Background/Aims: The purpose of this study was to investigate the role of autophagy in oxygen-glucose-deprivation/reoxygenation (OGD/R) injury in rat neurons. Methods and results: Cortical neurons were isolated from Sprague-Dawley rats and identified by immunofluorescence. The cortical neurons were randomly assigned to one of four groups: control group (I), experimental group (OGD/R group, II), JNK inhibitor pretreatment group (III) and JNK inhibitor pretreatment + OGD/R group (IV). Neuronal cell viability significantly decreased after 6h and 12h of reoxygenation in Group IV (P < 0.05). Electron microscopy showed the presence of many autophagic vacuoles and the formation of autolysosomes in the neurons; the number of autophagic vacuoles decreased transiently at 6h, while a new autophagic flux and a large number of empty autophagic vacuoles were observed at 12h. In Group IV, a large number of autophagic vacuoles were present at 0.5h and 2h of reoxygenation, which gradually decreased with increasing reoxygenation time. No significant differences in the expression of the LC3II protein were detected between the Group II and IV prior to 6h of reoxygenation, and LC3II expression showed an overall rise-decline pattern. However, LC3II protein expression increased in Group II at 12h of reoxygenation, whereas a continuous decline was observed in Group IV. The levels of phosphorylated JNK and Bcl-2 and the expression of Beclin-1 increased gradually as the reoxygenation time going in Group II, whereas they increased at 12h of reoxygenation in Group IV (P < 0.05). In addition, progressive dissociation of the Bcl-2/Beclin-1 complex was observed in the Group II, while JNK inhibitor suppressed this dissociation. Conclusion: The regulation of the JNK/Bcl-2/Beclin-1 signaling pathway may be one of the mechanisms underlying the OGD/R-induced autophagic cell death of neurons.

Despite the great increase in human lifespan with improved medical care, the physiological and pathological changes such as memory and cognitive disorders and associated anxiety and depression are major concern with aging. Molecular mechanisms underlying these changes are little known. The present study examined the differentially expressed genes (DEGs) and the genes with differentially expressed isoforms in three brain regions, anterior cingulate cortex (ACC), amygdala and hippocampus, throughout the lifespan of mice. Compared to 2-month old mice, both 12-and 24-month old mice displayed memory and cognitive impairments in the Morris water maze, Y-maze, and novel object recognition tests and depression-and anxiety-like behaviors in the tail suspension, forced swimming, open field, and elevated plus maze tests. RNA sequencing analysis identified 634 and 1078 DEGs in ACC, 453 and 1015 DEGs in the amygdala and 884 and 1054 DEGs in hippocampus in the 12-and 24-month old mice, respectively. Similarly, many genes with differentially expressed isoforms were also identified in these three brain regions in the 12-and 24-month old mice. Further functional analysis revealed that many DEGs and the genes with differentially expressed isoforms in the ACC and amygdala were mapped to depression-and anxiety-related genes, respectively and that a lot of DEGs and the genes with differentially expressed isoforms in hippocampus were mapped to cognitive dysfunction-related genes from both 12-and 24-month old mice. All of these mapped DEGs and the genes with differentially expressed isoforms were closely related to neuroinflammation. Our findings indicate that these neuroinflammation-related DEGs and the genes with differentially expressed isoforms are likely new targets in the management of memory/cognitive impairment and emotional disorders during the aging.

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Evidence reveals that propofol protects cells via suppressing excessive autophagy induced by hypoxia/reoxygenation (H/R). Previously, we found in a genome-wide microRNA profile analysis that several autophagy-related microRNAs were significantly altered during the process of H/R in the presence or absence of propofol posthypoxia treatment (P-PostH), but how these microRNAs work in P-PostH is still largely unknown. Here, we found that one of these microRNAs, microRNA-30b (miR-30b), in human umbilical vein endothelial cells (HUVECs) was downregulated by H/R treatment but significantly upregulated by 100 M propofol after H/R treatment. miR-30b showed similar changes in open heart surgery patients. By dual-luciferase assay, we found that Beclin-1 is the direct target of miR-30b. This conclusion was also supported by knockdown or overexpression of miR-30b. Further studies showed that miR-30b inhibited H/R-induced autophagy activation. Overexpression or knockdown of miR-30b regulated autophagy-related protein gene expression in vitro. To clarify the specific role of propofol in the inhibition of autophagy and distinguish the induction of autophagy from the damage of autophagy flux, we used bafilomycin A1. LC3-II levels were decreased in the group treated with propofol combined with bafilomycin A1 compared with the group treated with bafilomycin A1 alone after hypoxia and reoxygenation. Moreover, HUVECs transfected with Ad-mCherry-GFP-LC3b confirmed the inhibitory effect of miR-30b on autophagy flux. Finally, we found that miR-30b is able to increase the cellular viability under the H/R condition, partially mimicking the protective effect of propofol which suppressed autophagy via enhancing miR-30b and targeting Beclin-1. Therefore, we concluded that propofol upregulates miR-30b to repress excessive autophagy via targeting Beclin-1 under H/R condition. Thus, our results revealed a novel mechanism of the protective role of propofol during anesthesia. Clinical Trial Registration Number. This trial is registered with ChiCTR-IPR-14005470. The name of the trial register: Propofol Upregulates MicroRNA-30b to Repress Beclin-1 and Inhibits Excessive Autophagy and Apoptosis.

BackgroundSpinal cord ischemia reperfusion injury (SCIRI) is a complication of aortic aneurysm repair or spinal cord surgery that is associated with permanent neurological deficits. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have been shown to be potential therapeutic options for improving motor functions after SCIRI. Due to their easy access and multi-directional differentiation potential, adipose‐derived stem cells (ADSCs) are preferable for this application. However, the effects of ADSC-derived sEVs (ADSC-sEVs) on SCIRI have not been reported.ResultsWe found that ADSC-sEVs inhibited SCIRI induced neuronal apoptosis, degradation of tight junction proteins and suppressed endoplasmic reticulum (ER) stress. However, in the presence of the ER stress inducer, tunicamycin, its anti-apoptotic and blood-spinal cord barrier (BSCB) protective effects were significantly reversed. We found that ADSC-sEVs contain tumor necrosis factor-(TNF) stimulated gene-6 (TSG-6) whose overexpression inhibited ER stress in vivo by modulating the PI3K/AKT pathway.ConclusionsADSC-sEVs inhibit neuronal apoptosis and BSCB disruption in SCIRI by transmitting TSG-6, which suppresses ER stress by modulating the PI3K/AKT pathway.