Cross Tlak Between Innate Immune TLR4 Signaling And Angiotensin Ⅱ Induce Pathophysiological Changes In Human Tubular Epithelial Cells Under High Glucose Conditions

Objective:To investigate the cross talk between TLR4 and angiotensin Ⅱ by ob-serve the expression of TLR4 in tubular epithelial cells under high glucose conditions, and observe changes of fibrogenic factor and inflamamatory factors in human renal tubular epithelial cells. revealing the innate immune pathogenesis in diabetic nephrop-athy.Methods:Cells were divided into five groups after cultivated with normal gluco-se medium:1. normal-glucose group (5.5mmol/L)2. mannitol group 3. Ang Ⅱ group 4. high-glucose group(25mmol/L) 5. high-glucose+Irbesartan group, extract total RNA and total protein after 24 hours. Real time PCR was used to analyze the expressions of TLR4, MyD88, HSP47 mRNA, western blot was used to observe the expressions of TLR4, MyD88, NF-κB, CoⅣ, HSP47 protein, analyse the effect of high-glucose and Ang Ⅱ to TLR4/MyD88/NF-κB signaling pathway. Three TLR4-siRNA seque nces were designed and synthesized, BLOCK-IT Alexar Fluor with red fluorescence as negative control. The transfection efficiency was observed by fluorescence micros-cope after the cells were transfected for 24 hours, Real time PCR was used to analyze the expression of TLR4 mRNA, we choose the TLR4-siRNA which the expression of TLR4 were reduced most for the fllowed experiments. Cells were divided into sen-ven groups:1. normal-glucose group 2. high-glucose group 3. Ang Ⅱ+negative cont-rol group 4. high-glucose+negative control group 5. Ang Ⅱ group 6. Ang Ⅱ+siRNA group 7. High-glucose+siRNA group, Real time PCR was used to analyze the expre-ssion of TLR4, MyD88, Hsp47 mRNA, western blot was used to observe the express-ion of TLR4, MyD88, NF-kB, CoⅣ, HSP47 protein, collect supernate of the cells a-nd ELLISA was used to analyze the expression of inflammatory factors called IL-6 and MCP-1.Results:Compared with normal group, mannitol group were statistically signifi-cant (p>0.05), exclud the possibility that these effects were induced by high osmotic pressure. TLR4, Myd88, HSP47 mRNA and TLR4, Myd88, NF-kB, CoⅣ, HSP47 pr-otein were highly expressed under high glucose condition and after Ang Ⅱ stimulate (p<0.01). The expression of IL-6 and MCP-1 also increased (p<0.01). Compared wit-h high-glucose group, the expresssion of TLR4, MyD88, HSP47 mRNA and TLR4, MyD88, NF-kB, CoIV, HSP47 protein in the high-glucose+Irbesartan group were significantly reduced (p<0.01). The difference of above indicators between the negat-ve transfected group and high-glucose group and AngⅡ group were statistically signi-ficant (p>0.05). Compared with high glucose group, AngⅡ group, the expression of TLR4, MyD88, HSP47 mRNA and TLR4, MyD88, NF-kB, CoIV, HSP47 protein in the transfected group were obviously inhibited (p<0.01). the expression of IL-6 and MCP-1 in the group that cells were transfected with TLR4-siRNA also reduce (p< 0.01)Conclusion:High-glucose can activate the TLR4/MyD88/NF-kB signaling pat-hway, up-regulate the expression of inflammatory factors and fibrogenic factors. The presence of cross talk between angiotensin Ⅱ and TLR4 can up-regulate the expressi-on of inflammatory factors and fibrogenic factors. Irbesartan can block the activation of TLR4 signaling pathway induced by high glucose and angiotensin Ⅱ. specific gene silencing can block the activation of TLR4 signaling pathway induced by high gluco-se and angiotensin Ⅱ, reduced the release of inflammatory factors and fibrogenic fact-ors. TLR4 signaling pathway is the major pathway induce the release of inflammatory factors and fibrogenic factors in tubular epithelial cells under high glucose condition.