Sevoflurane Inhibited Cell Viability and Induced Neuronal Cell Death

To investigate the neurotoxicity of sevoflurane on neonatal neuronal cells, cellular viability was evaluated using the MTT assay after exposure of mouse hippocampal HT22 cells and rat primary hippocampal neurons to 2, 4, and 8% sevoflurane for 6, 12, and 24 h [10]. Mouse hippocampal HT22 cells are similar to undifferentiated neural cells and are particularly sensitive to oxidative stress. The primary hippocampal neurons of rats are immature neurons, which were isolated from neonatal Sprague‒Dawley rats. As shown in Supplementary Fig. 1a, sevoflurane exposure significantly reduced the viability of neuronal cells in a time- and concentration-dependent manner in contrast to those in the control group. Considering that the integrity of dying cell membranes was damaged, which produced a quantifiable amount of LDH, we used an LDH release test to determine whether sevoflurane induced neuronal cell death. Neuronal cells treated with 2% sevoflurane for 6 h showed an overt increase in cell death in contrast to cells in the control group, and this effect was exacerbated with increasing time and concentrations (Supplementary Fig. 1b). Consistently, microscopy showed that the majority of neuronal cells treated with 8% sevoflurane for 24 h were smaller in size and round in shape (Supplementary Fig. 1c). Thus, these findings demonstrated that exposure to sevoflurane reduced cell viability and resulted in neuronal cell death in a concentration- and time-dependent manner.

Sevoflurane Induced Neonatal Neuronal Cell Ferroptosis

To elucidate the mechanism of sevoflurane-induced neonatal neuronal cell death, we determined whether ferroptosis was implicated in neuronal death caused by sevoflurane. Ferroptosis is typically characterized by increases in intracellular ferrous iron and lipid peroxidation [12], and we examined sevoflurane-induced changes in ferrous iron and the lipid peroxidation product MDA. We found that the levels of intracellular iron and MDA were increased when neuronal cells were subjected to 2% sevoflurane for 12 h in contrast to those in the control group, and these effects were further enhanced with increasing time and concentrations (Fig. 1a, b). These results suggested that sevoflurane could increase intracellular ferrous iron and lipid peroxidation in a time- and concentration-dependent manner in neuronal cells.

Fig. 1

Sevofluraneinduced neonatal neuronal cell ferroptosis. (a) Iron assays revealed that sevoflurane increased intracellular ferrous iron in a concentration- and time-dependent manner. (b) The MDA assay showed that sevoflurane induced lipid peroxidation in a concentration- and time-dependent manner. (c) The iron assay demonstrated that DFO markedly suppressed the increase in intracellular iron in neonatal neuronal cells exposed to 4% or 8% sevoflurane for 24 h. (d) The MDA assay proved that lipid peroxidation caused by 8% sevoflurane exposure for 24 h in neuronal cells was alleviated in the presence of Fer-1 (50 μM), Lip-1 (10 μM), DFO (100 μM), VitE (100 μM), or GSH (2.5 mM). (e) The MTT assay proved that the reduction in neuronal cell viability induced by 8% sevoflurane for 24 h was significantly rescued by Fer-1, Lip-1, DFO, VitE, or GSH. (f) The LDH release assay revealed that neuronal cell death induced by 8% sevoflurane exposure for 24 h was markedly suppressed by Fer-1, Lip-1, DFO, VitE, or GSH. (g) Representative transmission electronic microscopy images showed that HT22 cells exposed to 8% sevoflurane for 24 h had irregularly shaped nuclei (N) with multiple nucleoli (Nu), a slightly widened perinuclear space (black arrows), shrunken mitochondria (M) with increased membrane density and expanded cristae, and local rough endoplasmic reticulum (RER) expansion compared to those in the control group. (h–l) Western blotting demonstrated that sevoflurane induced the upregulation of TFR and TF and downregulation of FPN in a time- and concentration-dependent manner, but there was no significant change in FTH or FTL. In comparison with the control group, ∗p < 0.05, ∗∗p < 0.01; compared with the group treated with sevoflurane, #p < 0.05, ##p < 0.01

