第一部分 肝脏代谢紊乱中代谢相关脂肪肝病对冠心病的预后价值
摘要
背景：冠心病与肝脏代谢紊乱密切相关。代谢功能障碍相关脂肪肝病（metabolic-(dysfunction) associated fatty liver disease, MAFLD），曾被称为非酒精性脂肪肝病（nonalcoholic fatty liver disease, NAFLD），是2020年提出的新概念，是指因代谢功能障碍引发的肝脏疾病。本研究旨在探讨低密度脂蛋白胆固醇（low density lipoprotein cholesterol, LDL-C）水平控制良好的冠心病人群中肝脏代谢紊乱与不良心脑血管事件的相关性。
方法：研究将LDL-C<1.8mmol/L的冠心病患者被分为MAFLD和非MAFLD组。倾向性评分匹配用于平衡MAFLD组和非MAFLD组之间的基线特征。主要终点为主要不良心脑血管事件。通过Kaplan-Meier方法分析MAFLD和非MAFLD组人群的累积生存率，Cox比例风险模型用于计算风险比和95%置信区间。
结果：倾向性评分匹配校正后，LDL-C<1.8mmol/L的MAFLD患者和非MAFLD患者的基线资料相似。在非MAFLD和MAFLD患者中主要不良心脑血管事件的发生率分别为6.6%和9.6%（p<0.05）。Cox比例风险回归分析发现，MAFLD患者发生主要不良心脑血管事件风险显著增加（HR 1.48，95%置信区间1.04-2.1，p<0.05）。进一步研究发现肝纤维化指数Fibrosis-4（FIB-4）是其中的预后影响因素。根据FIB-4指数的截止值（FIB-4<3.25或FIB-4≥3.25），将MAFLD患者进一步分为有无显著肝纤维化的两组。与不合并显著肝纤维化的MAFLD患者相比，合并显著肝纤维化的MAFLD患者发生主要不良心脑血管事件风险更高（HR 2.42，95%置信区间 1.12-5.22，p<0.05）。
结论：在LDL-C水平控制良好的冠心病患者中，MAFLD是主要不良心脑血管事件的独立危险因素。FIB-4指数升高与MAFLD患者发生主要不良心脑血管事件的风险升高密切相关。
 

第二部分 肝脏代谢紊乱中肝纤维化对冠心病的预后价值
摘要
背景：本论文第一部分研究发现代谢障碍脂肪肝与冠心病患者不良预后相关，同时脂肪肝患者的肝纤维化程度与不良事件的发生风险相关。肝纤维化是肝脏代谢紊乱中的重要病理过程。根据年龄、天冬氨酸转氨酶、丙氨酸氨基转移酶和血小板计数计算的Fibrosis-4（FIB-4）指数是一种无创性评估肝纤维化的工具，计算简单便捷，方便临床上进行使用。本研究的目标是进一步在急性冠脉综合征（Acute coronary syndrome, ACS）患者中探究FIB-4指数评估的肝纤维化对冠心病的预后价值。
方法：本研究的纳入了6563例患者，主要终点为主要心脑血管不良事件（major adverse cardiac and cerebrovascular events, MACCE）。根据既往文献中FIB-4指数的截止值将患者分为三组：FIB-4<1.45、1.45-3.25和≥3.25。通过Kaplan-Meier分析比较各组人群之间的累积无事件生存率。通过限制性立方样条分析FIB-4指数作为连续变量与结局事件之间的相关性。Cox比例风险回归模型用于评估FIB-4评分与结局事件之间的风险比和95%置信区间。
结果：本研究的中位随访时间为2.4年，共记录了270例主要终点事件MACCE。相比于FIB-4低分组，FIB-4中、高分组患者发生主要终点事件MACCE（HR 1.33，95%CI 1.02-1.74；HR 2.59，95%CI 1.21-4.23）的风险显著升高。FIB-4指数升高与发生全因死亡、心源性死亡和心肌梗死的风险升高相关。FIB-4指数与缺血性卒中和大出血风险无关。限制性立方样条显示，无论是否存在2型糖尿病，FIB-4作为连续变量与主要终点事件MACCE风险增加呈正相关。此外，将FIB-4纳入由传统风险因素构建的预后模型能够显著提高模型的预测价值。在2型糖尿病和非2型糖尿病人群中进行亚组分析，结果与总人群具有一致性。
结论：在肝脏代谢紊乱中FIB-4指数评估的肝纤维化程度加重与MACCE、全因死亡和心肌梗死风险增加相关。FIB-4指数可能有助于患者的风险分层，无论患者是否合并2型糖尿病。
 

