| Diabetic nephropathy is the main cause of end-stage kidney disease.Age,course of disease,blood pressure,obesity,blood lipids,uric acid,environmental pollutants and so on are the main risk factors for diabetic nephropathy.The pathogenesis of diabetic nephropathy is complex and not yet clear.The occurrence of diabetic nephropathy may involve a variety of factors,such as hemodynamic changes in the kidneys,inflammatory response,oxidative stress,internal environmental metabolic disorders,advanced glycation end products accumulation and cell autophagy changes.Sleep Disorder in type 2 diabetic patients were significantly higher than those of Non-diabetics.Sleep disorders are related to insulin resistance,impaired glucose tolerance,and abnormal secretion of the first phase of insulin,which affect the neuro-endocrine-metabolic pathway and affect the occurrence and development of T2 DM.Melatonin is widely distributed in nature.Melatonin is functionally active in single-celled organisms,plants,fungi and animals.Melatonin,as an antioxidant,can reduce oxidative stress in a number of ways: directly detoxifying reactive oxygen and active nitrogen,indirectly by stimulating antioxidant enzymes,and inhibition of promoting the activity of antioxidant enzymes.Studies have suggested that melatonin may help reduce kidney damage in diabetic nephropathy rats induced by Streptozotocin.Melatonin has also been shown to regulate glycolipid metabolism and improve islet function,improving insulin sensitivity.In recent years,the Glucagon-like peptide-1 Receptor Agonist was widely used in the clinical treatment of diabetes,with better safety.While regulating and controlling blood glucose,GLP-1 Receptor Agonists may also have a better role in improving kidney structure and function.It may be related toimproving blood flow in the kidneys,inflammatory response,oxidative stress,activation of autophagy,etc.At present,the protective mechanism of GLP-1 Receptor Agonists and melatonin on the kidney is not clear.At the same time,the interaction between GLP-1 Receptor Agonists and melatonin is not clear.The purpose of this study was to investigate the effects of melatonin and GLP-1 receptor agonists on glucose and lipid metabolism and renal disease,as well as their possible interactions.To provide a theoretical basis and strategy for the prevention and treatment of DM kidney disease.Part one Effects of Melatonin on Wnt/?-catenin Signaling Pathway,TGF-?-Smad Signaling Pathway and Renal Lesions in Diabetic RatsObjectives: To observe the effects of melatonin on superoxide dismutase,Catalase,glutathione peroxidase activity and Malondialdehyde levels,as well as Wnt/?-catenin signal ingress and TGF-?1-Smad2/3 signaling pathways.To understand the protective effects and possible mechanisms of melatonin on the kidneys of diabetic kidney disease rats.Methods:1.50 SD rats were included in this study.48 rats meeting the normal blood glucose standard were enrolled in the group,and 8 rats were randomly selected as NC group.The remaining 40 rats were given high fat diet combined with Streptozotocin subcutaneous injection to induce DM model.2.The 38 rats successfully modeled were randomly divided into the diabetes group(DM group,8 rats),the low-dose melatonin treatment group(MLT-L group,10 rats),and the medium-dose melatonin treatment group(MLT-M group,10 rats),high-dose melatonin treatment group(MLT-H group,10 rats).Different doses of melatonin were given by gavage in the MLT treatment groups(MLT-L group 5mg/kg,MLT-M group 15mg/kg,MLT-H group MLT 30mg/kg),for 4 weeks.3.The changes of renal pathology in experimental animals were observed.4.FBG,TG,TC,HDL-C,LDL-C,INS,BUN,SCR and other biochemical tests were detected.SOD,CAT,GSH-Px activity and MDA levels in experimental animal kidney tissue were tested.The levels of Wnt4,?