基于钙超载探讨自主神经调控对HFpEF大鼠心肌结构重塑、电重塑及纤维化的影响

doi:10.11958/20220897

天津医药 ›› 2023, Vol. 51 ›› Issue (1): 30-34.doi: 10.11958/20220897

基于钙超载探讨自主神经调控对HFpEF大鼠心肌结构重塑、电重塑及纤维化的影响

彭明(), 李玉凯, 王岚, 黄粮, 成忠, 肖杰()

武汉市第四医院心血管内科（邮编430022）

收稿日期:2022-06-08 修回日期:2022-07-06 出版日期:2023-01-15 发布日期:2023-01-17
通讯作者: 肖杰 E-mail:150672639@qq.com;puai_xj@163.com
作者简介:彭明（1986），男，主治医师，主要从事心血管内科相关疾病临床及基础方面研究。E-mail：150672639@qq.com
基金资助:
湖北省卫生健康委员会科研计划资助项目(WJ2019M041)

Effects of autonomic nervous regulation on myocardial structural remodeling, electrical remodeling and fibrosis in rats with ejection fraction preserved heart failure based on calcium overload

PENG Ming(), LI Yukai, WANG Lan, HUANG Liang, CHENG Zhong, XIAO Jie()

Department of Cardiology, Wuhan Fourth Hospital, Wuhan 430022, China

Received:2022-06-08 Revised:2022-07-06 Published:2023-01-15 Online:2023-01-17
Contact: XIAO Jie E-mail:150672639@qq.com;puai_xj@163.com

彭明, 李玉凯, 王岚, 黄粮, 成忠, 肖杰. 基于钙超载探讨自主神经调控对HFpEF大鼠心肌结构重塑、电重塑及纤维化的影响[J]. 天津医药, 2023, 51(1): 30-34.

PENG Ming, LI Yukai, WANG Lan, HUANG Liang, CHENG Zhong, XIAO Jie. Effects of autonomic nervous regulation on myocardial structural remodeling, electrical remodeling and fibrosis in rats with ejection fraction preserved heart failure based on calcium overload[J]. Tianjin Medical Journal, 2023, 51(1): 30-34.

摘要/Abstract

摘要：

目的探究自主神经调控对大鼠射血分数保留心力衰竭（HFpEF）模型结构重塑、电重塑及纤维化的影响。方法 SPF级12周龄SD大鼠44只，抽取10只作为对照组，余34只用于建立HFpEF模型。通过腹主动脉-下腔静脉瘘结扎的方式构建HFpEF模型。其中30只建模成功，4只死亡。建模成功的大鼠分为模型组、自主神经调控组（调控组）及自主神经调控+乙酰胆碱M₂受体拮抗剂组（调控+拮抗组），每组10只。调控组在模型组基础上经皮耳缘迷走神经刺激。调控+拮抗组在调控组基础上每天经尾静脉注射美索曲明（0.5 mg/kg）。通过心脏超声仪测量心脏舒张末期左心室后壁厚度（LVPWD-D）、舒张末期室间隔厚度（IVS-D）及二尖瓣舒张早期/舒张晚期血流速度最大峰值（E/A）。心脏电生理刺激仪获取心脏的有效不应期（ERP）、单向动作电位时程（MAPD）。酶联免疫吸附试验（ELISA）检测B型尿钠肽前体（NT-proBNP）水平。HE染色观察心肌细胞排列及炎性细胞浸润情况。Masson染色观察心肌纤维化程度。RT-PCR和Western blot检测心肌组织基质金属蛋白酶-9（MMP-9）、金属蛋白酶组织抑制因子-1（TIMP-1）、肌浆网Ca²⁺-ATP酶（SERCA2a）及其调节蛋白受磷蛋白（PLB）mRNA及蛋白的表达。结果 HE染色可见模型组心肌细胞排列紊乱，细胞间隙不够明显，伴有炎性细胞浸润。Masson染色可见模型组心肌纤维排列紊乱，大量胶原纤维生成。调控组上述病理改变与模型组比较均明显减轻，而调控+拮抗组与模型组相比无明显改善。与对照组比较，模型组NT-proBNP、LVPWD-D、IVS-D、ERP、MAPD、MMP-9 mRNA及蛋白、PLB mRNA及蛋白表达均升高，E/A、TIMP-1 mRNA及蛋白、SERCA2a mRNA及蛋白表达均降低（P<0.05）。与模型组比较，调控组NT-proBNP、LVPWD-D、IVS-D、ERP、MAPD、MMP-9 mRNA及蛋白、PLB mRNA及蛋白表达均降低，E/A、TIMP-1 mRNA及蛋白、SERCA2a mRNA及蛋白表达均升高（P<0.05）。而调控+拮抗剂组上述指标与模型组比较差异无统计学意义。结论自主神经调控可能通过钙超载途径减轻HFpEF大鼠的结构重塑、电重塑以及心肌纤维化状态，改善HFpEF预后。

