The Mechanism Study On The Prolongation Of Survival Period Effect Of The Combined Therapy With Tetramethylpyrazine And Aminoguanidine On Insulin Resistant Rats

Objective:To establish an animal modal of insulin resistant in newborn Wistar rats induced by streptozotocin(STZ)and to observe its survival rate in 32 weeks,to explore the mechanism on the prolongation of survival period effect of the combined therapy with tetramethylpyrazine and aminoguanidine.Methods:1.Animal and group divisionThe insulin resistant model of newborn Wistar rats was duplicated with streptozocin(90mg/kg).The rats were raised for 8 weeks,and those whose fasting plasma glucose higher than or equal to 7.0mmol/L or whose 2 hours postprandial blood glucose higher than or equal to 11.1 mmol/L in glucose tolerance test were selected.Rats were divided into 4 groups randomly: normal control(CN)group,insulin resistant(IR)group,the dimethybiguanide(MET)group,and the combination of tetramethylpyrazine and aminoguanidine(TMP+AG)group.2.The measurement of fasting blood glucose(FPG),fasting insulin(FINS) and glucosylated serum protein(GSP)MeasurementFPG,FINS and GSP were detected in 8^th,24^th,32^thweeks after treatment from the tail vein,insulin resistance index(IRI)was count by calculation formula of FPG×FINS/22.5.3.Measurement of blood biochemical indexAt the end of the experiment,the serum total cholesterol(TC), triglyceride(TG),blood urea nitrogen(BUN),creatinine(Cr)were detected.4.In situ hybridization Test Detection of Klotho and insulin like growth factol-1 receptor (IGF-1R)mRNA in the peripheral blood leukocytes by in situ hybridization.5.Histological and Morphological AnalysesThe hematoxylin-eosin(HE)staining and periodic acid Schiff(PAS) staining were applied in the pathological observation.6.Immunohistochemistry stainingKlotho and phosphated IGF-1R protein were detected by immunohistochemistry staining.7.Real-Time Fluorescent Quantitative Polymerase Chain Reaction (RT-PCR)The mRNA levels of advanced glycation end products receptors (RAGE),IGF-1R and Klotho were quantified by RT-PCR using SYBR Green technology.Results:1.Survival rate of each groupAt the end of the experiment,the Survival rate in IR group,MET group, TMP+ AG group and CN group were 35%,70%,85%and 100%respectively.The survival rate in CN group was significantly higher than that in the other three groups(P＜0.05),The survival rate in MET group and TMP+ AG group were higher than IR group(P＜0.05),but there was no statistically difference between the two treated groups.2.FPG,FINS and GSP ResultThe FPG,FINS,IRI and GSP in modal groups were higher than CN group at the age of 8 weeks(P＜0.05);The index above in MET group and TMP+ AG group were lower than IR group at the age of 16 weeks(P＜0.05),higher than CN group(P＜0.05);At the age of 32 weeks,The FPG and FINS in MET group and TMP+ AG group were more decreased compared with IR group at the age of 24 weeks(P＜0.05),the FPG had no significance with CN group,FINS and IRI in the two groups were higher than CN group(P＜0.05),there was no significant difference in GSP levels between TMP+ AG group and CN group.3.Blood biochemical index Result The differences of serum TG,TC and Cr among groups were insignificant, the BUN in MET group and TMP+AG group was decreased significantly compared to IR group.4.In situ hybridization ResultThe positive rate of IGF-1R mRNA of IR group was significantly lower than CN group(P＜0.05),which was higher in IR group than that in TMP+AG group(P＜0.05),and lower than that in MET group(P＜0.05).The positive rate of Klotho mRNA of IR group was significantly lower than CN group (P＜0.05).The expression of Klotho mRNA in both MET group and TMP+AG group were higher than IR group(P＜0.05),which is higher in TMP+AG group compared to MET group(P＜0.05).5.Histological and Morphological AnalysesHE staining showed that two cases appeared slight mesangial cells hyperplasia and the others showing no abnormal.The epithelial cells of renal tubule occurred a little of glycogen deposition in PAS staining in IR group.6.Immunohistochemistry ResultThe expression of IGF-1R in the distal convoluted renal tubule and collecting duct from IR group was significantly lower than CN group (P＜0.05),IGF-1R expression in both MET group and TMP+AG group was higher than IR group(P＜0.05).There was no significant difference between TMP+ AG group and CN group.The IGF-IR of MET group is higher than that of CN group.The Klotho expression from IR group was deceased compared with CN group(P＜0.05),which was increased in both MET group and TMP+AG group compared to IR group(P＜0.05),and no differences in the two treated groups were found.7.RT-PCR Analyses(1)The expression of RAGE mRNAThe expression of RAGE mRNA of IR group in renal tissues was increased significantly compared to CN group(P＜0.05),which was higher than that of TMP+ AG group(P＜0.05),and there was no statistical difference between TMP+ AG group and CN group.The RAGE mRNA of MET group was lower than that of IR group(P＜0.05),higher than that of CN group(P＜0.05).(2)The expression of IGF-1R mRNAThe expression of IGF-IR mRNA in brain,hepatic tissues and peripheral blood lymphocytes from IR group was significantly lower than CN group (P＜0.05),which was increased in brain and hepatic tissues of TMP+ AG group than that of IR group(P＜0.05),but decreased in blood lymphocytes than IR group(P＜0.05).No statistical differences in the expression of IGF-1R mRNA in renal tissues of CN group,MET group and TMP+ AG group were found.IGF-1R mRNA expression of MET group in renal,brain tissues and blood lymphocytes were significantly increased than IR group expression (P＜0.05).(3)The expression of Klotho mRNAThe Klotho mRNA in renal,brain tissues and peripheral blood lymphocytes from IR group was significantly lower than that of CN group (P＜0.05),which was increased in renal,brain tissues from TMP+AG group compared with IR group(P＜0.05).No differences of Klotho was detected in blood lymphocytes between TMP+ AG group and IR group.The Klotho mRNA in renal,brain tissues and peripheral blood lymphocytes from MT group was significantly increased than IR group(P＜0.05),which was higher in renal tissues than that in TMP+ AG group(P＜0.05),but lower in brain tissues than that in TMP+ AG group(P＜0.05).Conclusions:1.The insulin resistant modal of newborn was established with streptozotocin intraperitoneally.The level of FPG,FINS,IRI and GSP were increased with the time going on.Meanwhile the survival period was decreased.The expression of Klotho mRNA in several tissues were decreased in this model,which suggested that the high morality of IR rats was closely related with Klotho expression.2.Combination of tetramethylpyrazine and aminoguanidine can upregulate the expression of Klotho mRNA in several tissues of IR rats, inhabite the expression of RAGE mRNA of renal tissues,and significantly prolonge survival period.The results indicated that the mechanism of TMP+AG for the prolongation of survival period was related to regulating the mRNA level of Klotho and RAGE.The exact mechanism needs to be further explored.3.The expression of IGF-1R mRNA was decreased in several tissues,which indicated that the mechanism of shortening survival period of IR rats was associated with down-regulated of IGF-1R mRNA.The expression of IGF-1R mRNA changed differently in different tissues.It was not found that the mechanism of TMP+AG for the prolongation of survival period was directly related with regulating the mRNA level of IGF-1R.