第三部分 肝脏代谢紊乱的潜在治疗靶点SIMPLE的作用研究
摘要
背景：本论文第一和二部分的临床研究表明肝脏代谢紊乱与冠心病患者发生不良预后相关，因此治疗肝脏代谢紊乱对于冠心病的防治具有重要意义。本论文第三部分进一步通过基础研究探究肝脏代谢紊乱的潜在治疗靶点。溶酶体介导的蛋白质降解功能异常与多种代谢紊乱疾病密切相关，如动脉粥样硬化、2型糖尿病和脂肪肝。溶酶体/晚期内切体小整合膜蛋白（Small integral membrane protein of lysosome/late endosome, SIMPLE）是内体对溶酶体运输和细胞信号传导的调节者，但SIMPLE在肝脏代谢紊乱中的作用尚不清楚。本文研究了SIMPLE在肝脏代谢紊乱中的功能及其作用。
方法：研究构建了体外SIMPLE基因敲低的细胞模型，在给予代谢刺激的情况下，通过尼罗红检测细胞内脂质沉积情况，通过实时荧光定量PCR检测脂质合成转运和炎症相关基因表达水平。构建肝细胞特异性SIMPLE敲除小鼠（hepatocyte-specific Simple-knockout, Simple-HKO），建立高脂饮食、高脂高胆固醇饮食和蛋氨酸胆碱缺乏饮食诱导肝脏代谢紊乱模型。通过体重、肝重、血糖、葡萄糖耐量、血清脂质水平、血清肝酶水平等评估糖脂代谢情况。通过油红O染色、苏木精-伊红染色检测肝脏脂质沉积情况，通过天狼星红染色检测肝脏纤维化情况，通过cd11b染色检测肝脏炎症细胞浸润情况。通过转录组学分析Simple-HKO小鼠分子通路和基因表达的变化情况。通过免疫印迹、免疫共沉淀等分子生物学试验探索分子机制。
结果：本研究发现，体外敲低SIMPLE会显著加重代谢刺激引起的肝细胞内脂质沉积和炎症反应。体内实验得到了一致的结果，相比于对照组小鼠， Simple-HKO小鼠表现出更严重的高脂饮食、高脂高胆固醇饮食和蛋氨酸胆碱缺乏饮食诱导的肝脏脂肪变性、糖耐量异常、肝脏炎症和纤维化。同时，RNA测序显示，在代谢应激的情况下，与对照组小鼠相比，Simple-HKO小鼠肝脏样本中与脂质代谢、炎症和纤维化相关的信号通路活性增强，特征基因的表达水平明显上调。分子机制研究发现SIMPLE能够调控表皮生长因子受体（Epidermal growth factor receptor, EGFR）的溶酶体降解。SIMPLE缺乏会引起EGFR的溶酶体降解异常，EGFR磷酸化过度激活，进而促进肝脏代谢紊乱的进展。此外，我们还证明了使用EGFR抑制剂或者敲低EGFR表达可以改善SIMPLE基因敲低诱导的肝脏脂质沉积。
结论：SIMPLE能够通过促进EGFR的溶酶体降解改善肝脏代谢紊乱，可作为肝脏代谢紊乱的潜在治疗靶点。
 

综述 肝脏代谢紊乱与心血管疾病
摘要
心血管疾病是目前全球死亡的主要原因。肝脏代谢紊乱与心血管疾病密切相关，并可能增加心脏代谢风险。越来越多的研究表明，肝脏代谢紊乱与多种心血管疾病风险增加独立相关，包括冠心病、心肌病和心律失常。在这篇综述中，我们以常见的肝脏代谢紊乱疾病非酒精性脂肪肝病为例，讨论了肝脏代谢紊乱疾病与多种心血管疾病风险增加相关的证据，肝脏代谢紊乱疾病和心血管疾病之间共同的风险因素，以及两者之间潜在的病理生理机制。
 

