黄芪多糖降低脂多糖对鸡巨噬细胞促炎细胞因子和TLRs mRNA转录水平影响的效应分析  

Immunoregulation Effect of Astragalus Polysaccharide on Lipopolysaccharide Injuryed HD11 Cells and the Effect on TLRs mRNA Expression

作　　者：刘倩 李大鹏 张宏 刘琴 王学智[2] 李建喜[2] 杨孝朴[1] 张景艳[2] LIU Qian;LI Dapeng;ZHANG Hong;LIU Qin;WANG Xuezhi;LI Jianxi;YANG Xiaopu;ZHANG Jingyan(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Institute of Husbandry and Pharmaceutical Sciences,Chinese Academy of Agricultural Sciences,Lanzhou 730050,China;College of Chinese Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;The Monitoring Institute of Veterinary Drug and Feed of Gansu Province,Lanzhou 730000,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州畜牧与兽药研究所,兰州730050 [3]河北农业大学中兽医学院,保定071001 [4]甘肃省兽药饲料监察所,兰州730000

出　　处：《畜牧兽医学报》2022年第9期3251-3261,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31602101);兰州市人才创新创业项目(2020-RC-147);国家重点研发计划政府间国际科技创新合作重点专项(2017YFE0114400)。

摘　　要：黄芪多糖(APS)调节天然免疫的作用已被广为认知,但有关APS对鸡巨噬细胞(HD11)调节作用及其分子机制的研究报道鲜少。本试验以脂多糖(LPS)诱导损伤的HD11细胞为研究模型,旨在评价APS对HD11抵御LPS刺激损伤的保护作用及对信号通路中敏感TLRs mRNA转录水平的影响。采用CCK-8法确定LPS诱导细胞炎症损伤模型的最适浓度和APS最适工作浓度;结合显微观察、一氧化氮(NO)检测和乳酸脱氢酶(LDH)检测评价APS对LPS诱导损伤HD11细胞的干预作用;采用实时荧光定量PCR(RT-PCR)方法检测APS对LPS诱导损伤HD11细胞的炎性细胞因子及TLR1、TLR2、TLR4、TLR15 mRNA表达的调节作用。结果表明:当LPS浓度>9.76μg·mL^(-1)时,在2%鸡血清+1%双抗的完全培养液中培养的HD11细胞的增殖活力显著下降,且细胞的炎症损伤变化明显。当APS浓度>50μg·mL^(-1)时,HD11细胞增殖活性持续上升,其添加剂量与细胞增殖活性呈正相关性。100μg·mL^(-1)APS能显著提高HD11细胞中NO、LDH的释放(P<0.05),但极显著(P<0.01)、显著(P<0.05)降低9.76μg·mL^(-1)LPS诱导损伤HD11细胞中NO、LDH的释放;其能显著提高HD11细胞促炎因子(IL-1β、TNF-α及iNOS)mRNA水平(P<0.05),极显著抑制IL-10 mRNA水平(P<0.01),但能极显著抑制LPS损伤HD11细胞中IFN-α、TNF-α、IL-10 mRNA水平的升高(P<0.01),显著降低LPS诱导损伤HD11细胞中IL-6、IL-1β、iNOS mRNA的升高(P<0.05)。100μg·mL^(-1)APS能极显著降低HD11细胞TLR2、TLR4及TLR15 mRNA转录水平(P<0.01),极显著抑制LPS诱导损伤HD11细胞TLR2 mRNA转录水平升高(P<0.01),可极显著(P<0.01)、显著(P<0.05)降低LPS诱导损伤HD11细胞TLR4、TLR15 mRNA转录水平,但对HD11细胞和LPS诱导损伤HD11细胞TLR1 mRNA转录水平均无明显作用。综上可知,APS对HD11细胞的安全浓度范围大,可通过调节TLR2、TLR4及TLR15 mRNA水平,降低LPS诱导损伤HD11细胞过度炎症反应,发挥免疫双向调节作用。Astragalus polysaccharide(APS) has been widely recognized as a key medicine for supplementing qi and its role in regulating natural immunity. However few studies were related with the immunoregulation effect and molecular mechanism of APS on chicken macrophages. The purpose of this study was to evaluate the protective effect of APS on HD11 cell injuryed by LPS and the recognition of sensitive TLRs in the signaling pathway. The optimal concentration of LPS-injuryed HD11 cells and the working concentration of APS were decided by CCK-8 method. The effect of APS on LPS-injuryed HD11 cells was evaluated by microscopic observation, nitric oxide(NO) detection and lactate dehydrogenase(LDH) detection. The regulation of APS on inflammatory cytokines and mRNA expression of TLR1, TLR2, TLR4 and TLR15 in LPS-injuryed HD11 cells was detected by real-time fluorescence quantitative PCR(RT-PCR). The results showed that the proliferation activity of HD11 cells cultured in the complete medium of 2% chicken serum +1% penicillin-streptomycin decreased significantly and the changes of inflammatory damage were obvious when LPS concentration was greater than 9.76 μg·mL^(-1). The proliferation activity of HD11 cells continued to increase when APS concentration was greater than 50 μg·mL^(-1), and there was a positive correlation between APS dosage and proliferation activity. 100 μg·mL^(-1)APS significantly increased the release of NO and LDH in HD11 cells(P<0.05), but extremely significantly(P<0.01) and significantly(P<0.05) decreased the release of NO and LDH in HD11 cells induced by 9.76 μg·mL^(-1)LPS. 100 μg·mL^(-1)APS significantly increased the mRNA levels of pro-inflammatory factors(IL-1β, TNF-α and iNOS)(P<0.05) and extremely significantly inhibited the mRNA levels of IL-10(P <0.01) in HD11 cells, but extremely significantly inhibited the increasing of mRNA levels of IFN-α、TNF-α、IL-10(P<0.01) and significantly inhibited the increasing of mRNA levels of IL-6, IL-1β, iNOS(P<0.05) in LPS-injuryed HD11 cells. 100 μ

关 键 词：黄芪多糖 鸡巨噬细胞 炎症因子 TLRS 免疫调节 

分 类 号：S853.74[农业科学—临床兽医学]