Regulation Of Melatonin On Toll-like Receptor 4 Signaling In Diabetic Db/db Mice Kidneys

Objective:Diabetic nephropathy is one of the major microvascular complications of type 1 and type 2 diabetes mellitus.The pathogenesis of DN has been related to both oxidative stress and chronic renal inflammation,and Toll-like receptor 4(TLR4)signaling pathway mediates immune and inflammatory responses.Therefore numerous domestic and foreign researches have confirmed that it is related to DN.Melatonin(MT),also known as N-acetyl-5-methoxytryptamine is produced by the circadian rhythm of the pineal gland,and it is the most known one of the effective antioxidants,and it also has anti-inflammatory properties.The effects of melatonin(MT)on diabetic mice db/db mice were measured.To investigate regulation of melatonin on TLR4 signaling in diabetic db/db mice kidneys We detected the general indexes such as blood glucose,body weight,kidney weight and 24 h urinary albumin excretion rate(UAER),TLR4 and downstream pathway protein such as myeloid differentiation factor 88(My D88),TIRdomain-containing adaptor inducing interferon-?(TRIF),interferon regulatory factor3(IRF-3)and NF-?B p65 and macrophage(ED-1 positive cell),the relative expression of tumor necrosis factor-?(TNF-?)and monocyte chemoattractant protein 1(MCP-1)m RNA in renal tissue.Methods:The 48 10-week-old male db /db mice mice were randomly divided into db/db group,db/db + MT50?g / kg group,db/db + MT 100?g/kg group and db/db + MT 200?g / kg group,each consisting of 12 mice.These mice received i.p.injections of MT whichdissoved in phosphate buffer solution(PBS)/dimethylsulfoxide(DMSO)solution,given every day.Alternatively,12 db/m mice served as the control group.db/m and db / db group were injected i.p.with the same volume of PBS/DMSO solution.The animals were sacrificed after 12 weeks of dosage administration.Blood glucose(BG),body weight(BW),kidney weight(KW)and 24 h urinary albumin excretion rate(UAER)were measured at 12 weeks.Pathological changes were evaluated by renal pathological staining.Immunohistochemistry of renal TLR4,NF-kB P65,and ED-1 was performed to determine the immunoreactivity.Western blot was used detected renal TLR4,My D88,TRIF,IRF-3 ? NF-kB p65 expression,while renal TNF-?,MCP-1 m RNA level was evaluated by real-time PCR.Results:1.The blood glucose,body weight,kidney weight and 24 h UAER were significantly higher in the db/db group than in the db/m group(P <0.01),while KW in db/db+MT(100,200?g/kg)groups and UAER level in db/db+MT(50,100,200?g/kg)groups were distinctly decreased(P<0.01).In week 12 db/db mice,the glomerular mesangial expansion index and tubulointerstitial injury index were increased compared with that in db/m mice(P<0.01).The above kidney histopathologic lesions were distinctly ameliorated by 50,100,200?g/kg MT(P<0.05).2.Immunohistochemistry intensity of renal TLR4,NF-k B p65 and ED-1 displayed obvious differences between db/m mice and db/db mice(P<0.01),and that were remarkably decreased in db/db+MT(50,100,200?g/kg)mice compared with db/db mice(P<0.05).Western blot showed that the expression of renal TLR4,My D88,TRIF,IRF-3 and NF-?B p65 protein in db/db mice was significantly higher than that in db/m group(P <0.05),weaker in db/db+MT(50,100,200?g/kg)groups(P<0.05).3.Futhermore,real-time quantitative PCR showed that the renal m RNA the expression of MCP-1 and TNF-? was higher in control group(P<0.01)and weaker indb/db+MT(50,100,200?g/kg)groups(P<0.01).Conclusions:The expression of TLR4,My D88,TRIF,IRF-3 and NF-?B p65 in the mouse kidney tissues and the relative expression of TNF-? and MCP-1 increased,and MT could down-regulate the expression of TLR4 signaling pathway and inflammatory factor,suggesting that the occurrence and development of DN may be related to TLR4 signaling pathway,Melatonin may down regulate TLR4 signaling pathway to inhibit inflammatory response and alleviate kidney injury in diabetic db/db mice.