贝那普利通过下调心锚重复蛋白改善心肌梗死后心肌重塑

doi:10.3877/cma.j.issn.1674-6880.2023.04.005

目的

探讨贝那普利在改善大鼠心肌梗死后心肌重塑中与心锚重复蛋白（CARP）的相关性。

方法

将70只Sprague-Dawley大鼠分为假手术组（20只）、心肌梗死组（25只）和贝那普利组（25只）。术后7 d和28 d，采用随机数字表法每组各时间点分别纳入8只大鼠。心肌梗死组和贝那普利组大鼠按照结扎冠状动脉前降支的方法制作大鼠心肌梗死模型，假手术组麻醉开胸穿线不结扎。贝那普利组采用贝那普利10 mg·kg^-1·d^-1（溶于5 mL/kg等渗NaCl溶液）灌胃，假手术组和心肌梗死组分别采用等量等渗NaCl溶液灌胃。分别于喂养7 d和28 d后，处死大鼠后取出心脏，测定左心室重量指数（LVWI）和心肌细胞横截面积。采用苏木素-伊红（HE）染色法观察各组大鼠心肌组织病理学特征，最后分别采用酶联免疫吸附测定（ELISA）法和免疫组织化学法检测大鼠血清及心肌细胞中CARP和血管紧张素Ⅱ（AngⅡ）表达水平。

结果

HE染色结果显示：术后7 d和28 d，假手术组大鼠心肌结构均完整，细胞排列均整齐，细胞核均位于细胞中央，染色均匀，无肌丝断裂；心肌梗死组大鼠梗死区心肌细胞严重变性和坏死，肌丝排列紊乱，炎症细胞浸润，大量胶原纤维增生，非梗死区心肌细胞代偿性肥大；贝那普利组大鼠心肌细胞病变较轻，炎症细胞浸润较少。心肌梗死组和贝那普利组大鼠28 d时梗死区心肌纤维化均较7 d时更为明显。术后7 d和28 d，3组大鼠LVWI（F = 25.111，P<0.001）、心肌细胞横截面积（F = 566.062，P<0.001）以及血清和心肌细胞中CARP（F = 90.384、23.642，P均<0.001）、AngⅡ（F = 97.764、116.133，P均<0.001）水平比较，差异均有统计学意义。进一步两两比较发现，术后28 d，心肌梗死组心肌细胞横截面积以及血清CARP、AngⅡ和心肌细胞中AngⅡ水平均较假手术组和贝那普利组显著升高（P均<0.05）。免疫组织化学法结果显示，CARP和AngⅡ主要在细胞质中表达，假手术组大鼠心肌细胞中黄色和棕色较浅，CARP和AngⅡ阳性表达率较低；心肌梗死组大鼠满布黄色和棕色颗粒，表达较高；贝那普利组阳性表达率介于假手术组和心肌梗死组之间。Pearson相关分析结果显示，术后7 d和28 d，大鼠血清（r = 0.796、0.894，P均<0.001）和心肌（r = 0.880、0.807，P均<0.001）中CARP与AngⅡ均存在正相关性。

结论

心肌梗死后血清及心肌组织中CARP和AngⅡ明显升高，均参与了心肌梗死后心肌重塑，且二者存在正相关性。贝那普利改善心肌梗死后心肌重塑可能与下调CARP有关。

关键词: 心锚重复蛋白, 血管紧张Ⅱ, 心肌梗死, 心肌重塑

Objective

To investigate the relationship between the improvement of myocardial remodeling by Benazepril and the expression of cardiac ankyrin repeat protein (CARP) after myocardial infarction in rats.

Methods

A total of 70 Sprague-Dawley rats were randomly divided into a sham surgery group (n = 20), a myocardial infarction group (n = 25) and a Benazepril group (n = 25). Eight rats were included at 7 days and 28 days after surgery in each group using random number tables. Rats in the myocardial infarction group and Benazepril group were induced by ligating the anterior descending coronary artery, while rats in the sham surgery group were anesthetized with thoracotomy and threaded without ligation. In the Benazepril group, 10 mg·kg^-1·d^-1 Benazepril (dissolved in 5 mL/kg isotonic sodium chloride solution) was used for gavage. Rats in the sham surgery group and myocardial infarction group were treated with 10 mg·kg^-1·d^-1 isotonic sodium chloride solution. After feeding for 7 days and 28 days, the hearts were removed after the rats were sacrificed, and the left ventricular weight index (LVWI) and cardiomyocyte cross-sectional area were determined. The characteristics of myocardial histopathology were observed by hematoxylin-eosin (HE) staining, and the expression levels of CARP and angiotensin Ⅱ (AngⅡ) in the serum and cardiac myocytes were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry respectively.

