Tlr4 Gene Encoding Area Of Non-synonymous Snps To Lps Stimulation Reaction And Mfhas1 Regulation Tlr4 Signaling Pathways

Sepsis is a syndrome that is initiated by infection and is characterized by a systemic inflammatory response. It is a major cause of death in ICU. Toll like receptor4(TLR4) signaling pathway plays a pivotal role in imflamMatory response, thus its defectiveness may strongly impair the host anti-inflammatory immunity. TLR4initiated signaling pathway in cellular innate immune responses by recognizing microbial pathogens through pathogen associated molecular patterns (PAMPs). It is an essential pathophysiological mechanism in the development of sepsis.Based on current researches, we focused on two parts:1. We investigated all non-synonymous SNPs in coding region of TLR4gene on the LPS stimulation using bioinformatics and gene cloning approaches. It would be a molecular biology research to reveal the association with TLR4gene polymorphism and susceptibility to sepsis.2. We discussed the characteristics of a new protein MFHAS1in LPS/TLR4signaling pathway by molecular biology techniques. It would provide a new road for the therapy of sepsis.The main results and conclusions were summarized as follows:1. There are17non-synonymous SNPs of natural mutations in the coding region of human TLR4gene including14SNPs in extracellular and3SNPs in intracellular by bioinformatics approaches. pEGFP-huTLR4plasmid and17SNPs are constructed through PCR and site-directed mutagenesis technique. Dual-Luciferase Reporter assay shows that NF-κB reporter activity of A896G, C1196T, A2081G are lower than WT. There are no significant differences of NF-κB reporter activities in other SNPs. Real-time fluorescence quantitative PCR shows that the relative mRNA expressions of TNF-and IL6in these three SNPs are lower than in WT. It implies that these three SNPs may influence the normal transduction of TLR4signaling pathway.2. Real-time fluorescence quantitative PCR shows that the relative mRNA expression of MFHAS1in TLR4knowout mice is lower than in wild type mice. Knockdown using siRNA directed against MFHAS1in macrophages enhanced the expression of pP65、pP38、pJUN、pERK and the level of TNF-α IL-6and IL-12productions following LPS stimulation. Therefore, MFHAS1may play a negative regulatory role in TLR4signaling pathway.Together, the above results suggest that mutations of A896G, C1196T. A2081G may be three functional SNPs, which could play important roles in susceptibility to sepsis and MFHAS1may negative regulate TLR4signaling pathway.