-catenin,p-EGFR,EGFR,TGF?1,Smad 2,Smad 3,p-Smad 2,p-Smad 3 in rats were detected by westernblot.Results:1.The DM experimental model was successfully prepared.The FBG level in the DM group was significantly higher than that in the NC group(P<0.01).2.After 2 weeks of melatonin treatment,the FBG levels of the MLT-M and MLT-H groups were lower than those of the DM group(P<0.05,P<0.01).There was no significant difference in FBG levels between the MLT-L group and the DM group(P>0.05).The levels of FBG in MLT-L group,MLT-M group and MLT-H group was significantly different(P<0.05,P<0.01).In order,MLT-Hgroup <MLT-Mgroup <MLT-Lgroup.After 4 weeks of melatonin treatment,the FBG levels of the MLT-M and MLT-H groups were lower than those of the DM group(P<0.05,P<0.01).There was no significant difference in FBG levels between the MLT-L group and the DM group(P>0.05).The FBG levels among the MLT-L group,the MLT-M group and the MLT-H group were significantly different from each other(P<0.05,P<0.01).In order,MLT-Hgroup <MLT-Mgroup <MLT-Lgroup.3.At the end of 4th week,the degree of renal lesions was significantly reduced in all MLT treatment groups.The thickening degree of glomerular basement membrane was obviously improved.The degree of glomerular membrane matrix hyperplasia was significantly reduced.According to the order of lesion severity from light to heavy,the disorder in rats was NC group< MLT-H group < MLT-M group<MLT-L group< DM group.4.At the end of 4th week,the levels of TC,TG,and LDL-C in the DM group,MLT-L group,MLT-M group,and MLT-H group were significantly higher than those in the NC group(P<0.05,P<0.01).The levels of HDL-C in DM group and MLT-L group were lower than those in NC group(P<0.05).There was no significant difference in HDL-C levels among the MLT-M group, MLT-H group and the NC group(P>0.05).After 4 weeks of melatonin treatment,The levels of TC in MLT-L group,MLT-M group and MLT-H group were lower than those of DM group(P<0.05,P<0.01).There was no statistical difference in TC level between MLT-L group and DM group(P>0.05).The levels of TG in MLT-M group and MLT-H group were lower than those of DM group(P<0.05,P<0.01).The level of LDL-C in MLT-H group was lower than that of DM group(P<0.05).There was no significant difference in HDL-C levels between the MLT-L,MLT-M,MLT-H group and DM group(P>0.05).The level of TC in MLT-H group was lower than that in MLT-M group and MLT-L group(P<0.05,P<0.01).There was no significant difference in level of TC between MLT-M group and MLT-L group(P>0.05).The TG levels among the MLT-L group,the MLT-M group and the MLT-H group were significantly different from each other(P<0.05,P<0.01).In order,MLT-Hgroup <MLT-Mgroup <MLT-Lgroup.The level of HDL-C in MLT-H group was higher than that of MLT-L group(P<0.01).The levels of HDL-C in MLT-M group and MLT-H group were higher than those of MLT-L group(P<0.05,P<0.01).There was no statistical difference in HDL-C level between MLT-M group and MLT-H group(P>0.05).The LDL-C level of MLT-H group was lower than that of MLT-L group(P<0.05).There was no statistical difference in LDL-C levels between the MLT-H and MLT-M groups(P>0.05).Similarly,there was no statistical difference in LDL-C levels between the MLT-M and MLT-L groups(P>0.05).5.At the end of 4th week,the INS levels in the DM group,MLT-L group,MLT-M group and MLT-H group were higher than those in the NC group(P<0.01).In contrast,ISI levels in the DM group,MLT-L group,MLT-M group and MLT-H group were lower than those in the NC group(P<0.01).After 4 weeks of melatonin treatment,compared with DM group,the level of insulin was decreased significantly in MLT-M group and MLT-H group(P<0.01),but the insulin sensitivities index increased(P<0.01).There was no significant difference in INS level between MLT-L group and DM group(P>0.05).Similarly,there was no significant difference in ISI level between MLT-L group and DM group(P>0.05).There were significant differences among MLT-L group,MLT-M group,and MLT-H group in the levels of INS and ISI(P<0.05,P<0.01).Sort by INS level,MLT-H group <MLT-M group < MLT-Lgroup.Sort by ISI level,MLT-Lgroup <MLT-M group<MLT-H group.6.At the end of 4th week,the levels of BUN,SCR,UMA,CCRin DM group,MLT-L group,MLT-M group and MLT-H group were higher than those in NC group,and the differences were statistically significant(P<0.01).After 4 weeks of melatonin treatment,compared with DM group the levels of BUN,SCR,UMA,CCR were decreased significantly in the MLT-M group and MLT-H group(P<0.05).There