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Isoflurane, a common volatile anesthetic, has been widely used to provide general anesthesia in operations. However, exposure to isoflurane may cause widespread neurotoxicity in the developing animal brain. Fraxetin, a natural coumarin derivative extracted from the bark of Fraxinus rhynchophylla, possesses versatile pharmacological properties including anti-oxidative, anti-inflammatory, and neuroprotective effects. However, the effect and action mechanism of fraxetin on neurotoxicity induced by isoflurane are unknown. Reactive oxygen species (ROS) generation, cell viability, lactate dehydrogenase (LDH) release, and apoptosis were estimated by 2',7'-dichlorofluorescin-diacetate (DCFH-DA) staining, MTT, LDH release, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining assays, respectively. The protein levels of light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were detected by western blot analysis. Isoflurane induced ROS, LDH release, apoptosis, and autophagy, but inhibited the viability in HT22 cells, which were overturned by fraxetin or ROS scavenger N-acetyl-L-cysteine. Fraxetin suppressed isoflurane-induced PI3K/Akt inactivation in HT22 cells. PI3K/Akt inactivation by LY294002 resisted the effects of fraxetin on isoflurane-induced autophagy and autophagy-modulated neurotoxicity in HT22 cells. In conclusion, fraxetin suppressed ROS-dependent autophagy by activating the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.

Propofol, a general intravenous anesthetic, has been demonstrated to cause a profound neuroapoptosis in the developing brain followed by long-term neurocognitive impairment. Our study aimed to examine the neuroprotective effect of neuronal PAS domain protein 4 (NPAS4), an activity-dependent neuron-specific transcription factor, on propofol-induced neurotoxicity in hippocampal neuronal HT22 cells. The differentially expressed genes in HT22 cells after treatment with propofol were screened from Gene Expression Omnibus dataset GSE106799. NPAS4 expression in HT22 cells treated with different doses of propofol was investigated by qRT-PCR and Western blot analysis. Cell viability, lactate dehydrogenase (LDH) release, caspase-3 activity, and apoptosis were evaluated by MTT, a LDH-Cytotoxicity Assay Kit, a Caspase-3 Colorimetric Assay Kit, and TUNEL assay, respectively. The protein levels of LC3-I, LC3-II, Beclin 1, p62 and NPAS4 were detected using Western blot analysis. Propofol treatment concentration-dependently decreased NPAS4 expression in HT22 cells. Propofol treatment inhibited cell viability, increased LDH release and caspase-3 activity, and induced apoptosis and autophagy in HT22 cells. NPAS4 overexpression suppressed propofol-induced cell injury and autophagy in HT22 cells. Mechanistically, autophagy agonist rapamycin attenuated the neuroprotective effect of NPAS4 in propofol-treated HT22 cells. In conclusion, NAPS4 overexpression protected hippocampal neuronal HT22 cells against propofol-induced neurotoxicity by reducing autophagy.