摘要目的:评价YTH结构域家族蛋白2(YTHDF2)在糖尿病大鼠心肌缺血再灌注损伤(MIRI)中的作用及其与核因子E2相关因子2(NRF2)-铁自噬的关系。方法:动物实验　SPF级健康雄性大鼠，6～8周龄，体质量200～220 g。腹腔注射1%链脲佐菌素65 mg/kg制备1型糖尿病模型。取糖尿病大鼠36只，采用随机数字表法分为3组( n＝12)：糖尿病假手术组(DS组)、糖尿病心肌缺血再灌注组(DIR组)和YTHDF2敲低＋糖尿病心肌缺血再灌注组(AAV-Y＋DIR组)。另取非糖尿病大鼠36只，采用按随机数字表法分为4组：假手术组(NS组， n＝12)、心肌缺血再灌注组(NIR组， n＝12)、腺相关病毒对照组(AAV-N组， n＝6)和YTHDF2敲低组(AAV-Y组， n＝6)。采用结扎冠状动脉左前降支30 min、再灌注2 h的方法制备MIRI模型。采用腺相关病毒敲低YTHDF2。再灌注结束时，测定血清肌酸激酶同工酶(CK-MB)和心肌肌钙蛋白Ⅰ(cTnI)浓度，处死大鼠取心肌组织，光镜下观察病理学结果，确定心肌梗死面积，测定YTHDF2、NRF2和核受体辅激活因子4(NCOA4)表达。细胞实验　将H9c2细胞采用随机数字表法分为9组( n＝24)：对照组(NC组)、高糖组(HG组)、缺氧复氧组(HR组)、高糖缺氧复氧组(HHR组)、转染对照组(siN组)、YTHDF2敲低组(siY组)、YTHDF2敲低＋高糖缺氧复氧组(siY＋HHR组)、NRF2抑制剂ML385＋高糖缺氧复氧组(M＋HHR组)和YTHDF2敲低＋NRF2抑制剂ML385＋高糖缺氧复氧组(siY＋M＋HHR组)。使用siRNA转染细胞敲低YTHDF2。采用高糖48 h、缺氧4 h、复氧2 h的方法建立高糖缺氧复氧损伤模型。检测细胞活力和乳酸脱氢酶(LDH)活性，计数自噬小体，检测YTHDF2、NRF2和NCOA4表达。 结果:动物实验　与NS组比较，心肌缺血再灌注结束时，糖尿病大鼠和非糖尿病大鼠组血清CK-MB、cTnI水平和心肌梗死面积百分比升高，心肌组织YTHDF2和NCOA4表达上调，NRF2表达下调( P<0.05)；与NIR组比较，DIR组血清CK-MB、cTnI水平和心肌梗死面积百分比升高，心肌组织YTHDF2和NCOA4表达上调，NRF2表达下调( P<0.05)；与DIR组比较，AAV-Y＋DIR组血清CK-MB、cTnI水平和心肌梗死面积百分比降低，心肌组织YTHDF2和NCOA4表达下调，NRF2表达上调( P<0.05)，病理学损伤减轻。细胞实验　与HG和HR组比较，HHR组细胞活力降低，LDH活性升高，自噬小体计数增多，YTHDF2和NCOA4表达上调，NRF2表达下调( P<0.05)。与HHR组比较，M＋HHR组细胞活力降低，LDH活性升高，自噬小体计数增多，NRF2表达下调，NCOA4表达上调( P<0.05)；siY＋HHR组细胞活力升高，LDH活性降低，自噬小体计数减少，YTHDF2和NCOA4表达下调，NRF2表达上调( P<0.05)；siY＋M＋HHR组上述各指标差异无统计学意义( P>0.05)。 结论:YTHDF2可通过下调NRF2表达，增强铁自噬水平，参与糖尿病大鼠MIRI的过程。

abstractsObjective:To evaluate the role of YTH domain family protein 2 (YTHDF2) in myocardial ischaemia-reperfusion injury (MIRI) in diabetic rats and the relationship with the nuclear factor E2-related factor 2 (NRF2)-ferritinophagy.Methods:This experiment was performed in 2 parts. Part Ⅰ Animal experiment SPF healthy male rats, aged 6-8 weeks, weighing 200-220 g, were used. A type 1 diabetes mellitus (DM) model was established by intraperitoneal injection of 1% streptozotocin at a dose of 65 mg/kg. Thirty-six diabetic rats were divided into 3 groups ( n=12 each) using a random number table method: DM sham operation group (DS group), DM myocardial ischaemia-reperfusion group (DIR group), and YTHDF2 knockdown + DM myocardial ischaemia-reperfusion group (AAV-Y+ DIR group). Another 36 non-diabetic rats were selected and divided into 4 groups using the random number table method: sham operation group (NS group, n=12), myocardial ischaemia-reperfusion group (NIR group, n=12), adeno-associated virus control group (AAV-N group, n=6), and YTHDF2 knockdown group (AAV-Y group, n=6). The MIRI model was established by ligating the left anterior descending branch of the coronary artery for 30 min, followed by reperfusion for 2 h. Adeno-associated virus was employed to knock down YTHDF2. At the end of reperfusion, serum concentrations of creatine kinase isoenzyme MB(CK-MB) and cardiac troponin Ⅰ(cTnI) were measured using enzyme-linked immunosorbent assay. The animals were sacrificed, myocardial tissues were harvested, and the pathological changes were observed with a light microscope to assess the myocardial infarct size. The expression of YTHDF2, NRF2, and nuclear receptor coactivator 4 (NCOA4) was detected by Western