was no significant difference in BUN,SCR,UMA and CCR between MLT-L group and DM group(P>0.05).The differences in the levels of BUN,SCR,UMA,and CCR among the groups of MLT-L,MLT-M,and MLT-H were significant(P<0.05).In order,MLT-H group < MLT-M group< MLT-L group.7.At the end of 4th week,compared with NC group,the activity of SOD,CAT and GSH-Px in DM group,MLT-L group,MLT-M group and MLT-H group decreased significantly(P<0.01).The levels of MDA in DM group and MLT-L group were higher than those in NC group(P<0.01).There was no significant difference in MDA level among MLT-M group,MLT-H group and NC group(P>0.05).After 4 weeks of melatonin treatment,compared with DM group,the activity of CAT and GSH-Px in MLT-L group,MLT-M group,MLT-H group increased significantly(P<0.05,P<0.01).The levels of SOD in MLT-M group and MLT-H group were higher than those in DM group(P<0.05,P<0.01).There was no significant difference in SOD level between MLT-L group and DM group(P>0.05).The level of MDA in the MLT-L group,MLT-M group,and MLT-H group were lower than those in the DM group.SOD,CAT,GSH-Px levels were statistically different among MLT-L group,MLT-M group,and MLT-H group(P<0.05,P<0.01).In order,MLT-L group < MLT-M group< MLT-H group.The level of MDA in MLT-H group was lower than that in MLT-L group and MLT-M group(P<0.05,P<0.01).There was no significant difference in MDA level between MLT-L group and MLT-M group(P>0.05).8.At the end of 4th week,the levels of EGFR,Smad2,and Smad3 in DM group,MLT-L group,MLT-M group and MLT-H group were lower than those in NC group,with no statistical difference(P>0.05).The levels of Wnt4,?-catenin,TGF-?1,P-EGFR,P-Smad2,and P-Smad3 in DM group,MLT-L group,MLT-M group and MLT-H group were higher than those in the NC group(P<0.05,P<0.01).After 4 weeks of melatonin treatment,the levels of ?-catenin in MLT-L group,MLT-M group and MLT-H group were higher than those in DM group.The levels of Wnt4 in MLT-M group and MLT-H group were lower than those in DM group.There was no significant difference in Wnt4 level between MLT-L group and DM group(P>0.05).The differences in the levels of Wnt4 and ?-catenin among MLT-L group,MLT-M group,and MLT-H group were significant(P<0.05).In order,MLT-H group < MLT-M group< MLT-L group.The expressions of P-EGFR in MLT-L group,MLT-M group and MLT-H group were significantly lower than those in the DM group(P<0.01).The expressions of TGF-?1,P-Samd2,and P-Samd3 in MLT-M group and MLT-H group were significantly lower than those in the DM group(P<0.05,P<0.01).The level of P-EGFR in MLT-H group was lower than that in MLT-L group(P<0.01).The level of P-EGFR in MLT-M group was lower than that in MLT-L group(P<0.01).The P-EGFR level in MLT-H group was lower than that in MLT-M group,but there was no statistical difference(P> 0.05).The level of TGF-?1 in the MLT-H group was lower than that in the MLT-L group(P<0.01).The level of TGF-?1 in the MLT-H group was not significantly lower than that in the MLT-M group(P>0.05).The level of TGF-?1 in the MLT-M group was lower than that in the MLT-L group(P<0.01).In order,MLT-H group < MLT-M group< MLT-L group.The levels of P-Smad2 and P-Smad3 in MLT-H group were lower than those in MLT-L group(P<0.01).The levels of P-Smad2 and P-Smad3 in MLT-M group were lower than those in MLT-L group(P<0.01). There was no significant difference in P-Smad2 level between MLT-M group and MLT-H group(P>0.05).The difference of P-Smad3 level between MLT-M group and MLT-H group was no significant(P>0.05).Summary:1.Diabetic nephropathy rats induced by Streptozotocin were in a state of perioxygen stress.2.Melatonin increased insulin sensitivity and improve insulin resistance in DM rats.Melatonin treatment reduced oxidative stress in kidney tissue.3.The renal protective effect of melatonin was achieved by Wnt/?