研究背景与目的

长期的高血糖会增加心肌梗死风险，损害心功能。目前临床针对糖尿病合并心肌梗死患者并无理想的治疗策略。因此，有效减轻糖尿病心肌缺血造成的损伤对于提高患者的生命质量、改善其预后至关重要。

花生四烯酸（Arachidonic acid，AA）是一种ω-6 多不饱和脂肪酸，在维持细胞膜结构和功能完整性方面起着重要作用，是机体重要的必要脂肪酸之一。新近研究表明，AA及其代谢产物对心血管系统具有重要保护作用。然而，尚无研究报道AA在糖尿病心肌缺血性损伤中的作用及机制。本研究拟通过体外细胞实验、动物在体实验及临床研究，全面阐明AA抗糖尿病心肌缺血性损伤的作用及其机制。

研究方法

首先，通过一次性静脉注射大剂量链脲佐菌素（Streptozotocin, STZ）方法建立小型猪糖尿病模型（Diabetes mellitus, DM）。然后，利用冠状动脉左前降支结扎术建立小型猪糖尿病心肌梗死模型（Diabetes mellitus myocardial infarction，DM + MI）。术前和术后通过血液学、心电图、超声心动图、心脏磁共振成像（Magnetic resonance imaging，MRI）、扫描电镜及心肌组织2,3,5-三苯基氯化四氮唑（2,3,5-Triphenyltetrazolium chloride，TTC）染色等方法综合评估不同组别心肌损伤程度。使用非靶向的UPLC-qTOF-MS方法分析冠状动脉结扎术后0.5小时冠状静脉窦血液代谢特征，对比MI组（心肌梗死）和DM + MI组（糖尿病心肌梗死）代谢产物差异。

然后，对大鼠胚胎心肌细胞（H9C2）施加高糖（High glucose, HG）和缺氧（Oxygen   deprivation, OGD）刺激，体外建立高糖缺氧细胞模型，模拟糖尿病心肌缺血性病理变化，并用不同剂量AA对模型细胞进行处理。通过检测PINK1/Parkin介导的线粒体自噬途径关键蛋白、线粒体自我更新相关蛋白、细胞内活性氧（Reactive oxygen species，ROS）、线粒体活性氧（Mitochondrial ROS，mtROS）、线粒体跨膜电位（Mitochondrial transmembrane potential，ΔΨm）、线粒体与基因组DNA比例（mtDNA：gDNA），初步阐明AA的抗高糖缺氧性损伤效应及机制。此外，在HG + OGD + AA条件下，分别使用环氧酶抑制剂吲哚美辛、PGI₂受体抑制剂Cay10441、线粒体自噬轴关键分子PINK1的特异性siRNA（PINK1-siRNA）和自噬抑制剂3-甲基腺嘌呤（3-Methyladenine，3-MA）处理H9C2细胞，反向印证AA对心肌细胞保护作用依赖于AA/PGI₂代谢通路及与之相关的线粒体自噬途径。

其次，为验证细胞实验结论，本研究利用静脉注射STZ并结扎冠状动脉左前降支的方法建立大鼠糖尿病心肌梗死（DM + MI）模型，AA治疗后通过缺口末端标记法染色（TdT-mediated dUTP nick-end labeling，TUNEL）、HE染色、扫描电镜观察、Western blot、丙二醛（Malondialdehyde，MDA）和总抗氧化能力（Total antioxidant capacity，T-AOC）检测的方法，进一步阐明AA抗糖尿病心肌缺血性损伤的作用及机制，与细胞实验形成互证。

最后，收集健康受试者（Control）、心肌梗死患者（MI）和糖尿病心肌梗死患者（DM + MI），检测血清心肌肌钙蛋白I（cTnI）、AA浓度和超声心动图，分析各检测指标之间的关系，初步评估AA向临床转化的可行性。

研究结果

与MI组比较，DM + MI组实验猪的心肌梗死面积更大，左室射血分数（Left ventricular ejection fraction，LVEF）明显降低，心肌细胞中的线粒体受损更严重。冠状静脉窦血液代谢组学分析的结果表明DM + MI组和MI组之间有11种代谢物差异调节，AA及其代谢产物（PGI₂和6-酮-PGF1α）的浓度降低最为显著，AA降低约32倍，这可能与其梗死周边区磷脂酶A2（PLA₂）的活性显著降低有关。