blot. Part Ⅱ Cell experiment H9c2 cells were divided into 9 groups ( n=24 each) using the random number table method: control group (NC group), high-glucose group (HG group), hypoxia-reoxygenation group (HR group), high-glucose hypoxia-reoxygenation group (HHR group), transfection control group (siN group), YTHDF2 knockdown group (siY group), YTHDF2 knockdown + high-glucose hypoxia-reoxygenation group (siY + HHR group), NRF2 inhibitor ML385 + high-glucose hypoxia-reoxygenation group (M + HHR group), and YTHDF2 knockdown + NRF2 inhibitor ML385 + high-glucose hypoxia-reoxygenation group (siY + M + HHR group). The cells were transfected with siRNA to knock down YTHDF2, and a high-glucose, hypoxia and reoxygenation injury model was established by subjecting cells to 48 h of high glucose, followed by 4 h of hypoxia and 2 h of reoxygenation. The cell viability and lactic dehydrogenase(LDH) activity were determined, autophagic vesicles were counted, and the expression of YTHDF2, NRF2 and NCOA4 was detected by Western blot. Results:Part Ⅰ Animal experiment At the end of myocardial ischaemia-reperfusion, serum levels of CK-MB and cTnI and the percentage of myocardial infarct size were significantly increased, the expression of YTHDF2 and NCOA4 in myocardial tissues was up-regulated, and the expression of NRF2 was down-regulated in both diabetic and non-diabetic groups ( P<0.05). Compared with NIR group, serum levels of CK-MB and cTnI and the percentage of myocardial infarct size were significantly increased, the expression of YTHDF2 and NCOA4 in myocardial tissues was up-regulated, and the expression of NRF2 was down-regulated in DIR group ( P<0.05). Compared with DIR group, serum levels of CK-MB and cTnI and the percentage of myocardial infarct size were significantly decreased, the expression of YTHDF2 and NCOA4 in myocardial tissues was down-regulated, and the expression of NRF2 was up-regulated ( P<0.05), and the pathological damage was reduced in AAV-Y + DIR group. Part Ⅱ Cell experiment Compared with HG and HR groups, the cell viability was significantly decreased, the activity of LDH was increased, the counts of autophagic vesicle were increased, the expression of YTHDF2 and NCOA4 was up-regulated, and the expression of NRF2 was down-regulated in HHR group ( P<0.05). Compared with HHR group, the cell viability was significantly decreased, the activity of LDH was increased, the counts of autophagic vesicle were increased, the expression of YTHDF2 and NCOA4 was up-regulated, and the expression of NRF2 was down-regulated in M + HHR group, and the cell viability was significantly increased, the activity of LDH was decreased, the counts of autophagic vesicle were decreased, the expression of YTHDF2 and NCOA4 was down-regulated, and the expression of NRF2 was up-regulated in siY + HHR group ( P<0.05), and no statistically significant changes were found in the above indicators in siY + M + HHR group ( P>0.05) Conclusions:YTHDF2 can down-regulate the expression of NRF2, enhance the level of ferritinophagy, and participate in the process of MIRI in diabetic rats.