研究背景和目的

多囊卵巢综合征（Polycystic ovary syndrome, PCOS）是育龄期女性中常见的一种以代谢异常、排卵障碍为主要特征的妇科内分泌与生殖疾病，具有病因不清、机制不明、无法治愈的特点。随着年龄的增长，其血糖异常、胰岛素抵抗、血脂异常和代谢综合征等临床表现可能日益显著。在PCOS患者中，非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）与PCOS常同时出现，比例在34%至70%之间。NAFLD已成为全球慢性肝病的主要病因，对社会健康及经济造成了重大影响。研究显示，单纯的肝脏脂肪变性并不足以诱发 NAFLD，而是由于肥胖、胰岛素抵抗、炎症、高雄激素血症以及线粒体功能障碍等多种平行因素的“多重打击”作用，导致 NAFLD 发生。PCOS和NAFLD这两种代谢性疾病的发病机制和临床特征密切相关，PCOS和NAFLD的代谢紊乱均受到胰岛素抵抗、线粒体功能障碍的影响。肝脏富含线粒体，线粒体功能与NAFLD的起因及进展紧密相连。通过纠正线粒体功能障碍，有望在改善NAFLD上发挥积极作用。治疗PCOS合并NAFLD的首要目标为减重、改善胰岛素抵抗以及预防远期并发症，然而，在目前的PCOS诊治指南和NAFLD防治指南中均未提出PCOS合并NAFLD患者的首选药物治疗方式。二甲双胍作为治疗多囊卵巢综合征患者胰岛素抵抗的首选药物，其在预防PCOS患者非酒精性脂肪性肝病方面的潜在疗效尚未得到充分研究。因此，本研究旨在探讨二甲双胍对NAFLD在PCOS中的潜在保护作用和调控机制，揭露二甲双胍改善PCOS合并NAFLD的线粒体功能的可能分子通路，深入探讨二甲双胍在PCOS中改善肝脏代谢紊乱中的作用，为二甲双胍在临床PCOS患者NAFLD中的应用，提供新的依据和治疗策略。

研究方法

本研究采用在母鼠见栓日第16.5天至第18.5天期间腹腔注射抗苗勒氏管激素（Anti-Müllerian hormone, AMH）的方法诱导其子代雌鼠产生PCOS样表型，建立PCOS样模型小鼠。PCOS样模型小鼠和对照组小鼠在出生后30天开始监测体重，小鼠出生2个月后进行糖耐量实验、胰岛素耐受实验以及监测动情周期变化以评估PCOS模型构建效果。二甲双胍治疗组小鼠从小鼠出生后2个月开始每日灌胃治疗，根据二甲双胍剂量分为高、中、低3个剂量分组干预2个月，之后再重新对小鼠进行糖耐量实验、胰岛素耐受实验、监测动情周期、体重变化等评估，以明确二甲双胍的干预剂量和有效性。之后对对照组小鼠、PCOS模型组小鼠以及二甲双胍治疗的PCOS模型组小鼠进行卵巢组织形态学、肝脏组织形态学、血清性激素水平、血清血脂水平、血清肝功能检测、肝组织氧化应激水平检测、肝组织线粒体功能检测以及线粒体自噬水平以检验二甲双胍对PCOS模型小鼠肝脏组织中代谢和肝脏线粒体功能的影响。利用全转录组测序和蛋白质-蛋白质互作网络系统，通过生物信息学分析筛选得到关键蛋白——ETHE1（Ethylmalonic encephalopathy protein 1, 过硫化物双加氧酶）。

