兰州大学机构库 >第一临床医学院
SGLT2抑制剂通过调控自噬改善心脏重塑的作用及机制研究
Alternative TitleThe Effect and Mechanism of SGLT2 Inhibitors on Alleviating Cardiac Remodeling by Regulating Autophagy
何智余
Subtype博士
Thesis Advisor张钲
2023-05-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学博士
Degree Discipline内科学
KeywordSGL T2抑制剂 SGL T2 Inhibitor 心力衰竭 heart failure 心脏重塑 cardiac remodeling 自噬 autophagy SIRT1/PGC-1α通路 SIRT1/PGC-1α pathway
Abstract

背景 心力衰竭(HF)是各种心血管疾病发展的终末阶段,也是心血管疾病患者主要的死亡原因之一。据统计全球约有6430万人患有HF。心脏重塑是HF发生发展的核心环节之一。钠-葡萄糖转运蛋白2(SGLT2)抑制剂通过抑制肾小管对葡萄糖的重吸收发挥降糖作用,并且可降低HF患者的死亡率和再住院率(无论是否合并有糖尿病)。SGLT2抑制剂治疗HF的临床试验取得了显著进展,然而对于SGLT2抑制剂改善HF的具体作用机制及是否对心脏发挥直接作用仍缺乏认识。SGLT2抑制剂作为一类非常有前景的治疗HF的药物,进一步明确其作用机制,不仅有助于我们对SGLT2抑制剂药理学的理解,还有助于开辟新的治疗领域和策略。

目的 1. 明确SGLT2抑制剂改善心脏重塑的作用,同时行转录组测序(RNA-seq),以进一步明确其可能的作用机制。

2. 探讨自噬在SGLT2抑制剂改善心脏重塑作用中的调控机制。

3. 揭示SGLT2抑制剂调控自噬改善心脏重塑的分子机制。

方法 1. 构建主动脉缩窄(TAC)诱导的心脏重塑动物模型。采用小动物超声、苏木精-伊红染色、天狼星红染色、免疫荧光染色及RT-qPCR,明确SGLT2抑制剂达格列净(DAPA)改善心脏重塑的作用;同时行RNA-seq,明确其可能的机制。

2. 采用Ang II刺激H9C2细胞和原代心肌细胞(NVRMs)诱导的心肌细胞肥大和细胞外基质(ECM)沉积模型。行RT-qPCR、Western blot、免疫荧光、双荧光标记LC3B 的质粒转染心肌细胞,同时采用自噬抑制剂3-MA及SIRT1特异性抑制剂EX527明确自噬及SIRT1/PGC-1α信号通路在SGLT2抑制剂DAPA改善心肌细胞肥大和ECM分泌中的作用及机制。

3. 构建心肌梗死(MI)诱导的心脏重塑动物模型。行小动物超声、苏木素-伊红染色、Masson染色、免疫荧光、免疫组化、ELISA、RT-qPCR及Western blot,同时采用SIRT1特异性抑制剂EX527进一步明确自噬及SIRT1/PGC-1α信号通路在SGLT2抑制剂DAPA改善心脏重塑中的作用及机制。

4. 统计学分析及作图采用GraphPad Prism 9.4 软件。P < 0.05表示差异具有统计学意义。

结果 1. DAPA可改善TAC术后小鼠的左室功能、心脏肥厚及心脏纤维化。RNA-seq提示:DAPA主要参与脂分解代谢、糖异生等能量代谢的过程,同时对溶酶体及自噬体产生影响,主要的相关通路为SIRT1/PGC-1α信号通路。

2. 体内实验表明DAPA恢复TAC术后的自噬流。体外实验发现: DAPA改善Ang Ⅱ 诱导的心肌细胞肥大和ECM分泌,同时可改善Ang Ⅱ 诱导的心肌细胞自噬流降低,自噬抑制剂3-MA逆转了 DAPA 促进自噬、改善心肌细胞肥大和ECM分泌的作用。