To verify whether ferrous iron induces lipid peroxidation and contributes to iron-dependent cell death, neuronal cells were administered the iron chelator deferoxamine (DFO) at a concentration of 100 μM 1 h prior to sevoflurane exposure. We found that inhibiting ferrous iron with DFO significantly suppressed sevoflurane-induced MDA production and the decrease in the viability of neuronal cells (Fig. 1c–e). Moreover, the LDH release test showed that sevoflurane-induced neuronal cell death was attenuated in the presence of DFO (Fig. 1f). Furthermore, DFO pretreatment restored the sevoflurane-induced reduction in neuronal cell size, as seen under a light microscope (Supplementary Fig. 1c). These results suggested that sevoflurane induced neonatal neuronal lipid peroxidation and cell death through an increase in intracellular ferrous iron. Then, to further investigate the involvement of lipid peroxidation in sevoflurane-induced neuronal cell death, neuronal cells were exposed to sevoflurane in the presence of the lipophilic antioxidants ferrostatin-1 (Fer-1, 50 μM) and liproxstatin-1 (Lip-1, 10 μM). We found that Fer-1 or Lip-1 pretreatment reversed the sevoflurane-induced increase in MDA and mitigated neonatal neuronal cell death induced by sevoflurane (Fig. 1d, f). Consistently, pretreatment with 100 μM vitamin E or 2.5 mM GSH markedly suppressed the sevoflurane-induced increase in MDA and attenuated the lethal effect of sevoflurane on neuronal cells (Fig. 1d, f). These results indicated that lipid peroxidation contributed to sevoflurane-induced neonatal neuronal cell death. Additionally, transmission electron microscopy revealed that HT22 cells exposed to 8% sevoflurane for 24 h had irregularly shaped nuclei (N) with multiple nucleoli (Nu), a slightly widened perinuclear space (black arrows), shrunken mitochondria (M) with increased membrane density and expanded cristae, and local rough endoplasmic reticulum (RER) expansion compared to those in the control group (Fig. 1g), which was consistent with the morphological features of ferroptosis [40]. Thus, sevoflurane exposure induced ferroptosis in neonatal neuronal cells.

To elucidate the mechanism of the sevoflurane-induced aberrant increase in intracellular iron, we examined the changes in proteins that could regulate intracellular iron metabolism [41]. In this study, we found that transferrin receptor (TFR), which mediates ferric iron uptake by cells through the iron-TF-TFR complex, was significantly upregulated after sevoflurane exposure in a time- and concentration-dependent manner (Fig. 1h–j). Correspondingly, the sevoflurane-induced expression of TF was increased, and this effect was exacerbated with increasing time and concentrations (Fig. 1h, i, k). Moreover, ferroportin (FPN), which mediates intracellular iron export, was markedly downregulated by each sevoflurane concentration and time, but there was no significant change in ferritin (both FTH and FTL), which could bind to intracellular ferrous iron after sevoflurane exposure (Fig. 1h, i, l). Thus, our data suggested that sevoflurane improved intracellular iron levels by upregulating TFR and TF and downregulating FPN but did not suppress ferritin (FT).