为了探究二甲双胍的确切作用机制，本研究运用了脱氢表雄酮（Dehydroepiandrosterone, DHEA）及游离脂肪酸（Free fatty acid, FFAs，包括棕榈酸和油酸）对小鼠肝细胞（Alpha mouse liver 12, AML-12）进行体外实验，成功构建了非酒精性脂肪性肝病（NAFLD）的小鼠肝细胞模型。通过油红O染色法对细胞进行染色、检测细胞内甘油三酯含量、测量细胞内氧化应激程度、测定细胞内腺嘌呤核苷三磷酸（Adenosine triphosphate, ATP）水平、检测细胞内线粒体呼吸链复合体、测定细胞内线粒体拷贝数、检测细胞内线粒体活性氧、线粒体膜电位、进行细胞线粒体自噬共定位染色以及蛋白免疫印迹（Western Blot, WB）等技术，我们验证了所构建的NAFLD小鼠肝细胞模型能够复现PCOS模型小鼠肝脏组织中的相关特征。进一步的，利用免疫共沉淀（Co-immunoprecipitation, Co-IP）、转染小干扰RNA（Small interfering RNA, siRNA）等方法，验证二甲双胍调控线粒体功能的具体机制。在PCOS患者临床样本中进行验证，收集PCOS患者治疗前和治疗后以及健康对照组受试者的全血样本，进行WB实验，验证二甲双胍对PCOS患者的全血中线粒体自噬的改善。

研究结果

第一部分 多囊卵巢综合征小鼠肝脏脂肪蓄积伴随线粒体功能障碍

成功建立孕期AMH诱导PCOS小鼠模型。表现为性激素异常、小鼠动情周期改变、卵巢组织呈现PCOM样改变、小鼠糖耐量胰岛素耐量异常、小鼠呼吸和基础代谢降低以及小鼠肝脏脂肪蓄积，PCOS患者的代谢和生殖异常表现与PCOS患者临床特征高度一致；

PAMH诱导的PCOS模型小鼠呈现肝脏线粒体功能受损现象。具体表现为氧化应激水平升高，线粒体中mtDNA的拷贝数降低，ATP生成量减少，线粒体呼吸链复合物的数量减少，以及线粒体自噬过程的减少。

第二部分 二甲双胍调节线粒体功能参与减轻PCOS模型小鼠肝脏脂肪蓄积的机制

明确中等剂量的二甲双胍干预可以改善PCOS模型小鼠生殖相关功能异常、代谢异常、肝脏脂肪沉积、肝功能异常异常的表型；

明确中等剂量的二甲双胍干预可以减轻PCOS模型小鼠肝脏组织线粒体功能障碍和增强线粒体自噬；

依托全基因组转录测序以及蛋白质相互作用网络的研究手段，发现ETHE1基因有潜力成为二甲双胍治疗合并多囊卵巢综合征和非酒精性脂肪肝疾病的一个新靶标。ETHE1所介导的线粒体自噬机制在二甲双胍治疗中可能承担着不可或缺的功能。

第三部分 二甲双胍通过激活ETHE1/KEAP1/PINK1途径改善NAFLD肝细胞脂肪蓄积和线粒体自噬水平

成功构建由游离脂肪酸或脱氢表雄酮诱发的NAFLD肝细胞模型，其特征为肝细胞内脂肪堆积、甘油三酯含量上升、氧化应激反应加剧、线粒体膜电位降低、线粒体自噬障碍及功能障碍，这些特征与多囊卵巢综合征模型小鼠肝组织中的病理变化相吻合；

通过体外实验证实，二甲双胍可以改善NAFLD模型肝细胞的损伤，表现为肝细胞脂肪沉积减轻、甘油三酯含量下降、氧化应激减轻、线粒体自噬增强、线粒体功能得到部分挽救，这与经过二甲双胍治疗的PCOS模型小鼠肝组织中的改善现象基本一致；

基于蛋白免疫共沉淀结果，观察到二甲双胍作用靶点ETFDH与ETHE1之间的相互作用，验证了二甲双胍可以通过ETFDH调节ETHE1的表达；

基于体外实验敲低NAFLD模型肝细胞中ETHE1的结果，观察到细胞中敲低的ETHE1削弱了二甲双胍的治疗作用，具体表现为：在敲低ETHE1的NAFLD细胞中即使二甲双胍处理，仍然表现为细胞内脂质沉积和线粒体功能障碍、线粒体自噬减少，提示ETHE1可以影响下游线粒体自噬分子的表达和调控；