细胞实验结果表明，AA（25µM）可显著降低HG + OGD诱导的H9C2细胞凋亡指数，减少ROS的产量，改善ΔΨm，增加ATP产量；Western blot实验结果表明，AA逆转了HG + OGD诱导的LC3II/I表达下调、p62表达上调、PINK1和Parkin表达下调，使Drp1和FIS1表达水平升高，并阻止了细胞色素C从线粒体释放到细胞质中，降低了Caspase 3水平；升高mtDNA: gDNA比率，促进了线粒体的自我更新。然而，AA的高糖缺氧性损伤保护作用可分别被吲哚美辛、Cay10441、PINK1-siRNA和3-MA阻断，提示AA抗高糖缺氧性损伤的作用依赖于AA/ PGI₂代谢途径并与PINK1/Parkin介导的线粒体自噬相关。

AA治疗糖尿病心肌梗死大鼠后，DM + MI + AA组大鼠梗死面积显著减小；心肌组织TUNEL染色结果显示AA显著降低了细胞凋亡指数；扫描电镜观察到AA显著减轻了糖尿病心肌缺血性损伤造成的心肌纤维断裂、明暗带融合、线粒体肿胀；Western blot结果表明，DM + MI + AA组LC3II/LC3I表达上调，p62下调，PINK1和Parkin表达增加，Drp1和FIS1表达上调；AA使DM + MI组大鼠血清中MDA恢复正常；DM + MI + AA组的T-AOC显著高于DM + MI组。

临床研究数据表明，与非糖尿病急性心梗患者比较，糖尿病患者在心肌梗死4h后血清cTnI浓度更高，左心射血分数（LVEF）下降更加显著，血浆AA和6-酮-PGF1α水平更低。患者血浆AA浓度与空腹血糖水平呈负相关（r²=0.771）。

结论

本研究发现AA可以保护心肌细胞免受高糖和缺氧引起的损伤，并通过激活PINK1-Parkin线粒体自噬通路，增强心肌细胞线粒体的自我更新并减少细胞凋亡，此过程与AA-PGI₂代谢途径相关。因此，AA有望成为一种保护糖尿病心肌缺血性损伤的新策略。

关键词：花生四烯酸；心肌梗死；糖尿病；线粒体自噬

Background and objective

Long-term hyperglycaemia increase the risk of myocardial infarction and impair heart function. Currently, there is no ideal treatment strategy for patients with diabetic  myocardial ischemia. Therefore, effectively reducing diabetic ischemic damage is crucial for improving patients’ quality of life and prognosis.

Arachidonic acid (AA) is an ω-6 polyunsaturated fatty acid, one of essential fatty acids in the body. It plays an important role in maintaining the structural and functional integrity of cell membranes. Recent studies have shown that AA and its metabolites play an important protective role in the cardiovascular system. However, there are no studies reporting the role and mechanism of AA in diabetic myocardial ischemic injury. This study aims to analyze the changes in AA concentration before and after myocardial ischemia in diabetic patients using non-targeted metabolomics. Through in-vitro cell experiments, animal models, and clinical cohort trials, we aimed to comprehensively elucidate the effect and underlying mechanism of AA against diabetic myocardial ischemic injury.

Methods

First, a porcine diabetes model was established through a single intravenous injection of a high dose of Streptozotocin (STZ). Subsequently, the miniature pig model of diabetic myocardial infarction (DM + MI) was established by ligating the left anterior descending coronary artery. Preoperative and post-operative evaluations of myocardial damage were conducted using hematological, electrocardiographic, echocardiographic, cardiac magnetic resonance imaging (MRI), scanning electron microscopy, and 2,3,5-triphenyltetrazolium chloride (TTC) staining of myocardial tissue. The severity of myocardial injury in different groups was comprehensively assessed. To compare the differences in metabolites between the MI group (myocardial infarction) and the DM + MI group (diabetes mellitus + myocardial infarction), the coronary venous sinus blood of each group was detected at 0.5 hours after coronary artery ligation by non-targeted UPLC-qTOF-MS method.

Then, we stimulated rat embryonic cardiomyocytes (H9C2) with high glucose (HG) and oxygen deprivation (OGD) to established HG + OGD cell model in vitro that could mimick the pathological changes of diabetic myocardial ischemia. Then the the model cells were treated with different doses of AA. The effects of AA on the model cells were evaluated by measuring the expression level of PINK1/Parkin mediated mitophagy pathway key proteins and mitochondrial self-renewal related proteins, intracellular reactive oxygen species (ROS), mitochondrial ROS (mtROS), mitochondrial transmembrane potential (ΔΨm), and the ratio of mitochondrial to genomic DNA (mtDNA: gDNA). Furthermore, under HG + OGD + AA conditions, H9C2 cells were treated with the cyclooxygenase inhibitor indomethacin, the PGI₂ receptor inhibitor Cay10441, PINK1 specific siRNA (PINK1-sirna) and autophagy inhibitor 3-Methyladenine (3-MA).