H2O2 Was Implicated in Sevoflurane-Induced Neuronal Cell Ferroptosis via an Increase in Iron

To determine whether H2O2 causes the increase in intracellular iron that results in iron-dependent cell death following sevoflurane exposure, an H2O2 assay was conducted, and the role of H2O2 in the regulation of the increase in intracellular iron caused by sevoflurane was examined because H2O2 plays a crucial role in modulating the progression of ferroptosis [22]. We found that sevoflurane increased intracellular H2O2 in a concentration- and time-dependent manner (Fig. 2a), which was accompanied by a reduction in intracellular GSH (Fig. 2b). In contrast, inhibiting intracellular H2O2 with GSH (2.5 mM) markedly alleviated the increases in intracellular ferrous iron and lipid peroxidation, in addition to neuronal cell death caused by sevoflurane (Figs. 1d, f and 2c, d). Furthermore, we showed that GSH supplementation reversed the sevoflurane-induced upregulation of TF and TFR and downregulation of FPN (Fig. 2e–g). These data suggest that H2O2 is an upstream factor in the intracellular iron increase in sevoflurane-induced neuronal ferroptosis. To further confirm the role of H2O2 in the regulation of ferroptosis, neuronal cells were administered exogenous H2O2. We found that incubating neuronal cells with exogenous H2O2 (500 μM) for 24 h significantly promoted cell death, increased intracellular iron and lipid peroxidation, upregulated TFR and TF, and downregulated FPN (Fig. 2h–m). Conversely, we observed that pretreatment with the antioxidant NAC significantly mitigated neuronal cell death and lipid peroxidation caused by H2O2, resulting in a decrease in intracellular iron, the downregulation of TFR and TF, and the upregulation of FPN (Fig. 2h–m). These results showed that H2O2 contributed to the sevoflurane-induced increase in intracellular iron by upregulating TFR and TF and downregulating FPN, which led to iron-dependent lipid peroxidation and ferroptosis.

Fig. 2

H2O2 was implicated in sevoflurane-induced neuronal cell ferroptosis via an increase in iron. (a) Sevoflurane increased intracellular H2O2 in a time- and concentration-dependent manner. (b) Sevoflurane reduced GSH levels in a time- and concentration-dependent manner. (c) Supplementation with GSH (2.5 mM) prevented the increase in H2O2 caused by 8% sevoflurane exposure for 24 h. (d) The iron assay showed that 8% sevoflurane exposure for 24 h increased intracellular iron in neuronal cells, and this effect was suppressed by pretreatment with GSH. (e–g) Western blotting revealed that neuronal cells treated with 8% sevoflurane for 24 h had upregulated TFR and TF expression and downregulated FPN expression, and these effects were prevented by GSH. (h–j) Western blotting showed that NAC (5 mM) markedly suppressed the upregulation of TFR and TF and downregulation of FPN induced by treatment of neuronal cells with 500 μM H2O2 for 24 h, but had no significant effect on FTH or FTL. (k) The LDH release assay showed that treatment with H2O2 (500 μM) for 24 h markedly increased the rate of neuronal cell death, which was significantly alleviated by NAC. (l) The MDA assay demonstrated that lipid peroxidation caused by exposure of neuronal cells to 500 μM H2O2 for 24 h was mitigated by the administration of NAC. (m) The iron assay showed that neuronal cells treated with H2O2 at 500 μM for 24 h had increased levels of intracellular ferrous iron, and this effect was inhibited by pretreatment with NAC. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01

ER Stress-Mediated ATF3 Activation Promoted Sevoflurane-Induced Neuronal Cell Ferroptosis by Increasing H2O2

Given that ATF3 can improve intracellular H2O2 and promote ferroptosis [28, 29, 32], we investigated whether ATF3 activation was involved in sevoflurane-induced neuronal cell ferroptosis by increasing H2O2. As shown in Fig. 3A (a, e, g), each concentration of sevoflurane and time increased the expression and nuclear translocation of ATF3 in neuronal cells. Consistently, confocal microscopy verified that ATF3 clearly accumulated in the nuclei of neuronal cells following sevoflurane exposure (Fig. 3B (a)). To determine the role of ATF3 in neuronal ferroptosis caused by sevoflurane, siRNA was use to knock down ATF3 in HT22 cells and hippocampal neurons. We observed that knockdown of ATF3 expression suppressed the sevoflurane-induced increase in intracellular H2O2 and neuronal cell death (Fig. 3B (b, c)). Moreover, the overt increase in intracellular ferrous iron and lipid peroxidation levels, in addition to TF and TFR upregulation and the downregulation of FPN caused by sevoflurane, was mitigated by siRNA-mediated knockdown of ATF3 (Fig. 3B (d–i)). These results suggested that ATF3 activation contributed to sevoflurane-induced neuronal cell ferroptosis by increasing intracellular H2O2.

