Research On The Effect And Mechanism Of Puerarin On AGEs In Serum And Retina Of STZ-induced Diatetic Rats

ObjectiveNon enzymatic glycation of proteins is closely related to diabetes and diabetic microvascular complications.Advanced Glycation End Products(AGEs)is a mass of big molecular substances,which were usually formecd by non-enzymatic glycation reaction of proteins,lipids and nucleic acid,and play a vital role in the onset and development of diabetes mellitus(DM).A large body of researches indicates that Puerarin can decrease the expression of AGEs in DM rats and therefor protect it from being destroyed.In this study,we will analysis the impact of Puerarin on the expression of AGEs in serum and retina of streptozotocin(STZ)-induced diabetic rats as well as for the exploration of molecular mechanism for clinical diagnosis and treatment of Diabetic Retinopathy(DR).Methods1.150 male SD rats are randomly divided into 6 groups.Control groups(1mons,3mons);DM model groups(1mons,3mons)and Puerarin intervention groups(1mons,3mons),there are 25 rats in each group.2.With the administration of 2% STZ(55mg/kg)to rats by intraperitoneal injection,DM rats model are built.The control group only received the same dose of cirtic acid solution.Testing the fasting blood glucose concentration after the administration 72 h and 1w later.We define the STZ-induced diabetic rat as the concentration of fasting blood glucose ≥16.7mmol/L both 72 h and1w after the administration.Puerarin(80mg/kg)is given to the intervention group by oral administration per day.3.After the administration of 1mon and 3mons,all the rats are sacrificed.Taking blood from the heart and separating retina as quickly as possible under low temperature environment.Part of retinal tissue section is made for the observation under light microscope and electron microscope.The rest of retinal tissue and blood were store in refrigerator with-80℃。4.Making preparation for the retinal homogenate and purifying serum and then detecting the AGEs in them with the ELISA method.5.The concentration of total protein in retinal tissue and serum is detected with the method of BCA protein assay and analyzing the expression of AGEs in retinal tissue and serum of each groupvia Western-blot.At last,the expression of AGEs were analyzed with the method of ANOVA by the software of Image Lab(P < 0.05 means there is statistically significant difference).Results1.Compared with control group,the weight of rats in both DM group(D1,D3)and puerarin intervention group(P1,P3)were significantly decreased,and there was statistical significance(P <0.05);there was no statistical difference between some groups(D1 vs D3,P1 vs P3,DM group vs prerarin intervention group).2.Compared with control group,the blood glucose of rats in both DM group(D1,D3)and puerarin intervention group(P1,P3)were significantly increased,and there was statistical significance(P < 0.05).At the same time,the blood glucose increased higher in DM group than that in prerarin intervention group(D1vs P1:27.90±1.79 vs 25.39±2.45;D3vs P3:30.33±1.69 vs25.54±2.00,P <0.05).3.The pathological changes of retina:There was no significant difference among control group,group D1 and P1.The changes were the most severe in group D3.The number of retinal cells in each layer decreased,cell structure disordered and damaged severely;capillary dilatation and congestion phenomenon appeared in D3.All the pathological changes have relieved in group P3.4.The result of ELSA quantitation of AGEs in serum and retina:the concentration of AGEs was significantly higher in both DM group(D1,D3)and puerarin intervention group(P1,P3)than that in control group(N1,N3),and there was statistically significant difference(P < 0.05).The concentration of AGEs in D3 was the highest among all the groups(P < 0.05).The concentration of AGEs was higher in D1 than that in P1,but the difference was no significance(P > 0.05).The concentration of AGEs was significantly higher in D3 than that in P3(P < 0.05).5.1-DE/Western of serum:There were 5 kind of AGEs isolated from serum(strip1-5).The expression of AGEs was significantly higher in both DM group(D1,D3)and puerarin intervention group(P1,P3)than that in control group(N1,N3)among these 5 strips,and there was statistically significant difference(P < 0.05).The concentration of two kind of AGEs(strip1,3)were much higher in D1 than that in P1(P < 0.05);The concentration of three kind of AGEs(strip1,3,4)were much higher in D3 than that in P3(P < 0.05).6.1-DE/Western of retina:There were 4 kind of AGEs isolated from retina(strip1-4).The expression of AGEs was significantly higher in both DM group(D1,D3)and puerarin intervention group(P1,P3)than that in control group(N1,N3)among 3 kind of AGEs(strip1,3,4),and there was statistically significant difference(P < 0.05).The concentration of AGEs in D3 were much higher than that in P3 among all the AGEs(P < 0.05).There was no statistically significant difference about the expression of all the AGEs between D1 and P1(P >0.05).Conclusions1.The expression of AGEs was significantly up-regulated in both serum and retina in DM rats,which increased with the extension of the duration of diabetes.2.The intervention of puerarin can reduce the blood glucose of DM rats and relieve the pathological damage of retina in DM rats.3.The intervention of prerarin can reduce the total concentration of AGEs in both serum and retina of DM rats and down-regulate the expression of some of AGEs in serum and retina,and the intensity of it is closely related to its working time.