褪黑素通过PI3K/AKT通路减轻缺氧缺血性脑损伤新生大鼠大脑皮层神经细胞自噬的研究

Melatonin alleviates autophagy in cortical neurons of neonatal rats with hypoxic- ischemic brain damage via the PI3K/AKT pathway

摘要:

目的 观察褪黑素（melatonin, Mel）对缺氧缺血性脑损伤（hypoxic-ischemic brain damage, HIBD）新生大鼠大脑皮层神经细胞自噬的影响，并从PI3K/AKT信号通路探讨其机制，为Mel的临床应用提供依据。方法 将7日龄Sprague-Dawley新生大鼠随机分为假手术组、HIBD组及Mel组（各组n=9）。采用经典Rice-Vannucci法构建新生大鼠HIBD模型。采用苏木精-伊红和尼氏染色法观察新生大鼠大脑皮层神经细胞形态；免疫荧光染色和Western blot法检测自噬相关蛋白微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3, LC3）及Beclin-1蛋白表达水平；免疫组化和Western blot法检测磷酸化磷脂酰肌醇3-激酶（phosphorylated phosphoinositide 3-kinase, p-PI3K）、磷酸化蛋白激酶B（phosphorylated protein kinase B, p-AKT）蛋白表达水平；通过Pearson相关性分析探讨Mel组与HIBD组自噬与PI3K通路的相关性。结果 建模后24 h，假手术组新生大鼠大脑皮层神经细胞大小形态正常、排列规则，HIBD组神经细胞胞体肿胀、排列不规则，Mel组神经细胞排列较规整、形态相对正常。假手术组尼氏小体形态规整，HIBD组尼氏小体形态异常，Mel组尼氏小体形态尚规整。HIBD组LC3及Beclin-1平均荧光强度较假手术组增加，Mel组较HIBD组减少（P<0.05）。HIBD组p-PI3K⁺、p-AKT⁺细胞数较假手术组减少，Mel组较HIBD组增加（P<0.05）。HIBD组LC3、Beclin-1蛋白表达水平较假手术组增加，p-PI3K、p-AKT蛋白表达水平较假手术组减少（P<0.05）；Mel组LC3、Beclin-1蛋白表达水平较HIBD组减少，p-PI3K、p-AKT蛋白表达水平较HIBD组增加（P<0.05）。相关性分析显示，Mel组与HIBD组损伤侧大脑皮层LC3、Beclin-1蛋白平均荧光强度差值与p-PI3K⁺、p-AKT⁺细胞数差值均呈负相关（P<0.05）。结论 Mel可抑制HIBD新生大鼠大脑皮层神经细胞过度自噬，从而减轻HIBD，其机制与PI3K/AKT通路相关。

Abstract:

Objective To observe the effects of melatonin on autophagy in cortical neurons of neonatal rats with hypoxic-ischemic brain damage (HIBD) and to explore its mechanisms via the PI3K/AKT signaling pathway, aiming to provide a basis for the clinical application of melatonin.Methods Seven-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group, an HIBD group, and a melatonin group (n=9 each). The neonatal rat HIBD model was established using the classic Rice-Vannucci method. Neuronal morphology in the neonatal rat cerebral cortex was observed with hematoxylin-eosin staining and Nissl staining. Autophagy-related protein levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence staining and Western blot analysis. Phosphorylated phosphoinositide 3-kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) protein expression levels were measured by immunohistochemistry and Western blot. The correlation between autophagy and the PI3K pathway in the melatonin group and the HIBD group was analyzed using Pearson correlation analysis.Results Twenty-four hours post-modeling, neurons in the sham operation group displayed normal size and orderly arrangement. In contrast, neurons in the HIBD group showed swelling and disorderly arrangement, while those in the melatonin group had relatively normal morphology and more orderly arrangement. Nissl bodies were normal in the sham operation group but distorted in the HIBD group; however, they remained relatively intact in the melatonin group. The average fluorescence intensity of LC3 and Beclin-1 was higher in the HIBD group compared to the sham operation group, but was reduced in the melatonin group compared to the HIBD group (P<0.05). The number of p-PI3K⁺ and p-AKT⁺ cells decreased in the HIBD group compared to the sham operation group but increased in the melatonin group compared to the HIBD group (P<0.05). LC3 and Beclin-1 protein expression levels were higher, and p-PI3K and p-AKT levels were lower in the HIBD group compared to the sham operation group (P<0.05); however, in the melatonin group, LC3 and Beclin-1 levels decreased, and p-PI3K and p-AKT increased compared to the HIBD group (P<0.05). The correlation analysis results showed that the difference of the mean fluorescence intensity of LC3 and Beclin-1 protein in the injured cerebral cortex between the melatonin and HIBD groups was negatively correlated with the difference of the number of p-PI3K⁺ and p-AKT⁺ cells between the two groups (P<0.05).Conclusions Melatonin can inhibit excessive autophagy in cortical neurons of neonatal rats with HIBD, thereby alleviating HIBD. This mechanism is associated with the PI3K/AKT pathway.