Abstract：
BACKGROUND: Sepsis-associated encephalopathy (SAE) is a disease characterized by neuroinflammation and cognitive dysfunction caused by systemic infection. Inflammation-induced microglial activation is closely associated with neuroinflammation in SAE. It is widely understood that melatonin has strong anti-inflammatory and immunomodulatory properties beneficial for sepsis-related brain damage. However, the mechanism of melatonin action in SAE has not been fully elucidated.
METHODS: The SAE cell model and SAE mouse model were induced by lipopolysaccharide (LPS). Behavioral tests were performed to analyze cognitive function. Microglial markers and M1/M2 markers were measured by immunofluorescence. Mitophagy was assessed by western blot, mt-Keima and transmission electron microscopy experiments. Immunoprecipitation and co-immunoprecipitation assays investigated the interactions between AMP-activated protein kinase α2 (AMPKα2) and PTEN-induced putative kinase 1 (PINK1).
RESULTS: Melatonin suppresses LPS-induced microglia M1 polarization by enhancing mitophagy, thereby attenuating LPS-induced neuroinflammation and behavioral deficits. However, inhibition or knockdown of AMPKα2 can inhibit the enhancement of melatonin on mitophagy, then weaken its promotion of microglia polarization towards M2 phenotype, and eliminate its protective effect on brain function. Furthermore, melatonin enhances mitophagy through activating AMPKα2, promotes PINK1 Ser495 site phosphorylation, and ultimately regulates microglial polarization from M1 to M2.
CONCLUSIONS: Our findings demonstrate that melatonin facilitates microglia polarization towards M2 phenotype to alleviate LPS-induced neuroinflammation, primarily through AMPKα2-mediated enhancement of mitophagy.
METHODS: The SAE cell model and SAE mouse model were induced by lipopolysaccharide (LPS). Behavioral tests were performed to analyze cognitive function. Microglial markers and M1/M2 markers were measured by immunofluorescence. Mitophagy was assessed by western blot, mt-Keima and transmission electron microscopy experiments. Immunoprecipitation and co-immunoprecipitation assays investigated the interactions between AMP-activated protein kinase α2 (AMPKα2) and PTEN-induced putative kinase 1 (PINK1).
RESULTS: Melatonin suppresses LPS-induced microglia M1 polarization by enhancing mitophagy, thereby attenuating LPS-induced neuroinflammation and behavioral deficits. However, inhibition or knockdown of AMPKα2 can inhibit the enhancement of melatonin on mitophagy, then weaken its promotion of microglia polarization towards M2 phenotype, and eliminate its protective effect on brain function. Furthermore, melatonin enhances mitophagy through activating AMPKα2, promotes PINK1 Ser495 site phosphorylation, and ultimately regulates microglial polarization from M1 to M2.
CONCLUSIONS: Our findings demonstrate that melatonin facilitates microglia polarization towards M2 phenotype to alleviate LPS-induced neuroinflammation, primarily through AMPKα2-mediated enhancement of mitophagy.
摘要：
背景：脓毒症相关性脑病（SAE）是一种以全身性感染引起的神经炎症和认知功能障碍为特征的疾病。炎症诱导的小胶质细胞活化与SAE中的神经炎症密切相关。广泛理解的是,褪黑激素具有对于败血症相关的脑损伤有益的强抗炎和免疫调节特性。然而,褪黑素在SAE中的作用机制尚未完全阐明。
方法：采用脂多糖(LPS)诱导SAE细胞模型和SAE小鼠模型。进行行为测试以分析认知功能。通过免疫荧光测量小胶质细胞标志物和M1/M2标志物。通过蛋白质印迹评估线粒体自噬，MT-Keima和透射电子显微镜实验。免疫沉淀和共免疫沉淀测定研究了AMP激活的蛋白激酶α2（AMPKα2）与PTEN诱导的推定激酶1（PINK1）之间的相互作用。
结果：褪黑素通过增强线粒体自噬抑制LPS诱导的小胶质细胞M1极化，从而减轻LPS诱导的神经炎症和行为缺陷。然而,抑制或敲低AMPKα2可以抑制褪黑素对线粒体自噬的增强,然后减弱其促进小胶质细胞向M2表型的极化，并消除其对大脑功能的保护作用。此外，褪黑素通过激活AMPKα2增强线粒体自噬，促进PINK1Ser495位点磷酸化，并最终调节从M1到M2的小胶质细胞极化。
结论：我们的研究结果表明，褪黑素促进小胶质细胞向M2表型极化，以减轻LPS诱导的神经炎症，主要通过AMPKα2介导的线粒体自噬增强。
方法：采用脂多糖(LPS)诱导SAE细胞模型和SAE小鼠模型。进行行为测试以分析认知功能。通过免疫荧光测量小胶质细胞标志物和M1/M2标志物。通过蛋白质印迹评估线粒体自噬，MT-Keima和透射电子显微镜实验。免疫沉淀和共免疫沉淀测定研究了AMP激活的蛋白激酶α2（AMPKα2）与PTEN诱导的推定激酶1（PINK1）之间的相互作用。
结果：褪黑素通过增强线粒体自噬抑制LPS诱导的小胶质细胞M1极化，从而减轻LPS诱导的神经炎症和行为缺陷。然而,抑制或敲低AMPKα2可以抑制褪黑素对线粒体自噬的增强,然后减弱其促进小胶质细胞向M2表型的极化，并消除其对大脑功能的保护作用。此外，褪黑素通过激活AMPKα2增强线粒体自噬，促进PINK1Ser495位点磷酸化，并最终调节从M1到M2的小胶质细胞极化。
结论：我们的研究结果表明，褪黑素促进小胶质细胞向M2表型极化，以减轻LPS诱导的神经炎症，主要通过AMPKα2介导的线粒体自噬增强。