3. TAC诱导的心脏重塑模型表明:DAPA激活TAC术后小鼠心脏SIRT1/ PGC-1α 信号通路。体外实验表明:DAPA预处理促进了H9C2细胞 SIRT1和PGC-1α蛋白的表达; SIRT1特异性抑制剂EX527逆转了DAPA促进SIRT1和PGC-1α蛋白表达的作用,同时逆转了DAPA促进自噬、改善心肌细胞肥大和ECM分泌的作用。MI诱导的心脏重塑动物模型表明:DAPA通过SIRT1/PGC-1α信号通路调节自噬改善MI后大鼠的心功能、心脏纤维瘢痕的形成及MI边缘区心肌细胞肥大。

结论 1. SGLT2抑制剂DAPA可改善TAC术后小鼠的左室功能和心脏重塑。对心脏组织行RNA-seq提示DAPA主要参与脂分解代谢、糖异生等能量代谢的过程,同时对溶酶体及自噬体产生影响,主要的相关通路为SIRT1/PGC-1α信号通路。

2. SGLT2抑制剂DAPA通过恢复自噬流改善TAC术后小鼠的心脏重塑和Ang Ⅱ 诱导的心肌细胞肥大和ECM的分泌。且DAPA主要作用于自噬体的形成阶段而促进自噬。

3. SGLT2抑制剂DAPA可通过SIRT1/PGC-1α信号通路调节自噬进而改善TAC术后小鼠及MI术后大鼠的心脏重塑。

总之,TAC或MI诱导的心脏重塑中自噬流和SIRT1/PGC1-α通路受到抑制。SGLT2抑制剂DAPA可激活SIRT1/PGC-1α信号通路促进自噬体的形成,恢复自噬流,从而改善心脏重塑。

Other Abstract

Background: Heart failure (HF) is the end stage of various cardiovascular diseases, and it is also one of the main causes of death in patients with cardiovascular diseases. According to statistics, approximately 64.3 million people worldwide suffer from HF. Cardiac remodeling is the central process in the development of HF. Sodium-glucose co-transporter 2 (SGLT2) inhibitors inhibit the reabsorption of glucose in renal tubules, and reduce the mortality and readmission rate of HF patients regardless of presence or absence of diabetes. Clinical trials of SGLT2 inhibitors for the treatment of HF have made significant progress, but the specific mechanism of action of SGLT2 inhibitors in improving HF and whether they have a direct effect on the heart are still not well understood. SGLT2 inhibitors are a very promising class of drugs for the treatment of HF. Further clarifying their mechanism of action will not only help us to understand the pharmacology of SGLT2 inhibitors, but also help to open up new therapeutic fields and strategies.

Aims: 1. To clarify the role of SGLT2 inhibitor dapagliflozin (DAPA) in alleviating cardiac remodeling, and RNA sequencing (RNA-seq) was performed to further clarify its possible mechanism of action.

2. To explore the regulatory mechanism of autophagy in the effect of DAPA on improving cardiac remodeling.

3. To reveal the molecular mechanism of DAPA improving cardiac remodeling by regulating autophagy.

Methods: 1. A mice model of cardiac remodeling induced by transverse aortic constriction (TAC) was constructed. Echocardiography, hematoxylin-eosin staining, sirius red staining, immunofluorescence staining, and RT-qPCR were used to clarify the role of DAPA in improving cardiac remodeling. RNA-seq was also performed to clarify its possible mechanism.

2. An Ang II-induced cardiomyocyte hypertrophy and extracellular matrix (ECM) deposition model was established using H9C2 cells and primary neonatal rat ventricular myocytes (NVRMs). RT-qPCR, western blot, immunofluorescence, and dual fluorescence-labeled LC3B plasmid transfection of cardiomyocytes were performed. Autophagy inhibitor 3-MA and SIRT1 specific inhibitor EX527 were used to clarify the roles and mechanisms of autophagy and the SIRT1/PGC-1α signaling pathway in the improvement of cardiomyocyte hypertrophy and ECM deposition by SGLT2 inhibitor DAPA.

