第一部分：合并糖尿病的特发性肺动脉高压患者的临床特征和预后分析

摘    要

背景与目的：特发性肺动脉高压（Idiopathic Pulmonary Arterial Hypertension, IPAH）是一种罕见且严重的心血管疾病，其病程和预后可被合并疾病所影响，如糖尿病（Diabetes Mellitus, DM）。本研究旨在探讨合并DM的IPAH（IPAH Associated with DM, IPAH-DM）患者与单纯性IPAH患者在临床特征、心脏结构和功能、以及治疗反应方面的差异，以期为临床治疗提供指导。

方法：本研究为前瞻性、观察性队列的事后分析，前瞻性纳入2018年9月至2022年8月于我院就诊的IPAH患者161例。根据是否合并DM，将患者分为IPAH-DM组（N=19）和单纯IPAH组（N=142）。收集上述患者的基线数据、实验室指标、心脏超声指标和右心导管检查结果等。两组患者的生存比较采用Kaplan-Meier 曲线和Log-rank检验进行比较，Cox多因素回归分析进一步探究 DM是否能独立影响IPAH患者不良预后，影响的大小采用风险比（Hazard Ratio, HR）和 95% 置信区间（Confidence Interval, CI）表示。

结果：IPAH-DM组患者的平均年龄（48.05 ± 14.82岁 vs 34.83 ± 8.10岁，p = 0.001）、BMI（24.03 ± 1.46 kg/m² vs 22.84 ± 1.80 kg/m²，p = 0.003）、白细胞计数（6.94 [四分位距(Interquartile Range, IQR): 5.62, 8.57] ×10⁹/L vs 5.94 [IQR: 4.80, 6.85] ×10⁹/L，p = 0.017）、甘油三酯水平（1.57 ± 0.53 mmol/L vs. 1.20 ± 0.59 mmol/L, p = 0.010）和WHO肺高血压功能分级III级和IV级比例均显著高于显著高于单纯IPAH组（均满足p < 0.05）。高密度脂蛋白胆固醇水平显著低于单纯IPAH组（1.00 ± 0.26 mmol/L vs. 1.17 ± 0.33 mmol/L, p = 0.012）。IPAH-DM组6分钟步行距离的中位数为304米（IQR: 183，382），显著低于IPAH组的415米（IQR：297，523）米，差异具有统计学意义（p < 0.001）。在心脏超声参数方面，IPAH-DM组患者的左室舒张末前后径显著高于单纯IPAH组，而左室射血分数显著低于单纯IPAH组（均满足p < 0.05）。右心导管检查结果显示，IPAH-DM组的心输出量中位数为4.00升（IQR: 3.72, 4.64），显著低于单纯IPAH组的4.57升（IQR: 3.98, 5.09），差异有统计学意义（p = 0.036）；此外，IPAH-DM组的混合静脉血氧饱和度显著低于单纯IPAH组（60.02 ± 5.39 vs. 67.41 ± 4.43，p < 0.001）。生存分析显示，IPAH-DM组的生存较单纯IPAH组更差（p = 0.001）。此外，多因素Cox回归分析显示，DM 可能是影响IPAH患者预后的独立危险因素（HR = 5.413，95% CI：2.076-14.115，p < 0.001）。

结论：IPAH-DM患者与单纯IPAH患者患者相比，临床表型更加严重，预后更差；且DM是IPAH患者预后不良的独立危险因素，但本研究所纳入的样本量较小，需要更大样本量的多中心研究进一步证实该研究结果。