-catenin signaling pathway and TGF-?1-smad2/3 signaling pathway.The effects of melatonin on increasing insulin sensitivity,enhancing kidney function,improving renal oxidative stress was related to the dosage of melatonin.Part two Effects of liraglutide on glycolipid metabolism,NLRP3 inflammatory bodies and renal lesions in diabetic ratsObjectives: To observe the effect of liraglutide on glycolipid metabolism and renal function in diabetic nephropathy model rats,and to explore the possible renal protective mechanism of liraglutide and NLRP3.Methods:1.40 SD rats were included in this study.39 rats meeting the normal blood glucose standard were enrolled in the group,and 8 rats were randomly selected as NC group.The remaining 31 rats were given high fat diet combined with Streptozotocin to induce DM model.2.The 30 rats successfully modeled were randomly divided into the diabetes group(DM group,10 rats),the low-dose melatonin treatment group(Lira-L group,10 rats)and the high-dose melatonin treatment group(Lira-H group,10 rats).According to different doses of liraglutide,the experimental SD rats were divided into Lira-L group and Lira-H group.Different doses of liraglutide were administered subcutaneously to the model group animals(Lira-L group 125?g/Kg/d,Lira-H group 200?/kg/d),for 8 weeks.3.The changes of renal pathology in experimental animals were observed.4.FBG,TG,TC,HDL-C,LDL-C,INS,BUN,SCR,and 24-hour urine microalbuminwere detectedby automatic biochemical instrument.Enzyme linked immunosorbent assay was used to detect the glycogen content of liver and muscle in each group of rats.The levels of NLRP3 and ASC in rats were detected by westernblot.Results:1.The DM experimental model was successfully prepared.The FBG level in DM group was significantly higher than that in NC group(P<0.01).2.The degree of renal lesions was significantly reduced in all Liraglutide treatment groups.The thickening degree of glomerular basement membrane was obviously improved.The degree of glomerular membrane matrix hyperplasia was significantly reduced.According to the order of lesion severity from light to heavy,the disorder in rats was NC group <Lira-H group<Lira-L group < DM group.3.At the end of 2nd,4th and 8th week,the FBG levels of Lira-L group and Lira-H group decreased compared with DM group(P<0.01).At the end of2 nd week,there was no significant difference in FBG level between Lira-L group and Lira-H group(P>0.05).At the end of 4th and 8th week,the FBG level of Lira-H group was lower than that of Lira-L group(P<0.01).4.At the end of the 8th week,compared with NC group,fasting insulin level in DM group,Lira-L group and Lira-H group increased significantly(P<0.01),and insulin sensitivity index decreased significantly(P<0.01).The difference is especially obvious in DM group.After 8 weeks of liraglutide treatment,the fasting INS level in Lira-L group and Lira-H group were lower than that in DM group(P<0.01).The fasting INS level in Lira-H group was lower than that in Lira-L group(P<0.01).The ISI levels in Lira-L group and Lira-H group were higher than those in DM group(P<0.05).The fasting ISI level in Lira-H group was lower than that in Lira-L group(P<0.01).5.At the end of the 8th week,the levels of TC,TG,and LDL-C in the DM group,Lira-L group,and Lira-H group were significantly higher than those in the NC group(P<0.05,P<0.01).There was no significant difference in HDL-C levels among DM group,Lira-L group,Lira-H group and NC(P>0.05).After 8 weeks of liraglutide treatment,the levels of TC,TG and LDL-C in Lira-L group and Lira-H group were decreased,while HDL-C was increased.The TC level in Lira-H group decreased compared with DM group(P<0.01).While,there was no significant difference in TC level between Lira-L group and DM group(P>0.05).The level of TC in Lira-H group was lower than that in Lira-L group(P<0.01).The TG level in Lira-L group and Lira-H group were lower than those in DM group(P<0.05,P<0.01).The level of TG in Lira-H group was lower than that in Lira-L group(P<0.01).There was no significant difference in LDL-C level among Lira-L group,Lira-H group and DM group(P>0.05).There was no significant difference in LDL-C level between Lira-L group and Lira-H group(P>0.05).There was no significant difference in HDL-C among Lira-L group,Lira-H group and DM group(P>0.05).The difference of HDL-C between Lira-L group and Lira-H group was no significant(P>0.05).6.At the end of the 8th week,compared with NC group,the UMA levels in DM group,Lira-L group and Lira-H