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. 2022 Sep 11:16:3071-3085.
doi: 10.2147/DDDT.S378786. eCollection 2022.

Dihydromyricetin Attenuates Cerebral Ischemia Reperfusion Injury by Inhibiting SPHK1/mTOR Signaling and Targeting Ferroptosis

Jiangbo Xie  1   2 , Tingting Zhang  3 , Peichun Li  3 , Dong Wang  2 , Tao Liu  2 , Shunliang Xu  1
Affiliations

Dihydromyricetin Attenuates Cerebral Ischemia Reperfusion Injury by Inhibiting SPHK1/mTOR Signaling and Targeting Ferroptosis

Jiangbo Xie et al. Drug Des Devel Ther. .

Abstract

Background: Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury.

Methods: We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting.

Results: DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR).

Conclusion: This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.

Keywords: SPHK1; cerebral ischemia reperfusion injury; dihydromyricetin; ferroptosis; mTOR.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

None
Graphical abstract
Figure 1
Figure 1
Dihydromyricetin treatment alleviated cerebral I/R injury in rats. Rats were subjected to MCAO/R to induce cerebral I/R injury and then administered DHM treatment in increasing doses. Sham-operated rats served as controls. (A) The neurological scores of rats. (B) Brain water content of rats. (C) Infarct volume of brain tissues as assessed by TTC staining. (D) Apoptotic cells in brain tissues were examined by TUNEL staining. **P < 0.01, compared with the sham group. #P < 0.05, ##P < 0.01, compared with the MCAO/R group.
Figure 2
Figure 2
Dihydromyricetin repressed SPHK1 and p-mTOR expression and reduced ferroptosis in rats with cerebral I/R injury. (A–F) Rats were subjected to MCAO/R to induce I/R injury or followed by treatment with L-DHM, M-DHM, H-DHM. Sham-operated rats served as controls. Western blotting analysis of SPHK1, p-mTOR/mTOR, GPX4, ACSL4 and PEBP1 in the brain tissues of rats. **P < 0.01, compared with the sham group. #P < 0.05, ##P < 0.01, compared with the MCAO/R group.
Figure 3
Figure 3
Dihydromyricetin enhanced cell viability and repressed apoptosis in OGD/R-treated HT22 cells. HT22 cells were subjected to OGD/R or pretreated with 1, 10, 20 or 30 μM of DHM. CCK-8 assay (A) and flow cytometry (B) were performed to detect viability and apoptosis in HT22 cells. **P < 0.01, compared with the control group. #P < 0.05, ##P < 0.01, compared with the OGD/R group. @P < 0.05, @@P < 0.01, compared with the 20-DHM group.
Figure 4
Figure 4
Dihydromyricetin suppressed SPHK1 and p-mTOR expression and reduced ferroptosis in OGD/R-treated HT22 cells. HT22 cells were subjected to OGD/R or pretreated with 30 μM of DHM. The levels of lipid ROS (A) and intracellular iron (B) were detected. (C–H) Western blotting analysis of SPHK1, p-mTOR/mTOR, GPX4, ACSL4 and PEBP1 in HT22 cells. **P < 0.01, compared with the control group. #P < 0.05, ##P < 0.01, compared with the OGD/R group.
Figure 5
Figure 5
Dihydromyricetin reduced ferroptosis in OGD/R-treated HT22 cells by regulating GPX4 expression. HT22 cells were transfected with si-GPX4 or si-NC, followed by OGD/R or combined with 30 μM of DHM treatment. CCK-8 assay (A) and flow cytometry (B) were performed to detect cell viability and apoptosis in HT22 cells. The levels of lipid ROS (C) and intracellular iron (D) were detected. (E–H) Western blotting analysis of GPX4, ACSL4 and PEBP1 in HT22 cells. **P < 0.01, compared with the control group. ##P < 0.01, compared with the OGD/R group. @P < 0.05, @@P < 0.01, compared with the OGD/R+30-DHM+si-NC group.
Figure 6
Figure 6
Dihydromyricetin limited OGD/R-induced ferroptosis by inhibiting SPHK1/mTOR signaling pathway. HT22 cells were transfected with pcDNA3.1-SPHK1/Vector or treated with MHY1485, followed by OGD/R or combined with 30 μM of DHM treatment. CCK-8 assay (A) and flow cytometry (B) were performed to detect cell viability and apoptosis in HT22 cells. The levels of lipid ROS (C) and intracellular iron (D) were detected. (E–G) Western blotting analysis of SPHK1, p-mTOR/ mTOR in HT22 cells. **P < 0.01, compared with the control group. ##P < 0.01, compared with the OGD/R group. @P < 0.05, @@P < 0.01, compared with the OGD/R+30-DHM group.
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