| Background and purpose:Diabetic nephropathy(DN)continues to be the leading cause of chronic kidney disease(CKD)and end-stage renal disease(ESRD),and the pathogenesis and treatment of DN has been the focus of attention of scholars.Recently,numerous studies have suggested that the macrophage infiltration and activation in diabetic kidney initiate inflammation via the release of some related factors,as evidenced by increased levels of inflammatory cytokines,C-reactive protein and NF-?B activation,which subsequently lead to the development and progression of DN.However,little information is indicated about the signal transduction pathways of macrophage activation in diabetic kidney tissue.TLRs are germline-encoded innate immune receptors that has been utilized in the pathogenesis of diabetic nephropathy.The members of the family of TLR2 and TLR4 receptor,combinated with the pathological products of diabetes mellitus,can initiate downstream signaling pathways,causing macrophages recruitment and activation,which finally lead to a series of inflammatory response and sustained renal injury in diabetic nephropathy.DN patients have shown an increasing interest in the use of Chinese medicinal herbal therapies.Paeoniflorin(PF),a typical Chinese herbal medicine ingredient,is a main bioactive component of total glucosides of paeony(TGP).As a promising drug,PF is testified to have the anti-inflammatory,anti-oxidative,immune-regulatory effects.In the current study,we used PF in STZ-induced diabetic rats models and BMDMs stimulated by high glucose,to observe the TLR2 / 4 signal pathway and the expression of proinflammatory cytokines respectively from the integral,cellular and molecular level.We explore the role of TLR2 / 4 signal pathway in the pathogenesis of DN and the intervention effects of PF,thus add novel ideas for DN treatment.Methods Part 1The bone marrow cells extracted from C57BL6/J mice and TLR2/4 gene knockout mice(TLR2/4-/-)were induced into macrophages.The concentration and stimulated time of D-Glucose concentration,in addition to the end point of experiment and PF concentration,were optimized firstly.BMDMs were grouped as follows: normal glucose concentration control group(LG),normal glucose concentration +PF intervention group(LG+PF),high glucose stimulated group(HG),PF intervention group(HG+PF),normal glucose concentration of TLR2 knockout group(TLR2-/-),TLR2 knockout macrophages + high glucose stimulated group(TLR2-/-+HG),TLR2 knockout macrophages + high glucose stimulated group + PF intervention group(TLR2-/-+HG+PF),normal glucose concentration of TLR4 knockout group(TLR4-/-),.TLR4 knockout macrophages + high glucose stimulated group(TLR4-/-+HG),TLR4 knockout macrophages + high glucose stimulated group + PF intervention group(TLR4-/-+HG+PF).CCK-8 method was used to determine whether PF influenced macrophage viability.Cell migration was observed by a transwell method.The antigens expressed on cell surface including F4/80,CD11 b and CD11 c were detected by flow cytometry analyses.The expression of TNF-?,MCP-1,IL-1? and iNOS mRNA which extracted from cells were assayed by real-time quantitative PCR.Western blot wasused to detect the protein expression of TLR2,TLR4,MyD88,Trif,p-IRAK-1,p-IRF3,IRF3,NF-?B p65,NF-?B p-p65 and iNOS in each group.Expression of pro-inflammatory factor of TNF-?,MCP-1,IL-1? in cell supernatant were determined by ELISA.Part 2The models of healthy male C57BL/6J mice and TLR2/4 gene knockout mice was induced by 5 days intraperitoneal injection of STZ at the dose of 50mg/kg,whose random caudal vein blood glucose level were over 16.7mmol/l.The diabetic models were randomly divided into diabetic model group,administration of PF group(25,50,100mg/kg.d),TLR2-/-diabetic model group,TLR4-/-diabetic model group,each with the number of 12.In addition,we take healthy male C57 mice and TLR2/4 gene knockout mice as normal control groups,each with 12.Blood,urine and renal tissue samples were collected after 12 weeks of treatment to determine the blood glucose,body weight,kidney weight and 24 h urinary albumin excretion rate.Renal pathologic changes were observed by light microscopy.Protein expression of TLR2,TLR4,CD68,and NF-?B p65 were determined by immunohistochemistry.Detection in renal tissue of