group were significantly increased(P<0.01).After 8 weeks of liraglutide treatment,the UMA levels in Lira-L group and Lira-H group were significantly lower than those in DM group(P<0.01).The UMA level in Lira-H group was significantly lower than that in Lira-L group(P<0.01).7.At the end of the 8th week,compared with NC group,the levels of BUN and SCR in the DM group,Lira-Lgroup and Lira-H group were significantly increased(P<0.05,P<0.01).After 8 weeks of liraglutide treatment,compared with DM group,the BUN and SCR levels in Lira-L group and Lira-H group decreased significantly(P<0.05,P<0.01).The levels of BUN and SCR in Lira-H group were lower than those in Lira-L group(P<0.05,P<0.01).The levels of BUN and SCR in Lira-H group were lower than those in Lira-L group(P<0.05,P<0.01).8.At the end of the 8th week,compared with NC group,the hepatic glycogen levels decreased in DM group and lilalutide treatment group(P<0.01).On the contrary,the levels of muscle glycogen increased(P<0.01).After 8 weeks of liraglutide treatment,the level of glycogen in the liver of rats in each lilalutide treatment groups increased,and the difference between the lilalutide treatment groups and the DM group was statistically significant(P<0.01).The liver glycogen level of Lira-H group was higher than that of Lira-L group(P<0.01).The level of muscle glycogen in each lilalutide treatment groups decreased,and the difference between the lilalutide treatment groups and the DM group was statistically significant(P<0.01).There was no significant difference in muscle glycogen between Lira-L group and Lira-H group(P>0.05).9.At the end of the 8th week,compared with NC group,renal NLRP 3and ASC levels were significantly increased in DM group and lilalutide treatment groups(P<0.05,P<0.01).After 8 weeks of liraglutide treatment,compared with DM group,the levels of NLRP3 and ASC decreased in Lira-L group and Lira-H group(P<0.01).The levels of NLRP3 and ASC in Lira-H group were lower than those of Lira-L group(P<0.05,P<0.01).Summary:1.GLP-1 receptor agonists reduced TC,TG level and renal tissue NLRP3,ASC protein level.These effects were related to the improvement of kidney function in DM animal models.2.The improvement of fasting glucose and insulin resistance in liraglutide treatment groups were associated with inhibited liver glycogen output and increased muscle glycogen utilization.Part three Effect of short-term liraglutide therapy on serum melatonin secretion in early stage type 2 diabetic nephropathy patients with sleep disturbance and its protective effect on kidneyObjectives: To investigate the effects of short-term liraglutide treatment on melatonin secretion in early type 2 diabetic nephropathy patients with sleep disorders and its possible protective effect on the kidneys.Methods:1.In this study,134 patients with early diabetic nephropathy who met the enrollment criteria were recruited.The subjects were randomly divided into control group(DN Group)and treatment group(T Group).2.In DN group,the original treatment plan was maintained from the beginning of the experiment.In T group,liraglutide was given subcutaneously once daily on the basis of the original basic treatment regimen.The therapeutic dose of lilarutide was 0.6mg/d at week 1,1.2mg/d at week 2,1.8mg/d at week 3 and remained at 1.8mg/d thereafter.The study lasted 6months.In all patients' basic treatment regimens,the oral hypoglycemic drugs used include metformin,acarbose,and reglinide,and the types of insulin used include glargine,lispro,aspart,aspart 30.Adjust the dose of hypoglycemic drugs according to blood glucose,without adding the types of hypoglycemic drugsand other additionaldrugs besides basic treatment.The dose of hypoglycemic drugs in DN groupcan be adjusted in both positive and negative directions.When hypoglycemia occurred in T group,basic hypoglycemic drugs were reduced.3.Collect general information of patients,such as age,gender,course of disease,height,weight,calculation of body mass index,etc.4.The PSQI score,sleep duration,sleep quality score of patients before and after treatment were evaluated by PSQI scale.5.The FPG,Hb A1 c,FC-P,FINS,TG,TC,LDL-C,HDL-C,MLT,Cys