右美托咪定通过AMPK/Sirt1信号通路对糖尿病大鼠心肌缺血再灌注损伤的作用及其机制研究

Effects and mechanisms of dexmedetomidine on myocardial ischemia/reperfusion injury in diabetic rats via the AMPK/Sirt1 signaling pathway

摘要:

目的 探讨右美托咪定通过调控腺苷酸活化蛋白激酶（AMPK）/沉默信息调节因子-1（Sirt1）信号通路对糖尿病大鼠心肌缺血再灌注（I/R）损伤的作用和机制研究。方法 选取48只健康雄性SD大鼠，应用链脲佐菌素成功复制糖尿病模型，按照随机数字表法分为假手术组（Sham组）、心肌缺血/再灌注组（I/R组）、右美托咪定+I/R组（DEX组）、右美托咪定+I/R+AMPK抑制剂Compound C组（CC组），每组12只。I/R组、DEX组、CC组结扎糖尿病大鼠冠状动脉左前降支缺血30 min、再灌注120 min复制心肌I/R模型，Sham组仅开胸不结扎。通过酶联免疫吸附试验检测大鼠血浆肌酸激酶同工酶（CK-MB）、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平，TTC染色检测心肌梗死情况，苏木精-伊红染色观察心肌组织病理学变化，Western blotting检测心肌组织p-AMPK、AMPK、Sirt1、凋亡相关蛋白（Bcl-2、Bax）的表达。结果 I/R组CK-MB、TNF-α和IL-6较Sham组高（P <0.05），DEX组CK-MB、TNF-α和IL-6较I/R组低（P <0.05），CC组TNF-α、IL-6较DEX组高（P <0.05）。TTC染色结果示，Sham组心肌组织大部分被染成砖红色，I/R组、DEX组、CC组心肌组织被染成不同程度的灰白色，以I/R组最为明显。I/R组心肌梗死面积占比较Sham组增大（P <0.05），DEX组较I/R组减小（P <0.05），DEX组与CC组比较，差异无统计学意义（P >0.05）。HE染色结果示，Sham组心肌细胞形态、结构完整，排列整齐，未见明显的细胞变性、坏死和炎症浸润，心肌纤维排列规整，结构清晰。与Sham组比较，I/R组心肌细胞排列不规则，细胞水肿明显，可见心肌细胞坏死，胞核碎裂或溶解，部分胞质溶解呈嗜酸性均质状，周围伴有出血，可见少量炎症细胞浸润，肌纤维不连续且间隙不等。与I/R组比较，DEX组和CC组心肌细胞排列较为整齐，细胞水肿程度减小，未见明显的炎症细胞浸润，心肌纤维排列相对规整，其中DEX组改善较为明显。I/R组心肌组织p-AMPK/AMPK、Sirt1较Sham组升高（P <0.05），Bcl-2/Bax较Sham组降低（P <0.05）；DEX组较I/R组升高（P <0.05），CC组较DEX组降低（P <0.05）。结论 右美托咪定可能通过激活AMPK/Sirt1信号通路减轻糖尿病大鼠心肌I/R损伤。

Abstract:

Objective To investigate the effects and mechanisms of dexmedetomidine on myocardial ischemia/reperfusion (I/R) injury in diabetic rats via the AMPK/Sirt1 signaling pathway.Methods Forty-eight healthy male SD rats were used to successfully establish diabetic models using streptozotocin, and were divided into the sham operation group (sham group), myocardial I/R group (I/R group), dexmedetomidine + I/R group (DEX group), and dexmedetomidine + I/R + AMPK inhibitor Compound C group (CC group) by the random number table method, with 12 rats in each group. The myocardial I/R model was established by ligation of the left anterior descending coronary artery of diabetic rats in the I/R group, DEX group and CC group for 30 min of ischemia and 120 min of reperfusion, while the rats in the sham group only underwent thoracotomy without artery ligation. The levels of creatine kinase isoenzyme (CK-MB), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in plasma of rats were detected by enzyme-linked immunosorbent assay. TTC staining was used to detect the myocardial infarction area. Hematoxylin-eosin staining was used to observe the pathological changes of myocardial tissues, and Western blotting was used to detect the protein expressions of p-AMPK, AMPK, Sirt1 and apoptosis-related proteins (Bcl-2, Bax) in myocardial tissues.Results The levels of CK-MB, TNF-α and IL-6 in the I/R group were higher than those in the sham group (P < 0.05), those in the DEX group were lower compared with the I/R group (P < 0.05), and those in the CC group were higher compared with the DEX group (P < 0.05). TTC staining results showed that most of the myocardial tissues in the sham group were stained brick red, while those in the I/R, DEX, and CC groups were stained various shades of gray, with the I/R group being the most obvious. The myocardial infarction area in the I/R group was larger than that in the sham group (P < 0.05), that in the DEX group was smaller compared with the I/R group (P < 0.05), and that was no different between the DEX group and the CC group (P > 0.05). The HE staining results revealed intact morphology and structure as well as orderly arrangement of myocardial cells, no obvious cellular degeneration, necrosis or inflammatory infiltration, and well-organized myocardial fibers with clear structures. Compared to the sham group, myocardial cells in the I/R group showed irregular arrangement, marked cellular edema, evident myocardial cell necrosis with nuclear fragmentation or karyolysis, and partial cytoplasmic disintegration with eosinophilic and homogenous changes, accompanied by hemorrhages and mild inflammatory cell infiltration as well as discontinuous myocardial fibers with variable intercellular spaces. Compared to the I/R group, myocardial cells in both the DEX and CC groups showed more regular arrangement, reduced cellular edema, and no significant inflammatory cell infiltration. Myocardial fibers were arranged relatively orderly, with the DEX group showing more marked improvement. The protein expressions of p-AMPK/AMPK and Sirt1 in myocardial tissues were increased but those of Bcl-2/Bax were decreased in the I/R group compared with the sham group (P < 0.05), in the DEX group compared with the I/R group (P < 0.05), and in the CC group compared with the DEX group (P < 0.05).Conclusions Dexmedetomidine may alleviate I/R injury in diabetic rats by activating the AMPK/Sirt1 signaling pathway.