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Ischemic stroke (IS) has a high mortality and disability rate worldwide. NAF-1 is involved in the occurrence and development of nervous system diseases, but its involvement in the pathophysiology of IS is unclear. In this study, we investigated the role of NAF-1 in IS. Middle cerebral artery occlusion and reperfusion in Sprague-Dawley rats was used as an in vivo model of IS. Moreover, oxygen-glucose deprivation and reoxygenation of PC12 cells was used for in vitro study. Intracellular NAF-1 protein expression was influenced by LV-NAF-1 or siRNA. Quantitative real-time PCR and western blot analysis showed that NAF-1 was significantly downregulated following IS. Overexpression of NAF-1 reduced mitophagy in PC12 cells, and it alleviated tissue damage in vivo in rats and cell injury in vitro. In contrast, knockdown of NAF-1 increased mitophagy and worsened cell damage. These findings suggest that NAF-1 may have potential as a therapeutic target for IS.

The aim of this study was to investigate the role and underlying mechanism of the long non-coding RNA ANRIL (antisense noncoding RNA in the INK4 locus, ANRIL) in ischemia stroke (IS) injury. Downregulation of ANRIL by right intracerebroventricular injected si-ANRIL in middle cerebral artery occlusion-reperfusion (MCAO/R) C57/BL6 mice and by transferring si-ANRIL in oxygen glucose deprivation/reperfusion (OGD/R) HT22 cells. The results showed that ANRIL levels increased in IS model, downregulation of ANRIL reduced infract area, neurological deficit scores and injured cells, and prolong fall latency time in MCAO/R mice, improved cell viability and reduced cell cytotoxicity in OGD/R cells. Fluorescence in Situ Hybridization detected that there were both ANRIL and miR-671-5p in neurons; miranda v3.3a and dual luciferase reporter assay demonstrated that miR-671-5p was one of direct target of ANRIL; and our previously published research demonstrated that NF-κB was one of direct target of miR-671-5p. Downregulation of ANRIL alleviated neuroinflammation and reduced p-NF-κB, NF-κB, pro-inflammatory cytokines (IL-1β, IL-6, TNF-a), and iNOS, which diminished by miR-671-5p antagomir both in in vivo and in vitro IS models. Downregulation of ANRIL alleviated disruption of blood brain barrier, and protected against tight junction (ZO-1, occludin and claudin 5) disorder in MCAO/R mice. This work clarified that downregulation of ANRIL reduced neuroinflammation by negatively regulating miR-671-5p to inhibit NF-κB in IS models, which provided a theoretical foundation for the protective effect of downregulating ANRIL for IS patients.

Increasing evidence suggests that long noncoding RNAs can exert neuroprotective effects in cerebral ischemia-reperfusion injury. Levels of the long noncoding RNA ANRIL (ANRIL) are reportedly altered in ischemic stroke (IS) patients, but its role in IS requires further clarification. This study was designed to explore the mechanistic function of ANRIL in IS. In vitro, HT22 cells was treated with an oxygen-glucose deprivation/reperfusion (OGD/R). In vivo, brain ischemia/reperfusion was induced by 60-minute transient middle cerebral artery occlusion/ reperfusion (MCAO/R) IS model in C57/BL6 mice. Additionally, cells were transfected with si-ANRIL, pcDNA3.1-ANRIL, pcDNA3.1-NF-κB, or appropriate negative controls, and si-ANRIL and pcDNA3.1-NF-κB were administered into the lateral ventricles in MCAO/R model mice. Cell viability and apoptosis were detected via MTT and flow cytometry assays. mRNA and protein expression of NF-κB were detected via qRT-PCR and Western blotting. IL-1β, IL-6, TNF-a, and iNOS levels were detected via ELISA. In addition, infarcted area and neuronal injury were evaluated via TTC, Nissl, and immunofluorescent staining. We found that ANRIL knockdown increased cell viability and reduced apoptosis in vitro. Additionally, we found that ANRIL knockdown decreased p-P65, P65, IL-1β, IL-6, TNF-a, and iNOS levels, whereas these effects were reversed by NF-κB overexpression both in vitro and in vivo. Our results suggest that ANRIL knockdown attenuates neuroinflammation by suppressing the expression of NF-κB both in vitro and vivo model of IS, sugguesting that ANRIL might be a potentially viable therapeutictarget to diminish neuroinflammation in IS patients.