Fig. 3

A Sevoflurane induced changes in ER stress marker proteins in neuronal cells. (a–g) Western blotting analysis shown that sevoflurane not only upregulated the levels of ER stress markers proteins GRP78, PERK, ATF4, and ATF3 in neuronal cells in a concentration- and time-dependent manner, but also promoted the nuclear translocation of ATF4 and ATF3. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01. (B) ATF3 activation promoted sevoflurane-induced neuronal cell ferroptosis by increasing H2O2. (a) Representative confocal microscopy images showed that ATF3 accumulated clearly in the nuclei of HT22 cells following 8% sevoflurane exposure for 24 h in contrast to that in the control group. (b) The increase in intracellular H2O2 in neuronal cells induced by 8% sevoflurane for 24 h was attenuated when ATF3 was knocked down by siRNA. (c) The LDH release assay proved that ATF3 silencing suppressed neuronal cell death induced by 8% sevoflurane exposure for 24 h. (d) The iron assay demonstrated that ATF3 silencing prevented the increase in ferrous iron in neuronal cells induced by 8% sevoflurane for 24 h. (e) MDA assay showed that lipid peroxidation in neuronal cells induced by 8% sevoflurane exposure for 24 h was inhibited by knockdown of ATF3 expression. (f–i) Western blotting showed that 8% sevoflurane exposure for 24 h not only increased ATF3 expression in the cytoplasm and nucleus of neuronal cells but also upregulated the levels of TFR and TF and downregulated the level of FPN, and these effects were markedly suppressed when neuronal cells were transfected with siRNA ATF3. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01

Considering that ER stress is a major source of H2O2 and that ATF3 expression can be induced by ER stress through the PERK/ATF4-mediated pathway [28, 29], we examined the role of ER stress in sevoflurane-induced ATF3 activation and neuronal ferroptosis. The results showed that neuronal cells treated with 4% and 8% sevoflurane for 6, 12, and 24 h exhibited increases in proteins representing ER stress markers, including GRP78, PERK, and ATF4, and the effect was dependent on time and concentration (Fig. 3A (a–d, f)); these effects were abrogated by the ER stress inhibitor 4-PBA or the PERK inhibitor GSK2606414 (Fig. 4A (a–d)). Moreover, we observed that neuronal death caused by sevoflurane was ameliorated when the cells were pretreated with 4-PBA and GSK2606414 or siRNA targeting ATF4 (Fig. 4B (a, b)). These results suggested that the ER stress-related PERK/ATF4 pathway participated in sevoflurane-induced neuronal cell death. Furthermore, the sevoflurane-induced upregulation of ATF3 in the cytoplasm and nucleus was markedly suppressed by 4-PBA, GSK2606414, or knockdown of ATF4 expression (Fig. 4A (a, e, f, j)), indicating that sevoflurane-induced ATF3 activation was regulated by the PERK/ATF4 pathway in response to ER stress. In addition, we found that the sevoflurane-induced increases in intracellular H2O2, iron, and lipid peroxidation, as well as the upregulation of TFR and TF and downregulation of FPN, were significantly alleviated by GSK2606414 or ATF4 knockdown (Fig. 4A (a–c, f–h) and 4B (c–h)). Collectively, these findings revealed that ER stress and PREK/ATF4-mediated ATF3 activation promoted sevoflurane-induced neuronal cell ferroptosis by increasing intracellular H2O2.