第二部分：糖尿病、血糖相关指标、SGLT2抑制剂与动脉性肺动脉高压风险的关系：一项孟德尔随机化研究

摘    要

目的：本研究旨在通过生物信息分析的方法，探究糖尿病（Diabetes Mellitus, DM）、血糖相关指标及钠-葡萄糖共转运体2（Sodium-Glucose Co-Transporter-2, SGLT2）抑制在动脉性肺动脉高压（Pulmonary Arterial Hypertension, PAH）发病风险中的作用。方法：通过采用双样本双步骤的孟德尔随机化（Mendelian Randomization, MR）研究，首先，确定DM和血糖相关指标（包括胰岛素抵抗、糖化血红蛋白以及空腹胰岛素和葡萄糖）对PAH发病风险的影响；其次，进一步预测了SGLT2抑制剂对PAH的潜在治疗效应。本研究将与SLC5A2基因（编码SGLT2蛋白的基因）表达水平及血红蛋白A1c均有显著关联的单核苷酸多态位点作为SGLT2抑制剂的工具变量。采用比值比（Odds Ratio, OR）和95% 置信区间（Confidence Interval, CI）表示。结果：通过系统的MR分析，本研究发现基因预测的DM与PAH风险增加具有因果关联，DM的患者相比于非DM的患者，发生PAH的风险增加43.2%（OR = 1.432，95% CI = 1.040-1.973，p = 0.028），且在分别矫正吸烟、体重指数、心衰、高血压等潜在混杂因素，以及同时矫正上述混杂因素之后（OR = 1.469，95% CI = 1.021-2.115，p = 0.038），上述因果推断仍然成立。此外，进一步MR分析提示基因预测的SGLT2抑制剂与PAH风险降低存在因果关联（OR = 1.681*10^-7，95% CI = 7.059*10^-12-0.004，p = 0.002）。结论：DM与PAH发病风险增加之间具有潜在的因果效应，SGLT2抑制可能是PAH患者的潜在治疗靶点。

第三部分 恩格列净对动脉性肺动脉高压治疗作用的基础研究

摘  要

背景与目的：钠-葡萄糖共转运体2（Sodium-Glucose Co-Transporter-2, SGLT2）抑制剂作为一类新型降糖药物，已显示出显著的心血管益处，可改善心力衰竭患者的预后，显著降低糖尿病患者和左心衰患者的心血管事件发生风险和全因死亡率。然而，作为SGLT2抑制剂的恩格列净在动脉性肺动脉高压（Pulmonary Arterial Hypertension, PAH）和右心室（Right Ventricular, RV）功能障碍方面的疗效尚不明确。此外，恩格列净是否通过改善PAH失衡的BMP/TGF-β等经典通路而发挥作用，有待进一步研究。本研究旨在系统探究恩格列净对PAH模型的治疗作用及其对BMP/TGF-β通路的影响，并探讨其潜在的作用机制。

方法：本研究采用了三种不同的动物模型来模拟PAH的发病机制：野百合碱（Monocrotaline, MCT, 60 mg/kg）诱导的PAH大鼠模型、低氧+Sugen（SuHx）诱导的PAH大鼠模型和低氧诱导的PAH小鼠模型。在每种模型内部，动物随机分为三组：模型组（未经治疗）、恩格列净治疗组（于造模当天开始，灌胃给予SGLT2抑制剂恩格列净，10 mg/kg/天）和健康对照组（未诱导PAH），实验持续4周。实验终点时，采用超声心动图评估心脏结构和功能，通过右心导管术测量肺动脉平均压和右心室收缩压，以评估PAH的发展情况及恩格列净的治疗效果。随后，收集肺组织样本进行组织病理学分析，以评估肺血管的重构程度（WA%），在此基础上进一步探究恩格列净治疗对PAH大鼠模型经典通路BMP/TGF-β的影响。此外，为了探究恩格列净治疗PAH的潜在机制，本研究还对人源肺动脉平滑肌细胞（Human Pulmonary Arterial Smooth Muscle Cells, HPASMC）进行了转录组测序分析。通过比较血小板衍生生长因子（Platelet-derived Growth Factor, PDGF）处理组和“PDGF+恩格列净”处理组细胞的基因表达差异，筛选出潜在的与恩格列净治疗PAH相关的关键基因和信号通路。结合文献调研和生物信息学分析，进一步探讨恩格列净对这些靶点的调节作用，以阐明其治疗PAH的分子机制。

