基于AVP-V2R-AQP2通路探讨益气活血方干预慢性心力衰竭水潴留的实验研究

Experimental study on the intervention of Yiqi Huoxue Prescription on water retention in chronic heart failure based on AVP-V2R-AQP2 pathway

关键词:  益气活血方  慢性心力衰竭  水通道蛋白2  水钠潴留

Key words:  Yiqi Huoxue Prescription  chronic heart failure  aquaporin 2  water and sodium retention

摘要:

[目的]观察益气活血方对慢性心力衰竭（CHF）大鼠心脏及肾脏结构的影响，并从水的重吸收角度开展机制研究。[方法]对Sprague-Dawley（SD）大鼠进行麻醉处理后，通过左侧冠状动脉前降支结扎方法建立CHF模型。造模4周后，对大鼠进行分组，随机分为假手术组、模型组、卡托普利组及益气活血方低、中、高剂量组。卡托普利组给予卡托普利片13.5 mg（/kg· d），益气活血方各剂量组分别给予益气活血颗粒剂2.5、5、10 g（/kg· d），假手术组及模型组给予等量蒸馏水灌胃，持续灌胃8周。干预结束后采用小动物彩色多普勒超声分析大鼠心脏结构及功能；采集血清检测氨基末端脑利钠肽前体（NT-proBNP）表达水平；采用苏木精-伊红（HE）染色观察大鼠的心肌纤维化水平及肾脏上皮细胞形态；染色后对大鼠肾小管上皮细胞进行分析；通过免疫组织化学方法分析大鼠肾脏水通道蛋白2（AQP2）、pS256-AQP2分布和表达水平；通过酶联免疫吸附（ELISA）法检测血清精氨酸加压素（AVP）水平；通过蛋白免疫印迹（Western Blot）法检测肾脏AQP2、pS256-AQP2、抗利尿激素V2型受体（V2R）表达水平。[结果]与假手术组比较，模型组大鼠射血分数（EF）降低（P<0.01），血清NT-proBNP水平上升（P<0.01）；模型组大鼠的心肌形态不规则，出现明显的炎性浸润，肾小管管腔狭窄，模型组AQP2、pS256-AQP2阳性面积高于假手术组，AQP2、AVP、V2R表达上升（P<0.01）。益气活血方各剂量组大鼠EF高于模型组（P<0.01），血清NT-proBNP水平较模型组降低（P<0.01）；心肌组织基本保持规则，对应的坏死和凋亡不明显；各给药组大鼠肾小管未见明显异常，肾细胞轻微水肿。卡托普利组、益气活血方中、高剂量组中AQP2、pS256-AQP2阳性表达面积降低（P<0.01或P<0.05），各给药组均能不同程度下调AQP2、pS256-AQP2、AVP、V2R蛋白表达水平。[结论]益气活血方能够通过改善肾脏调节机制，进而调控肾脏对水的重吸收效应，改善心脏结构及功能，延缓CHF进程，其机制可能与调节AVP-V2R-AQP2信号通路的相关因子表达相关。

Abstract:

[Objective] To observe the effects of Yiqi Huoxue Prescription on the structure of heart and kidney in rats with Chronic heart failure（CHF），and to study the mechanism from the perspective of water reabsorption. [Methods] Sprague-Dawley（SD） rats were anesthetized and the CHF model was established by ligation of the anterior descending branch of the left coronary artery，4 weeks later，the rats were randomly divided into sham operation group，model group，captopril group and Yiqi Huoxue Prescription low-dose，medium-dose and high-dose groups. Sham operation group and model group were given the same amount of distilled water by instillation. In the administration group，captopril tablets were given 13.5 mg/（kg·d），Yiqi Huoxue Prescription 2.5，5，10 g/（kg·d）， and continued gavage for 8 weeks，respectively. After the intervention，the heart structure and function of rats were analyzed by color Doppler ultrasound；serum was collected to detect the expression level of N-terminal pro brain natriuretic peptide（NT-proBNP）；Hematoxylin-eosin（HE） staining was used to observe the level of myocardial fibrosis and the morphology of renal epithelial cells. The rat renal tubular epithelial cells were analyzed after staining. The distribution and expression levels of aquaporin 2（AQP2） and pS256-AQP2 in rat kidney were analyzed by immunohistochemistry；serum argininevasopressin AVP levels were detected by Enzym-Linked ImmunoSorbent Assay（ELISA）；the expression levels of renal AQP2，pS256-AQP2 and V2 vasopressin receptor（V2R） were detected by Western Blot. [Results] Comparedwith sham operation group，ejection fraction（EF） in model group was significantly decreased（P<0.01），and serum NT-proBNP level was increased（P<0.01）. The myocardial morphology of the model group was irregular，with obvious inflammatory infiltration and renal tubule stenosis，the positive area of AQP2 and pS256-AQP2 in the model group was higher than that in the sham operation group，and the expressions of AQP2， AVP and V2R were significantly increased（P<0.01）. EF in Yiqi Huoxue groups was higher than that in model group （P<0.01），and serum NT-proBNP level was lower than that in model group（P<0.01）；the myocardial tissue kept regular， and the corresponding necrosis and apoptosis were not obvious；the positive expression area of AQP2 and pS256- AQP2 in captopril group，Yiqi Huoxue medium dose group and Yiqi Huoxue high dose group decreased（P<0.01 or P<0.05），and the protein expression levels of AQP2，pS256-AQP2，AVP and V2R in each administration group could be reduced to different degrees. [Conclusion] Yiqi Huoxue Prescription can regulate the renal reabsorption of water，improve the cardiac structure and function，and delay the progression of CHF by improving the renal regulatory mechanism. The mechanism may be related by regulating the expression of related factors in the AVP- V2R-AQP2 signaling pathway.