Fig. 4

A Sevoflurane-induced ATF3 activation was regulated by the ER stress via the PERK/ATF4-mediated pathway. (a–e) Western blotting analysis revealed that neuronal cells treated with 8% sevoflurane for 24 h markedly induced apparent GRP78, PERK, ATF4, ATF3, TFR, and TF upregulation and FPN downregulation, which were all significantly mitigated by pretreatment with ER stress inhibitor 4-PBA (250 μM) or PERK inhibitor GSK2606414 (1 μM). (f–j) Western blotting shown that knockdown of ATF4 prevented the PERK, ATF4, ATF3, TFR, and TF upregulation and FPN downregulation induced by 8% sevoflurane for 24 h. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01. (B) ER stress-induced PERK/ATF4 pathway participated in sevoflurane-induced neuronal cell ferroptosis. (a, b) The LDH release assay proved that neuronal cell death induced by 8% sevoflurane exposure for 24 h was alleviated by 4-PBA and GSK2606414 or knockdown of ATF4 expression. (c, d) The increase in intracellular H2O2 in neuronal cells induced by 8% sevoflurane for 24 h was reversed by pretreatment with GSK2606414 or siRNA targeting ATF4. (e, f) The iron assay showed that GSK2606414 pretreatment or ATF4 knockdown suppressed the increase in iron caused by 8% sevoflurane exposure for 24 h. (g, h) The MDA assay showed that lipid peroxidation in neuronal cells caused by 8% sevoflurane exposure for 24 h was inhibited by GSK2606414 or ATF4 knockdown. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01

ATF3 Promoted the Sevoflurane-Induced Increase in H2O2 by Activating NOX4 and Suppressing Catalase, GPX4, and SLC7A11

To examine why ATF3 could promote the sevoflurane-induced increase in intracellular H2O2, we investigated the role of ATF3 in regulating NADPH oxidase 4 (NOX4) because NOX4 is mainly located in the endoplasmic reticulum and can generate superoxide, which is subsequently transformed into H2O2 during ER stress [42]. The level of sevoflurane-induced intracellular superoxide was detected by using dihydroethidium (DHE). Fluorescence microscopy revealed that the levels of superoxide, as indicated by the intensity of DHE fluorescence, were significantly increased in neuronal cells after 4% sevoflurane exposure for 24 h and were further elevated when neuronal cells were subjected to 8% sevoflurane (Fig. 5A (a)). Consistently, our results showed that sevoflurane exposure was linked with the upregulation of NOX4 in neuronal cells (Fig. 5A (b, c)). In contrast, pretreatment with the NOX4 inhibitor GKT137831 markedly counteracted the sevoflurane-induced upregulation of NOX4 and increase in superoxide and H2O2 in neuronal cells (Fig. 5A (a) and 5B (a–c, g)), suggesting that NOX4 promoted the sevoflurane-induced increase in intracellular H2O2. Moreover, the increases in iron and lipid peroxidation, as well as the upregulation of TFR and TF and downregulation of FPN induced by sevoflurane, were markedly alleviated by GKT137831 (Fig. 5B (a–c, h, i)). This further confirmed that H2O2 contributed to sevoflurane-induced iron-dependent neuronal cell death. Considering that catalase is a known intracellular H2O2 scavenging enzyme [43], we then verified the role of catalase in the increase in intracellular H2O2 induced by sevoflurane. We observed that catalase levels were significantly decreased by sevoflurane (Fig. 5A (b, d)), suggesting that catalase was involved in the sevoflurane-induced increase in H2O2. Notably, we found that ATF3 silencing markedly prevented the sevoflurane-induced increase in NOX4 and decrease in catalase, as well as the generation of superoxide and H2O2 (Fig. 5A (a) and 5B (d–f)). Thus, our findings indicated that ATF3 promoted the sevoflurane-induced increase in H2O2 by upregulating NOX4 and downregulating catalase.

