Cross-talk Between Angiotension ? And Toll-like Receptor 4 In Rat Mesangial Cells Under High Glucose Conditions And The Innate Immune Inflammatory Responses

Objective To investigate the effects of Angiotensin II and high glucose on the TLR4 expression and the related innate immune responses in rat mesangial cells(MCs).Applying TLR4 specific Si RNA and angiotensin ?receptor blocker(ARB)to interfere the MCs that were stimulated by angiotensin II and/or high glucose,detected the changes of TLR4 and downstream signal moleculers,demonstrating the cross-talk relationship between Ang II and TLR4 signaling under hyperglycemia conditions in MCs and revealing the novel innate immune-related pathogenesis of diabetic nephropathy(DN)which may have clinical implication.Methods We did the experiments by two steps,firstly,after synchronization,MCs were divided into four groups :1.5.5 m M glucose group(NG)2.25 m M glucose group(HG)3.Ang group 4.19.5 m M mannitol was added along with 5.5 m?M glucose as anosmotic control.Extract total protein after 24 hours,western blotting was used to observe the protein expression of TLR4?My D88 and NF-k B,analysis the influence to TLR4/My D88 signaling that cased by osmotic effect.Three TLR4-si RNA sequences were designed and synthetized.After transfection,we select the most effctive si RNA to use for further expriments.Divided cells into two groups: The first sub-group is: 1.low-glucose group 2.high-glucose group 3.high-glucose + Ang?group 4.high-glucose +irbesartan group 5.high-glucose +TLR4si RNA group 6.high-glucose + Sc Si RNA group.The second sub-group is: 1.low-glucose group 2.Ang ?group 3.high-glucose + Ang?group 4.Ang ?+irbesartan group 5.Ang ?+TLR4si RNA group 6.Ang ?+ Sc Si RNA group.Real time PCR was used to analyze the expression of TLR4 and Myd88 m RNA;western blot was used to detect the expression of TLR4?Myd88 and NF-k B protein.ELISA was used to detect the expression of MCP-1 and IL-6.Results Compared with NG,TLR4,My D88 m RNA and TLR4,My D88,NF-?B protein were highly expressed under high glucose and Ang?conditions(p<0.05),the expression level of IL-6 and MCP-1 also increased significantly(p<0.01);the above parameters were significantly further increased under co-stimulated with high glucose and Ang II.Compared with HG,Ang II and co-stimulated with high glucose and Ang II group,the above indicators were obviously inhibited in the Irbesartan and TLR4 Si RNA groups(p<0.05);Among the blank vector transfected and HG as well as Ang II group,these indicators were not statistically significant(p>0.05).Conclusion The TLR4/Myd88 signal was activated under high-glucose and Ang?conditions which result in the upregulation of inflammatory responses in rat MCs.Specific TLR4 Si RNA inhibited the signaling activation significantly that were induced by ang?and hyperglycemia in MCs,similar effects were detected in cells that were treated with Irbesartan.High glucose synergized with Ang II strengthen the TLR4-mediated innate immune inflammatory response.These data demonstrated the cross-talk relationship between TLR4 and Ang ?,indicating that Ang ?can act as an innate immune signal peptide under high glucose conditions in MC.These evidences provide an strongly theory basis that with joint using TLR4 si RNA and angiotensin ?receptor blockers may be a promising intervention strategy for the treatment of DN.