The Mechanism Study On The Effects Of The Combined Treatment Of Tetramethylpyrazine And Aminoguanidine On Renal Functions In N0-STZ Wistar Rats

Aim:To establish an animal model of nO-strepozotocin wiswar rats(nO-STZ),neonatal wistar rats were injected intraperitoneally of streptozotocin(STZ);To investigate the mechanism and the effects on renal function of the combined treatment of tetramethylpyrazine and aminoguanidine by compared with the management of metformin.Methods:1. Animal models and group divisionsThe nO-STZ wistar rats were intraperitoneally injected with a single dose of streptozotocin(STZ) at a dose of90mg/kg. The nO-STZ wistar rats were assesed by measuring the fasting serum glucose level at8th week,whose level above7.Ommol/L were selected for the experiment.The animal models were then divided into4groups:normal control group (NC),insulin resistant group(IR), metformin treatment group(MET) and tetramethylpyrazine+andaminouguanidine treatment group(TMP+AG).2. Measurement of blood parametersFasting blood glucose(FPG),fasting insulin (FIN),Glucosylated serum protein(GSP) were measured at the8th,32nd week. According to the equation that the multiplication of FPG and FIN,which was divided into22.5,was equal to insulin resistance index(IRI),by which IRI was determined.3. Assessment of renal functionAt the32nd week.rats were shut in the metabolic cages to collect twenty-four-hour urine.Urine protein was detected with biuret test.Urine creatinine was measured by the use of automatic biochemistry analysing equipment. With creatinine clearance to represent glomerular filtration rate (GFR), GFR was attained by the equation that GFR is equal to the division with multiplication of urine creatinine and urinary volume by serunm creatinine.Then the rats were deprived of the right eye to get blood.which were centrifugated in order to obtain serum. Serum BUN and creatinine were assessed using enzymology test.4. Determination of NO concentrations,iNOS and TNOS activity in kidney homogenate.10%kidney homogenate was blended with reagents orderly under the instruction,incubated at37℃for30min.Finally the absorbance in samples were determined at490nm(for NO) and530nm(for iNOS and TNOS).5. Morphologic features of kidneyThe histological sections of kedney were dyed by hematoxylin and eosin stain to observe the changes of morphology.6. Immunohistochemical staining of iNOS、3-NTThe primary antibody against iNOS(1:50) or3-NT(1:100)with PBS as a negative control.7. Reverse transcription-polymerase chain reactionAbsorbance of amplification products of iNOS mRNA and β-actin mRNA was measured,than calculate its relative value.8. Western-blot test to detect iNOS and3-NTPrimary antibody(iNOS1:200,3-NT1:500) was incubated under4℃overnight,and secondary antibody(1:5000) was incubated under37℃1hour.Visualization was developed in a dark chamber.Results:1.Survial rates of each groupAt the32th week,the survival rates of IR group, MET group and TMP+AG group were lower than NC group(P<0.05),but MET group and TMP+AG group were higher than IR group(P<0.05).There is no difference between MET group and TMP+AG group(P>0.05).2.Results of blood parametersAt the8th week,IR group had a higher FPG,FINS,IRI and GSP than NC group(P<0.05).At the32nd week, FPG,FINS,IRI and GSP in MET and TMP+AG group was lower than IR group(P<0.05),but TMP+AG was superior to improve insulin sensitivity and had an advantage in reducing GSP(P<0.05).3.Results of renal functionCompared to NC group,IR group had a significant higher serum BUN(P<0.05),serum creatinine(P<0.05),Urine protein(P<0.05),lower GFR (P<0.05).Both MET and TMP+AG can improve renal function in n0-STZ rats (P<0.05).With contrary to MET group,serum BUN and serum creatinine in TMP+AG was even lower(P<0.05),and GFR was more powerful,which had a significant difference(P<0.05).4. Changes of NO concentration and iNOS,TNOS activityNO concentration and iNOS, TNOS activity in IR group tended to be higher compared with NC group(P<0.05).TMP+AG is better than MET at reducing NO concentration and iNOS,TNOS activity(P<0.05).5. Morphologic features of kidneyHE.staining gave the evidence that nephric tubule and glomcrulus in NC group were completed and the morphology was normal.Compared to NC group,In IR group nephric tubule was depauperateing;the range was degenerating;interstitial fibers were hyperplasying;glomcrulus were larger,the amount of mesenterium was increasing.The treatment group had a obvious improvement to IR group.Besides,the morphology was comparable between TMP+AG and NC group.6. Immunohistochemical tests of iNOS、3-NT in kidneyiNOS:The expression of iNOS in NC group was potty.IR group had a more expression than NC group(P<0.05),MET group was less compared to NC group(P<0.05).TMP+AG group was further less(P<0.05).There was no expression at glomcrulus in all four groups.3-NT:There was a little expression in nephric tubule of NC group, IR group had more and bigger fuscescent granulations than NC group (P<0.05).MET group had significantly less fuscescent granulations compared to IR group(P<0.05),the staining in TMP+AG group was less than MET group(P<0.05).7. Semi-determination of iNOS mRNA of RT-PCRIn contrast to IR group,TMP+AG group had a less expression of iNOS mRNA(P<0.05),but the distinction between MET and TMP+AG group was no significance(P>0.05).8. Western-blot of iNOS and3-NTAlthough both metformin and the combined treatment could reduce the generation of iNOS and3-NT(P<0.05),the reduction of TMP+AG were more than MET group(P<0.05),suggested the combined treatment had an advantage in decreasing the expression of iNOS and3-NT.Conclusions:l.The nO-STZ wistar rats model were successfully established by a single injection of STZ.The level of FPGsFINS、IRI and GSP was significantly increasing as the prolongation of the time,meanwhile the survival period was decreasing, renal function was severely damaged.The expression of NO,iNOS,TNOS,3-NT in renal tissues were increasing during the process of the experiment,which suggested that NO,iNOS.TNOS,3-NT played a major role in the damagement of renal function.2.The combined treatment of tetramethylpyrazine and aminoguanidine had the ability of reducing NO expression,repressing the activity of iNOS and TNOS.and decreasing3-NT production,significantly improved renal function in nO-STZ wistar rats.which implies that TMP+AG improved the renal function perhapse through accommodating the formation of NO,iNOS,TNOS,3-NT.The exact mechanism needed further investigation.3. Comparison between the combined treatment of tetramethylpyrazine and aminoguanidine and metformin gave a evidence that combined treatment was better at decreasing FPG、 FINS、 IRI and GSP,strikingly improving renal function in the condition of insulin resistance.