基于体外实验敲低NAFLD模型肝细胞中ETHE1的结果，观察到在ETHE1敲低细胞中即使二甲双胍处理，仍然存在KEAP1/NRF2的功能失衡，表现为NRF2入核比例显著减少，同时蛋白免疫共沉淀结果显示，ETHE1和KEAP1之间存在相互作用关系，提示ETHE1可能通过调节KEAP1/NRF2通路发挥抵抗氧化应激和调节线粒体自噬的作用；

基于体外实验敲低NAFLD模型肝细胞中NRF2的结果，观察到敲低NRF2降低了下游自噬分子PINK1/Parkin的表达，从而削弱了二甲双胍通过ETHE1/KEAP1/NRF2途径的线粒体保护作用；

之前的研究结果提示NRF2入核后与PINK1启动子特定位点结合，激活NRF2/PINK1通路，基于体外实验敲低NAFLD模型肝细胞中PINK1的结果，观察到敲低PINK1减弱了二甲双胍对于受损肝细胞的线粒体保护作用，提示二甲双胍通过ETHE1/KEAP1/NRF2/PINK1/Parkin途径发挥调节线粒体自噬和肝细胞脂质沉积的作用。

第四部分 二甲双胍联合饮食运动改善多囊卵巢综合征女性的代谢异常和线粒体功能障碍的初探

基于临床基线资料分析结果，观察到二甲双胍和生活方式干预治疗改善了多囊卵巢综合征患者的糖代谢异常和肝功能异常等临床表型；

基于蛋白免疫印迹结果，观察到在多囊卵巢综合征患者的全血蛋白中存在调节线粒体自噬关键分子的表达异常，而二甲双胍和生活方式干预治疗可以部分挽救线粒体自噬关键分子的异常表达。

研究结论

二甲双胍可以改善孕期AMH诱导的PCOS模型小鼠生殖相关功能异常、代谢异常、肝脏脂肪沉积、肝功能异常等表型，有效缓解PCOS模型小鼠肝组织中线粒体功能异常并促进线粒体自噬作用；

ETHE1在PCOS模型小鼠肝脏组织中显著下调，在二甲双胍治疗后的PCOS模型小鼠肝脏组织中显著上调，在二甲双胍调节肝细胞线粒体功能和代谢平衡中有重要作用；

二甲双胍可以通过激活ETHE1/KEAP1/NRF2/PINK1/Parkin通路，恢复PCOS肝脏中异常的线粒体自噬，减轻PCOS肝脏代谢功能障碍增强线粒体自噬，改善PCOS模型小鼠的NAFLD/MAFLD；

二甲双胍联合饮食运动调节可能是通过增强线粒体自噬改善PCOS患者代谢异常以及肝脏脂肪蓄积，这为二甲双胍在PCOS合并NAFLD/MAFLD患者的临床应用提供了依据。

Background and Objective:

Polycystic ovary syndrome （PCOS） is the most prevalent reproductive endocrine disorder characterized by metabolic abnormalities among women of reproductive age, marked by unclear etiology, ambiguous pathogenesis, and incurability. As patients with PCOS age, their metabolic complications progressively exacerbate, manifesting as a doubling or even exponential increase in metabolic syndrome, insulin resistance, dysglycemia, and dyslipidemia. PCOS frequently coexists with non-alcoholic fatty liver disease （NAFLD）, with 34–70% of PCOS patients exhibiting NAFLD. NAFLD has become the leading cause of chronic liver disease in China, imposing significant health and economic burdens. Research indicates that simple hepatic steatosis alone is insufficient to induce NAFLD; instead, it arises from "multiple hits," including obesity, insulin resistance, inflammation, hyperandrogenemia, and mitochondrial dysfunction. The pathogenesis and clinical features of PCOS and NAFLD are closely linked, with both disorders influenced by insulin resistance and mitochondrial dysfunction. As the organ richest in mitochondria, the liver’s mitochondrial dysfunction plays a pivotal role in NAFLD progression, making mitochondrial-targeted therapies promising for NAFLD management. While lifestyle interventions and weight loss remain first-line strategies for PCOS-NAFLD comorbidity, current clinical guidelines lack specific pharmacological recommendations for this population. Metformin, a first-line insulin-sensitizing agent for PCOS, shows potential for NAFLD prevention through mitochondrial autophagy enhancement, yet its efficacy in PCOS-related NAFLD remains underexplored. This study aims to investigate metformin’s protective effects and regulatory mechanisms in PCOS-associated NAFLD, elucidate its molecular pathways in improving mitochondrial function, and provide clinical evidence for metformin as a therapeutic strategy for PCOS patients with NAFLD.

