽医学院,扬州225009 [3]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国畜牧兽医》2024年第8期3643-3651,共9页China Animal Husbandry & Veterinary Medicine

基　　金：泰州市科技支撑(农业)项目(TN202218);江苏高校优势学科建设工程资助项目;江苏省职业院校学生创新创业培育计划项目(G-2023-0726);江苏农牧科技职业学院农牧产业核心技术创新项目(NSF2022ZR05)。

摘　　要：[目的]探究桑叶多糖(mulberry leaf polysaccharide,MLP)对猪肺泡巨噬细胞免疫功能的影响及其作用机制。[方法]以猪肺泡巨噬细胞为研究对象,通过CCK-8方法检测不同浓度MLP作用后猪肺泡巨噬细胞的增殖率,筛选MLP的适宜作用浓度。将细胞分成空白对照组(BC组)、阳性对照组(LPS组)和不同浓度MLP组,通过Griess法检测猪肺泡巨噬细胞中一氧化氮(NO)释放量;利用中性红吞噬试验检测猪肺泡巨噬细胞吞噬能力;使用ELISA法测定猪肺泡巨噬细胞中白细胞介素-10(IL-10)、IL-6和肿瘤坏死因子-α(TNF-α)含量;利用实时荧光定量PCR检测TNF-α、Toll样受体4(TLR4)、IL-6、IL-10以及核转录因子κB(NF-κB)的mRNA表达量;通过Western blotting检测猪肺泡巨噬细胞中TLR4和p65蛋白表达量。[结果]与0μg/mL MLP组相比,MLP浓度为20、40和80μg/mL时猪肺泡巨噬细胞增殖率极显著升高(P<0.01),且呈剂量依赖性,后续选择20、40和80μg/mL MLP进行试验。与BC组相比,不同浓度MLP组猪肺泡巨噬细胞吞噬率和NO释放量极显著升高(P<0.01)。ELISA检测结果显示,MLP浓度为80μg/mL时,猪肺泡巨噬细胞中IL-6和TNF-α含量显著或极显著高于BC组(P<0.05;P<0.01)。实时荧光定量PCR结果显示,MLP浓度为40和80μg/mL时,猪肺泡巨噬细胞中TNF-α、IL-6和TLR 4基因mRNA表达量均极显著高于BC组(P<0.01)。Western blotting检测结果显示,MLP浓度为80μg/mL时,猪肺泡巨噬细胞中p65和TLR4蛋白表达量极显著高于BC组(P<0.01)。[结论]MLP能激活猪肺泡巨噬细胞,显著促进NO的释放以及IL-6、TNF-α的分泌,具有良好的免疫调节活性,其免疫调节机制与巨噬细胞表面TLR4的激活相关。[Objective]The purpose of this study was to investigate the effects of mulberry leaf polysaccharide(MLP)on the immune function of porcine alveolar macrophages and its mechanism.[Method]The experiment took porcine alveolar macrophages as the study object,CCK-8 method was used to detect the proliferation rate of porcine alveolar macrophages treated with different concentrations of MLP,and screen the appropriate concentration of MLP.The cells were divided into blank control group(BC group),positive control group(LPS group)and MLP groups with different concentrations.The release of nitric oxide(NO)in porcine alveolar macrophages was detected by Griess method.The phagocytosis ability of porcine alveolar macrophages was detected by neutral red phagocytosis assay.The contents of interleukin-10(IL-10),IL-6 and tumor necrosis factor-α(TNF-α)in porcine alveolar macrophages were determined by ELISA.The mRNA expression levels of TNF-α,Toll-like receptor 4(TLR4),IL-6,IL-10 and nuclear transcription factorκB(NF-κB)were detected by Real-time quantitative PCR.The expression levels of TLR4 and p65 proteins were detected by Western blotting.[Result]Compared with 0μg/mL MLP group,the proliferation rate of porcine alveolar macrophages in 20,40 and 80μg/mL MLP groups was extremely significantly increased(P<0.01),and showed a dose-dependent effect.Therefore,20,40 and 80μg/mL MLP were selected for follow-up tests.Compared with BC group,the phagocytosis rate and NO release of porcine alveolar macrophages in different concentration of MLP groups were extremely significantly increased(P<0.01).ELISA results showed that the contents of IL-6 and TNF-αin porcine alveolar macrophages were significantly or extremely significantly higher than those in BC group when MLP concentration was 80μg/mL(P<0.05 or P<0.01).Real-time quantitative PCR results showed that the mRNA expressions of TNF-α,IL-6 and TLR 4 genes in porcine alveolar macrophages at MLP concentrations of 40 and 80μg/mL were extremely significantly higher than those in BC gr

关 键 词：桑叶多糖 猪肺泡巨噬细胞 免疫调节 细胞因子 

分 类 号：S852.2[农业科学—基础兽医学]