The Role And Mechanism Of Melatonin Regulated Autophagy In Early Brain Injury After Subarachnoid Hemorrhage

BackgroundSubarachnoid hemorrhage (SAH) accounts for5%-10%of all strokes. Usually occurred secondary to the rupture of intracranial aneurysms. Considerable advances have been made in endovascular techniques, diagnostic methods and surgical and perioperative management paradigms, the rate of aneurysms’rebleeding is well controlled. However, treatments aims to the intracranial aneurysm do not stop the pathophysiological process of SAH, as a result, the outcome for patients with SAH remains poor. For decades, delayed vasospasm after subarachnoid hemorrhage has traditionally been considered the most important determinant of poor outcome. However, theory targeting vasospasm is being questioned increasingly.Early brain injury refers to immediate injury lasts72h after SAH. EBI is the primary cause of mortality and morbidity in SAH patients. The treatment of EBI is a major goal in the management of patients surviving SAH. The possible mechanisms of EBI after SAH involve the rapid rise of intracranial pressure and the reduction of cerebral perfusion pressure, BBB disruption, brain edema, oxidative and nitrosative stress, and neural apoptosis.However, the exact molecular mechanism of EBI still remains obscure.Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Recently, the crosstalk between autophagy and inflammasome has been researched widely. The result of the studies till now suggest that autophagy may lead to swollen mitochondria and the release of its DNA (mtDNA), as a result, the activation of NLRP3inflammasome is blocked and IL-1β secretion is impaired. There also is interplay between autophagy and apoptosis. The regulation of autophagy activation is thought to be related to protection from mitochondrial dysfunction and may play an important role in neurodegenerative disorders. Our earlier research demonstrated the protective role of autophagy in EBI after SAH and these protective effects may be related to the fact that autophagy is a clearance route for damaged mitochondria.Some studies suggest that melatonin reduces autophagy in some experimental models, while many other studies support an active role for melatonin in autophagy. Previous studies have shown that melatonin protects against ischemia/reperfusion-induced brain injury via autophagy induction. However, no study has investigated the influence of melatonin on autophagy and the related neuro-inflammation and neural apoptosis in EBI after SAH. In the current study, we focused on the role of melatonin in autophagy modulation and its relationship to anti-apoptotic and neuro-inflammation mechanisms to increase the understanding of the neuroprotective effect of melatonin treatment in SAH models.MethodsPart11. SAH model was performed using endovascular perforation technique. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH6h group, SAH12h group, SAH24h group, SAH48h group, and SAH72h group. Neurological deficit scores and brain edema were evaluated. The levels of Proteins (LC3, Atg5, Beclinl, and IL-1β) were evaluated by western blot analysis. The LC3cellular localization is performed by double fluorescence labeling. Immunohistochemistry against ED-1is performed for inflammation related cells.2. Adult male Sprague Dawley rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors3-methyladenine (3-MA) or inducer rapamycin before the onset of SAH.Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group, SAH+3MA group, and SAH+RAP group. Neurological deficit scores and brain edema were evaluated. The levels of Proteins (LC3, Atg5, Beclinl, IL-1β, NLRP3, ASC, and Caspasel) were evaluated by western blot analysis.Part21. Adult male Sprague Dawley rats were treated with intraperitoneal injection of melatonin2h after SAH. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group and SAH+MEL group. Neurological deficit scores and brain edema were evaluated.2.(1) Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group and SAH+MEL group. BBB permeability was quantitatively evaluated by Evans blue extravasation and IgG staining. The levels of Proteins (ZO-1, occluding, claudin5, MMP9, VEGF, IL-1β, IL-6, and TNF-a) were evaluated by western blot analysis.The activity of MMP9and MMP2were detected by gel zymography.(2) Adult male Sprague Dawley rats were treated with pretreated with intracerebral ventricle infusion of the autophagy inhibitors3-methyladenine (3-MA) and intraperitoneal injection of melatonin2h after SAH. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group, SAH+MEL group and SAH+MEL+3-MA group. The levels of Proteins (LC3, Atg5, Beclinl, IL-1β, NLRP3, ASC, and Caspasel) were evaluated by western blot analysis.3.(1) Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group and SAH+MEL group. Apoptosis were evaluated by TUNEL and immunohistochemistry against Caspase3.(2) Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group, SAH+MEL group and SAH+MEL+3-MA group. The levels of Proteins (LC3, Caspase3, Bax, and cytochrome c) were evaluated by western blot analysis.ResultPart11. Western blot analysis showed that SAH resulted in increased ratio of LC3-II/LC3-I, Atg5, Beclinl, and IL-1β levels. These changes peaked at24h after SAH. Immunofluorescence double-staining demonstrated that LC3was co-located with Iba-1, indicated LC3were expressed in microglia.2. Western blot analysis showed that3-MA treatment decreasedthe ratio of LC3-Ⅱ/LC3-Ⅰ, the levelsof Atg5,Beclinl, IL-1β, NLRP3, ASC, and Caspasel, increased brain edema and neurological deficits after SAH. Conversely, RAP treatment reversed these changes and decreased neurological deficits.Part21. Melatonin treatment improved neurological score and reduced water content in early brain injury after SAH.2.(1) Treatment with melatonindecreased EB extravasation and IgG staining. Western blot analysis showed that melatonin treatment repressedthe levelsof ZO-1, occludin, claudin5, MMP9, VEGF, IL-1β,IL-6, and TNF-a which were increased after SAH. Gel zymography showed that melatonin treatment repressedthe activity of MMP9which was increased after SAH(2) Western blot analysis showed that melatonin treatment significantly increasedthe ratio of LC3-Ⅱ/LC3-Ⅰ, the levelsof Atg5, Beclinl, IL-1β, NLRP3, and Caspasel after SAH. Conversely,3-MA treatment reversed these changes.3.(1) Treatment with melatonin decrease the number of TUNEL-positive cells and Caspase3-positive cells.(2) Western blot analysis showed that melatonin treatment increasedthe ratio of LC3-Ⅱ/LC3-Ⅰ, decreased Bax translocation to the mitochondria, the sequent cytochrome c release from the mitochondria to the cytosol, and the level of Caspase3. Conversely,3-MA treatment reversed these changes.ConclusionPart11. The level of IL-1β and autophagy activation increase and both peaked at24h after SAH. Autophagic related protein expresses on microglial. These indicated the autophagy activation may play a key role in inflammation after SAH.2. Rapamycin enhanced autophagy decreases the level of IL-1β while3-MA treatment reversedsthese changes. These changes are possibly associated with regulation of NLRP3inflammasome by autophagy activation.Part21. Melatonin treatment improves neurological score and attenuate water edema in early brain injury after SAH.2. Melatonin could prevent disruption of tight junction proteins which might play a role in attenuating BBB dysfunctions by repressing the inflammatory response in EBI after SAH. These changes are possibly associated with melatonin enhanced autophagy regulate proinflammatory cytokines via its effects of NLRP3inflammasome.3. Melatonin enhanced autophagy plays a protective role against apoptosis in EBI after SAH, which is possibly associated with a mitochondrial pathway that reduced Bax translocation and cytochrome c release from the mitochondria.