Part 1 The prognostic value of metabolic related fatty liver disease in liver metabolic disorders for coronary heart disease 
Abstract
Background: Coronary heart disease (CHD) is closely related to liver metabolic disorders. Metabolic (dysfunction)-associated fatty liver disease (MAFLD), formerly known as nonalcoholic fatty liver disease (NAFLD), is a newly proposed concept, emphasizing the liver diseases caused by metabolic dysfunction. MAFLD shares common metabolic risk factors with cardiovascular disease. This study aims to investigate the association between liver metabolic disorders and adverse cardiovascular outcomes in CHD patients with well-controlled LDL-C.
Methods: CHD patients with LDL-C<1.8mmol/L were divided into MAFLD and non-MAFLD groups. Propensity score matching (PSM) balanced baseline differences between the two groups. The primary endpoint was major adverse cardiac and cerebrovascular events (MACCE). The cumulative event-free survival rates of MAFLD and non-MAFLD groups were analyzed with Kaplan-Meier analysis. Cox proportional risk model was used to calculate hazard ratio and 95% confidential interval.
Results: After PSM, the baseline characteristics of MAFLD patients and non-MAFLD patients had no significant difference. MAFLD patients exhibited a significantly higher cumulative incidence of MACCE than those without MAFLD (9.6% versus 6.6%, p<0.05). Consistently, Cox proportional hazards regression analysis found MAFLD patients had a significantly increased risk for MACCE (HR 1.48, 95% confidential interval (CI) 1.04-2.1, p<0.05). Further research found that Fibrosis-4 (FIB-4) was a prognostic factor. MAFLD patients were divided into two groups with and without advanced liver fibrosis according to FIB-4≥3.25 or FIB-4<3.25. Advanced liver fibrosis staged by the FIB-4 index was associated with an elevated risk for MACCE (HR 2.42, 95% CI 1.12-5.22, p<0.05) compared to those without advanced liver fibrosis.
Conclusion: MAFLD was an independent risk factor for MACCE among CHD patients with well-controlled LDL-C levels. Advanced liver fibrosis evaluated by FIB-4 index was associated with increased risk of MACCEs in MAFLD patients. 
 

Part 2 The prognostic value of liver fibrosis in liver metabolic disorders for coronary heart disease
Abstract
Background: The first part of the study found that metabolic disorder is associated with poor prognosis in patients with coronary heart disease, and liver fibrosis stage of patients with fatty liver disease is associated with the risk of adverse events. Liver fibrosis is an important pathological process in liver metabolic disorders. Fibrosis-4 (FIB-4) index, calculated by age, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and platelet count, is a well-recognized, noninvasive tool to evaluate liver fibrosis. The first part of the study found that the FIB-4 index may be the main prognostic indicator for patients with coronary heart disease and liver metabolic disorders. We aim to further explore the value of the FIB-4 index for coronary heart disease in patients with acute coronary syndrome (ACS). 
Methods: This study included 6563 patients, with the primary endpoint being the major adverse cardiac and cerebrovascular event (MACCE), composed of all-cause death, myocardial infarction (MI), and ischemic stroke (IS). Patients were divided into three groups according to literature-based FIB-4 cut-offs: < 1.45, 1.45-3.25 and ≥3.25. Kaplan-Meier analysis was used to compare the cumulative event-free survival rates among groups. Restricted cubic spline (RCS) was used to analyze the correlation between FIB-4 index as a continuous variable and outcome events. The Cox proportional risk regression model was used to evaluate the hazard ratio and 95% confidence interval between the FIB-4 index and the outcomes.
Results: During the median 2.4-year follow-up, a total of 270 MACCEs were recorded. Compared to patients with low FIB-4 scores, patients with intermediate and high FIB-4 scores were significantly associated with elevated MACCE risks (HR 1.33, 95%CI 1.02-1.74; HR 2.59, 95%CI 1.21-4.23). Elevated FIB-4 index was associated with increased risks of all-cause death, cardiac death and MI. FIB-4 index was not associated with an elevated risk of major bleeding. RCS showed that FIB-4 index as continuous variables was positively associated with increased adjusted risk of MACCE regardless of the presence of type 2 diabetes (T2DM). Furthermore, incorporating FIB-4 index into a risk prediction model constructed by traditional risk factors significantly raised the predictive performance. The results were consistent in subgroups with and without T2DM as the similar analyses were performed in subgroups.
Conclusions: Liver fibrosis staged by FIB-4 index was correlated with an increased risk of MACCE, mortality, and MI in ACS. FIB-4 index may help risk stratification of patients independent of T2DM status.
 