结果：在本研究中，通过右心导管检查发现，在建模后，与健康对照组相比，右室收缩压（RV Systolic Pressure, RVSP）在三种不同的PAH模型组均显著升高，平均肺动脉平均压（mean Pulmonary Artery Pressure, mPAP）在 MCT 和SuHx模型组显著升高，差异具有统计学意义（p < 0.05），提示成功构建了PAH动物模型。经过恩格列净治疗后，三种模型中的RVSP较模型组均显著降低，大鼠模型中的mPAP较模型组也显著降低（p<0.05）；与模型组相比，恩格列净治疗组的肺动脉重构指数（WA%）和超声心动图指标显著改善（p < 0.05），表明恩格列净对PAH具有显著的治疗效果。从分子机制角度，恩格列净能够调节BMP/TGF-β信号通路的平衡，这是其治疗PAH的重要作用机制之一。HPASMC的转录组测序分析和文献调研揭示，Top2a和Plk1在PDGF处理组显著升高（p < 0.05），经过恩格列净治疗后，相应的表达下降，可能是恩格列净治疗PAH的潜在靶点。在PAH大鼠模型中，与模型组相比，恩格列净治疗组的肺组织中Top2a和Plk1蛋白及mRNA的表达显著下降（p < 0.05），这一结果提示恩格列净可能通过抑制Top2a和Plk1的表达发挥治疗作用。

结论：（1）SGLT2抑制剂恩格列净在三种不同的PAH动物模型中均能显著降低RVSP和降低大鼠模型的mPAP，减轻肺动脉重构和右室肥厚，表明恩格列净能够有效减轻PAH的发展；（2）恩格列净治疗能够调节BMP/TGF-β信号通路的平衡，这可能是其治疗PAH的重要机制之一；（3）HPASMC的转录组测序分析和文献调研揭示，Top2a和Plk1可能是恩格列净治疗PAH的潜在靶点。在PAH动物模型中，恩格列净治疗能够显著降低肺组织中Top2a和Plk1蛋白及mRNA的表达。上述发现为SGLT2抑制剂在PAH治疗中的应用提供了新的见解，并为未来的研究指明了方向。然而，该研究并未对恩格列净治疗PAH的潜在靶点和机制展开进一步验证，Top2a和Plk1在恩格列净治疗PAH中的具体作用有待于进一步的研究。

Part I: Clinical Characteristics and Prognostic Analysis of Patients with Idiopathic Pulmonary Arterial Hypertension Complicated by Diabetes Mellitus

Abstract

Background and Objective: Idiopathic Pulmonary Arterial Hypertension (IPAH) is a rare and severe cardiovascular disorder whose course and prognosis can be influenced by comorbid conditions such as Diabetes Mellitus (DM). This study aims to investigate the differences in clinical characteristics, cardiac structure and function, and therapeutic responses between IPAH patients with DM (IPAH-DM) and those with IPAH alone, to provide guidance for clinical treatment.

Methods: This study is a retrospective analysis of a prospective, observational cohort. A total of 161 IPAH patients treated at our hospital between September 2018 and August 2022 were included. Patients were categorized into two groups based on the presence of DM: the IPAH-DM group (N=19) and the IPAH-only group (N=142). Baseline data, laboratory indicators, echocardiographic parameters, and right heart catheterization results were collected. Survival comparisons between the two groups were performed using Kaplan-Meier curves and Log-rank tests. Cox multivariate regression analysis was used to explore whether DM independently influences the adverse prognosis of IPAH patients, with the effect size expressed as Hazard Ratio (HR) and 95% Confidence Interval (CI).

