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. 2024 Mar 5;16(5):4670-4683.
doi: 10.18632/aging.205622. Epub 2024 Mar 5.

Echinatin mitigates sevoflurane-induced neurotoxicity through regulation of ferroptosis and iron homeostasis

Yanqiu You  1 , Xudong Zhou  1 , Qiuqin Tang  2 , Tianshou Zhao  2 , Juan Wang  2 , Hanqin Huang  2 , Jibing Chen  2 , Zhongquan Qi  1 , Fujun Li  2
Affiliations

Echinatin mitigates sevoflurane-induced neurotoxicity through regulation of ferroptosis and iron homeostasis

Yanqiu You et al. Aging (Albany NY). .

Abstract

Surgery and anesthesia are vital medical interventions, but concerns over their potential cognitive side effects, particularly with the use of inhalational anesthetics like sevoflurane, have surfaced. This study delves into the neuroprotective potential of Echinatin against sevoflurane-induced neurotoxicity and the underlying mechanisms. Echinatin, a natural compound, has exhibited anti-inflammatory, antioxidant, and anticancer properties. Sevoflurane, while a popular anesthetic, is associated with perioperative neurocognitive disorders (PND) and neurotoxicity. Our investigation began with cellular models, where Echinatin demonstrated a significant reduction in sevoflurane-induced apoptosis. Mechanistically, we identified ferroptosis, a novel form of programmed cell death characterized by iron accumulation and lipid peroxidation, as a key player in sevoflurane-induced neuronal injury. Echinatin notably suppressed ferroptosis in sevoflurane-exposed cells, suggesting a pivotal role in neuroprotection. Expanding our research to a murine model, we observed perturbations in iron homeostasis, inflammatory cytokines, and antioxidants due to sevoflurane exposure. Echinatin treatment effectively restored iron balance, mitigated inflammation, and preserved antioxidant levels in vivo. Behavioral assessments using the Morris water maze further confirmed Echinatin's neuroprotective potential, as it ameliorated sevoflurane-induced spatial learning and memory impairments. In conclusion, our study unveils Echinatin as a promising candidate for mitigating sevoflurane-induced neurotoxicity. Through the regulation of ferroptosis, iron homeostasis, and inflammation, Echinatin demonstrates significant neuroprotection both in vitro and in vivo. These findings illuminate the potential for Echinatin to enhance the safety of surgical procedures involving sevoflurane anesthesia, minimizing the risk of cognitive deficits and neurotoxicity.

Keywords: ALOX12; Echinatin; ferroptosis; neurotoxicity; sevoflurane.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest related to this study.

Figures

Figure 1
Figure 1
Echinatin mitigates sevoflurane-induced apoptosis in HT22 cells. HT22 cells were subjected to Echinatin treatment (0-40 μM) for a duration of 24 hours, followed by exposure to sevoflurane or control conditions. (A) Cell vitality was measured by MTT assay. (B) Cell apoptosis was measured by flow cytometry. (C) LDH release was measured by LDH assay kits. (D) Protein levels of cleaved-caspase3, Bcl-2, and Bax were measured by western blot. The data are presented as the mean ± SD. Ech, Echinatin; Sevo, sevoflurane. Compared with the Sevo+ Ech (0 μM) group, **P<0.01, ***P<0.001.
Figure 2
Figure 2
Echinatin restrains sevoflurane-induced oxidative stress and inflammatory cytokines. HT22 cells were subjected to Echinatin treatment (0-40 μM) for a duration of 24 hours, followed by exposure to sevoflurane or control conditions. (A) Protein levels of GCL, NQO1, and Prx1 were measured by western blot. (B–D) The levels of MDA, GSH, TNF-α and IL-1β were measured by commercial kits. The data are presented as the mean ± SD. Ech, Echinatin; Sevo, sevoflurane. Compared with the Sevo+ Ech (0 μM) group, **P<0.01, ***P<0.001.
Figure 3
Figure 3
Echinatin inhibits sevoflurane-induced ferroptosis in HT22 cells. HT22 cells were subjected to Fer-1 (1 μM) treatment or Echinatin (40μM) treatment for 24 hours, followed by exposure to sevoflurane or control conditions. (A) Cell apoptosis was measured by flow cytometry. (B) Intracellular Fe2+ detected by FerroOrange. (C) Protein levels of FPN, TFR1, and DMT1 were measured by western blot. The data are presented as the mean ± SD. Compared with the Sevo group, *P<0.05, **P<0.01.
Figure 4
Figure 4
The co-differentially expressed genes in Echinatin and ferroptosis. (A) The Venn graph of overlap genes in both Echinatin and ferroptosis. (B) PPI network and key functional modules of Echinatin against ferroptosis. The enrichment of overlap genes using the Metascape database. Network of enriched terms: (C) colored by cluster ID, where nodes that share the same cluster ID are typically close to each other; (D) colored by p-value, where terms containing more genes tend to have a more significant p-value.
Figure 5
Figure 5
Echinatin inhibits sevoflurane-induced ferroptosis via ALOX12. (A) HT22 cells were exposed to either sevoflurane or control conditions. Protein levels of p53, p21, and SLC7A11 were measured by western blot. Compared with the Ctrl group, **P<0.01. (B) HT22 cells were subjected to Echinatin treatment (40 μM) for a duration of 24 hours, followed by exposure to sevoflurane or control conditions. Protein level of ALOX12 was measured by western blot. (C) ALOX12 activity was measured by detecting 12-HETE levels by ELISA. Compared with the Sevo group, *P<0.05, **P<0.01. (D) HT22 cells infected with shRNA-control (si-NC) lentivirus or shRNA-ALOX12 (si-ALOX12) lentivirus, followed by exposure to sevoflurane or control conditions. Protein levels of ALOX12, FPN, TFR1, and DMT1 were measured by western blot. Compared with the Sevo+si-NC group, *P<0.05, **P<0.01. (E) HT22 cells were infected with lentiviruses carrying either the overexpression control (OE-NC) or ALOX12 overexpression (OE-ALOX12) constructs, and subsequently subjected to Echinatin treatment (40 μM) followed by exposure to sevoflurane. Cell apoptosis and ROS levels were measured using flow cytometry. Intracellular Fe2+ detected by FerroOrange. Compared with indicated group, **P<0.01, ***P<0.001. The data are presented as the mean ± SD. Ech, Echinatin; Sevo, sevoflurane.
Figure 6
Figure 6
Echinatin enhances learning and memory capabilities of mice exposed to sevoflurane. (A) Changes in mice body weight after injection of different concentrations of Echinatin. For in vivo experiments, mice exposed to sevoflurane (3%) were intraperitoneally administered Echinatin at a dose of 20 mg/kg. (B) Protein levels of FPN, TFR1, and DMT1 in hippocampal tissue were assessed using western blot analysis. (C–E) Expression levels of TNF-α, IL-1β, IL-6, GSH, and SOD in hippocampal tissue were measured by commercial kits. (F) Escape latency during Morris water maze testing. (G) Swimming distance during Morris water maze testing. (H) Time in target quadrant during Morris water maze testing. (I) Swimming velocity during Morris water maze testing. The data are presented as the mean ± SD. Ech, Echinatin; Sevo, sevoflurane. Compared with the Sevo group, **P<0.01, ***P<0.001.
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