Fig. 5

A Sevoflrane induced the increase in superoxide and upregulation of NOX4 and downregulation of calatase, GPX4, and SLC7A11 in neuronal cells. (a) Fluorescence microscopy revealed that the levels of superoxide, as shown by the intensity of dihydroethidium (DHE) fluorescence, were significantly increased in neuronal cells following 4% sevoflurane exposure for 24 h and were further elevated when neuronal cells were subjected to 8% sevoflurane for 24 h, and these effects were counteracted by the NOX4 inhibitor GKT137831 (50 μM) or knockdown of ATF3 expression. (b–f) Western blotting demonstrated that sevoflurane upregulated NOX4 and downregulated catalase, GPX4, and SLC7A11 in a time- and concentration-dependent manner. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01. (B) ATF3 promoted the sevoflurane-induced increase in H2O2 by activating NOX4 and suppressing catalase, GPX4, and SLC7A11. (a–c) Western blotting revealed that neuronal cells treated with 8% sevoflurane for 24 h had upregulated NOX4, TFR, and TF expression and downregulated FPN expression, and these effects were prevented by GKT137831. (d–f) Western blotting showed that ATF3 silencing markedly suppressed the upregulation of NOX4 and downregulation of catalase, GPX4, and SLC7A11 in neuronal cells caused by 8% sevoflurane for 24 h. (g) Supplementation with GKT137831 prevented the increase in H2O2 caused by 8% sevoflurane exposure for 24 h. (h) MDA assay showed that lipid peroxidation induced by 8% sevoflurane for 24 h in neuronal cells was mitigated in the existence GKT137831. (i) The iron assay showed that 8% sevoflurane exposure for 24 h increased intracellular iron in neuronal cells, and this effect was suppressed by pretreatment GKT137831. (j) Sevoflurane induced a concentration- and time-dependent decrease in cysteine in neuronal cells. (k, l) The depletion of intracellular cysteine and GSH induced by 8% sevoflurane for 24 h was markedly suppressed when neuronal cells were transfected with siRNA ATF3. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01

Intracellular H2O2 can be scavenged by glutathione peroxidase 4 (GPX4) through the consumption of GSH, which is synthesized mainly from cysteine that can be converted from cystine [32, 44]. To examine the role of GPX4 in H2O2 accumulation induced by sevoflurane, we examined sevoflurane-induced alterations in GSH and GPX4. The results showed that sevoflurane decreased GPX4 in neuronal cells, and there was a decrease in GSH and an increase in intracellular H2O2 (Figs. 2b, c and 5A (b, e)). Then, we examined the expression of SLC7A11, which is a unique subunit of amino acid antiporter system Xc − that facilitates the exchange between internal glutamate and extracellular cysteine [32, 45]. Our results showed that sevoflurane exposure decreased SLC7A11 and cysteine levels in neuronal cells in a time- and concentration-dependent manner (Fig. 5A (b, f) and 5B (j)). Moreover, overexpression of GPX4 and SLC7A11 not only significantly suppressed the increase in intracellular H2O2 and lipid peroxidation induced by sevoflurane but also markedly attenuated the increase in ferrous iron and the decrease in cellular viability after sevoflurane exposure (Fig. 6a–f). These results further confirmed that the inhibition of GPX4 and SLC7A1 in iron-dependent neuronal cell death processes is the main mechanism by which sevoflurane induces an increase in intracellular H2O2. Notably, the sevoflurane-induced downregulation of GPX4 and SLC7A11, as well as the depletion of cysteine and GSH, was markedly alleviated when neuronal cells were transfected with siRNA against ATF3 (Fig. 5B (d–f, k, l)). These data demonstrated that ATF3 promoted the sevoflurane-induced increase in H2O2 by suppressing GPX4 and SLC7A11.

