褪黑素调节DNA甲基化相关酶减轻大鼠脑缺血再灌注损伤

Melatonin modulates DNA methylation-related enzymes and attenuates cerebral ischemia reperfusion injury in rats

摘要:

目的 观察褪黑素(Mel)对脑缺血再灌注损伤(CIRI)大鼠神经细胞凋亡的影响，并从DNA甲基化相关酶的角度探讨其分子机制。方法 健康成年雄性Sprague-Dawley大鼠35只，随机分为：假手术组(Sham组，n=5)、模型组(CIRI组，n=15)、褪黑素组(Mel组，n=15)。CIRI及Mel组大鼠采用改良的Zea-Longa线栓法建立大鼠CIRI模型，Sham组仅分离颈总动脉。Mel组于造模前后30 min经腹腔注射Mel（5 mg/kg体质量），Sham组于造模前后注射等量的生理盐水。CIRI后24 h，行苏木素－伊红(HE)染色观察大鼠缺血侧大脑皮质神经细胞形态学的变化；CIRI后6 h、24 h及72 h，TUNEL染色法观察Mel对大鼠缺血侧大脑皮质凋亡细胞的影响；免疫组织化学染色法观察Mel对大鼠缺血侧大脑皮质DNA甲基化转移酶1(DNMT1)、DNA甲基化转移酶3a(DNMT3a)蛋白表达的影响。结果 HE染色结果发现，Sham组大鼠大脑皮质神经细胞排列整齐、形态规则；CIRI组大鼠缺血侧大脑皮质神经细胞排列紊乱、形态不规则；Mel组大鼠缺血侧大脑皮质神经细胞排列较整齐、形态较规则。TUNEL染色结果显示，CIRI后6 h、24 h、72 h，与CIRI组相比，Mel组大鼠缺血侧大脑皮质TUNEL⁺细胞数量减少，差异均具有统计学意义(P<0.01)。免疫组织化学染色结果显示，CIRI后6 h、24 h及72 h，Mel组大鼠DNMT1⁺、DNMT3a⁺细胞数量较CIRI组减少，差异均具有统计学意义(P<0.01)。结论 Mel可抑制CIRI大鼠神经细胞凋亡，发挥神经保护作用，其机制可能与Mel降低DNMT1、DNMT3a蛋白表达，下调CIRI后DNA甲基化水平有关。 [引用格式:国际神经病学神经外科学杂志, 2021, 48(4): 333-338.]

Abstract:

Objective To investigate the effect of melatonin （Mel） on neural cellapoptosis in rats with cerebral ischemia reperfusion injury （CIRI） and its molecular mechanism from the perspective of DNA methylation-related enzymes.Methods A total of 35 healthy adult male Sprague-Dawley rats were randomly divided into sham-operation group （Sham group with 5 rats）， model group （CIRI group with15 rats）， and Mel group with 15 rats. The rats in the CIRI group and the Mel group were used to establish a rat model of CIRI using the modified Zea-Longa suture method， and those in the Sham group were given isolation of the common carotid artery alone. The rats in the Mel group were given intraperitoneal injection of Mel （5 mg/kg body weight） at 30 minutes before and after modeling， and those in the Sham group were given injection of an equal volume of normal saline before and after modeling. HE staining was performed at 24 hours after CIRI to observe the change in neural cell morphology at the ischemic side of cerebral cortex； at 6， 24， and 72 hours after CIRI， the TUNEL staining method was used to observe the effect of Mel on apoptotic cells at the ischemic side of cerebral cortex； immunohistochemistry was performed to observe the effect of Mel on the expression of DNA methyltransferase 1 （DNMT1） and DNA methyltransferase 3a （DNMT3a） at the ischemic side of cerebral cortex.Results HE staining showed that in the Sham group， the neural cells in the cerebral cortex had ordered arrangement and regular morphology； in the CIRI group， the neural cells at the ischemic side of cerebral cortex had disordered arrangement and irregular morphology； in the Mel group， the neural cells at the ischemic side of cerebral cortex had relatively ordered arrangement and regular morphology. TUNEL staining showed that at 6， 24， and 72 hours after CIRI， the Mel group had a significant reduction in the number of TUNEL⁺ cells at the ischemic side of cerebral cortex compared with the CIRI group （P<0.01）. Immunohistochemical staining showed that at 6， 24， and 72 hours after CIRI， the Mel group had a significant reduction in the number of DNMT1 and DNMT3a positive cells compared with the CIRI group （P<0.01）.Conclusions Mel can inhibit the apoptosis of nerve cells in CIRI rats and thus exert a neuroprotective effect， possibly by down regulating the protein expression of DNMT1 and DNMT3a and reducing the level of DNA methylation after CIRI. ［Citation:Journal of International Neurology and Neurosurgery, 2021, 48(4): 333-338.］