Long non-coding RNA ZFAS1 alleviates bupivacaine-induced neurotoxicity by regulating the miR-421/zinc finger protein564 (ZNF564) axis.

This research aimed to explore the biological role of long non-coding RNA (lncRNA) ZFAS1 in bupivacaine-induced neurotoxicity. The levels of lncRNA ZFAS1, miR-421, and zinc finger protein 564 (ZNF564) were detected by RT-qPCR. MTT and TUNEL assays were utilized to evaluate cell viability and apoptosis, respectively. Caspase-3 activity was measured by the caspase-3 activity assay kit. The binding ability between miR-421 and ZFAS1 or ZNF564 was confirmed by Rip and dual-luciferase reporter assays. In this study, it was found that the levels of ZFAS1 and ZNF564 were gradually upregulated and miR-421 expression was downregulated with increasing concentrations of bupivacaine. Functional assays indicated that the silencing of ZFAS1 suppressed cell viability and facilitated cell apoptosis of SH-SY5Y cells, while overexpression of ZFAS1 had the opposite effects. Moreover, it was identified that miR-421 was a target of ZFAS1, and ZFAS1 regulated the bupivacaine-induced neurotoxicity via miR-421. In addition, we confirmed that ZNF564 was a downstream target of miR-421. The upregulation of miR-421 decreased the cell viability, and increased the cell apoptosis rate and caspase-3 activity, while the upregulation of ZND564 partially abolished these effects. Finally, it was demonstrated that ZFAS1 could upregulate the expression of ZNF564 by targeting miR-421. In conclusion, our results demonstrated that ZFAS1 alleviated bupivacaine-induced neurotoxicity through the miR-421/ZNF564 axis, suggesting a new strategy for the amelioration of bupivacaine-induced neurotoxicity.

Preactivation of Notch1 in remote ischemic preconditioning reduces cerebral ischemia-reperfusion injury through crosstalk with the NF-κB pathway.

BACKGROUND: Remote ischemic preconditioning (RIPC) initiates endogenous protective pathways in the brain from a distance and represents a new, promising paradigm in neuroprotection against cerebral ischemia-reperfusion (I/R) injury. However, the underlying mechanism of RIPC-mediated cerebral ischemia tolerance is complicated and not well understood. We reported previously that preactivation of Notch1 mediated the neuroprotective effects of cerebral ischemic preconditioning in rats subjected to cerebral I/R injury. The present study seeks to further explore the role of crosstalk between the Notch1 and NF-κB signaling pathways in the process of RIPC-induced neuroprotection. METHODS: Middle cerebral artery occlusion and reperfusion (MCAO/R) in adult male rats and oxygen-glucose deprivation and reoxygenation (OGD/R) in primary hippocampal neurons were used as models of I/R injury in vivo and in vitro, respectively. RIPC was induced by a 3-day procedure with 4 cycles of 5 min of left hind limb ischemia followed by 5 min of reperfusion each day before MCAO/R. Intracerebroventricular DAPT injection and sh-Notch1 lentivirus interference were used to inhibit the Notch1 signaling pathway in vivo and in vitro, respectively. After 24 h of reperfusion, neurological deficit scores, infarct volume, neuronal apoptosis, and cell viability were assessed. The protein expression levels of NICD, Hes1, Phospho-IKKα/ß (p-IKK α/ß), Phospho-NF-κB p65 (p-NF-κB p65), Bcl-2, and Bax were assessed by Western blotting. RESULTS: RIPC significantly improved neurological scores and reduced infarct volume and neuronal apoptosis in rats subjected to I/R injury. OGD preconditioning significantly reduced neuronal apoptosis and improved cell viability after I/R injury on days 3 and 7 after OGD/R. However, the neuroprotective effect was reversed by DAPT in vivo and attenuated by Notch1-RNAi in vitro. RIPC significantly upregulated the expression of proteins related to the Notch1 and NF-κB pathways. NF-κB signaling pathway activity was suppressed by a Notch1 signaling pathway inhibitor and Notch1-RNAi. CONCLUSIONS: The neuroprotective effect of RIPC against cerebral I/R injury was associated with preactivation of the Notch1 and NF-κB pathways in neurons. The NF-κB pathway is a downstream target of the Notch1 pathway in RIPC and helps protect focal cerebral I/R injury.