DMDD Extracted From The Root Of Averrhoa Carambola L. Ameliorates Diabetic Nephropathy By Inhibiting TLR4/MyD88/NF-?B Signaling Pathway

Background:Diabetic nephropathy(DN)is one of complications of diabetic mellitus(DM),but the pathogenesis of DN is not clear.Researches have demonstrated that TLR4 signaling pathway play an important role in the development of DN.In previous studies,we have successfully extracted a monomeric compound named 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione(DMDD)from the root of Averrhoa carambola L.,and found that DMDD obviously decreased the blood glucose,declined urinary protein and protected the injury of kidney in diabetic mice.However,the mechanism of DMDD alleviated renal damage in the model of diabetic mouse need to be required.In recent studies,we have demonstrated that DMDD markedly inhibited the inflammation and cell apoptosis of MIN6 exposed to palmitic acid and protected insulin resistance of diabetic mice induced by high fat diet by suppression TLR4/My D88/NF-?B signaling pathway.Therefore,combining with preliminary report,we speculated that DMDD might ameliorate diabetic nephropathy by inhibiting TLR4/My D88/NF-?B signaling pathway.Objective:In this study,we mainly investigate the protection of DMDD on the kidney damage of diabetic mice and podocyte injury induced by high glucose,and the effect of DMDD on TLR4/My D88/NF-?B signaling pathway,hoping to reveal the mechanism of DMDD prevent diabetic nephropathy.Methods:In vivo experimentThe mouse model of diabetic was prepared through a diet of high-sugar and high-fat accompany injected intraperitoneally with Streptozocin(STZ,100mg.kg^-1)in the mice of TLR4 wild type(WT)and TLR4 knockout type(KO)after the mice fasted for 8 hours.The diabetic mouse model was considered successfully established when the fasting blood glucose?11.1mmol/L after administration of STZ for three days.The WT and KO mice were distributed into 6 groups:NC group(normal control group),DN group(diabetic nephropathy group),G group(gliquidone group,10mg.kg^-1.d^-1),H group(DMDD high dosage group,50 mg.kg^-1.d^-1),M group(DMDD middle dosage group,25 mg.kg^-1.d^-1),L group(DMDD low dosage group,12.5mg.kg^-1.d^-1),respectively.The mice of NC group and DN group were accepted with equivalent normal saline.After administration of gliquidone and DMDD for 4weeks,the blood of the mice was obtained by pulled the eyes,and then sacrificed the mice,exfoliation the kidneys promptly.The content of serum creatine(Scr),blood urea nitrogen(BUN),total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL)and low density lipoprotein(L-DL)were measured by biochemical analyzer,and the levels of IL-1?,IL-6,IL-10,TNF-?and MCP-1 were detected by ELISA kits.The changes of pathological and ultrastructural of kidney tissue were evaluated by HE staining and electron microscopy,respectively.The cell apoptosis of renal tissue was examined by TUNEL.Furthermore,the expression of m RNA and protein of TLR4,My D88,NF-?B in renal tissue were observed by PCR,immunohistochemistry and Western Blot.In vitro experimentThe cell of podocyte was damage induced by high glucose(30mmol/L).The viability and cytotoxicity of DMDD on podocyte cells were measured by CCK-8assay,which would confirm the IC₅₀and the treatment dosage of DMDD on podocyte injury.The apoptosis of MPC5 cells were monitored by AO/EB and flow cytometry and the inflammation cytokines of IL-6 and TNF-a were detected by ELISA method.Meanwhile,the m RNA and protein levels of TLR4/My D88/NF-?B signaling pathway were also assessed by PCR and Western Blot.ResultsIn vivo experimentThe model of diabetic was smoothly duplicated through high sugar and high fat dietary integrated with low dose STZ injection.After intervention DMDD for4 weeks,we found that DMDD significantly reduced the blood glucose,kidney weight,and protected renal function by decreasing the contents of serum creatine,blood urea nitrogen,cystatin-C and urine protein.Moreover,DMDD successfully improved dyslipidemia,suppressing the generation of TC,TG and LDL,and augment HDL level.However,in comparison to the same group of WT mice,the KO mice were not obviously difference in those indicators of above after administration DMDD.HE staining and electron microscopy showed that the glomerular volume of diabetic mice apparently was enhanced and the basement membrane significantly was thickened,and hyaline lesion was appeared.After DMDD treatment,the renal tissue damage of diabetic mice was improved.On the other hand,the electron microscopy observed the ultrastructure of kidney podocytes in mice,founding that the renal basement membrane of diabetic mice was thickened,the foot processes were fused,and the number of Sertoli cell was decreased.After DMDD supplementary,the number of podocytes was increased and the foot processes fusion was decreased.Meanwhile,TUNEL results showed that renal cell apoptosis was significantly increased in diabetic model mice,and the apoptosis was notably reduced after DMDD intervention.Relative to diabetic mice,the mice supplied with high dosage of DMDD were markedly blocked the secretions of IL-6,IL-1?,TNF-?,and MCP-1,and simultaneously facilitated the content of IL-10.Besides,the concentrations of IL-6 and TNF-?were higher in the WT mice than those in KO mice after administration 50 mg.kg^-1.d^-1DMDD.The protein levels of TLR4,My D88,NF-?B,IL-6 and TNF-a of kidney tissue in diabetic mice were higher than those in NC group,and those proteins were restrained by treatment with DMDD.Comparing with the same group of WT mice,the TLR4 protein level was prominently descended in KO mice.In addition,contrast with the diabetic mice,the protein and m RNA expressions of TLR4 signaling pathway were dramatically inhibited by supplement of DMDD which measured by the methods of PCR and Western Blot.What's more,the downstream factors My D88 and NF-?B were lower in KO diabetic mice than those in WT diabetic mice after administration 50 mg.kg^-1.d^-1DMDD.In vitro experimentThe half maximal inhibitory concentration(IC₅₀)of DMDD on podocyte cells was 60?mol/L that was estimated by detecting CCK-8 enzyme activity.On this finding,the optimum concentration of DMDD on MPC5 cells was 1.875?mol/L,3.75?mol/L and 7.5?mol/L,considering as low,medium and high dosage of DMDD to treat podocyte insult.Comparison with the low glucose(5.5mmol/L)group,the podocyte cell apoptosis was evidently rise in high glucose(30 mmol/L),but this cell apoptosis was effectively reversed after addition DMDD treatment.On the other hand,the flow cytometry results also verified that the apoptosis rate of podocytes exposed to high glucose was significantly higher than that of the low-glucose group.However,after the treatment of DMDD,the apoptosis of podocytes was significantly reduced.The levels of the inflammatory factors IL-6 and TNF-a were significantly reduced and TAK-242an inhibitor of TLR4 signaling was obviously down-regulated the production of IL-6 and TNF-a in high glucose lured podocyte injury by DMDD treatment.Detection by PCR and Western Blot,the m RNA and protein expressions of TLR4,My D88 and NF-?B were lower in low glucose group than those in high glucose group,and those levels were obviously declined through exposed with DMDD.Conclusion:DMDD relieves diabetic nephropathy and protects podocyte damage through inhibiting TLR4/My D88/NF-?B signaling pathway.