Results: Compared to the IPAH-only group, patients in the IPAH-DM group had a higher mean age (48.05 ± 14.82 years vs 34.83 ± 8.10 years, p = 0.001), BMI (24.03 ± 1.46 kg/m² vs 22.84 ± 1.80 kg/m², p = 0.003), white blood cell count (6.94 [Interquartile Range, IQR: 5.62, 8.57] ×10⁹/L vs 5.94 [IQR: 4.80, 6.85] ×10⁹/L, p = 0.017), triglyceride levels (1.57 ± 0.53 mmol/L vs. 1.20 ± 0.59 mmol/L, p = 0.010), and a higher proportion of WHO functional class III and IV (all p < 0.05). The IPAH-DM group also had significantly lower high-density lipoprotein cholesterol levels (1.00 ± 0.26 mmol/L vs. 1.17 ± 0.33 mmol/L, p = 0.012). The median six-minute walk distance was significantly shorter in the IPAH-DM group (304 meters [IQR: 183, 382] vs 415 meters [IQR: 297, 523], p < 0.001). Echocardiographic parameters showed that the left ventricular end-diastolic diameter was significantly larger, and the left ventricular ejection fraction was significantly lower in the IPAH-DM group (both p < 0.05). Right heart catheterization revealed that the median cardiac output in the IPAH-DM group was 4.00 liters (IQR: 3.72, 4.64), significantly lower than in the IPAH-only group (4.57 liters [IQR: 3.98, 5.09], p = 0.036), and mixed venous oxygen saturation was significantly lower (60.02 ± 5.39 vs. 67.41 ± 4.43, p < 0.001). Survival analysis indicated poorer survival in the IPAH-DM group (p = 0.001). Furthermore, Cox multivariate regression analysis showed that DM might be an independent risk factor for adverse prognosis in IPAH patients (HR = 5.413, 95% CI: 2.076-14.115, p < 0.001).

Conclusion: Compared to IPAH-only patients, IPAH-DM patients exhibit more severe clinical phenotypes and poorer prognosis. DM is an independent risk factor for adverse prognosis in IPAH patients. However, the small sample size of this study necessitates further validation through larger, multicenter studies.

Part II: The Relationship Between Diabetes, Glycemic Indices, SGLT2 Inhibitors, and the Risk of Pulmonary Arterial Hypertension: A Mendelian Randomization Study

Abstract

Objective: This study aims to investigate the roles of diabetes mellitus (DM), glycemic indicators, and Sodium-Glucose Co-Transporter-2 (SGLT2) inhibitors in the risk of developing pulmonary arterial hypertension (PAH) through bioinformatic analysis.

Methods: A two-sample, two-step Mendelian Randomization (MR) approach was used. First, we assessed the impact of DM and glycemic indicators (including insulin resistance, glycated hemoglobin, fasting insulin, and glucose) on the risk of PAH. Second, we predicted the potential therapeutic effects of SGLT2 inhibitors on PAH. Single nucleotide polymorphisms significantly associated with the expression levels of the SLC5A2 gene (encoding the SGLT2 protein) and hemoglobin A1c were used as instrumental variables for SGLT2 inhibitors. Odds Ratios (OR) and 95% Confidence Intervals (CI) were used to record the results.

Results: Systematic MR analysis revealed a causal relationship between genetically predicted DM and increased PAH risk. Patients with DM had a 43.2% higher risk of developing PAH compared to non-DM patients (OR = 1.432, 95% CI = 1.040-1.973, p = 0.028). This causal inference remained significant after adjusting for potential confounders such as smoking, body mass index, heart failure, and hypertension (OR = 1.469, 95% CI = 1.021-2.115, p = 0.038). Further MR analysis indicated a causal relationship between genetically predicted SGLT2 inhibitor use and a reduced risk of PAH (OR = 1.68110^-7, 95% CI = 7.05910^-12-0.004, p = 0.002).

Conclusion: There is a potential causal effect of DM on an increased risk of PAH. SGLT2 inhibition may serve as a potential therapeutic target for PAH patients.

Part III: Basic Research on the Therapeutic Effect of Empagliflozin on Pulmonary Arterial Hypertension

Abstract

Background and Purpose: Sodium-Glucose Co-Transporter-2 (SGLT2) inhibitors, a class of novel antidiabetic agents, have shown significant cardiovascular benefits, improving the prognosis of heart failure patients and significantly reducing the risk of cardiovascular events and all-cause mortality in patients with diabetes and left heart failure. However, the efficacy of empagliflozin, an SGLT2 inhibitor, on pulmonary arterial hypertension (PAH) and right ventricular (RV) dysfunction remains unclear. Additionally, whether empagliflozin exerts its effects by modulating the imbalanced BMP/TGF-β signaling pathways in PAH warrants further investigation. This study aims to systematically explore the therapeutic effects of empagliflozin on PAH models and its impact on the BMP/TGF-β pathway, as well as to elucidate its potential mechanisms of action.

Methods: Three different animal models were used to simulate the pathogenesis of PAH: monocrotaline (MCT, 60 mg/kg)-induced PAH rat model, hypoxia + Sugen (SuHx)-induced PAH rat model, and hypoxia-induced PAH mouse model. Within each model, animals were randomly divided into three groups: model group (untreated), empagliflozin treatment group (administered orally with 10 mg/kg/day of empagliflozin starting on the day of modeling), and healthy control group (not induced with PAH). The experiments lasted for 4 weeks. At the end of the experiment, echocardiography was used to assess cardiac structure and function, and right heart catheterization was performed to measure mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) to evaluate the development of PAH and the therapeutic effects of empagliflozin. Lung tissue samples were collected for histopathological analysis to assess pulmonary vascular remodeling (WA%). Additionally, the study further explored the impact of empagliflozin on the BMP/TGF-β pathway in PAH rat models. To investigate the potential mechanisms of empagliflozin in treating PAH, transcriptome sequencing analysis of human pulmonary arterial smooth muscle cells (HPASMCs) was conducted. By comparing the gene expression differences between the PDGF group and the "PDGF + empagliflozin" group, key genes and signaling pathways potentially related to empagliflozin's treatment of PAH were identified. Literature review and bioinformatics analysis were combined to further investigate the regulatory effects of empagliflozin on these targets to elucidate its molecular mechanisms in treating PAH.

Results: In this study, right heart catheterization revealed that, post-modeling, RVSP significantly increased in all three different PAH models compared to the healthy control group, and mPAP significantly increased in the MCT and SuHx models, indicating successful construction of PAH animal models (p < 0.05). After empagliflozin treatment, RVSP significantly decreased in all three models, and mPAP significantly decreased in the rat models compared to the model group (p < 0.05). Additionally, empagliflozin treatment significantly improved pulmonary artery remodeling indices (WA%) and echocardiographic parameters compared to the model group (p < 0.05), indicating a significant therapeutic effect of empagliflozin on PAH. On a molecular level, empagliflozin was able to regulate the balance of the BMP/TGF-β signaling pathway, which is one of the important mechanisms underlying its therapeutic effects on PAH. Transcriptome sequencing analysis of HPASMCs and literature review revealed that Top2a and Plk1 were significantly upregulated in the PDGF-treated group (p < 0.05), and their expression decreased after empagliflozin treatment, suggesting potential targets for empagliflozin in treating PAH. In the PAH rat models, empagliflozin treatment significantly reduced the expression of Top2a and Plk1 proteins and mRNA in lung tissue compared to the model group (p < 0.05), indicating that empagliflozin might exert its therapeutic effects by inhibiting the expression of Top2a and Plk1.

Conclusion: (1) The SGLT2 inhibitor empagliflozin significantly reduces RVSP in three different PAH animal models and mPAP in PAH Rat models, alleviating pulmonary artery remodeling and RV hypertrophy, indicating that empagliflozin effectively mitigates the progression of PAH. (2) Empagliflozin treatment can regulate the balance of the BMP/TGF-β signaling pathway, which may be one of the important mechanisms underlying its therapeutic effects on PAH. (3) Transcriptome sequencing analysis of HPASMCs and literature review suggest that Top2a and Plk1 may be potential targets for empagliflozin in treating PAH. In PAH animal models, empagliflozin treatment significantly reduces the expression of Top2a and Plk1 proteins and mRNA in lung tissue. These findings provide new insights into the application of SGLT2 inhibitors in PAH treatment and indicate directions for future research. However, this study did not further validate the potential targets and mechanisms of empagliflozin in treating PAH, and the specific roles of Top2a and Plk1 in empagliflozin treatment of PAH require further investigation.