6-Shogaol Alleviates Neuronal Cell Excessive Autophagy and Calcium Overload in Cerebral Ischemia-Reperfusion Injury via Modulating miRNA-26a-5p/DAPK1 Signaling Axis
Authors: Rao Ouyang , Li Shixin , Zhu Ning , Zhou Hangxiang , Tao Junling , LI Yehong , Liu Ying 
Published: June 2024

Objectives：Clarifying the Potential of 6-shogaol (6-SH) in Reducing Excessive Autophagy and Calcium Overload in Neuronal Cells during Cerebral Ischemia-Reperfusion Injury via Regulation of the miRNA-26a-5p/DAPK1 Signaling Axis.
Methods：In this study, oxygen-glucose deprivation/reoxygenation (OGD/R) in HT22 cells was used to create an in vitro model of cerebral ischemia-reperfusion Injury (CIRI). The following groups of cells were created：normal control (NC), model (OGD/R), model+6-shogaol treatment (6-SH+OGD/R), model+6-shogaol treatment+miRNA-26a-5p inhibitor negative control (inhibitorNC+6-SH+OGD/R) and model+6-shogaol treatment+miRNA-26a-5p inhibitor (inhibitor+6-SH+OGD/R).Cell morphology was observed under an inverted microscope, cell viability was assessed using the CCK8 assay, neuronal ultrastructure was examined via transmission electron microscopy, intracellular calcium ion concentration was measured using flow cytometry, and the fluorescence intensity of LC3 and Beclin1 was detected by immunofluorescence. Western blotting was performed to evaluate the expression levels of proteins related to autophagy and calcium ion channels, and Quantitative real-time PCR（RT-qPCR）was conducted to measure the gene expression levels of miRNA-26a-5p and DAPK1. The Sprague Dawley rat CIRI model was established in vivo utilizing the suture-occluded approach. Three groups were used: sham, model (I/R), and model+6-shogaol therapy (6-SH+I/R).TTC staining was used to observe cerebral infarction, HE staining to assess brain tissue pathology, immunofluorescence to detect the fluorescence intensity of LC3 and Beclin1, Western blotting to measure protein expression levels related to autophagy and calcium ion channels, and RT-qPCR to determine gene expression levels of miRNA-26a-5p and DAPK1. Additionally, molecular docking techniques were employed to validate the spontaneous binding of 6-SH to miRNA-26a-5p, and dual luciferase reporter gene assays were conducted to confirm the targeting relationship between miRNA-26a-5p and DAPK1.
Results：The molecular docking study showed that 6-SH spontaneously bound to miRNA-26a-5p, and the dual luciferase reporter gene assay confirmed that miRNA-26a-5p can target and regulate DAPK1. According to the outcomes of in vitro experiments, 6-SH dramatically improved HT22 cell viability during OGD/R, reduced pathological damage, elevated miRNA-26a-5p, downregulated DAPK1, and reduced excessive autophagy and calcium overload. The ability of 6-SH to downregulate DAPK1-mediated neuronal excessive autophagy and calcium overload was greatly diminished when miRNA-26a-5p expression was blocked. Results of in vivo experiments showed that 6-SH markedly decreased the size of the cerebral infarct area in SD rats following MCAO/R, mitigated excessive autophagy and calcium overload, relieved pathological damage, and partially recovered neuronal function. It also elevated miRNA-26a-5p and downregulated DAPK1.
Conclusion：6-SH modulates miRNA-26a-5p to target and inhibit DAPK1, thereby attenuating the neurons excessive autophagy and calcium overload caused by cerebral ischemia-reperfusion Injury.