关键词: 心室重构, 纤维化, 肌浆网钙转运ATP酶类, 迷走神经刺激术, 射血分数保留性心力衰竭, 结构重塑, 电重塑, 钙超载

Abstract:

Objective To explore the effects of autonomic nervous regulation on structural remodeling, electrical remodeling and fibrosis in rats with ejection fraction preserved heart failure (HFpEF). Methods A total of 44 SPF grade 12-week-old SD rats were selected, 10 of them were selected as the control group, and the remaining 34 rats were used to establish the HFpEF model. The rat HFpEF model was established by ligation of abdominal aorta-inferior vena cava fistula. Among them, 30 rats were successfully modeled, and 4 rats died. The successfully established rats were divided into 3 groups with 10 rats in each group: the model group, the autonomic nerve regulation group and the autonomic nerve regulation+ acetylcholine M₂ receptor antagonist group. The autonomic nerve regulation group received percutaneous auricular marginal vagus nerve stimulation on the basis treatment of the model group. In the autonomic regulation + acetylcholine M₂ receptor antagonist group, mesotramine (0.5 mg/kg) was injected into the caudal vein every day on the basis treatment of the autonomic nervous regulation group. Left ventricular posterior wall end-diastolic dimension (LVPWD-D), interventricular ventricular septum end-diastolic dimension (IVS-D) and the peak value of mitral flow velocity in the early diastole/peak value of mitral flow velocity in late diastole (E/A) were measured by echocardiography.The effective refractory period (ERP) and monophasic action potential duration (MAPD) of rat hearts were obtained by cardiac electrophysiological stimulator in each group. The value of NT-proBNP was detected by ELISA. The disordered arrangement of cardiomyocytes and the infiltration of inflammatory cells were observed by HE staining. The degree of myocardial fibrosis was observed by Masson staining. The expression levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), sarcoplasmic reticulum Ca²⁺-ATP enzyme (SERCA2a) and its regulatory protein phosphoprotein (PLB) mRNA and protein were detected by RT-PCR and Western blot assay. Results HE staining showed that the arrangement of cardiomyocytes was disordered, the intercellular space was not obvious enough, accompanied by the infiltration of inflammatory cells in the model group. Masson staining showed that the myocardial fibers were disordered, and a large number of collagen fibers were produced in the model group. The above pathological changes were significantly less in the autonomic nervous regulation group than those in the model group. The above pathological changes were not significantly improved in the autonomic nervous regulation + acetylcholine M₂ receptor antagonist group compared with those in the model group. Compared with the control group, the values of NT-proBNP, LVPWD-D, IVS-D, ERP, MAPD, MMP-9 mRNA and protein, PLB mRNA and protein were all increased in the model group, and levels of E/A, TIMP-1 mRNA and protein, SERCA2a mRNA and protein decreased significantly (P<0.05). Compared with the model group, values of NT-proBNP, LVPWD-D, IVS-D, ERP, MAPD, MMP-9 mRNA and protein, PLB mRNA and protein decreased in the autonomic nerve regulation group, and levels of E/A, TIMP-1 mRNA and protein, SERCA2a mRNA and protein were all increased (P<0.05). There were no significant differences in the above-mentioned indexes between the autonomic nervous regulation + acetylcholine M₂receptor antagonist group and the model group. Conclusion Autonomic nervous regulation may reduce structural remodeling, electrical remodeling and myocardial fibrosis in HFpEF model rats and improve the prognosis of HFpEF through reducing the level of calcium overload.

Key words: ventricular remodeling, fibrosis, sarcoplasmic reticulum calcium-transporting ATPases, vagus nerve stimulation, heart failure with preserved ejection fraction, structural remodeling, electrical remodeling, calcium overload

中图分类号:

R541.6

图/表 8

参考文献 18

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基因名称	引物序列（5′→3′）	产物大小（bp）
MMP-9	上游：AGGTGCCTCGGATGGTTATCG	208
	下游：TGCTTGCCCAGGAAGACGAA	208
TIMP-1	上游：GGCGAACCGGAAACCTGT	188
	下游：GCGCCCTTTGCATCTCTGG	188
SERCA2a	上游：CACCTG?GAA?GAT?TCT?GCG?AAC?T	226
	下游：CAT?CCT?CAT?CCT?GCC?CAA?AG	226
PLB	上游：GGA?GCC?TGT?GTC?CTT?GTG?TC	159
	下游：GAA?GTC?ATC?CCT?GGT?GTC?GT	159
GAPDH	上游：TGCCACTCAGAAGACTGTGG	227
	下游：GGATGCAGGGATGTTCT	227

