Impact Of TLR4-siRNA Transfection On The Expression Of TLR4/Myd88Signaling In Rat Tubular Epithelial Cells Under High Glucose And Angâ…¡ Condition

Objective:To observe the expresstion of toll like receptor(TLR4) and myeloiddifferentiation88(MyD88) Signaling and the release of inflammation factors in rattublar epithelial cell(NRK-52E) under high glucose and AngⅡ condition afterTLR4-siRNA transfection.Methods: Divided cells into three groups after cultivated with normal-glucosemedium:1. normal-glucose group2. high-glucose group3. AngⅡ group;extract totalRNA and total protein after12hours. Real time PCR was used to analyze TLR4andMyd88mRNA expression; western blot was used to observe the expression ofTLR4、Myd88and NF-kB protein expression,analyse the influence to NRK-52E ofhigh-glucose and AngⅡ.Three TLR4-siRNA sequences (siRNA-1, siRNA-2,siRNA-3) were designed and synthesized, BLOCK-IT Alexar Fluor with redfluorescence as negative control.The transfection efficiency was observed byfluorescence microscope after transfection, divided cells into six groups:1. normal-glucose group2. high-glucose+AngⅡ group3. high-glucose+AngⅡ+negativecontrol group4. high-glucose+AngⅡ+siRNA1group5. high-glucose+AngⅡ+siRNA2group6. high-glucose+AngⅡ+siRNA3group,and the expression ofTLR4mRNA change was detectd by real time PCR. The most effctive siRNA wasselected to be used for forward expriments. Divided cells into six groups again：1.normal-glucose group2. high-glucose+AngⅡ group3. high-glucose+AngⅡ+negative control group4. high-glucose+AngⅡ+siRNA group5. high-glucose+AngⅡ+irbesartan group6. high-glucose+AngⅡ+siRNA+irbesartan group.Aftertransfection for24hours, cells were stimulated with25mmol/l glucose and10-7mmol/l Angiotension(AngⅡ) for12hours,cells without stimulation were as nomalcontrol. Real time PCR was used to analyze TLR4and Myd88mRNA expression;western blot was used to observe the expression of TLR4、Myd88and NF-kB proteinexpression,collect supernate of the cells and ELISA was used to analyze theexpression of inflammation factors called IL-6and MCP-1.Results: TLR4/Myd88mRNA and TLR4/Myd88/NF-kB protein were highly expressed under high glucose or AngⅡ environment(p<0.01).The expression ofTLR4can be reduced by siRNA interference (RNAi) technology,siRNA3is the mosteffective one. TLR4/Myd88mRNA and TLR4/Myd88/NF-kB protein were highlyexpressed under high glucose and AngⅡ coincubated NRK-52E(p<0.01),the IL-6and MCP-1levels were also increased markly compared with normal group(p<0.01).TLR4/Myd88mRNA and TLR4/Myd88/NF-kB protein expression were obviouslyinhibited in cells that were transfected with TLR4-siRNA or intervened withirbesartan. Compared with high glucose group(p<0.01), IL-6and MCP-1prouctiondecreased remarkably. Compared with high glucose and AngⅡ coincubated group(p<0.01),there are synergies in siRNA and irbesartan(p<0.01).Conclusion: Both High-glucose and AngⅡ can lead to the activation of TLR4/Myd88/NF-kB signaling pathway. High-glucose and AngⅡ coincubate can lead tothe activation of TLR4/Myd88/NF-kB signaling pathway and the secretion ofinflammation factors in NRK-52E.The effect can be blocked efficiently by specificsiRNA gene silience and irbesartan. TLR4/Myd88signaling pathway plays a pivotalrole in the innate-immune infammatory reaction in NRK-52E. The siRNA genesiliencing technology provide another pathway for finding a cure for the nephropathy.