Experimental Studies Of The Effects Of Puerarin On The Diabetic Vascular Complications And Its Mechanisms

Part1 Effects and mechanisms of puerarin on Diabetic VascularComplications ratsObjective: In order to explore the effects and mechanisms of puerarin on Diabetic vascular complications(DVC) rats. The fasting blood glucose (FBG), fasting insulin (FINs) and insulin sensitivity index(ISI); fructosamine(FMN) and urinary microalbumin(MAU); nitric oxide (NO) and endothelin (ET); intercellular adhension molecule-l(sICAM-1), tumor necrosis factor-a (TNF-a) and oxidized-LDL (ox-LDL); the formation of AGEs and the transcription of RAGE mRNA in aorta, the aspect of morphology in diabetic vascular complications rats induced by streptozotocin were investigated. Method: Rats that range from 180g to 220g were administered after injecting streptozotocin into intraperitonel with a dose of 60mg kg-1. Four days later, blood glucose was measured by glucose oxygen kit. On the basis of present documents, the rats that blood glucose was over 16.7mM were accepted as diabetic ones, and then were divided randomly into five groups: diabetic mellitus(DM), aminoguanidine(AG 0.1g kg-1, ig.), puel(0.5g kg-1, ig.), pue2(0.25g kg-1, ig.), pue3(0.125g kg-1, ig.) and a normal group additionally. In order to set up a chronic model, we observed 12 weeks according to our past experiments. The FBS, FINs, ISI, FMN and MAU were measured at the eighth and twelfth week using the test kit. NO and NOS in serum were determined by checking their OD values, ET and ANF in plasma were detected by radio-immunization. SICAM-1 and ox-LDL were measured by ELISAkit, and TNF-a was measured by radio-immunization. According to the characteristic that AGEs could emit fouorescence, typeV collagenase was used to digest aorta. After a series of washing, centrifugation, and some other steps, the digestion was used to detect the fluorescence intensity on spectrofiuorometer with exciting wavelength at 370nm and emission wavelength at 440nm. For the quantitative of AGEs, content of hydroxyproline was measured by the text kit. An arbitrary unit was specified for the fluorescence intensity every milligram hydroxyproline. Then the AGEs were expressed by AU.mg-1 hydroxyproline. RAGE mRNA transcription level was determined by semi-quantitativeRT-PCR. For observing the pathological changes of thoracic aorta endothelium and kidney, light microscope and transmission electron microscope (TEM) was adopted to study its permeability. Result: After 12 weeks duration, every sample of the DVC rats showed apparent pathological changes as follows: vascular endothelial cells swelled and disorderly arranged, uneven surface of endothelium, increased adherence of blood cells to the endothelium. As to the kidney, the damage of renal glomeruli was found to be significant. Its volume decreased accompanied with diffused mesangial proliferation. The GBM turned to be a stria medullaris-like thickened shape. The glomus capillary appeared to be occlusive but the sacculus dilated. The renal tubule exhibited to be hyaline degeneration. The small artery and afferent arteriole also showed thickened appearance and hyaline degeneration, but those treated with all dosage puerarins had improved diabetic symptoms and ISI, decreased FBG, FINs, inhibited formation of FMN and the excretion of urinary microalbumin obviously(P<0.05). AG and Puel, 2 groups all showed an increase of NO and NOS levels and a decrease of ET and ANF values, while puerarin 1 and AG could down-regulate all of them significantly(P<0.01). Pue 1 and 2 lowered AGEs accumulation in the aorta significantly(P<0.01). High RAGE mRNA transcription level in the aorta of DVC rats was also down-regulated by pue 1. And all the pathological changes above were ameliorated by puerarin, which suggested that the component could protect the endothelium from damaging by the diabetic state. Conclusion: Through decreasing blood glucose,improving insulin sensitivity; restoring partially the dynamic equilibrium between NO and ET, ET and ANF, decreasing too high serum levels of sICAM-1, TNF-a and ox-LDL, down-regulating excessive gene expression of...