Results

HE staining showed that at 7 days and 28 days after surgery, the myocardial structures of rats in the sham surgery group were intact, the cells were neatly arranged, and the nuclei were located in the center of the cells, with uniform staining and no myofilament break. In the myocardial infarction group, cardiomyocytes were severely degenerated and necrotic in the infarct area with disordered myofilament arrangement, inflammatory cell infiltration and massive collagen fiber hyperplasia, and cardiomyocytes were compensatorily hypertrophied in the non-infarcted area. Rats in the Benazepril group had milder cardiomyocyte lesions and less inflammatory cell infiltration. Myocardial fibrosis in the infarct zone was more obvious in the myocardial infarction group and Benazepril group at 28 days than at 7 days after surgery. After 7 days and 28 days postoperatively, the levels of LVWI (F = 25.111, P < 0.001), cardiomyocyte cross-sectional area (F = 566.062, P < 0.001), and CARP (F = 90.384, 23.642; both P < 0.001) and AngⅡ (F = 97.764, 116.133; both P < 0.001) in serum and cardiomyocytes were compared among the three groups, and the differences were statistically significant. After 28 days after surgery, the levels of cardiomyocyte cross-sectional area, serum CARP and AngⅡ, and cardiomyocyte AngⅡ in the myocardial infarction group were significantly higher than those in the sham surgery group and Benazepril group (all P < 0.05). The immunohistochemistry showed that CARP and AngⅡ were mainly expressed in the cytoplasm. In the sham surgery group, the myocardial cells were lighter in yellow and brown, and the positive rates of CARP and AngⅡ were lower. Rats in the myocardial infarction group were covered with cloth yellow and brown granules and had higher positive expression. The positive expression rate in the Benazepril group was intermediate between the sham surgery and myocardial infarction groups. In addition, the expression levels of CARP and AngⅡ in cardiomyocytes at 7 days in the myocardial infarction group and Benazepril group were higher than those at 28 days. Pearson correlation analysis showed that there were positive correlations between CARP and AngⅡ in serum (r = 0.796, 0.894; both P < 0.001) and myocardium (r = 0.880, 0.807; both P < 0.001) at 7 days and 28 days postoperatively.

Conclusions

After myocardial infarction, CARP and AngⅡ are significantly elevated in serum and myocardial tissue and both are involved in its myocardial remodeling, and there is a positive correlation between them. Improvement of myocardial remodeling after myocardial infarction by Benazepril may be related to the down-regulation of CARP.

Key words: Cardiac ankyrin repeat protein, Angiotensin Ⅱ, Myocardial infarction, Cardial remodeling

图1 各组大鼠心肌组织病理图（HE染色　× 100）注：HE.苏木素-伊红；a图为术后7 d假手术组，可见大鼠心肌细胞排列整齐，未见异常；b图为术后7 d心肌梗死组，可见梗死区心肌细胞严重变性坏死，炎症细胞浸润，大量胶原纤维沉积，非梗死区心肌细胞代偿性增生肥大；c图为术后7 d贝那普利组，可见梗死区心肌细胞变性坏死较心肌梗死组轻，部分胶原纤维沉积；d图为术后28 d假手术组，可见大鼠心肌细胞排列整齐，偶见炎症细胞；e图为术后28 d心肌梗死组，可见梗死区心肌细胞严重变性坏死，胶原纤维沉积较7 d时更严重；f图为术后28 d贝那普利组，可见梗死区心肌细胞变性坏死，胶原沉积，且较7 d时明显

表1 各组大鼠间LVWI和心肌细胞横截面积的比较（ ± s）

组别	只数	LVWI（mg/g）		心肌细胞横截面积（× 10⁴ μm²）
组别	只数	7 d	28 d	7 d	28 d
假手术组	8	2.34 ± 0.09	2.30 ± 0.10	13.3 ± 0.7	14.6 ± 0.8
心肌梗死组	8	2.96 ± 0.15^a	3.13 ± 0.18^a	32.8 ± 1.0^a	28.8 ± 1.0^a
贝那普利组	8	2.64 ± 0.62^a	2.81 ± 0.30^a	18.7 ± 1.1^ab	21.9 ± 0.8^ab
F值		25.111		566.062
P值		<0.001		<0.001

表1 各组大鼠间LVWI和心肌细胞横截面积的比较（ ± s）

组别	只数	LVWI（mg/g）		心肌细胞横截面积（× 10⁴ μm²）
组别	只数	7 d	28 d	7 d	28 d
假手术组	8	2.34 ± 0.09	2.30 ± 0.10	13.3 ± 0.7	14.6 ± 0.8
心肌梗死组	8	2.96 ± 0.15^a	3.13 ± 0.18^a	32.8 ± 1.0^a	28.8 ± 1.0^a
贝那普利组	8	2.64 ± 0.62^a	2.81 ± 0.30^a	18.7 ± 1.1^ab	21.9 ± 0.8^ab
F值		25.111		566.062
P值		<0.001		<0.001

