ABSTRACT
Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) or high glucose on the toll-like receptor 4 (TLR4) expression,inflammatory cytokines and fibrotic factors in human tubular epithelial cells (HK-2),revealing the innate immune-related pathogenesis of diabetic nephropathy (DN) which may have clinical implications.Methods Three TLR4 siRNA sequences were designed and synthetized.After transfection,the most effective siRNA was selected to use for further expriments.The experiment consisted of 2 parts.Part 1:Cells were divided into three groups:normal-glucose group (NG,5.5mmol/L glucose),mannose group (M,5.5 mmol/L glucose + 19.5 mmol/L mannose),High-glucose group (HG,25 mmol/L glucose),preliminary validated the effects of high glucose and high osmotic pressure.Part 2:Cells were divided into seven groups:NG group,HG group,Ang Ⅱ group,Ang Ⅱ + negative group,HG+ negative group,Ang Ⅱ + siRNA group and HG+ siRNA group.Real time PCR was used to analyze the mRNA expression of TLR4,myeloid differentiation factor 88 (MyD88),heat shock protein 47 (HSP47).Western blotting was used to observe the protein expression of TLR4,MyD88,HSP47,NF-κB,type Ⅳ collagen (ColⅣ).ELISA was used to detect the expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6).Results Compared with NG group,TLR4,MyD88,HSP47 mRNA and TLR4,MyD88,NF-κB,ColⅣ,HSP47 protein were highly expressed under high glucose or Ang Ⅱconditions (P ＜ 0.01),and the expression levels of MCP-1 and IL-6 also increased significantly (P ＜ 0.01).Compared with HG or Ang Ⅱ group,the above indicators were obviously inhibited in the TLR4 siRNA groups (P＜0.01).Comparison between blank vector transfected groups and HG group as well as Ang Ⅱ group indicated no statistic significance (P ＞ 0.05).Conclusions Both Ang Ⅱ and high glucose stimulate TLR4 expression,which result in the up-regulation of inflammatory and fibrotic factors in HK-2.Specific target of TLR4 gene silencing can block the TLR4 pathway that is activated by high glucose and Ang Ⅱ,and thus reduce the inflammatory and fibtogenic factors' release.TLR4 signal is the common innate immune response pathway which induces the release of inflammatory and fibrotic factors in HK-2 under high glucose or high angiotension conditions.