基因名称	引物序列（5′→3′）	产物大小（bp）
MMP-9	上游：AGGTGCCTCGGATGGTTATCG	208
	下游：TGCTTGCCCAGGAAGACGAA	208
TIMP-1	上游：GGCGAACCGGAAACCTGT	188
	下游：GCGCCCTTTGCATCTCTGG	188
SERCA2a	上游：CACCTG?GAA?GAT?TCT?GCG?AAC?T	226
	下游：CAT?CCT?CAT?CCT?GCC?CAA?AG	226
PLB	上游：GGA?GCC?TGT?GTC?CTT?GTG?TC	159
	下游：GAA?GTC?ATC?CCT?GGT?GTC?GT	159
GAPDH	上游：TGCCACTCAGAAGACTGTGG	227
	下游：GGATGCAGGGATGTTCT	227

组别	NT-proBNP（ng/L）	E/A	LVPWD-D（mm）	IVS-D（mm）	LVEF	ERP（ms）	MAPD（ms）
对照组	45.22±6.26	1.85±0.06	7.59±0.06	1.51±0.02	0.58±0.04	155.43±4.32	172.04±4.85
模型组	301.25±16.07^a	1.28±0.06^a	7.99±0.22^a	2.14±0.08^a	0.57±0.06	183.11±5.83^a	216.95±4.76^a
调控组	79.33±5.63^b	1.66±0.05^b	7.74±0.04^b	1.70±0.02^b	0.57±0.06	167.49±2.23^b	179.86±2.98^b
调控+拮抗组	297.73±18.77	1.28±0.04	7.97±0.20	2.14±0.07	0.58±0.05	182.88±4.90	216.22±4.40
F	1 112.080^＊＊	255.841^＊＊	16.429^＊＊	371.247^＊＊	0.049	87.549^＊＊	301.465^＊＊

组别	NT-proBNP（ng/L）	E/A	LVPWD-D（mm）	IVS-D（mm）	LVEF	ERP（ms）	MAPD（ms）
对照组	45.22±6.26	1.85±0.06	7.59±0.06	1.51±0.02	0.58±0.04	155.43±4.32	172.04±4.85
模型组	301.25±16.07^a	1.28±0.06^a	7.99±0.22^a	2.14±0.08^a	0.57±0.06	183.11±5.83^a	216.95±4.76^a
调控组	79.33±5.63^b	1.66±0.05^b	7.74±0.04^b	1.70±0.02^b	0.57±0.06	167.49±2.23^b	179.86±2.98^b
调控+拮抗组	297.73±18.77	1.28±0.04	7.97±0.20	2.14±0.07	0.58±0.05	182.88±4.90	216.22±4.40
F	1 112.080^＊＊	255.841^＊＊	16.429^＊＊	371.247^＊＊	0.049	87.549^＊＊	301.465^＊＊

组别	MMP-9 mRNA	MMP-9 蛋白	TIMP-1 mRNA	TIMP-1 蛋白
对照组	1.00±0.02	0.22±0.04	1.00±0.02	0.80±0.07
模型组	5.78±0.81^a	0.69±0.06^a	0.13±0.03^a	0.31±0.03^a
调控组	1.37±0.03^b	0.49±0.05^b	0.72±0.16^b	0.71±0.04^b
调控+拮抗组	5.40±1.62	0.70±0.04	0.13±0.02	0.30±0.06
F	79.460^＊＊	220.176^＊＊	296.185^＊＊	269.906^＊＊

基于钙超载探讨自主神经调控对HFpEF大鼠心肌结构重塑、电重塑及纤维化的影响

Effects of autonomic nervous regulation on myocardial structural remodeling, electrical remodeling and fibrosis in rats with ejection fraction preserved heart failure based on calcium overload

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组别	SERCA2a mRNA	SERCA2a 蛋白	PLB mRNA	PLB 蛋白
对照组	0.99±0.01	0.71±0.08	1.01±0.02	0.24±0.02
模型组	0.14±0.03^a	0.19±0.05^a	5.76±0.99^a	0.74±0.03^a
调控组	0.80±0.06^b	0.52±0.03^b	1.95±0.12^b	0.44±0.03^b
调控+拮抗组	0.11±0.03	0.23±0.04	5.68±0.75	0.76±0.05
F	1 439.914^＊＊	215.837^＊＊	156.731^＊＊	539.140^＊＊

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