Study On The Mechanism Of Dmdd, Isolated From The Root Of Averrhoa Carambola L., In Improving Diabetic Nephropathy Through Tlr4/tgf? Signaling Pathway

Background:Diabetic kidney disease?DKD?has a high incidence and great harmfulness.It is of great significance to explore the target of DKD and its medicationtherapy.Ourpreviousstudiesfoundthat2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione?DMDD?,isolated from the root of Averrhoa Carambola L.,was able to improve DKD,but its mechanism was unclear.Objective:In this study,we aim to investigate the effects of DMDD on TLR4/TGF?signaling pathway and associated protein expression.Furthermore,we explore the mechanism of DMDD in prevention and treatment of DKD.Methods:In vivo experiment:DKD model was induced by high sugar and high fat diet combined with low dose streptozocin?STZ?in wild type mice and TLR4-knockout(TLR4^-/-)mice.All mice were randomly divided into 6 groups in wild type and TLR4^-/-mice respectively.They were blank control group?NC?,model group?DM?,positive control group?GQ?and three DMDD groups?high dosage,medium dosage and low dosage?.The mice in the NC group were given distilled deionized water and the other groups'mice were administrated high sugar and high fat diet.After 4 weeks,all the mice except that in the NC group were received an intraperitoneal injection of 110mg/kg streptozotocin?STZ?.When diabetes model was successfully duplicated,the mice in the positive control group was given 10mg/kg/day of Qliquidone.The three DMDD groups were received DMDD at a dose of 50,25 and 12.5mg/kg/day,respectively.In addition,besides the blank control group,high glucose and high fat diet were continued until the end of the experimental period in all the other mice.After 4 weeks of intervention,the mice were sacrificed to determine the change of renal function and the other common measure.Pathologic changes of kidney tissue were viewed with HE staining method.The changes of fibrosis were observed by Masson staining,and the morphology of renal tubules and fibrosis were observed by transmission electron microscopy?TEM?.The localization of TLR4,BAMBI,TGF?1 and Smad2/3 protein were examined by immunohistochemistry.Furthermore,the expression leves of mRNA and protein TLR4,BAMBI,TGF?1and Smad2/3 were evaluated by RT-PCR and Western-blot,respectively.In vitro experiment:High glucose?60mmol/L?induced renal proximal tubular epithelial cell line?HK-2 cells?was cultured and treated with DMDD.The cell viability and DMDD cytotoxicity were assessed by CCK8.The changes of associated protein involved in TLR4-TGF?/Smad signaling pathway were detected by immunofluorescence,RT-PCR and Western-blot methods.Furthermore,HK-2cells were transfected with lentivirus codifying for BAMBI?BMP and activin membrane bound inhibitor?and interfering RNA for determination of the effect of BAMBI over-expression and silencing,respectively.TLR4-BAMBI-Smad2/3pathway was analyzed by means of RT-PCR and Western-blot.In addition,the mRNA and protein expression of E-cadherin and Vimentin,the primary indicator of epithelial-to-mesenchymal transition?EMT?,were also observed by RT-PCR and Western-blot.Results:1.In vivo experiment:?1?Diabetic kidney disease model was successfully duplicated by high sugar and high fat diet combined with STZ stimulation.Furthermore,DMDD had a significant hypoglycemic and hypolipidic effects on diabetic kidney disease in mice.Meanwhile,DMDD was able to improve insulin resistance,glucose tolerance and insulin tolerance.?2?Serum cystatin C?CysC?and urinary protein levels could be used as sensitive markers of renal dysfunction.DMDD significantly improved the CysC and urinary protein levels in DKD mice.?3?The results of HE staining showed that the glomeruli of diabetic model mice was obviously enlarged and the basement membrane was abnormally thickened in the wild-type mice.Besides,inflammatory infiltration and fibrosis were also occured in the renal interstitium.After the intervention of DMDD,the volume of glomeruli decreased obviously and the basement membrane became thinner.The infiltration of inflammation and fibrosis also decreased gradually,and the pathological improvement was positively correlated with the dose of DMDD.In TLR4^-/-mice,the degree of pathological changes in the diabetic model group was significantly less than that in the wild type group.After DMDD intervention,the pathological changes also showed a trend of improvement.The results of Masson staining showed that the collagen deposition in renal tissue of diabetic mice was significantly increased,and the renal fibrosis was obviously alleviated after DMDD intervention in the wild-type mice.However,the situation was not improvement in the positive control group.Compared with the wild-type mice,the degree of fibrosis in TLR4^-/-mice was significantly reduced.TEM showed that the renal interstitial components in the model group were significantly increased,and more collagen fibers appeared in the model group.When treated with DMDD,the renal interstitial components and collagen fibers were significantly decreased in the different DMDD groups.The positive control drug Qliquidone did not have this effect.There was no obvious fibrosis in the TLR4^-/-mice.?4?The results of immunohistochemistry showed that the expression of TLR4 was significantly increased by high glucose and high fat diet and STZ stimulation in the wild-type mice.However,the expression of TLR4 decreased in a dose-dependent manner after DMDD treatment.In the TLR4^-/-mice,no expression of TLR4 was detected,and high glucose,high fat and STZ stimulation did not increase the expression of TLR4.The expression of TGF?1 was significantly increased in the wild-type model group.When treated with DMDD,the expression of TGF?1 decreased progressively in a dose-dependent manner.After the knockout of TLR4 gene,the expression of TGF?1 protein in the model group was significantly lower than that in the wild type group.As a downstream protein regulated by TGF?1,the expression of Smad2/3 in the model group showed the same trend as that of TGF?1.Compared with TGF?1 and Smad2/3,the opposite trend of BAMBI expression was observed by immunohistochemistry assay.The expression of BAMBI protein was found in the blank control group in wild type or TLR4knockout mice.When stimulated with high glucose,high fat and STZ,the expression of BAMBI protein in the model group decreased significantly.The expression of BAMBI in all DMDD groups increased in a dose-dependent manner.As a positive control drug,Qliquidone had little effect on the expression of TLR4,TGF?1,Smad2/3 and BAMBI in renal tissue,suggesting that gliaquinone has no protective effect on fibrosis of diabetic nephropathy.?5?.The results of RT-PCR and Western-blot showed that there were no expression of TLR4 mRNA and protein in TLR4^-/-mice.In the wild-type mice,the expression of TLR4 mRNA and protein in the model group were significantly increased,and the level of TLR4 decreased in a dose-dependent manner after the treatment of DMDD.There were no significant difference in mRNA and protein expression of BAMBI among TLR4^-/-mice.However,the mRNA and protein expression levels of BAMBI were significantly decreased after high glucose and high fat combined with STZ stimulation in wild-type mice.When DMDD was given,the BAMBI expression was increased especially in the medium and high dose DMDD groups.The expression of mRNA and protein of TGF?1 and Smad2/3 showed a similar trend.In the TLR4 knockout mice,there were no significant change in mRNA and protein of TGF?1 and Smad2/3 in the model group,and the DMDD intervention groups.In the wild type mice,the expression of mRNA and protein of TGF?1 and Smad2/3 in the model group increased significantly.After DMDD treatment,the mRNA and protein of TGF?1 and Smad2/3 decreased in a dose-dependent manner.In the positive control group,Gliquantel had no significant effect on the expression of mRNA and protein of TLR4,TGF?1,Smad2/3 and BAMBI,suggesting that the protective mechanism of Gliquantel on diabetic nephropathy was not related to the TLR4-TGF?-Smad2/3 signaling pathway.2.In vitro experiment:?1?The IC₅₀ of DMDD on HK-2 cells was 80?mol/L which was determined by measuring CCK8 enzyme activity.Based on this finding,the optimal concentration of DMDD on HK-2 cells was screened.Finally,the high,medium and low concentrations of DMDD were 40,20,10?mol/L,respectively.?2?The scratch test showed the migration ability of HK-2 cells,the characteristics of fibroblasts,was enhanced after stimulated by high glucose.In addition,the results of RT-PCR and Western-blot demonstrated the occurrence of EMT which is characterized by the decreased expression of the epithelial surface marker E-cadherin,and the increased expressions of the mesenchymal markers Vimentin.Specifically,the expression of E-cadherin mRNA and protein were decreased and increased for Vimentin under the stimulation of high glucose.When treated with DMDD,the relative expression of E-cadherin increased significantly and Vimentin decreased markedly.?3?Immunofluorescence showed that the expression of TLR4 and pSmad2/3 were remarkably increased in HK-2 cells after high glucose stimulation.Conversely,BAMBI was decreased in this circumstance.After the intervention with DMDD,the expression of TLR4 was down-regulated.?4?After the intervention with TAK-242?a TLR4 signaling inhibitor?,HK-2 cells was stimulated with high glucose medium.Then the expression of E-cadherin and Vimentin were observed in HK-2 cells by RT-PCR and Western-blot.The results described that E-cadherin had a clear rise in HK-2cells,and Vimentin had a huge decline.?5?The degree of EMT was decreased evidently in HK-2 cells after overexpression of BAMBI,and EMT enhanced apparently after BAMBI gene silencing.There was no significant change of EMT in HK-2 cells which high glucose and DMDD were given in the situation of BAMBI gene silencing.?6?As the downstream protein of BAMBI regulation,the expression of Smad2/3 was negatively correlated with BAMBI.When BAMBI was overexpression,Smad2/3 was down-regulated.By comparison,the expression of mRNA and protein of Smad2/3 were up-regulated clearly after BAMBI gene silencing.Furthermore,the expression of Smad2/3 had no obvious change after hyperglycemic and DMDD intervention in the circumstance of BAMBI gene silencing.Conclusions:?1?DMDD is able to improve the renal function of diabetic nephropathy mice.?2?DMDD is able to decrease the protein expression of TLR4 and TGF?-Smad2/3 in diabetic nephropathy mice.?3?There is a signal crossconnected between TLR4 and TGF?-Smad2/3signal pathway through BAMBI.?4?DMDD ameliorates diabetic nephropathy by regulating the TLR4-TGF?/Smad2/3 signaling pathway to slow down the occurrence of EMT.