Ischemic stroke is a complex brain disease regulated by several cell death processes, including apoptosis, autophagy, and ferroptosis. β-Caryophyllene (BCP), a natural bicyclic sesquiterpene abundantly found in essential oils, has been demonstrated to have potential pharmacological benefits in many diseases, including ischemic stroke.This research was to determine the existence of ferroptosis in the pathogenesis of acute ischemic stroke and investigate whether BCP can inhibit ferroptosis to improve cerebral ischemia injury by activating the NRF2/HO-1 signaling pathway in rats.First, we verified ferroptosis by assessing proferroptotic changes after middle cerebral artery occlusion reperfusion (MCAO/R), along with protein and lipid peroxidation levels. Then male rats were divided randomly into Sham, MCAO/R, ML385 (NRF2-specific inhibitor) and BCP groups. The effects of BCP on cerebral injury were detected by the modified neurological severity score, TTC staining, and hematoxylin-eosin staining. We conducted western blotting analyzes of proteins, including those involved in ferroptosis and related signaling pathways. To demonstrate the neuroprotective effect of BCP in vitro, primary astrocytes were pretreated with different concentrations of BCP (10, 20, and 40 μM) for 24 h before oxygen-glucose deprivation/re-oxygenation (ODG/R).We concluded that ferroptosis was engaged in the process of I/R-induced neurological damage, implying that this novel type of cell death might provide new therapeutic options for the clinical treatment of ischemic stroke. In vivo study proved that BCP improved neurological scores, infarct volume, and pathological features after MCAO/R. We demonstrated that BCP evidently enhanced NRF2 nuclear translocation, activated the NRF2/HO-1 pathway, which protected against ferroptosis. In vitro investigation revealed the same results. BCP decreased OGD/R-induced ROS generation and iron accumulation. Furthermore, the neuroprotective effects of BCP were reversed by the NRF2 inhibitor ML385.Our results indicated the critical role of ferroptosis in cerebral I/R injury. For the first time, we showed that the significant neuroprotective effects of BCP in attenuating ischemic stroke injury are correlated with ferroptosis regulation, and its mechanism is associated with activation of the NRF2/HO-1 axis.

This study was designed to investigate the role of miR-671-5p in in vitro and in vivo models of ischemic stroke (IS). Middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6 mice as well as oxygen-glucose deprivation and reoxygenation (OGD/R) in a mouse hippocampal HT22 neuron line were used as in vivo and in vitro models of IS injury, respectively. miR-671-5p agomir, miR-671-5p antagomir, pcDNA3.1-NF-κB, and negative controls were transfected into cells using riboFECT CP reagent. miR-671-5p agomir, pcDNA3.1-NF-κB, and negative vectors were administered into MCAO/R mice via intracerebroventricular injection. The results showed that miR-671-5p was significantly downregulated and that miR-671-5p agomir alleviated injury and neuroinflammation induced by ischemic reperfusion. A dual-luciferase reporter assay confirmed that NF-κB is a direct target of miR-671-5p. Reverse experiments showed that miR-671-5p agomir reduced neuroinflammation via suppression of NF-κB expression in both in vitro and in vivo models of IS. Our data suggest that miR-671-5p may be a viable therapeutic target for diminishing neuroinflammation in patients with IS.

Experimental studies have shown that β-caryophyllene (BCP) improved neurological deficits of cerebral ischemia-reperfusion injury (CIRI) rats resulting from Middle Cerebral Artery Occlusion (MCAO). However, research on targets of BCP on CIRI has not been completed. In this study, the mRNA sequencing was used to distinguish various therapeutic multiple targets of BCP on CIRI. Differentially expressed genes (DEGs) were identified from RNA-seq analysis. CIRI induced up-regulated genes (CIRI vs. Sham) and BCP -induced down-regulated genes (BCP vs CIRI) were identified. Significant DEGs were identified only that expressed in each of all samples. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of significant DEGs were determined by cluster Profiler. Protein interactive network (PPI) was analyzed using the String tool and Hub genes was identified by cytoHubba. Transcription factor (TF) regulatory network for the potential Hub genes was constructed. Western blot and ELISA were used to verified hub genes and relative inflammatory cytokines. After mRNA sequencing, a total of 411 DEGs were filtered based on the 2 series (CIRI vs. Sham and CIRI vs. BCP), with Pax1, Cxcl3 and Ccl20 are the most remarkable ones reversed by BCP. GO analysis was represented by DEGs involved in multiple biological process such as extra-cellular matrix organization, leukocyte migration, regulation of angiogenesis, reactive oxygen species metabolic process, etc. KEGG analysis showed that DEGs participated several signaling pathways including MAPK signaling pathway (rno04010), Cytokine-cytokine receptor interaction (rno04060), JAK-STAT signaling pathway (rno04630), and others. The protein-protein interaction (PPI) network consisted of 339 nodes and 1945 connections, and top ten Hub genes were identified by cytoHubba such as TIMP1, MMP-9, and STAT3. Subsequently, a TFs-miRNAs-targets regulatory network was established, involving 6 TFs, 5 miRNAs, and 10 hub genes, consisting of several regulated models such as Brd4 - rno-let-7e - Mmp9, Brd4 - rno-let-7i - Stat3, and Hnf4a- rno-let-7b -Timp1. Finally, western blot demonstrated that BCP could inhibit the increased TIMP1, MMP-9 and STAT3 expression in rat brains after I/R. ELISA represented that BCP could suppress inflammatory cytokines caused by CIRI and present anti-oxidative property. In conclusion, this study shows that the intervention of BCP can significantly reduce neurologic deficit, improve the cerebral ischemia, and a total of ten hub genes were found closely related to the treatment of BCP on CIRI. Prudent experimental validation suggests that the BCP might have the neuro-protective effects in CIRI by decreasing the expression of MMP-9 and TIMP-1, STAT3. In a sense, this study reveals that the MMP-9/TIMP-1 signaling pathway may be involved in the injury after CIRI and thus provides a new treatment strategy as well as a researching method for stroke.