Fig. 6

Overexpression of GPX4 and SLC7A11 alleviated sevoflurane-induced ferroptosis in HT22 cells. (a, b) The representative Western blot bands and semiquantitative analysis of GPX4 and SLC7A11 in each group. (c–f) Overexpression of GPX4 and SLC7A11 not only significantly suppressed the increase in intracellular H2O2 and lipid peroxidation induced by 8% sevoflurane for 24 h but also markedly attenuated the increase in ferrous iron and the decrease in cellular viability after sevoflurane exposure.  Compared with the control group, ∗p＜0.05, ∗∗p＜0.01

ER Stress-Mediated ATF3 Activation Contributed to Sevoflurane-Induced Neuronal Ferroptosis in the Hippocampus of Neonatal Mice

Previous studies have shown that exposing neonatal mice to 3% sevoflurane for 2 h every day for three consecutive days could result in neuronal cell death and persistent cognitive impairment [36, 37]. Thus, neonatal mice were treated with 3% sevoflurane for 2 h per day on postnatal days (P) 6, P7, and P8 to determine whether ferroptosis was involved in sevoflurane-induced neonatal neuronal death. Consistent with previous reports [46], the O2 partial pressure, pH, and CO2 partial pressure in neonatal mice exposed to sevoflurane for 2 h were not significantly different from those in the control group (Table 1). Pathological examination of the hippocampus on day 7 after sevoflurane anesthesia by HE staining showed that sevoflurane exposure markedly increased hippocampal neuronal death or injury, which was characterized by sparsely and morphologically pink cytoplasm, disordered neuronal structure, cell shrinkage, and pyknotic nuclei (Fig. 7a). Statistical analysis revealed that on day 7 after sevoflurane exposure, 80% of pyramidal neurons in the hippocampal CA1 region were alive (Fig. 7b). These results suggested that repeated sevoflurane exposure induced visible hippocampal neuronal death in neonatal mice. Moreover, we observed that repeated sevoflurane exposure increased ferrous iron and MDA levels in the hippocampi of neonatal mice (P8) at 6 h, and these effects were diminished by pretreatment with the iron chelator deferiprone (DFP) (Fig. 7c, d). Furthermore, DFP pretreatment markedly attenuated sevoflurane-induced morphological changes and significantly improved the number of pyramidal neurons that were still alive in the hippocampal CA1 region (Fig. 7a, b). These results showed that repeated sevoflurane exposure induced hippocampal neuronal cell ferroptosis in the developing brain.

Table 1 Arterial blood gas after 2 h of 3% sevoflurane or no anesthesia in P8 mice pups (n = 6 per group)Fig. 7

ATF3 contributed to sevoflurane-induced neuronal ferroptosis in the hippocampus of neonatal mice. (a) Representative images of hippocampal neurons stained with HE on day 7 after anesthesia, and the mice had received 3% sevoflurane exposure for 2 h on postnatal day (P) 6, P7, and P8. Scale bar = 20 µm. Compared with that in the control group, sevoflurane caused pyramidal neuron death (green arrow) in the hippocampal CA1 region, which was characterized by sparse and disordered neuronal structures, cell shrinkage, morphologically pink cytoplasm, and pyknotic nuclei, and these effects were ameliorated by GSK2606414 (50 mg/kg) and DFP (75 mg/kg) or ATF3 silencing. (b) Statistical analysis revealed that the percentage of living pyramidal neurons in the hippocampal CA1 region was 80% on day 7 after sevoflurane exposure, and pretreatment with GSK2606414 and DFP or ATF3 silencing markedly rescued the sevoflurane-induced decrease in living neurons. (c, d) Repeated sevoflurane exposure significantly increased intracellular iron and MDA levels in the hippocampi of neonatal mice, and these effects were prevented by GSK2606414 and DFP or ATF3 silencing. (e–g) Western blotting showed that sevoflurane exposure upregulated ATF3, TFR, TF, and NOX4 and downregulated FPN, catalase, GPX4, and SLC7A11, and these effects were reversed by the PERK inhibitor GSK2606414 or ATF3 silencing. (h) GSK2606414 pretreatment or ATF3 silencing significantly prevented the sevoflurane-induced accumulation of H2O2 in the hippocampi of neonatal mice. (i, j) Repeated sevoflurane exposure caused the depletion of GSH and cysteine in the hippocampi of neonatal mice, and this effect was mitigated by pretreatment with GSK2606414 or ATF3 silencing. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01

