Urolithin A alleviates early brain injury after subarachnoid hemorrhage by regulating the AMPK/mTOR pathway-mediated autophagy.

OBJECTIVE: Unfavorable outcomes in patients with subarachnoid hemorrhage (SAH) are mainly attributed to early brain injury (EBI). Reduction of neuronal death can improve the prognosis in SAH patients. Autophagy and apoptosis are critical players in neuronal death. Urolithin A (UA) is a natural compound produced by gut bacteria from ingested ellagitannins and ellagic acid. Here, we detected the role of UA in EBI post-SAH. METHODS: We established an animal model of SAH in rats by endovascular perforation, with administration of UA, 3-methyladenine (3-MA) and Compound C. SAH grading, neurological function, brain water content, western blotting analysis of levels of proteins related to apoptosis, autophagy and pathways, blood-brain barrier (BBB) integrity, TUNEL staining, and immunofluorescence staining of LC3 were evaluated at 24h after SAH. RESULTS: SAH induction led to neurological dysfunctions, BBB disruption, and cerebral edema at 24h post-SAH in rats, which were relieved by UA. Additionally, cortical neuronal apoptosis in SAH rats was also attenuated by UA. Moreover, UA restored autophagy level in SAH rats. Mechanistically, UA activated the AMPK/mTOR pathway. Furthermore, inhibition of autophagy and AMPK limited UA-mediated protection against EBI post-SAH CONCLUSION: UA alleviates neurological deficits, BBB permeability, and cerebral edema by inhibiting cortical neuronal apoptosis through regulating the AMPK/mTOR pathway-dependent autophagy in rats following SAH.

MiR-191-5p alleviates microglial cell injury by targeting Map3k12 (mitogen-activated protein kinase kinase kinase 12) to inhibit the MAPK (mitogen-activated protein kinase) signaling pathway in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Multiple reports have elucidated that microRNAs are promising biomarkers for AD diagnosis and treatment. Herein, the effect of miR-191-5p on microglial cell injury and the underlying mechanism were explored. APP/PS1 transgenic mice were utilized to establish mouse model of AD. Amyloid-ß protein 1-42 (Aß1-42)-treated microglia were applied to establish in vitro cell model of AD. MiR-191-5p expression in hippocampus and microglia was measured by reverse transcription quantitative polymerase chain reaction. The viability and apoptosis of microglia were evaluated by Cell Counting Kit-8 assays and flow cytometry analyses, respectively. The binding relationship between miR-191-5p and its downstream target mitogen-activated protein kinase kinase kinase 12 (Map3k12) was determined by luciferase reporter assays. Pathological degeneration of hippocampus was tested using hematoxylin-eosin staining and Nissl staining. Aß expression in hippocampus was examined via immunohistochemistry. In this study, miR-191-5p was downregulated in Aß1-42-stimulated microglia and hippocampal tissues of APP/PS1 mice. MiR-191-5p overexpression facilitated cell viability and inhibited apoptosis rate of Aß1-42-treated microglia. Mechanically, miR-191-5p targeted Map3k12 3'-untranslated region to downregulate Map3k12 expression. MiR-191-5p inhibited Aß1-42-induced microglial cell injury and inactivated the MAPK signaling by downregulating Map3k12. Overall, miR-191-5p alleviated Aß1-42-induced microglia cell injury by targeting Map3k12 to inhibit the MAPK signaling pathway in microglia.

Protective effect of microRNA­340­5p against oxygen­glucose deprivation/reperfusion in PC12 cells through targeting neuronal differentiation 4.

The expression levels of microRNA (miR)­340­5p are reportedly decreased in the peripheral blood during acute ischemic stroke; however, the direct effect and mechanism of action of miR­340­5p in ischemic stroke remains largely unknown. The present study aimed to investigate the effects of miR­340­5p, and its mechanism of action, on PC12 cells following oxygen­glucose deprivation/reperfusion (OGD/R) induction. OGD/R­induced PC12 cells served as the cellular model and subsequently, mRNA expression levels of miR­340­5p and neuronal differentiation 4 (Neurod4) were analyzed using reverse transcription­quantitative PCR. Tumor necrosis factor­α, interleukin (IL)­1ß and IL­6 expression levels were detected using ELISA kits, and flow cytometry was used to determine the rate of cellular apoptosis. In addition, a nitric oxide (NO) synthase activity assay kit was used to detect NO levels and a NADPH assay kit was used to measure NADPH levels. Western blotting was also performed to analyze protein expression levels of bax, bcl­2, cleaved caspase 3 and phosphorylated endothelial NOS (eNOS), and the target gene of miR­340­5p was predicted using TargetScan software and verified using a dual­luciferase reporter assay. The expression levels of miR­340­5p were decreased in PC12 cells following OGD/R induction and Neurod4 was identified as a target gene of miR­340­5p. In addition, miR­340­5p overexpression reduced inflammation, apoptotic rate, NO production and NADPH levels, in addition to increasing eNOS expression in PC12 cells following OGD/R induction. Notably, the overexpression of Neurod4 reversed the aforementioned effects of miR­340­5p on PC12 cells following OGD/R induction. In conclusion, the findings of the present study suggested that miR­340­5p may protect PC12 cells against OGD/R through targeting Neurod4, which could provide important implications for the treatment of ischemia­reperfusion injury based on miR­340­5p expression levels in vivo.

MicroRNA-27a-3p Downregulation Inhibits Inflammatory Response and Hippocampal Neuronal Cell Apoptosis by Upregulating Mitogen-Activated Protein Kinase 4 (MAP2K4) Expression in Epilepsy: In Vivo and In Vitro Studies.

BACKGROUND This study aimed to discover the effect and mechanism of microRNA-27a-3p (miR-27a-3p) in epilepsy. MATERIAL AND METHODS To perform our investigation, in vivo and in vitro models of epilepsy were induced using kainic acid (KA). Expression of miR-27a-3p in the hippocampus of epileptic rats or normal rats or neuronal cells was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Racine score was used to assess seizures in epileptic rats. Cell viability and cell apoptosis were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was performed to detect inflammatory factors expression. RESULTS Significantly higher expression of miR-27a-3p in the hippocampus of epileptic rats and in KA-induced neurons was observed. We found that miR-27a-3p inhibitor alleviated seizures in epileptic rats. miR-27a-3p inhibitor also inhibited apoptosis of hippocampal neurons in epileptic rats, promoted Bcl2 expression, and decreased Bax and Caspase3 expression. The results showed that miR-27a-3p inhibitor effectively reduced the expression levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-alpha) in hippocampal tissues of epileptic rats. Dual luciferase reporter assay showed that mitogen-activated protein kinase 4 (MAP2K4) was a direct target of miR-27a-3p. miR-27a-3p inhibitor significantly promoted the cell viability of KA-induced neurons, inhibited cell apoptosis, promoted the expression of Bcl-2, and decreased Bax and Caspase3 expression, and all these changes were abolished by MAP2K4-siRNA co-transfection. CONCLUSIONS Our preliminary findings indicated that miR-27a-3p inhibitor protected against epilepsy-induced inflammatory response and hippocampal neuronal apoptosis by targeting MAP2K4.