Effect Of QianYangYunYin Granulars On AngⅡ-mediated Endothelial Inflammatory Response And Apoptosis

AIM:To investigate the effect of QianYangYuYin(QYYY) granules on the levels of Angiotensin Ⅱ in the blood and renal tissues of Spontaneously Hypertensive Rats(SHRs) and discuss the effect of the medicated serum of QYYY granules on Angiotensin Ⅱ-induced apoptosis and inflammatory reaction in Human Umbilical Vein Endothelial cells(HUVECs). Elaborate the influence and possible protective mechanism of QYYY granules by curing the metabolic disturbance of arechidonic aid. To look forward a experimental evidence of inchoate intervention for the Chinese medicine.Methods:(1)40of SHRs were randomly divided into model group(the same amount of distilled water), group of Valsartan(0.013g/kg/d), low dose group of QYYY granules(2.5g/kg/d), medium dose group of QYYY granules(5g/kg/d) and high dose group of QYYY granules(10g/kg/d),8only each group.They are all14weeks old male rats. Choose another8of the same aged male Wister rats as control group (the same amount of distilled water).Rats were filled into the stomach with different medicines according to the different groups, once daily for6weeks. The kidneys and blood serum were taken out1hour later after the last administration in rats of each group. The non-invasive measurement of blood pressure by caudal artery of rats before treatment, after3weeks of administration and after6weeks of administration. Urinary micro-albumin, urinary β2-micro-globulin and urinary N-acetyl-glucosaminidase were determined by full automatic biochemical analyzer and radioimmunoassay. The pathologic changes of kidneys were tested by hematoxylin-eosin staining. The levels of Angiotensin Ⅱ in the blood and renal tissues of SHRs were determined by enzyme linked immune-sorbent assay.(2)SD rats were randomly divided into normal group, group of Valsartan, group of QYYY granules,8only each group. Rats were filled into the stomach with different medicines according to the different groups. Then the medicated serum of each group was prepared. HUVECs were divided into5groups. Except the normal group, the damaged model of cultured HUVECs were induced by10-6mol/L Angiotensin Ⅱ. At the same time, each medicated serum was used to intervene.①NADPH were determined by enzyme linked immune-sorbent assay, and the content of MDA was determined by the method of thibabituric acid.②The gene expression of PPARy-1was determined by reverse transcription polymerase chain reaction. Western blotting technology was used to detect the protein expression of PPARa-1, PPARy-1. ③DNA binding activity of NF-κB was tested by electrophoretic mobility shift assay.④TThe gene expression of AKT was determined by reverse transcription polymerase chain reaction. Western blotting technology was used to detect the protein expression of AKT-1, p-AKT-1, IκBα-1.⑤) The gene expression of VCAM-1was determined by reverse transcription polymerase chain reaction. Western blotting technology was used to detect the protein expression of VCAM-1. TNF-α, IL-1β and IL-6were determined by enzyme linked immune-sorbent assay.⑥The apoptosis rate of endothelial cells was assessed by flow cytometry with Annexin V/PI staining. Western blotting technology was used to detect the protein expression of Fas and FasL. The protein expression of caspase3was detected by immunocytochemistry. The apoptotic morphology of endothelial cells was tested by TUNEL.Results:(1) QYYY granules could make systolic blood pressure and diastolic blood pressure of SHRs decreased significantly.It could reduce urinary micro-albumin and urinary N-acetyl-glucosaminidase of SHRs significantly. Light microscopy revealed QYYY granules could reduce atrophic fibrosis of glomerulus, granular degeneration of epithelia of renal tubular and pathological changes of renal arteriole. The levels of Angiotensin Ⅱ in the blood and renal tissues of SHRs were higher than Wisters’. QYYY granules could decrease the content of Angiotensin Ⅱ significantly.(2) After HUVECs were damaged by10-6mol/L Angiotensin Ⅱ, the content of MDA and NADPH could be decreased by the medicated serum of QYYY granules. The expression of PPARa-1, PPARy-1were obvious increased by QYYY granules. DNA binding activity of NF-κB could be reduced obviously by the medicated serum of QYYY granules. It was also can decrease the expression of AKT, p-AKT-1and IκBα-1, then it reduce the expression of inflammatory factor as TNF-α, IL-1β, IL-6, VCAM-1. QYYY granules decreased apoptosis-positive rate and the expression of Fas, FasL, caspase3.It also make the apoptotic morphology of endothelial cells changed obviously.Conclusion:QYYY granules have a therapeutic and protective effect on early renal damage of hypertension to some extent. QYYY granules could significantly inhibit inflammatory reaction of HUVECs induced by Angiotensin II, and could decrease the expression of inflammatory factor as TNF-α, IL-1β, IL-6, VCAM-1. It may be related to the activation of NF-κB. These results indicate that QYYY granules could significantly inhibit apoptosis of HUVECs induced by Angiotensin Ⅱ, it may be related to lower protein expression of fas, fasL, caspasse3.