多模态小动物PET/CT分子显像评估二甲双胍对大鼠心肌缺血再灌注损伤的保护作用及其分子机制

Myocardial Ischemia Reperfusion Injury (MIRI) triggers inflammatory responses which play an important ‘double-edged sword’ role in the process of MIRI. Increasing evidence has shown that metformin (MF)-a non-expensive medication for the treatment of type 2 diabetes can improve clinical outcomes of congestive heart failure. The cardioprotective effect of MF might be attributed to suppression of toll-like receptor 4 (TLR4) signaling. In line with previous studies, our preliminary experiments with 18F-FDG microPET/CT imaging also showed the cardioprotective role of MF treatment in a rat model of MIRI. .The aims of this study are thus to investigate the underlying mechanisms that lead to MIRI and the cardioprotective role of MF in MIRI, especially in relation to inflammatory responses and TLR4-associated mechanisms. .We propose several experiments applying in vivo imaging techniques (micro PET/CT) to follow the myocardial injury longitudinally as well as ex vivo histologic and biochemical methodologies to examine key factors that are involved in myocardial injury and inflammatory responses. .(1) First, a MIRI model will be established by ligation of the left coronary artery for 30 mins followed by reperfusion. Then, several micro PET radioligands will be used to study myocardial perfusion (13N-NH3), myocardial glucose metabolism (18F-FDG), myocardial inflammation TSPO (18F-FDPA), and cardiac sympathetic denervation and innervation (11C-HED) in rats 1 day before surgery and at day 7 (acute phase), and week 4 (sub-acute phase) and 8 (chronic phase) after surgery. (2) The details of proposed experiments are presented as follows: The imaging data will be used to identify the size and the degree of ischemic injury, myocardial viability, the extent of myocardial scar (13N-NH3, 18F-FDG, 11C-HED) as well as myocardial inflammation (18F-FDPA). Left ventricular function (LVEF) and remodeling (ESV, EDV) was assessed by gated perfusion and FDG PET studies. To strengthen our study, histologic and biochemical techniques will be applied to investigate the dynamic changes of myocardial injury-associated biomarkers as well as inflammation-associated factors, such as TLR4 mRNA, TNF-α, and IL-6 in heart tissues and serum. Further, the correlation between in vivo imaging and ex vivo biomarkers will be analyzed. The outcome results would be beneficial for understanding biochemical mechanisms, especially TLR4-associated inflammatory responses underlying MIRI. In addition, based on the experimental data, MIRI rats with high risk of remodeling and heart failure will be selected and involved in the next step experiment to test the effect of MF treatment. (3) Next, the effect and mechanisms of MF on MIRI will be studied. In this experiment, MIRI rats will be divided into several groups to test the effect of different doses (50 mg/kg/12 h vs. 100 mg/kg/12 h) and regiments (prior vs. after injury) of MF treatment on MIRI. Micro PET/CT imaging as well as biochemical and histological experiments will be carried out to judge myocardial function and inflammatory responses in MIRI. It would be very important to determine the effectiveness and dosage of MF treatment in MIRI and unravel the mechanisms of such treatment in TLR-related signaling pathways and overall cardiovascular functions. .In summary, our proposed study sought to resolve 2 major questions in MIRI: 1). How does inflammation evolve after MIRI; 2). What are the efficacy and mechanisms of MF treatment in MIRI. .To the best of our knowledge, this is the first study attempting to combine in vivo imaging method and ex vivo histological and biochemical techniques to evaluate a drug candidate in MIRI. In combination of these techniques, our research questions can be investigated at functional, cellular, and molecular levels. Our study will shed light on the inflammatory mechanisms of MIRI and provide new insights into therapeutic strategies against MIRI.

炎症反应对心肌缺血再灌注损伤(MIRI)有“双刃剑”作用。我们前期研究证明二甲双胍（MF）对MIRI有保护作用，文献报道MF抑制心肌toll样受体4（TLR4）活性和心肌炎症，保护MIRI。本研究拟建立大鼠MIRI模型，MF治疗，于术后1，4，8周行多模态小动物PET/CT心肌显像：门控灌注(13N-NH3)、门控代谢(18F-FDG)、炎症特异性TSPO (18F-FDPA)、神经受体(11C-HED)，定量评价MF对缺血心肌的灌注、代谢、细胞活性、炎症、神经受体活性的作用，随时间变化规律以及对心室重构和心功能的影响。结合活体显像、组织病理学和分子生物学，从多角度、多层面（细胞、局部和整体），综合系统评价MIRI中炎症反应的规律和MF保护缺血心肌作用；揭示MF抑制TLR4活性与心肌炎症的分子机制，为临床选择高危患者，给予MF治疗，预防心室重构发生发展，提供理论依据，为研发新药物提供新思路。

心肌缺血再灌注（I/R）损伤与全身氧化应激、心肌线粒体稳态和心肌细胞凋亡相关。二甲双胍（Met）通过AMPK受体感受细胞内能量变化，调节心肌细胞活性。然而，Met对I/R后的长效心肌保护作用及相关机制尚不清楚。本研究建立大鼠I/R模型，分为I/R模型组（Vehi组）和Met治疗组（Met组），Met组大鼠灌胃Met（150mg/kg/24h）。另设对照组（Sham组）。分别在I/R后1周、4周和8周，行13N-NH3·H2O心肌血流灌注，18F-FDG门控心肌代谢和18F-FDPA 线粒体TSPO受体显像，评估大鼠心肌损伤（TPD、VM和NM）、左心室功能（LVEDV、LVESV和LVEF）和左心室在体TSPO（global TBR，infarct territory TBR和 remote territory TBR）。分析线粒体Hsp60与TSPO的相关性。结合不同时期I/R分子生物学和病理组织学特征，分析自由基氧化特点（SOD、MDA和GSH-Px）；揭示梗死区和远端区心肌细胞Bcl-2/Bax和p-AMPK/AMPK变化规律；分析心肌梗死区和远端区细胞活性、TSPO与CD68（+）共定位情况。结果显示（1）与Vehi组比较，1周、4周和8周Met组TPD无变化、NM减低，8周Met组VM增加。与Sham组比较，8周Vehi组LVEF显著降低，Met组无变化。Vehi组大鼠远端区线粒体Hsp60明显高于梗死区。与Vehi组大鼠相比，8周Met组大鼠Global TBR，Infarct territory TBR和Remote territory TBR均减少。（2）与Vehi组比较，Met组SOD、GSH-Px随时间延长而增加，MDA减少。与Vehi组比较，Met组1周、8周不同部位Bcl-2/Bax均增加。与Sham组比较，Vehi组心肌梗死区p-AMPK/AMPK在1周增加，Met组无变化；与Vehi组比较，Met组8周远端区p-AMPK/AMPK增加。I/R后1周，Vehi组梗死区TSPO高表达与CD68（+）共定位；I/R后8周，远端区TSPO高表达且无CD68（+）共定位。综上所述，（1）长期服用二甲双胍提高慢性期心肌活性、改善左心室功能，延缓左心室重塑进程。（2）二甲双胍通过“时间-空间”效应，维持线粒体稳态、保护细胞活性。