芪参益气滴丸通过调控IRE1/XBP1信号通路对高血压心肌损伤的保护作用

Protective effect of Qishen Yiqi Dropping Pills on hypertensive myocardial injury by regulating IRE 1/XBP 1 signaling pathway

关键词:  芪参益气滴丸  “两肾一夹”高血压大鼠  心肌损伤  IRE1/XBP1通路

Key words:  Qishen Yiqi Dripping Pill  “two kidneys and one clip” hypertensive rat  myocardial injury  IRE1/XBP1 pathway

摘要:

[目的] 探讨芪参益气滴丸对高血压大鼠心肌的保护作用及机制。[方法] 通过“两肾一夹法”构建高血压大鼠模型，术后4周，将50只SD雄性大鼠随机分成假手术（Sham）组（手术但不狭窄左肾动脉），模型（Model）组（造模成功后灌胃等量生理盐水），芪参益气滴丸低剂量（QS-L），高剂量组（QS-H）[造模成功后分别灌胃270、540 mg/（kg·d）芪参益气滴丸]，4-苯基丁酸组[4-PBA组，造模成功后灌胃500 mg/（kg·d）4-PBA]，每组10只。每日1次，连续给药8周。术前及术后每2周通过BP2000鼠尾血压仪测量大鼠血压指标：收缩压、舒张压，每2周监测血压及心率。给药结束后测定心脏体质指数（HWI）。通过小动物超声心动图检测各组大鼠的心脏收缩功能指标：左室射血分数（LVEF）、左室短轴缩窄率（LVFS）、左室收缩末期内径（LVESD）、左室舒张末期内径（LVEDD）。酶联免疫吸附实验（ELISA）法检测各组大鼠血清中血管紧张素Ⅱ（Ang-Ⅱ）、可溶性瘤变抑制因子（sST2）、N末端B型脑钠肽前体（NT-pro BNP）、葡萄糖调节蛋白78（GRP78）含量。流式细胞术检测心肌组织活性氧（ROS）水平。苏木精-伊红（HE）染色观察各组大鼠心肌细胞形态变化，马松（Masson）染色观察心肌胶原组织结构变化，计算心肌胶原容积分数（CVF），蛋白质免疫印迹（Western blot）检测各组大鼠心肌组织IRE1、内质网应激受体蛋白（XBP1）、GRP78相关蛋白表达情况。[结果] 与Sham组相比，Model组大鼠HWI升高（P＜0.05）；收缩压、舒张压、LVEF、LVFS、LVESD、LVEDD、Ang-Ⅱ、sST2、NT-pro BNP、GRP78、ROS水平升高（P＜0.05）；HE染色可见心肌细胞排列紊乱，有炎症细胞浸润，Masson染色可见心肌细胞排列不规则、纤维组织增生、有大量蓝色胶原组织表达等病理变化，CVF增高；心肌组织P-IRE1/IRE1、XBP1、GRP78蛋白水平表达升高，差异均有统计学意义（P＜0.05）。与Model组相比，QS-H组、4-PBA组、HWI下降（P＜0.05）；4-PBA组收缩压、舒张压下降；QS-H组、4-PBA组LVEF、LVFS、LVESD、LVEDD以及血清Ang-Ⅱ、sST2、NT-pro BNP、ROS、GRP78水平下降（P＜0.05），心肌组织P-IRE1/IRE1、XBP1、GRP78蛋白表达降低，差异均有统计学差异（P＜0.05）。[结论] 芪参益气滴丸可以减轻心肌组织损伤，降低氧化应激、内质网应激水平，可能与抑制IRE1/XBP1信号通路有关。

Abstract:

[Objective] To investigate the protective effect and mechanism of Qishen Yiqi Dripping Pills on the myocardium of hypertensive model rats. [Methods] A hypertensive rat model was established by the “two kidneys one clip”（2K1C） method. Four weeks after surgery，SD male rats were randomly divided into a sham operation group（Sham group，operated but without narrowing the left renal artery），a model group（Model group，administered an equal amount of physiological saline by gavage after successful modeling），a low-dose Qishen Yiqi Dropping Pills（QS-L） group，a high-dose group（QS-H） group[administered 270 and 540 mg/（kg·d） Qishen Yiqi Drop Pills by gavage after successful modeling]，and a 4-phenylbutyric acid group[4-PBA group，administered 500 mg/（kg·d） 4-PBA by gavage after successful modeling]，with 10 rats in each group. Administer once daily for 8 consecutive weeks. HWI of rats were measured. Blood pressure indicators of rats were measured by BP2000 tail blood pressure monitor every 2 weeks before and after surgery，including systolic blood pressure（SBP） and diastolic blood pressure（DBP）. Blood pressure and heart rate were also monitored every 2 weeks. After the end of drug administration，the cardiac systolic function indicators of each group of rats were detected by small animal echocardiography：left ventricular ejection fraction（LVEF），left ventricular fractional shortening（LVFS），left ventricular end systolic diameter（LVESD），and left ventricular end diastolic diameter（LVEDD）. Enzyme linked immunosorbent assay（ELISA） was used to detect angiotensin Ⅱ（Ang-Ⅱ），soluble suppressor of tumorigenicity factor（sST2），N-terminal B-type natriuretic peptide precursor（NT-proBNP），and glucose regulated protein 78（GRP78） content in the serum of each group of rats. Flow cytometry was used to detect the levels of reactive oxygen species（ROS） in myocardial tissue. HE staining was used to observe the morphological changes of myocardial cells in each group of rats. Masson staining was used to observe the structural changes of myocardial collagen tissue. And the collagen volume fraction（CVF） was calculated. Western blot was used to detect the expression of IRE1，XBP1，and GRP78 related proteins in the myocardial tissue of each group of rats. [Results] Compared with the Sham group，the HWI in the Model rats increased significantly（P＜0.05）；the systolic and diastolic blood pressure，LVEF，LVFS，LVESD，LVEDD，Ang-Ⅱ，sST2，NT pro BNP，GRP78，and ROS levels in the Model rats increased significantly（P＜0.05）；HE staining shows disordered arrangement of myocardial cells with infiltration of inflammatory cells，while Masson staining shows pathological changes such as irregular arrangement of myocardial cells，fibrous tissue proliferation，and abundant expression of blue collagen tissue，with increased CVF. The protein levels of P-IRE1/IRE1，XBP1，and GRP78 in myocardial tissue were elevated，and the differences were statistically significant（P＜0.05）. Compared with the model group，HWI decreased in the QS-H group and 4-PBA group（P＜0.05）；the 4-PBA group showed a decrease in systolic and diastolic blood pressure；the levels of LVEF，LVFS，LVESD，LVEDD，serum Ang-Ⅱ，sST2，NT-pro BNP，ROS，and GRP78 decreased in the QS-H group and 4-PBA group（P＜0.05），and the protein expression of P-IRE1/IRE1，XBP1，and GRP78 in myocardial tissue decreased，with statistical differences（P＜0.05）. [Conclusion] Qishen Yiqi Dripping Pills can alleviate myocardial tissue damage，reduce oxidative stress and endoplasmic reticulum stress levels，which may be related to the inhibition of the IRE1/XBP1 signaling pathway.