C,HIF-1?,VEGF,UACR,and L-FABP levels were measured in the study subjects.The HOMA-IR of the study subjects were calculated.Results:1.The baseline were the same for both groups of patients with good comparability(P>0.05).2.The overall composition of the two groups of patients' basic treatment regimens was no different and was comparable(P>0.05).3.In the two groups of patients,there was no difference in the average daily dose of basic treatment regimens and was comparable(P>0.05).4.After 6 months of liraglutide treatment,PSQI and sleep qualityscore in T group decreased compared with DN group(P<0.01).On the contrary,sleep duration and melatonin level in T group increased compared with DN group(P<0.05,P<0.01).In DN group,PSQI decreased after treatment(P<0.01),and sleep duration increased after treatment(P<0.05).However,there was no significant difference in sleep quality score and MLT levels before and after treatment in DN group(P>0.05).In T group,PSQI and sleep quality score decreased after treatment(P<0.01),sleep duration and MLT increased after treatment(P<0.01).5.After 6 months of liraglutide treatment,FBG,Hb A1 c and HOMA-IR in T group were lower than those in DN group(P<0.05).Compared with DN group,FINS and FC-P were significantly higher in T group(P<0.05).In DN group,FBG and Hb A1 c decreased after treatment(P<0.05,P<0.01).There was no significant difference about FINS,FC-P and HOMA-IR before and after treatment(P>0.05).In T group,FBG,Hb A1 c and HOMA-IR decreased after treatment(P<0.05,P<0.01),whereas FINS and FC-P increased after treatment(P<0.05,P<0.01).6.After 6 months of liraglutide treatment,the levels of TC,TG,and LDL-C in T group decreased compared with DN group(P<0.05,P<0.01).There was no statistical difference in HDL-C level between T Group and DN group(P>0.05).There was no significant difference in TC,LDL-C and HDL before and after treatment in DN group(P>0.05).The TG level in DN group was lower than that before treatment(P<0.05).The levels of TC,TG and LDL-C in T group were lower than those before treatment(P<0.05,P<0.01).There was no significant difference in HDL-C level before and after treatment in T group(P>0.05).7.After 6 months of liraglutide treatment,serum Cys C,HIF-1?,and VEGF levels in T group decreased compared with DN group(P<0.01).The levels of Cys C,HIF-1 ? and VEGF in DN group were significantly lower than those before treatment(P<0.01).The levels of Cys C,HIF-1 ? and VEGF in T group were significantly lower than those before treatment(P<0.01).8.After 6 months of liraglutide treatment,the urine UACR and L-FABP levels in T group decreased compared with DN group(P<0.01).The UACR and L-FABP levels of urine in DN group was lower than those before treatment(P<0.05,P<0.01).The UACR and L-FABP levels of urine in T group were significantly lower than those before treatment(P<0.01).Summary:1.After short-term liraglutide treatment,the sleep quality of patients with early diabetic nephropathy improved.2.After short-term liraglutide treatment,the serum melatonin level in patients with early diabetic nephropathy increased.3.Melatonin improved sleep quality.4.Melatoninalleviated sleep disorders,improved PSQI,improved islet function,reduced insulin resistance.5.Short-term liraglutide treatment reduced kidney damage in patients with early diabetic nephropathy.Conclusion:1.Melatonin improved the oxidative stress state of renal tissue and improved renal function.This effect was related to TGF-?1-smad2/3 signal pathway,Wnt/?-Catenin signal pathway,and the treatment dose of the melatonin.2.GLP-1 receptor agonists had renal protective effect,which was related to regulation of glycolipid metabolism,inhibition of NLRP3 inflammatory bodies and reduction of ASC protein level.3.GLP-1 receptor agonists improved insulin resistance and increased insulin sensitivity by inhibiting liver glycogen output and increasing muscle glycogen utilization.4.Melatonin improved sleep qualityand reduced sleep disorders.5.After short-term liraglutide treatment,the sleep quality of patients with early Diabetic nephropathy improved,and the serum MLT level increased. |
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