To validate the results from the above cell experiments, diabetic myocardial ischemic rats modeled by STZ intravenous injection and ligation of the left anterior descending coronary artery were treated with AA, and the effects and mechanism of AA against diabetic myocardial ischemic injury were further elucidated through the following tests on myocardial tissue: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, hematoxylin and eosin (HE) staining, scanning electron microscopy, Western blot, and measurements of malondialdehyde (MDA) and total antioxidant capacity (T-AOC). The data from animal experiments will be mutually corroborated with the results of cell experiments.

Finally, in order to preliminarily assess the feasibility of clinical translation of AA, blood samples from 10 healthy controls (Control), 10 patients with myocardial infarction (MI), and 10 patients with diabetic myocardial infarction (DM + MI) were collected within 4h after infarction onset to detect serum cardiac troponin I (cTnI), AA concentrations and left ventricular ejection fraction (LVEF), and analyze their correlation with each other.

Results

Compared to the MI group, the DM + MI group’s pigs showed larger myocardial infarction area, a significantly decrease in left ventricular ejection fraction (LVEF), and the more severely mitochondrial damaged in myocardial cells. Metabolomic analysis of coronary sinus blood showed 11 differential metabolites between the DM + MI and MI groups. AA and its metabolites (PGI₂ and 6-keto-PGF1α) reduced most significantly, and AA was decreased by approximately 32-fold. This might be related to the significant decrease in PLA₂ activity in the peri-infarct region.

The results of the cell experiments showed that AA (25 µM) significantly reduced the apoptosis index of H9C2 cells induced by HG + OGD, decreased ROS production, improved ΔΨm, and increased ATP efficiency. Western blot results indicated that AA reversed the downregulation of LC3II/I, the upregulation of p62, the downregulation of PINK1 and Parkin in the context of HG + OGD. In addition, AA also increased levels of mitochondrial self-renewal associated proteins (Drp1 and FIS1), prevented the release of cytochrome C from mitochondria to the cytoplasm, reduced Caspase 3 levels, and increased the mtDNA: gDNA ratio. However, the protective effects of AA against high glucose and oxygen deprivation damage could be blocked by indomethacin, Cay10441, PINK-siRNA and 3-MA, suggesting that the protective effect of AA against high glucose and oxygen deprivation damage is related to AA/PGI₂ metabolic pathways and PINK1/Parkin mitochondrial autophagy.

In rat experiments, after the treatment of AA, the infarct area in the DM + MI + AA group significantly decreased. TUNEL staining showed that AA significantly reduced the apoptosis index of cardiomyocytes in the periphery of infarction. Scanning electron microscopy revealed that AA significantly alleviated myocardial fiber rupture, light and dark bands fusion, and mitochondrial swelling caused by diabetic myocardial ischemic injury. Western blot results showed that the expression of LC3II/LC3I was upregulated, p62 was downregulated, PINK1 and Parkin were increased in the DM + MI + AA group, and the expression of Drp1 and FIS1 were also upregulated. AA normalized MDA levels in the serum of rats in the DM + MI group. The T-AOC in the DM + MI + AA group was significantly higher than that in the DM + MI group.

In clinical trial, compared to non-diabetic patients with myocardial infarction (MI), diabetic patients had higher serum concentrations of cTnI and more significant reductions in LVEF at 4 h after myocardial infarction. Additionally, diabetic patients had lower plasma levels of AA and 6-keto-PGF1α. There was a negative correlation between plasma AA concentration and fasting blood glucose levels in patients (r²=0.771).

Conclusions

In summary, we report that ischemic injury under diabetic condition decreases cardiac release of AA, and AA supplementation protects cardiomyocytes from hyperglycema- and ischemia- induced injury in vitro and in vivo. This is attributable to PINK-parkin mediated mitochondrial autophagy, enhanced mitochondrial turnover and decreased cell apoptosis, which is PGI2 dependent. AA might be a new strategy for protecting against diabetic myocardial ischemic injury.

Keywords: Arachidonic acid; Myocardial ischemic injury; Diabetes mellitus; Mitophagy