To verify whether ER stress-mediated ATF3 activation was involved in sevoflurane-induced neonatal hippocampal neuronal ferroptosis, wild-type (WT) mice treated with GSK2606414 or ATF3-knockout (KO) mice were exposed to 3% sevoflurane for 2 h daily from P6 to P8. We observed that repeated sevoflurane exposure increased PERK, ATF4, and ATF3 levels in the hippocampi of neonatal mice (Fig. 7e–g), indicating that sevoflurane promoted ER stress and PERK/ATF4/ATF3 pathway activation in the hippocampi of neonatal mice. Moreover, pretreatment with GSK2606414 or ATF3 silencing not only significantly reduced sevoflurane-induced neuronal cell death and the increase in ferrous iron and MDA levels but also suppressed the upregulation of TF and TFR and downregulation of FPN in the hippocampi of neonatal mice (Fig. 7a–g). These results suggested that ER stress-mediated ATF3 activation participated in sevoflurane-induced hippocampal neuronal ferroptosis in the developing brain. Furthermore, we found that the sevoflurane-induced increase in H2O2, upregulation of NOX4 and downregulation of SLC7A11, GPX4, and catalase, as well as the reductions in GSH and cysteine in the hippocampi of neonatal mice were all significantly mitigated by GSK2606414 treatment or ATF3 silencing (Fig. 7e–j). These results further confirmed that ER stress-mediated ATF3 activation contributed to sevoflurane-induced hippocampal neuronal ferroptosis via H2O2 accumulation and resultant iron overload in neonatal mice.

ATF3-Mediated Ferroptosis Participated in Sevoflurane-Induced Spatial Memory Impairment in Neonatal Mice

Given the important role of the hippocampus in spatial learning and memory, we used the Morris water maze test to evaluate spatial learning and memory performance on postnatal day 31 in mice that had received repeated sevoflurane exposures on the P6, P7, and P8. As training proceeded, the mice had visibly reduced latencies to detect the hidden platform, suggesting that the mice were learning from daily practice. The typical swimming tracks suggested that mice exposed to sevoflurane had markedly prolonged escape latencies in the spatial training tests on days 3, 4, and 5 and significantly reduced times in the indicated quadrant in the probing experiment on day 6 compared with those in the control group (Fig. 8a–c). In contrast, the mice treated with GSK2606414 and DFP or ATF3 silencing searched for the hidden platform in a more appropriate way, resulting in a shorter delay in the time to find the hiding platform and more time in the target quadrant in contrast to mice in the sevoflurane group (Fig. 8a–c). Therefore, our findings suggested that ER stress and ATF3 activation-mediated ferroptosis accounted for the spatial memory impairment caused by repeated sevoflurane exposure in neonatal mice.

Fig. 8

ER stress and ATF3 activation-mediated ferroptosis participated in sevoflurane-induced spatial memory impairment in neonatal mice. (a, b) Escape latency was longer in the sevoflurane group in the spatial training tests on days 3, 4, and 5 than in the control group, but mice treated with GSK2606414 and DFP or ATF3 silencing had shorter latencies than those in the sevoflurane group. (c) Statistical analysis revealed that mice exposed to sevoflurane spent markedly less time in the target quadrant than mice in the control group during the probe trial on day 6, whereas mice treated with GSK2606414 and DFP or ATF3 silencing spent more time in the target quadrant. Compared with the control group, ∗p < 0.05, ∗∗p < 0.01; compared with the sevoflurane group, #p < 0.05, ##p < 0.01. Data are represented as the mean ± SD (n = 6 mice per group)