3. A myocardial infarction (MI)-induced cardiac remodeling model was constructed. Echocardiography, hematoxylin-eosin staining, masson staining, immunofluorescence, immunohistochemistry, ELISA, RT-qPCR, and Western blot were performed. SIRT1 specific inhibitor EX527 was used to further clarify the roles and mechanisms of autophagy and the SIRT1/PGC-1α signaling pathway in the improvement of cardiac remodeling by SGLT2 inhibitor DAPA.

4. Statistical analysis and graphing were performed using GraphPad Prism 9.4 software. P < 0.05 indicates a statistically significant difference.

Results: 1. DAPA improved left ventricular function, cardiac hypertrophy, and cardiac fibrosis in mice after TAC surgery. RNA-seq indicated that DAPA is mainly involved in energy metabolism processes such as lipid metabolism and gluconeogenesis, and it also affects lysosomes and autophagosomes. The main related pathway is the SIRT1/PGC-1α signaling pathway.

2. In vivo experiments showed that DAPA restored autophagic flux after TAC surgery. In vitro experiments found that DAPA alleviated Ang II-induced cardiomyocyte hypertrophy and ECM deposition, and improved the autophagy flux. The autophagy inhibitor 3-MA reversed the effect of DAPA on promoting autophagy and alleviating cardiomyocyte hypertrophy and ECM deposition.

3. The TAC-induced cardiac remodeling model showed that DAPA activated the SIRT1/PGC-1α signaling pathway in mice after TAC surgery. In vitro experiments showed that DAPA pretreatment promoted the expression of SIRT1 and PGC-1α proteins in H9C2 cells. The SIRT1-specific inhibitor EX527 reversed the effect of DAPA on promoting the expression of SIRT1 and PGC-1α proteins, and also reversed the effect of DAPA on promoting autophagy and alleviating cardiomyocyte hypertrophy and ECM deposition. The MI-induced cardiac remodeling model showed that DAPA improved cardiac function, alleviated cardiac fibrotic scar formation, and reduced cardiomyocyte hypertrophy in the marginal zone of MI by regulating autophagy through the SIRT1/PGC-1α signaling pathway.

Conclusion 1. SGLT2 inhibitor DAPA alleviates TAC-induced left ventricular dysfunction and cardiac remodeling. RNA-seq analysis suggests that DAPA is mainly involved in energy metabolism processes such as lipid metabolism and gluconeogenesis, and it also affects lysosomes and autophagosomes. The main related pathway is the SIRT1/PGC-1α signaling pathway.

2. SGLT2 inhibitor DAPA alleviates cardiac remodeling in mice after TAC surgery and reduces Ang II-induced cardiomyocyte hypertrophy and ECM deposition by regulating autophagy. DAPA acts on the formation stage of autophagosomes to promote autophagy.

3. SGLT2 inhibitor DAPA alleviates TAC-induced and MI-induced cardiac remodeling by regulating autophagy through the SIRT1/PGC-1α signaling pathway.

In conclusion, autophagy flux and SIRT1/PGC1-α pathway are inhibited in TAC-induced and MI-induced cardiac remodeling. The SGLT2 inhibitor DAPA activates the SIRT1/PGC-1α signaling pathway to promote the formation of autophagosomes, restore autophagy flux, and thereby alleviates cardiac remodeling.

MOST Discipline Catalogue医学 - 临床医学 - 内科学(含:心血管病、血液病、呼吸系病、消化系病、内分泌与代谢病、肾病、风湿病、传染病)
URL查看原文
Language中文
Other Code262010_120190901751
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/537427
Collection第一临床医学院
Affiliation
兰州大学第一临床医学院
Recommended Citation
GB/T 7714
何智余. SGLT2抑制剂通过调控自噬改善心脏重塑的作用及机制研究[D]. 兰州. 兰州大学,2023.
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