Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction

Yu TAN; Yu-long BIE; Li CHEN; Yi-han ZHAO; Lei SONG; Li-na MIAO; Yan-qiao YU; Hua CHAI; Xiao-juan MA; Da-zhuo SHI

doi:10.1007/s11655-021-2881-0

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Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction

Original Article | Updated：2022-03-29

- Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction
- Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction
- Chinese Journal of Integrative Medicine 2022年28卷第4期页码：330-338
- Affiliations：
  
  1.National Clinical Research Center for Chinese Medicine Cardiology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing (100091), China
  2.Peking University Traditional Chinese Medicine Clinical Medical School (Xiyuan Hospital),Beijing (100191), China
  3.Department of Graduate School, Beijing University of Chinese Medicine, Beijing (100029), China
- Author bio：
  
  Dr. MA Xiao-juan, E-mail: abc_mxj@aliyun.com
- Funds：
  
  the National Natural Science Foundation of China(81774141;82074418)
- DOI：10.1007/s11655-021-2881-0
  中图分类号：
- 纸质出版日期：2022-04，
  
  网络出版日期：2021-11-26，
  
  录用日期：2021-08-04
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Yu TAN, Yu-long BIE, Li CHEN, 等. Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction[J]. Chinese Journal of Integrative Medicine, 2022,28(4):330-338.

Yu TAN, Yu-long BIE, Li CHEN, et al. Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction[J]. Chinese Journal of Integrative Medicine, 2022,28(4):330-338.
Yu TAN, Yu-long BIE, Li CHEN, 等. Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction[J]. Chinese Journal of Integrative Medicine, 2022,28(4):330-338. DOI： 10.1007/s11655-021-2881-0.

Yu TAN, Yu-long BIE, Li CHEN, et al. Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF-κB Pathways in Rat Model of Acute Myocardial Infarction[J]. Chinese Journal of Integrative Medicine, 2022,28(4):330-338. DOI： 10.1007/s11655-021-2881-0.

摘要

目的:

探究灵宝护心丹是否能通过SIRT1介导的FOXO1和NF-κB信号通路调控凋亡和炎症

保护急性心肌梗死大鼠 (AMI) 梗死边界区 (IBZ) 心肌组织.

方法:

将6周龄大鼠根据随机数字表法随机分配到假手术组、模型组、倍他乐克组(0.9 mg/kg 每天)、灵宝护心丹低剂量组(0.45 mg/kg 每天)、灵宝护心丹中剂量组(0.9 mg/kg 每天)、灵宝护心丹高剂量组(1.8 mg/kg 每天)、灵宝护心丹中剂量+EX527组(0.9 mg/kg 每天)

每组13只. 本研究通过结扎大鼠心脏冠状动脉左前降支构建AMI模型并通过2

5-三苯基氯化四氮唑染色法测量心肌梗死面积. 运用原位末端转移酶标记技术 (TUNEL) 测量IBZ心肌细胞凋亡指数. 同时分别采用Heidenhain、MASSON和HE染色法观察IBZ心肌组织病理改变. 使用酶联免疫吸附试验 (ELISA) 检测大鼠血清肿瘤坏死因子α、白介素6、白介素1β和细胞间黏附分子1的表达情况. 运用实时荧光定量PCR技术检测IBZ心肌组织中SIRT1和FOXO1mRNA表达情况. 使用蛋白印迹法检测IBZ中SIRT1

FOXO1

SOD2

BAX和NF-κB p65蛋白表达情况.

结果:

LBHX预给药缩小了AMI大鼠心肌梗死面积 (

0.01) . TUNEL染色显示LBHX可以抑制IBZ心肌细胞凋亡. 同时

病理染色结果显示LBHX预给药减小了IBZ心肌组织缺血范围 (

0.05) 、减轻了心肌纤维化程度 (

0.05) 和炎症反应. 此外

ELISA结果表明LBHX降低了AMI大鼠血清中炎症因子表达 (

0.05或

0.01) . WB结果表明LBHX预给药上调了大鼠心肌组织中SIRT1、FOXO1、SOD2蛋白表达水平 (

0.05)

降低了BAX和NF-κB p65蛋白表达水平 (

0.05) . RT-qPCR结果显示LBHX上调了SIRT1和FOXO1mRNA表达 (

0.05) . 在LBHX中剂量基础上应用SIRT1抑制剂EX527后

LBHX的抗凋亡、抗炎等心肌保护作用被部分逆转.

结论:

灵宝护心丹能够抑制AMI大鼠心肌凋亡和炎症

其保护机制与SIRT1介导的FOXO1和NF-κB信号通路有关.

Abstract

Objective:

To investigate whether Lingbao Huxin Pill (LBHX) protects against acute myocardial infarction (AMI) at the infarct border zone (IBZ) of myocardial tissue by regulating apoptosis and inflammation through the sirtuin 1 (SIRT1)-mediated forkhead box protein O1 (FOXO1) and nuclear factor-κB (NF-κB) signaling pathways.

Methods:

Six-week-old Wistar rats with normal diet were randomized into the sham

the model

Betaloc (0.9 mg/kg daily)

LBHX-L (0.45 mg/kg daily)

LBHX-M (0.9 mg/kg daily)

LBHX-H (1.8 mg/kg daily)

and LBHX+EX527 (0.9 mg/kg daily) groups according to the method of random number table

13 in each group. In this study

left anterior descending coronary artery (LADCA) ligation was performed to induce an AMI model in rats. The myocardial infarction area was examined using a 2

5-triphenyltetrazolium chloride solution staining assay. A TdT-mediated dUTP nick-end labeling (TUNEL) assay was conducted to assess cardiomyocyte apoptosis in the IBZ. The histopathology of myocardial tissue at the IBZ was assessed with Heidenhain

Masson and hematoxylin- eosin (HE) staining assays. The expression levels of tumor necrosis factor α (TNF-α)

interleukin (IL)-6

IL-1β

and intercellular adhesion molecule-1 were measured using enzyme-linked immunosorbent assays (ELISAs). The mRNA expressions of SIRT1 and FOXO1 were detected by real-time qPCR (RT-qPCR). The protein expressions of SIRT1

FOXO1

SOD2

BAX and NF-κB p65 were detected by Western blot analysis.

Results:

The ligation of the LADCA successfully induced an AMI model. The LBHX pretreatment reduced the infarct size in the AMI rats (

0.01). The TUNEL assay revealed that LBHX inhibited cardiomyocyte apoptosis at the IBZ. Further

the histological examination showed that the LBHX pretreatment decreased the ischemic area of myocardial tissue (

0.05)

myocardial interstitial collagen deposition (

0.05) and inﬂammation at the IBZ. The ELISA results indicated that LBHX decreased the serum levels of inflammatory cytokines in the AMI rats (

0.05 or

0.01). Furthermore

Western blot analysis revealed that the LBHX pretreatment upregulated the protein levels of SIRT1

FOXO1 and SOD2 (

0.05) and downregulated NF-κB p65 and BAX expressions (

0.05). The RT-qPCR results showed that LBHX increased the SIRT1 mRNA and FOXO1 mRNA levels (

0.05). These protective effects

including inhibiting apoptosis and alleviating inﬂammation in the IBZ

were partially abolished by EX527

an inhibitor of SIRT1.

Conclusion:

LBHX could protect against AMI by suppressing apoptosis and inﬂammation in AMI rats and the SIRT1- mediated FOXO1 and NF-κB signaling pathways were involved in the cardioprotection effect of LBHX.

关键词

Keywords

Lingbao Huxin PillChinese medicineinfarct border zoneacute myocardial infarctionSIRT1

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