表2 各组大鼠血清中CARP和AngⅡ水平比较（ ± s）

组别	只数	CARP（ng/L）		AngⅡ（ng/L）
组别	只数	7 d	28 d	7 d	28 d
假手术组	8	15.4 ± 3.0	14.2 ± 1.2	11.1 ± 1.3	8.7 ± 0.9
心肌梗死组	8	34.0 ± 3.5^a	26.6 ± 1.1^a	21.2 ± 1.7^a	17.0 ± 0.7^a
贝那普利组	8	21.7 ± 1.9^b	18.4 ± 1.6^ab	15.0 ± 1.5^ab	13.9 ± 1.0^ab
F值		90.384		97.764
P值		<0.001		<0.001

表2 各组大鼠血清中CARP和AngⅡ水平比较（ ± s）

组别	只数	CARP（ng/L）		AngⅡ（ng/L）
组别	只数	7 d	28 d	7 d	28 d
假手术组	8	15.4 ± 3.0	14.2 ± 1.2	11.1 ± 1.3	8.7 ± 0.9
心肌梗死组	8	34.0 ± 3.5^a	26.6 ± 1.1^a	21.2 ± 1.7^a	17.0 ± 0.7^a
贝那普利组	8	21.7 ± 1.9^b	18.4 ± 1.6^ab	15.0 ± 1.5^ab	13.9 ± 1.0^ab
F值		90.384		97.764
P值		<0.001		<0.001

表3 各组大鼠心肌细胞中CARP和AngⅡ的累积吸光度值比较（ ± s）

组别	只数	CARP（ng/L）		AngⅡ（ng/L）
组别	只数	7 d	28 d	7 d	28 d
假手术组	8	0.057 ± 0.011	0.058 ± 0.011	0.215 ± 0.021	0.179 ± 0.031
心肌梗死组	8	0.117 ± 0.022^a	0.107 ± 0.017^a	1.232 ± 0.117^a	1.173 ± 0.210^a
贝那普利组	8	0.082 ± 0.011^ab	0.075 ± 0.011^b	0.544 ± 0.058^ab	0.495 ± 0.158^ab
F值		23.642		116.133
P值		<0.001		<0.001

表3 各组大鼠心肌细胞中CARP和AngⅡ的累积吸光度值比较（ ± s）

组别	只数	CARP（ng/L）		AngⅡ（ng/L）
组别	只数	7 d	28 d	7 d	28 d
假手术组	8	0.057 ± 0.011	0.058 ± 0.011	0.215 ± 0.021	0.179 ± 0.031
心肌梗死组	8	0.117 ± 0.022^a	0.107 ± 0.017^a	1.232 ± 0.117^a	1.173 ± 0.210^a
贝那普利组	8	0.082 ± 0.011^ab	0.075 ± 0.011^b	0.544 ± 0.058^ab	0.495 ± 0.158^ab
F值		23.642		116.133
P值		<0.001		<0.001

图2 各组大鼠心肌组织CARP表达情况（免疫组织化学法染色　× 400）注：CARP.心锚重复蛋白；a ~ c图分别为假手术组、心肌梗死组和贝那普利组7 d时心肌细胞CARP的免疫组织化学法染色结果，其中a图中细胞淡染，阳性表达较低；b图阳性表达较高，集中在梗死区细胞的细胞质中；c图阳性表达介于a图和b图之间；d ~ f图分别为假手术组、心肌梗死组和贝那普利组28 d时心肌细胞中CARP的免疫组织化学法染色结果，其中d图细胞淡染，阳性表达较低；e图阳性表达较高，大部分表达于细胞质中；f图阳性表达介于图d和图e之间（棕黄色颗粒为阳性表达）

图3 各组大鼠心肌组织AngⅡ表达情况（免疫组织化学法染色　× 400）注：AngⅡ.血管紧张素Ⅱ；a ~ c图分别为假手术组、心肌梗死组和贝那普利组7 d时心肌细胞AngⅡ的免疫组织化学法染色结果，其中a图棕黄色颗粒较少，主要在细胞质中表达；b图满布棕黄色颗粒，大部分集中表达在坏死区域的细胞质中；c图棕黄色颗粒较b图少，较a图多；d ~ f图分别为假手术组、心肌梗死组和贝那普利组28 d时心肌细胞AngⅡ的免疫组织化学法染色结果，其中d图棕黄色颗粒较少，分布均匀；e图棕黄色颗粒集中在梗死区域的细胞质中；f图棕黄色颗粒介于d图和e图之间

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Benazepril improves myocardial remodeling after myocardial infarction by down-regulating cardiac ankyrin repeat protein

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Benazepril improves myocardial remodeling after myocardial infarction by down-regulating cardiac ankyrin repeat protein