阿利克仑对AGT-REN双转基因高血压小鼠肾损伤的作用研究

发布时间：2018-06-03 22:19

  本文选题：肾素-血管紧张素系统 + 非肽类肾素抑制剂　； 参考：《河北联合大学》2014年硕士论文

【摘要】：目的肾素-血管紧张素系统（Renin-angiotensin system，RAS）失调在高血压及高血压肾脏损伤的发生发展中起重要作用。肾素（renin）是启动RAS第一步的限速酶，对RAS的活化有至关重要的作用。本研究通过观察阿利克仑（Aliskiren）对人血管紧张素原-肾素（Angiotensinogen-renin，AGT-REN）双转基因高血压小鼠血压、肾损伤指标以及循环和肾脏局部RAS成分表达的变化，探讨非肽类肾素抑制剂阿利克仑对高血压及肾脏损伤的影响及其作用机制。 方法1动物分组及给药方法：12只10月龄雄性AGT-REN双转基因高血压小鼠随机分为高血压对照组（HT组）和阿利克仑治疗组（HT+Aliskiren组），每组6只，另选择背景相同的6只野生型C57B6雄性小鼠作为正常对照组（WT）。给药方法：单次给药采用灌胃经口途径，分别给予5mg/kg、10mg/kg、20mg/kg阿利克仑，监测给药后24h内血压；持续给药通过皮下埋置微量渗透泵，以20mg/kg/d对HT+Aliskiren组小鼠给予阿利克仑处理28d，其余两组给予生理盐水作为空白对照，期间监测血压，28d后取材，进行各项指标检测。2动脉血压监测：采用无创尾套法测量各组小鼠在清醒与安静状态下给药前后的平均动脉压（MAP），观察阿利克仑单次给药后24h内血压以及持续给药28d内隔日血压的变化。3肾功能损伤指标检测：①考马斯亮蓝（CBB）结合法测定24h尿蛋白含量；②全自动生化分析仪测定血清肌酐、尿素；③光镜（HE、Masson染色）和电镜观察肾脏组织病理形态改变。4氧化应激反应指标检测：化学发光法测定肾组织匀浆超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量以及蛋白免疫印迹法（Western blot, WB）检测肾组织p47phox蛋白表达变化。5RAS成分表达变化检测：ELISA法测定血清和肾组织匀浆AngⅡ和Ang(1-7)含量；WB检测肾组织ACE、ACE2蛋白表达。 结果1阿利克仑的降压效果：与WT小鼠比较，HT组小鼠MAP明显升高；单次和持续给药实验均显示，HT+Aliskiren组小鼠MAP均较HT组明显降低。2HT小鼠24h尿蛋白、血清尿素、肌酐均较WT小鼠明显升高；在HT+Aliskiren组小鼠上述肾功能损伤指标较HT组明显降低。3病理形态学结果显示：光镜下HT组小鼠肾脏实质厚度较WT组明显变薄，HT+Aliskiren组上述病理损伤得到改善。与WT小鼠比较，HT组小鼠肾小球硬化面积评分和肾间质损伤评分明显增加，HT+Aliskiren组评分结果较HT组降低。电镜下，HT小鼠肾小球基底膜增厚，足突融合甚至消失，肾小管上皮细胞胞浆内线粒体水肿、空泡变性，间质可见大量胶原纤维沉积，；HT+Aliskiren组小鼠肾脏上述病变较HT组明显减轻。4与WT小鼠比较，HT小鼠肾脏组织SOD活性降低，MDA生成增加，p47phox蛋白表达明显增多；与HT组小鼠比较，HT+Aliskiren组小鼠肾脏组织SOD活性增加，MDA的生成减少，p47phox蛋白表达明显减少。5与WT组比较，HT组小鼠ACE蛋白表达增加，ACE2蛋白表达下降，AngⅡ/Ang(1-7)比值增加；与HT组小鼠相比，阿利克仑可下调HT小鼠肾组织ACE蛋白表达，上调ACE2表达，降低血浆和肾组织AngⅡ/Ang(1-7)比值。 结论1阿利克仑具有良好的降压效果及肾脏保护作用。2阿利克仑通过抑制AngⅡ的生成，降低肾脏局部AngⅡ/Ang(1-7)比值，维持肾脏局部RAS稳态平衡。3肾素抑制剂从源头上抑制RAS激活，但对ACE2-Ang(1-7)无明显抑制作用，且可上调肾脏ACE2蛋白表达，这一作用可能与减少了AngⅡ生成有关。4阿利克仑可通过抑制NADPH氧化酶表达，，降低氧化应激水平而发挥肾脏保护作用。
[Abstract]:Objective Renin - angiotensin system ( RAS ) imbalance plays an important role in the development of hypertension and hypertensive kidney injury . Renin is a limiting enzyme for the first step of RAS , which plays an important role in the activation of RAS .

Method 1 Animal grouping and administration were divided into two groups : hypertension control group ( HT group ) and aliskiren group ( HT + Aliskiren group ) .
The mean arterial pressure ( MAP ) before and after administration of 5 mg / kg / d to HT + Aliskiren group was monitored by subcutaneous implantation of micro osmotic pump . The average arterial pressure ( MAP ) before and after administration of the rats in conscious and quiet state was measured by noninvasive method .
( 2 ) measuring serum creatinine and urea by a full - automatic biochemical analyzer ;
The activity of superoxide dismutase ( SOD ) , the content of malondialdehyde ( MDA ) and the expression of p47phox protein were detected by Western blot and WB .
The expression of ACE and ACE2 protein in renal tissue was detected by WB .

Results Compared with WT mice , MAP in HT group increased significantly compared with WT mice .
Both single and continuous administration showed that the MAP in HT + Aliskiren group was significantly lower than that in HT group . 24h urinary protein , serum urea and creatinine of 2HT mice were significantly higher than those in WT mice .
The results showed that the renal parenchyma thickness of HT group mice was significantly lower than that in WT group and HT + Aliskiren group was significantly higher than that in HT group .
Compared with WT mice , the activity of SOD in renal tissue of HT mice decreased , MDA formation increased , and the expression of p47phox protein increased significantly .
Compared with the HT group , the activity of SOD in kidney of HT + Aliskiren group increased , the expression of MDA was decreased , the expression of p47phox protein decreased significantly . Compared with WT group , the expression of ACE protein in HT group increased , the expression of ACE2 protein decreased , and the ratio of Ang 鈪

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