Methods:

This study first employed prenatal intraperitoneal injection of anti-Müllerian hormone （AMH） to induce PCOS-like phenotypes in female offspring mice, establishing a PCOS-like murine model. Body weight was monitored in both PCOS model and control mice from birth. At postnatal month 2, intraperitoneal glucose tolerance tests （ipGTT）, intraperitoneal insulin tolerance tests （ipITT）, and estrous cycle assessments were performed to evaluate model efficacy. For the metformin intervention, mice in the treatment groups received daily gavage at high-, medium-, and low-dose regimens starting at postnatal month 2 for 2 months. Post-intervention, IPGTT, ITT, estrous cycle monitoring, and body weight measurements were repeated to determine optimal metformin dosing and efficacy. Subsequently, ovarian and hepatic histomorphology, serum sex hormone levels, lipid profiles, hepatic function parameters, oxidative stress markers, mitochondrial function, and mitochondrial autophagy levels were analyzed in control, PCOS model, and metformin-treated PCOS mice to assess metformin’s impact on hepatic metabolism and mitochondrial function. Transcriptome-wide sequencing and protein-protein interaction （PPI） network analysis identified ​ETHE1 （ethylmalonic encephalopathy protein 1, persulfide dioxygenase） as a key regulatory protein via bioinformatics screening.

To elucidate metformin’s mechanisms, an in vitro NAFLD hepatocyte model was established using dehydroepiandrosterone （DHEA） and free fatty acids （FFAs; palmitic acid and oleic acid） in Alpha mouse liver 12 （AML-12） cells. Oil Red O staining, intracellular triglyceride quantification, oxidative stress assays, ATP content measurement, mitochondrial respiratory chain complex activity analysis, mitochondrial copy number assessment, mitochondrial reactive oxygen species （ROS） detection, mitochondrial membrane potential evaluation, colocalization of mitochondria-autophagy markers, and Western blot （WB） confirmed that the in vitro model replicated hepatic phenotypes observed in PCOS mice. Co-immunoprecipitation （Co-IP） and siRNA-mediated gene silencing further validated metformin’s regulatory effects on mitochondrial function. Finally, clinical validation was conducted using pre-treatment and post-treatment whole blood samples from PCOS patients and healthy controls. WB analysis demonstrated metformin’s ameliorative effects on systemic mitochondrial autophagy in PCOS patients.

Results:

Part I: Hepatic Lipid Accumulation Accompanied by Mitochondrial Dysfunction in PCOS Mice

A prenatal AMH-induced PCOS mouse model was successfully established, characterized by hormonal abnormalities, altered estrous cycles, polycystic ovary morphology （PCOM）-like ovarian changes, impaired glucose and insulin tolerance, reduced basal metabolic rate, and hepatic lipid accumulation, consistent with clinical manifestations of PCOS.

Mitochondrial dysfunction was observed in the livers of PCOS mice, evidenced by increased oxidative stress, decreased mitochondrial DNA (mtDNA) copy number, reduced ATP production, impaired mitochondrial respiratory chain complex activity, and suppressed mitochondrial autophagy.

Part II: Mechanisms Underlying Metformin-Mediated Attenuation of Hepatic Lipid Accumulation via Mitochondrial Regulation in PCOS-like Mice

Moderate-dose metformin intervention ameliorated reproductive abnormalities, metabolic disturbances, hepatic lipid deposition, and liver dysfunction in PCOS mice.