TLR2,TLR4,MyD88,Trif,p-IRAK-1,p-IRF3,IRF3,NF-?B p65,NF-?B p-p65 were analyzed by western blot and the mRNA of TNF-?,MCP-1,IL-1? and iNOS were determined by real-time quantitative PCR.Results Part 1?Compared with normal control,PF had no obvious effect on viability of HG cultured BMDMs(P>0.05).?Migration assay indicated that both PF and TLR2/4deficiency blocked HG-induced migration of macrophages(P<0.01).?Results of flow cytometry analyses showed that the percentage of M1 macrophages in HG group wassignificantly higher than that in untreated macrophages,the intervention of PF and TLR2/4 gene knockout could make the M1 type macrophage surface marker expression decrease(P<0.01).?The mRNA levels of iNOS,TNF-?,IL-1?,MCP-1in BMDMs were largely prevented in absence of TLR2/4 or in presence of PF(P<0.01).?Western blot analysis showed that PF intervention and knockout of TLR4 could make the expression of TLR4,MyD88,Trif,p-IRAK-1,p-IRF3,IRF3,NF-?B p-p65,NF-?B p65,iNOS remarkably weaker(P<0.01).TLR2-/-+HG have lower levels of TLR2,MyD88,p-IRAK1,NF-?B p-p65,NF-?B p65 and iNOS compared with that in HG group(P < 0.01),bu the knockout of TLR2 had no effect on the protein levels of HG-stimulated Trif,p-IRF3 and IRF3(P> 0.05).?Elisa detection results showed that PF treatment and knockout of TLR2/4 could inhibit the HG-induced secretion of TNF-?,IL-1? and MCP-1(P<0.05,P<0.01).Part 2?Compared with the control group,the blood glucose,body weight,kidney weight and 24 h urinary albumin excretion rate of 12 week diabetic mice significantly increased(P<0.01).However,PF intervention group and TLR2/4 knockout group had lower body weight,kidney weight and 24 h urinary albumin excretion rate than that of diabetic group(P<0.01).?At 12 weeks,we could observe the increased glomerular volume,mesangial cells,extracellular matrix and thickened basement membrane in diabetic model mice with light microscope(P<0.01).?Immunohistochemistry showed that compared with the control group,the expression of TLR2,TLR4,CD68 and NF-?B p65 protein in 12 weeks diabetic model mice significantly elevated(P<0.01),which significantly reduced with PF intervention and TLR2/4 gene knockout(P < 0.05;P<0.01).?Western blot analyses demonstrated that compared with the control group,the expression of TLR2,TLR4,MyD88,Trif,p-IRAK-1,p-IRF3,IRF3,NF-?B p-p65,NF-?B p65,iNOS protein in 12 weeks diabetic model mice significantly elevated(P<0.01),which significantly reduced with PF intervention and TLR4 geneknockout(P<0.01),However,the expression of Trif and p-IRF3,IRF3 in TLR2-/-diabetic group was not significantly decreased(P> 0.05).?Real-time quantitative PCR found that diabetic model mice group had higher expression of TNF-?,MCP-1,IL-1?and iNOS mRNA than that of control group(P<0.01),however,the expression of TNF-?,MCP-1,IL-1?and iNOS mRNA efficiently inhibited in PF intervention group and TLR2/4-/-group compared to diabetic model mice group(P<0.01).Conclusions1 Under high glucose condition,the activation of BMDMs lead to the up-regulated expression of intracellular TLR2/4 and downstream signaling pathway,as well as the high expression of proinflammatory cytokines and iNOS.In addition,PF intervention and TLR2/4 gene knockout could inhibit the activation of signaling pathway,contributing to the downregulation of the expression of inflammatory factor on macrophage.2 In vivo experiments confirmed that the expression of TLR2 / 4 enhanced in diabetic state in renal tissue,accompanied by chronic inflammation pathological kidney change,such as mesangial cell proliferation,extracellular matrix accumulation and activated macrophage infiltration,suggesting that TLR2 / 4 may play a role in the pathogenesis of diabetic nephropathy.3 The expression of TLR2/4 and downstream signaling pathway proteins up-regulated and the synthesis of proinflammatory cytokines and iNOS increased in STZ-induced diabetic mice kidney,which could be down regulated by PF intervention and TLR2/4gene knockout,indicating that the mechanism of diabetic nephropathy is related to the activation of TLR2/4 signaling pathway and PF can improve the inflammatory state of diabetes through inhibiting the activation of TLR2/4 signaling pathway,whose specific ways and targets need to be further studied. |
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