Part 3 Study on the potential therapeutic target SIMPLE for liver metabolic disorders

Abstract

Background: The first and second parts of this paper indicate that liver metabolic disorders are associated with poor prognosis in patients with coronary heart disease. Therefore, treating liver metabolic disorders is of great significance for the prevention and treatment of coronary heart disease. The third part of this paper further explores potential therapeutic targets for liver metabolic disorders through basic research. Impaired endo-lysosome-mediated protein degradation is observed in a variety of metabolic disorders, such as atherosclerosis, type 2 diabetes mellitus and NAFLD. Small integral membrane protein of lysosome/late endosome (SIMPLE) is a regulator of endosome-to-lysosome trafficking and cell signaling. However, the role that SIMPLE plays in liver metabolic disorders remains unknown. Here we investigated SIMPLE function in liver metabolic disorders.

Methods: In vitro Simple-knockdown cell model was constructed and received metabolic stimulation. Cellular lipid deposition was detected by Nile red staining. Lipid synthesis/transport and inflammation related gene expression levels were detected by quantitative real-time PCR. Hepatocyte-specific Simple-knockout mice (Simple-HKO) were constructed, liver metabolic disorders mouse models were induced with high-fat (HFD) diet, high-fat-high-cholesterol (HFHC) diet, and methionine-choline-deficient (MCD) diet. Glycolipid metabolism was evaluated by body weight, liver weight, blood glucose, glucose tolerance, serum lipid levels, and serum liver enzyme levels. Hepatic lipid deposition was detected by oil red O staining and hematoxylin & eosin staining. Liver fibrosis was detected by picrosirius red staining. Hepatic inflammatory cell infiltration was detected by CD11b staining. The changes of cellular signaling pathways and gene expression levels in Simple-HKO mice were analyzed by transcriptome. The molecular mechanism was explored with western blot, immune precipitation and other molecular biology experiments.

Results: This study found in vitro knockdown of SIMPLE significantly aggravated lipid accumulation, inflammation in hepatocytes treated with metabolic stimulation. Consistently, in vivo experiments showed that liver-specific Simple-knockout (Simple-HKO) mice exhibited more severe HFD-, HFHC-, and MCD-induced steatosis, glucose intolerance, inflammation, and fibrosis than those fed with normal-chow diet. Meanwhile, RNA-sequencing demonstrated the up-regulated signaling pathways and signature genes involved in lipid metabolism, inflammation and fibrosis in Simple-HKO mice compared to control mice under metabolic stress. Mechanically, we found SIMPLE regulate the lysosomal degradation of epidermal growth factor receptor (EGFR). SIMPLE deficiency results in dysregulated degradation of EGFR, subsequently hyperactivated EGFR phosphorylation, exaggerating liver metabolic disorders development. Moreover, we further demonstrated that using EGFR inhibitor or silencing EGFR expression could ameliorate lipid accumulation induced by the knockdown of SIMPLE. 

Conclusions: SIMPLE ameliorated liver metabolic disorders by prompting lysosomal degradation of EGFR and can also be a potential therapeutic candidate for liver metabolic disorders. 

Liver metabolic disorders and cardiovascular disease
Abstract
Cardiovascular disease is the leading cause of death worldwide, liver metabolic dysfunction is closely associated with cardiovascular disease and may increase the risk of cardiometabolic risk. A growing number of studies showed that liver metabolic dysfunction is independently related to an increased risk of various cardiovascular diseases, including coronary heart disease, cardiomyopathy, and arrhythmia. In this review, we take non-alcoholic fatty liver disease as an example to discuss the evidence that liver metabolic dysfunction disease is associated with an increased risk of multiple cardiovascular diseases, the common risk factors between liver metabolic dysfunction and cardiovascular diseases, and the underlying pathophysiological mechanisms between liver metabolic dysfunction disease and cardiovascular diseases.