Moderate-dose metformin effectively alleviated mitochondrial dysfunction and enhanced mitochondrial autophagy in the livers of PCOS mice.

Transcriptome-wide sequencing and PPI network analysis identified ​ETHE1 as a potential therapeutic target of metformin in PCOS-NAFLD comorbidity, with ETHE1 activation likely playing a critical role in mediating metformin’s effects.

Part III: Metformin Ameliorates NAFLD Hepatic Lipid Accumulation and Mitochondrial Autophagy via the ETHE1/KEAP1/PINK1 Pathway

The NAFLD hepatocyte model was successfully established using free fatty acids FFAs or DHEA in AML-12 cells, demonstrating lipid droplet accumulation, elevated intracellular triglycerides, increased oxidative stress, reduced mitochondrial membrane potential, impaired mitochondrial autophagy, and mitochondrial dysfunction, mirroring the hepatic phenotypes observed in PCOS mice.

Metformin treatment significantly improved hepatocyte lipid deposition, reduced intracellular triglycerides, mitigated oxidative stress, enhanced mitochondrial autophagy, and partially restored mitochondrial function in the in vitro NAFLD model, recapitulating the effects observed in PCOS mice.

Co-IP results revealed an interaction between ETFDH (the metformin-targeted enzyme) and ETHE1, demonstrating that metformin regulates ETHE1 expression through ETFDH modulation.

Knockdown of ETHE1 in NAFLD hepatocytes attenuated metformin’s therapeutic effects, as evidenced by persistent lipid deposition, mitochondrial dysfunction, and reduced autophagy, indicating ETHE1’s essential role in regulating downstream mitochondrial autophagy pathways.

ETHE1 knockdown disrupted the KEAP1/NRF2 balance, leading to diminished nuclear translocation of NRF2. Co-IP further confirmed an interaction between ETHE1 and KEAP1, suggesting ETHE1 modulates oxidative stress and mitochondrial autophagy via the KEAP1/NRF2 pathway.

Knockdown of NRF2 in NAFLD cells reduced the expression of autophagy-related molecules PINK1/Parkin, impairing metformin’s mitochondrial protective effects through the ETHE1/KEAP1/NRF2 axis.

Knockdown of PINK1 attenuated metformin’s mitochondrial protection in damaged hepatocytes, confirming that metformin exerts its effects via the ETHE1/KEAP1/NRF2/PINK1/Parkin pathway to regulate mitochondrial autophagy and lipid metabolism.

Part IV: Preliminary Exploration of Metformin Combined with Lifestyle Interventions in Improving Metabolic and Mitochondrial Dysregulation in PCOS Women

Clinical analysis demonstrated that metformin combined with lifestyle interventions improved glycemic control and hepatic function in PCOS patients.

Western blot analysis revealed aberrant expression of key mitochondrial autophagy regulators in PCOS patient whole blood, which were partially rescued following metformin and lifestyle treatments.

Conclusions:

Metformin ameliorated reproductive dysfunction, metabolic abnormalities, hepatic lipid accumulation, and liver dysfunction in PCOS mice induced by prenatal AMH exposure, while alleviating mitochondrial dysfunction and enhancing mitochondrial autophagy in the liver tissue of PCOS mice.

ETHE1 was significantly downregulated in the liver tissue of PCOS mice but upregulated following metformin treatment, indicating its critical role in mediating metformin’s regulation of mitochondrial function and metabolic homeostasis in hepatocytes.

Metformin activated the ​ETHE1/KEAP1/NRF2/PINK1/Parkin pathway, restoring abnormal mitochondrial autophagy in PCOS-related hepatic dysfunction, mitigating metabolic disturbances, and improving NAFLD/metabolic-associated fatty liver disease (MAFLD) in PCOS mice.

Metformin combined with dietary and exercise interventions may improve metabolic abnormalities and hepatic lipid accumulation in PCOS patients via enhanced mitochondrial autophagy, providing a rationale for its clinical application in managing PCOS comorbid with NAFLD/MAFLD.