镇肝熄风汤对原发性高血压血管重构致脑组织病理改变的影响

【作者】 孟云辉；

【导师】 凃晋文；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2007， 博士

【摘要】 目的：通过动物和细胞培养实验，观察镇肝熄风汤对自发性高血压大鼠（SHR）脑组织及其中动脉病理改变和镇肝熄风汤含药血清对体外培养血管紧张素Ⅱ（AngⅡ）刺激所致内皮细胞凋亡和平滑肌细胞增殖的影响，探讨镇肝熄风汤改善原发性高血压脑组织病理和血管重构的机理，为该方应用于原发性高血压及其继发性损伤提供科学依据。方法：将32只14周龄的雄性SHR随机分为4组（SHR组、镇肝熄风汤高剂量组、镇肝熄风汤低剂量组、依那普利组），同时以同源雄性WKY大鼠8只作为对照组。灌胃给药8周后，采用16导生理记录系统记录收缩压；HE染色观察脑组织及其中血管病理改变；放射免疫法检测血浆和脑组织中AngⅡ、内皮素-1（ET-1）含量；逆转录-多聚酶链反应（RT-PCR）法检测脑组织中过氧化物酶体增殖物激活受体γ（PPARγ）mRNA表达。采用酶消化法体外培养人脐静脉内皮细胞（HUVECs），镇肝熄风汤含药血清作用于10^-6mol／L AngⅡ刺激的HUVECs 24h。倒置显微镜下观察HUVECs形态、密度；流式细胞术Annexin V-FITC法测定HUVECs的凋亡；酶联免疫法测定HUVECs上清肿瘤坏死因子α（TNF-α）含量；RT-PCR法测定人脐动脉平滑肌细胞（HUASMCs）PPARγmRNA的表达。采用组织贴块法培养HUASMCs，镇肝熄风汤含药血清作用于10^-6mol／L AngⅡ刺激的HUASMCs 24h。用MTT法测定HUASMCs的增殖；RT-PCR法测定HUASMCs骨桥蛋白（OPN）mRNA的表达。结果：与WKY组相比，SHR组大鼠收缩压升高（P＜0.05），脑组织神经细胞排列紊乱，弥散，失去明显分层，神经细胞数目减少，结构模糊或消失，细胞核固缩，小胶质细胞增生，个别区域可见噬神经现象，脑中小动脉管腔／管壁面积降低。镇肝熄风汤高、低剂量可降低SHR收缩压，可明显改善脑组织缺血，血管重构。与WKY组相比，SHR组大鼠血浆和脑组织中AngⅡ含量显著升高（P＜0.05）；脑组织中ET-1含量显著升高（P＜0.05）。镇肝熄风汤高、低剂量均可降低SHR血浆AngⅡ含量，脑组织中ET-1含量（P＜0.05）。镇肝熄风汤高剂量可降低SHR脑组织AngⅡ含量（P＜0.05）。与WKY组相比，SHR组大鼠脑组织中PPARγmRNA表达减弱（P＜0.05）。镇肝熄风汤高、低剂量均可使SHR脑组织中PPARγmRNA表达增强（P＜0.05）。10^-6 mol／L AngⅡ可导致HUVECs密度降低，凋亡率增加，TNF-α的分泌增加，PPARγmRNA的表达降低（P＜0.05）。镇肝熄风汤含药血清可降低HUVECs凋亡率，减少TNF-α的分泌，增强PPARγmRNA表达（P＜0.05）。10^-6 mol／L AngⅡ可促进HUASMCs A₄₉₀升高，OPN mRNA表达增强（P＜0.05）。镇肝熄风汤含药血清可降低HUASMCs A₄₉₀，抑制OPN mRNA的表达（P＜0.05）。结论：镇肝熄风汤具有降压效应，可改善高血压脑组织血管重构病理改变。推测镇肝熄风汤可能通过①增强血管内皮细胞PPARγmRNA表达，减少TNF-α释放，抑制细胞凋亡，从而保护血管内皮；②抑制血管平滑肌细胞OPN mRNA表达，抑制细胞增殖；③增强脑组织PPARγmRNA表达，降低脑组织中AngⅡ、ET-1分泌，改善由于长期血压升高、过度激活神经内分泌系统引起的脑组织血管重构病理改变。更多还原

