miR-146b过表达对脂多糖刺激仔猪肠上皮细胞Toll样受体4基因表达和促炎因子产生的影响  

Effects of miR-146b Over-Expression on Toll-Like Receptor 4 Gene Expression and Generation of Pro-Inflammatory Cytokines in Intestinal Epithelial Cells of Piglets Stimulated by Lipopolysaccharide

作　　者：陶新[1] 邓波[1] 门小明[1] 刘淑杰[1] 徐子伟[1] TAO Xin;DENG Bo;MEN Xiaoming;LIU Shujie;XU Ziwei(Institute of Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China)

机构地区：[1]浙江省农业科学院畜牧兽医研究所,杭州310021

出　　处：《动物营养学报》2023年第1期623-631,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：浙江省重点研发计划(2021C02007,2021C02068-3);国家现代农业产业技术体系(CARS-35)。

摘　　要：本研究旨在探讨miR-146b过表达对脂多糖(LPS)刺激仔猪肠上皮细胞(IPEC-J2细胞)Toll样受体4(TLR4)基因表达和促炎因子产生的影响。首先利用LPS刺激IPEC-J2细胞,然后使用荧光定量PCR和酶联免疫吸附试验(ELISA)方法分别检测LPS刺激对IPEC-J2细胞中TLR4和促炎因子白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)和干扰素-γ(IFN-γ)等的mRNA表达以及对分泌到细胞培养液中促炎因子含量的影响。在此基础上,通过miR-146b mimic细胞转染试验,探讨miR-146b过表达对LPS刺激的IPEC-J2细胞中TLR4的mRNA表达和促炎因子产生的抑制作用。结果表明:1)与未刺激处理相比,LPS刺激处理的IPEC-J2细胞中TLR4、IL-1β、TNF-α、IL-8和IFN-γ的mRNA相对表达量极显著升高(P<0.01),细胞培养液中TNF-α、IL-8和IFN-γ含量极显著升高(P<0.01)。2)与LPS处理相比,LPS+miR-146b mimic处理的miR-146b的相对表达量极显著升高(P<0.01),IPEC-J2细胞中TLR4、TNF-α、IL-8和IFN-γ的mRNA相对表达量分别显著或极显著降低(P<0.05或P<0.01),细胞培养液中TNF-α、IL-8和IFN-γ含量极显著降低(P<0.01)。由此可见,miR-146b在LPS刺激的IPEC-J2细胞中可通过抑制促炎因子的产生发挥抗炎作用。This study was aimed to explore the effects of mir-146b over-expression on Toll-like receptor 4(TLR4)gene expression and generation of pro-inflammatory cytokines in intestinal epithelial cells of piglets(IPEC-J2 cells)stimulated by lipopolysaccharide(LPS). Firstly,the IPEC-J2 cells were stimulated by LPS,then,the mRNA expressional of TLR4 and pro-inflammatory cytokines of interleukins-1β(IL-1β),tumor necrosis factor-α(TNF-α),interleukins-6(IL-6),interleukins-8(IL-8)and interferon-γ(IFN-γ)in IPEC-J2cells stimulated by LPS and the protein contents of pro-inflammatory cytokines in cultured supernatant were detected by the methods of real-time PCR and enzyme linked immunosorbent assay(ELISA),respectively.Based on the above results,the inhibition of miR-146b over-expression on TLR4 gene expression and generation of pro-inflammatory cytokines in IPEC-J2 cells stimulated by LPS were studied by cell transfection test with miR-146b mimic. The results showed as follows:1)compared with the non-stimulated treatment,the mRNA relative expression levels of TLR4,IL-1β,TNF-α,IL-8 and IFN-γ in IPEC-J2 cells of LPS stimulated treatment were significantly increased(P<0.01),and the protein contents of TNF-α,IL-8 and IFN-γin cultured supernatant were significantly increased(P<0.01). 2)Compared with the LPS treatment,the miR-146b relative expression level of LPS+miR-146b mimic treatment was significantly increased(P<0.01),the mRNA relative expression levels of TLR4,TNF-α,IL-8 and IFN-γ in IPEC-J2 cells of LPS+miR-146b mimic treatment were significantly decreased(P<0.05 or P<0.01),and the protein contents of TNF-α,IL-8 and IFN-γin cultured supernatant were significantly decreased(P<0.01). In conclusion,the miR-146b play an anti-inflammatory role by inhibiting the generation of pro-inflammatory cytokines in IPEC-J2 cells stimulated by LPS.[Chinese Journal of Animal Nutrition,2023,35(1):623-631]

关 键 词：脂多糖 miR-146b 仔猪 肠上皮细胞 促炎因子 

分 类 号：S828[农业科学—畜牧学]