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This study aims to investigate the effects of Naotaifang (NTF) on neuronal ferroptosis and pyroptosis in cerebral ischemia-reperfusion injury (CIRI), with a focus on nuclear factor erythroid 2-related factor 2 (Nrf2). A rat model of CIRI was established by middle cerebral artery occlusion/reperfusion (MCAO/R). Sprague-Dawley rats were randomly assigned to a sham group, a model group, low/medium/high-dose (4.5, 9, 18 g·kg-1, respectively) NTF groups, and a butylphthalide (60 mg·kg-1) group (n=15 per group). After modeling, the treatment groups received corresponding agents by gavage once daily for 7 days, and the sham and model groups received an equal volume of normal saline. After treatment, the neurological function was assessed based on the Zea-Longa score, the infarct volume by TTC staining, and cellular morphology by HE staining. The levels of Fe2+, interleukin-1β (IL-1β), and interleukin-18 (IL-18) were measured by ELISA, and those of malondialdehyde (MDA) and glutathione (GSH) by biochemical assays. Western blot was employed to examine the expression levels of Nrf2, glutathione peroxidase 4 (GPX4), NOD-like receptor family pyrin domain-containing 3 (NLRP3), and the N-terminal fragment of gasdermin D (GSDMD-N). In the cell experiment, oxygen-glucose deprivation/reperfusion (OGD/R) was used to model CIRI in SH-SY5Y cells. siRNA-mediated Nrf2 knockdown (si-Nrf2) was employed to probe into the role of Nrf2 in OGD/R-induced ferroptosis and pyroptosis and the intervention effect of NTF. Membrane damage was evaluated by Hoechst/propidium iodide staining and LDH release, and GPX4 and GSDMD immunofluorescence was employed to indicate ferroptosis and pyroptosis, respectively. Fe2+, MDA, and GSH were quantified biochemically, and the levels of IL-1β and IL-18 by ELISA. The expression of Nrf2, GPX4, NLRP3, and GSDMD-N was quantified by Western blot. The cellular ultrastructure was observed by transmission electron microscopy. The results of the animal experiment showed that NTF promoted the expression of Nrf2 to alleviate neuronal ferroptosis and pyroptosis, thus producing significant neuroprotection against CIRI in a dose-dependent manner. The results of the cell experiment showed that the neuroprotective effects of NTF after CIRI were mediated by Nrf2-dependent suppression of neuronal ferroptosis and pyroptosis. Taken together, NTF attenuates CIRI by suppressing neuronal ferroptosis and pyroptosis through Nrf2-mediated regulation.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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