【作者】 陈艺；

【导师】 方祝元；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：研究潜阳育阴颗粒对SHR大鼠血液及肾脏局部AngⅡ含量的影响,以及潜阳育阴颗粒含药血清对AngⅡ诱导HUVEC炎症反应及凋亡的影响,阐述潜阳育阴颗粒与AngⅡ导致的花生四烯酸代谢紊乱相关的防治机制,以期为中医药临床干预高血压早期肾损害提供实验依据。方法：(1)动物实验：40只SHR大鼠随机分为模型组(等量蒸馏水)、缬沙坦组(0.013g/kg/d)、潜阳育阴颗粒低剂量组(2.5g/kg/d)、潜阳育阴颗粒中剂量组(5g/kg/d)、潜阳育阴颗粒高剂量组(10g/kg/d),每组8只,均为14周龄雄性大鼠；另选8只同周龄的雄性Wister大鼠设为正常组(等量蒸馏水)。灌胃给药,每日1次,连续6周。末次给药1h后,取大鼠血清及肾脏。采用无创伤性尾动脉测压法测量各组大鼠给药前、给药3周及给药6周后的血压,全自动生化仪和放免法检测大鼠给药6周后的尿m-ALB、尿β2-MG、尿NAG含量,HE染色检测肾脏病理,ELISA法检测血液和肾脏局部的AngⅡ含量。(2)细胞实验：将SD大鼠随机分为正常组、缬沙坦组、潜阳育阴颗粒组,每组8只,灌胃给药,制备含药血清。HUVEC分为5组,除正常组外,其余组均采用10-6mol/L AngⅡ作用于对数生长期的HUVEC建立细胞凋亡损伤模型。造模后,分别给予相应含药血清干预：正常组(16%空白血清培养液)、模型组(16%空白血清培养液)、缬沙坦组(16%缬沙坦血清培养液)、潜阳育阴颗粒低剂量组(8%潜阳育阴颗粒血清培养液+8%空白血清培养液)、潜阳育阴颗粒高剂量组(16%潜阳育阴颗粒血清培养液)。①ELISA法检测NADPH氧化酶,TBA法检测MDA含量；②RT-PCR检测PPARγ-1mRNA表达,Western Blot检测PPARα-1、PPARγ-1蛋白表达；③EMSA法检测NF-κB的DNA结合活性；④RT-PCR检测AKT mRNA表达,Western Blot检测AKT-1、p-AKT-1、IκBα-1蛋白表达；⑤RT-PCR检测VCAM-1mRNA表达,Western Blot检测VCAM-1蛋白表达,ELISA法检测TNF-α、 IL-1β、IL-6;⑥流式细胞术检测HUVEC凋亡率,Western Blot检测Fas、FasL蛋白表达,免疫细胞化学法检测caspase3表达,TUNEL法检测细胞凋亡形态。结果：(1)动物实验：①潜阳育阴颗粒可以降低SHR大鼠的SBP、DBP;②潜阳育阴颗粒显著降低尿m-ALB、尿NAG;③HE染色结果显示潜阳育阴颗粒可以减轻肾小球的萎缩性纤维化及肾小管上皮细胞颗粒变性,减轻肾小动脉、细动脉的玻璃样变；④SHR大鼠血液及肾脏局部的AngⅡ含量较Wister大鼠的明显升高,潜阳育阴颗粒可以显著降低SHR大鼠血液及肾脏局部的AngⅡ含量。(2)细胞实验：①潜阳育阴颗粒含药血清显著降低AngⅡ诱导HUVEC损伤后增加的NADPH氧化酶及MDA含量,增加其损伤后低表达的PPARα-1、PPARγ-1蛋白以及PPARy-1mRNA,显著降低增加的NF-κB的DNA结合活性,降低AKT mRNA、p-AKT-1及IicBa-1蛋白的高表达,降低TNF-α、IL-1β、IL-6、VCAM-1炎症因子的释放；②潜阳育阴颗粒含药血清显著降低AngⅡ诱导HUVEC损伤后的细胞凋亡率,降低Fas、FasL、caspase3蛋白的高表达,改善细胞凋亡形态。结论：①潜阳育阴颗粒对高血压引起的早期肾脏损害具有一定的治疗及保护作用,可能与其降低血液及肾脏局部AngⅡ含量有关：②潜阳育阴颗粒可以抑制AngⅡ诱导HUVEC损伤的炎症反应,降低TNF-α、IL-1β、IL-6、VCAM-1炎症因子的表达,其机制可能与抑制NF-κB的激活有关；同时,其拮抗AngⅡ诱导HUVEC的凋亡效应,机制可能与下调fas、 fasL、caspase3蛋白表达有关。更多还原

【Abstract】 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.更多还原