【Abstract】 Objective: To provide the scientific basis of Zhengan Xifeng decoction on essential hypertension and following impairment, we observed the effects of Zhengan Xifeng decoction on morphological alternations of brain in spontaneously hypertensive rats （SHR） and the effects of serum containing metabolic ingredients of Zhengan Xifeng decoction on the injury of Human umbilical vein endothelial cells （HUVECs） and the proliferation of human umbilical artery smooth muscle cells （HUASMCs） stimulated by angiotensinⅡ（AngⅡ） and explored the mechanism of Zhengan Xifeng decoction on the injury of brain and vascular remolding due to essential hypertension.Methods: Thirty-two male SHR （fourteen-week-old） were divided into four groups at random: SHR group, high dose Zhengan Xifeng decoction group, low dose Zhengan Xifeng decoction group, enalapril group, compared with eight congeneric male Wistar-Kyoto rats （WKY）. After eight weeks, the systolic blood pressure was measured by sixteen leads physiologic record system. The morphological alternations of brain were observed with HE. The concentrations of AnglI and ET-1 in plasma and brain were detected by radioimmunoassay. The expression of peroxisome proliferator-activated receptorγ（PPARγ） mRNA in brain was detected by the means of reverse transcription polymerase chain reaction （RT-PCR）.The serum containing metabolic ingredients of Zhengan Xifeng decoction was taken from rats. HUVECs were cultured in vitro by collagenase digestive method. HUVECs were stimulated with 10^-6mol/L AngⅡfor twenty four hours. The morphology and density of HUVECs were observed with invert microscope. The rate of apoptosis of HUVECs was analysed by the method of flow cytometry. The content of tumor necrosis factor-alpha （TNF-α） in supernatant medium was detected with enzyme-linked immunoadsorbent assay. The expression of PPARγmRNA in HUVECs was detected by the means of RT-PCR. HUASMCs were cultured by tissue explant method. HUASMCs were stimulated with 10^-6mol/L AngⅡfor twenty four hours. The proliferation of HUASMCs was observed by MTT assay. The expression of osteopontin （OPN） mRNA was detected by the means of RT-PCR.Results: Compared with WKY group, the systolic blood pressure of SHR elevated significantly （P＜0.05） and the brain of SHR had the ischemic and vascular remolding manifestation. High or low dose Zhengan Xifeng decoction could decrease systolic blood pressure of SHR significantly, but systolic blood pressure didn’t get the normal level. High or low dose Zhengan Xifeng decoction could improve the pathological changes significantly. Compared with WKY group, the concentrations of AngⅡin plasma and brain and ET-1 in brain of SHR increased significantly （P＜0.05）. High or low dose Zhengan Xifeng decoction could decrease the concentrations of AngⅡin plasma and ET-1 in brain of SHR significantly （P＜0.05）. Compared with WKY group, the expression of PPARγmRNA in brain of SHR decreased significantly （P＜0.05）. High or low dose Zhengan Xifeng decoction could increase the expression of PPARγmRNA in brain of SHR significantly （P＜0.05）.10^-6mol/L AngⅡcould increase the rate of apoptosis and the secrection of TNF-α, decrease expression of PPARγmRNA in HUVECs （P＜0.05）. The serum containing metabolic ingredients of Zhengan Xifeng decoction could decrease the rate of apoptosis and the secretion of TNF-αin HUVECs, increase the expression of PPARγmRNA （P＜0.05）. 10^-6mol/L AngⅡcould elevate A₄₉₀ and increase the expression of OPN mRNA in HUASMCs （P＜0.05）. The serum containing metabolic ingredients of Zhengan Xifeng decoction could reduce A490 and decrease the expression of OPN mRNA in HUASMCs（P＜0.05）.Conclusion: Zhengan Xifeng decoction decreases the systolic blood pressure and improves the pathological changes and vascular remolding in brain of SHR significantly.①Zhengan Xifeng decoction could increase the expression of PPARγmRNA, decrease the releasion of TNF-αand the rate of apoptosis to protect vascular endothelial cells.②Zhengan Xifeng decoction could inhibit the expression of OPN mRNA and the prolifertion of vascular smooth muscle cells.③Zhengan Xifeng decoction could increase the expression of PPARγmRNA and decrease the concentration of ET-1 and AngⅡto improve vascular remolding and protect the brain.更多还原