Effects of porcine MyD88 knockdown on the expression of TLR4 pathway-related genes and proinflammatory cytokines

被引：21

作者：

Dai, Chaohui ^{[1
]}

Sun, Li ^{[1
]}

Yu, Lihuai ^{[1
]}

Zhu, Guoqiang ^{[2
]}

Wu, Shenglong ^{[1
]}

Bao, Wenbin ^{[1
]}

机构：

[1] Yangzhou Univ, Coll Anim Sci & Technol, Key Lab Anim Genet Breeding Reprod & Mol Design J, Yangzhou 225009, Jiangsu, Peoples R China

[2] Yangzhou Univ, Coll Vet Med, Yangzhou 225009, Jiangsu, Peoples R China

来源：

BIOSCIENCE REPORTS | 2016年 / 36卷

关键词：

gene silencing; myeloid differentiation protein 88 (MyD88); pig; proinflammatory cytokines; Toll-like receptor (TLR)/Interleukin (IL)-1R pathway; TOLL-LIKE-RECEPTORS; SIGNALING PATHWAY; INNATE IMMUNITY; KAPPA-B; MICE; INTERLEUKIN-1; TRANSDUCTION; INHIBITION; ACTIVATION; IRAK-4;

D O I：

10.1042/BSR20160170

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

As a critical adapter protein in Toll-like receptor (TLR)/Interleukin (IL)-1R signalling pathway, myeloid differentiation protein 88 (MyD88) plays an important role in immune responses and host defence against pathogens. The present study was designed to provide a foundation and an important reagent for the mechanistic study of MyD88 and its role TLR/IL-1R signalling pathways in porcine immunity. Lentivirus-mediated RNAi was used to generate a porcine PK15 cell line with a silenced MyD88 gene and quantitative real-time PCR (qPCR) and Western blotting were used to detect changes in the expression of critical genes in the Toll-like receptor 4 (TLR4) signalling pathway. ELISA was used to measure the levels of seven proinflammatory cytokines - interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta -in cell culture supernatants after MyD88 silencing. We successfully obtained a PK15 cell line with 61% MyD88 mRNA transcript down-regulated. In PK15 cells with MyD88 silencing, the transcript levels of TLR4 and IL-1 beta were significantly reduced, whereas there were no significant changes in the expression levels of cluster of differentiation antigen 14 (CD14), interferon-alpha (IFN-alpha) or TNF-alpha. The ELISA results showed that the levels of most cytokines were not significantly changed apart from IL-8 without stimulation, which was significantly up-regulated. When cells were induced by lipopolysaccharide (LPS) (0.1 mu g/ml) for 6 h, the global level of seven proinflammatory cytokines up-regulated and the level of IL-1 beta, TNF-alpha, IL-6, IL-8 and IL-12 of Blank and negative control (NC) group up-regulated more significantly than RNAi group (P<0.05), which revealed that the MyD88 silencing could reduce the TLR4 signal transduction which inhibited the release of proinflammatory cytokines and finally leaded to immunosuppression.

引用

页数：10

共 30 条

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