Eight male and sixteen female specific pathogen free (SPF)-grade sprague dawley (SD) rats (aged 7–8 weeks) were provided by Chongqing Ensville Biotechnology Co., Ltd. (Chongqing, China). All the rats were maintained under standardized conditions with a temperature of 23 $\pm$ 2 °C, a humidity range of 35–60% and a 12-hour light-dark cycle. Those sixteen female rats were evenly assigned to two groups, comprising eight rats per group. Each group of female rats was co-housed with four male rats, and the vaginal plugs were removed as successful pregnancy. Anesthesia was administered on the 18th day of pregnancy. Rats of experimental group were placed in a self-made anesthesia box (50 cm $\times$ 50 cm $\times$ 30 cm) and connected to an anesthesia machine. 3% sevoflurane concentration was delivered with an oxygen flow rate of 2 L/min and an oxygen concentration of 50%, and inhalation was performed for four hours. The rats in the control group were only placed in the anesthesia box and given the same oxygen flow for four hours. All pregnant rats received continuous treatment for three days. After the third day of anesthesia, rats were euthanized by intraperitoneal injection of 3% sodium pentobarbital at a dose of 150 mg/kg, and then the fetal rats were taken for subsequent experiments.

Carefully remove the skull using straight and curved forceps, exposing the brain. Under a stereomicroscope, gently dissect the cerebral cortex using straight forceps to expose the hippocampal tissue. Carefully separate the hippocampal tissue from the surrounding brain tissue. Carefully remove the hippocampal tissue using curved forceps. Place the extracted hippocampal tissue in a container with phosphate buffered saline (PBS) buffer for washing and subsequent experiments.

All aninmal experiments were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Medical Research Ethics Committee of Children’s Hospital of Chongqing Medical University (No. (142) in 2023).

Extraction of hippocampal neurons from fetal rats was followed by their cultivation, with all procedures executed as detailed below: Hippocampus obtained from the fetal rat were cut up fully with ophthalmic scissors and were added to the 0.125% of trypsin (S310KJ, Beyotime, Shanhai, China) for incubation for 30 min at 37 °C. The digestion was terminated by the addition of dulbecco’s modified eagle medium (DMEM) (L110KJ, Basalmedia, Shanghai, China) containing 10% fetal bovine serum (FBS; AC03L055, Life-iLab, Shanghai, China). The cells were centrifuged, resuspended, and filtered until a uniform cell suspension was formed. Extracted cells were cultured in Neuronbasa medium (21103049, Gibco, Grand Island, NY, USA) supplemented with 2% B27 and 10% FBS (AC03L055, Life-iLab) at 37 °C with 5% CO₂. On day 3 of inoculation, 5 µmol/L cytarabine was added to the culture plates to inhibit growth of non-neural cells. The medium was changed every 3 days and neuron cell growth was observed under an inverted microscope (BLD-200, BJCOSSIM, Beijing, China). Flow cytometry analysis was applied to detect and identify fetal rat hippocampal neuron cells (Supplementary Fig. 1). Testing of the neuron cells for mycoplasma was negative.

Healthy fetal rat hippocampal neurons were harvested and digested using 0.25% trypsin solution to collect cells and prepare a cell suspension. Cell density was adjusted to 2 $\times$ 10⁵ cells/mL for plating according to experimental requirements. After cells adhered, they were divided into groups as follows: (A) Control group: Cells were cultured in a 95% air and 5% carbon dioxide environment for 24 hours. (B) 3% sevoflurane group: Cells were cultured normally for 24 hours followed by exposure to 3% sevoflurane for 12 hours. (C) 3% sevoflurane + SIRT1 inhibitor group: Cells were cultured in complete medium containing 38 nM SIRT1 inhibitor (abs42027291, Absin, Shanghai, China) for 24 hours, followed by exposure to 3% sevoflurane for 12 hours. (D) 3% sevoflurane + SIRT1 inhibitor + rapamycin group: Cells were cultured in complete medium containing 38 nM SIRT1 inhibitor for 24 hours, then exposed to a medium containing 3% sevoflurane and 12.5 nM rapamycin for 12 hours. After completing the treatments, samples were collected for analysis.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of fetal rat hippocampal tissue was performed upon application of 3,3’-Diaminobenzidine (Streptavidin-Horseradish Peroxidase) Tunel Cell Apoptosis Detection Kit (G1507, Servicebio, Wuhan, China) according to the instructions of the manufacturer. Briefly, those tissue slices were dewaxed and penetrated through Proteinase K working solution at 37 °C for 20 min. Each sample was added into Equilibration Buffer and incubated in Terminal deoxynucleotidyl transferase (TdT) incubation buffer at 37 °C for 1 h. Subsequently, pre-diluted streptavidin-Horseradish Peroxidase (HRP) reaction solution was added into each tissue. Following sample washing, the slides were dyed by Diaminobenzidine (DAB) chromogenic solution and were added into hematoxylin. The slides were sealed with a neutral resin. Finally, images were obtained through the inverted microscope (MF53, Mshot, Guangzhou, China).

Concentration and purity of RNA were measured in each sample via spectrophotometry after total RNA extraction. Subsequently, a reverse transcription kit (TSK302M, Tsingke, Beijing, China) was used for cDNA synthesis, and fluorescence quantitative PCR detection was performed through 2 $\times$T5 Fast qPCR Mix (SYBR Green I) (TSE002, Tsingke, Beijing, China) based on the instructions of the manufacturer. Primer sequences were presented in Table 1. Relative quantitative analysis was conducted with the 2^{-Δ⁢Δ⁢CT} method with GAPDH as an internal reference.

After extracting 500 µg of total proteins from the samples and mixing them with 5 $\times$ SDS loading buffer at 4:1 ratio resulting in a concentration of approximately 3.3 µg/µL, proteins were denatured by heating in a metal bath, followed by loading 60 µg of denatured total proteins onto the gel for electrophoresis. After electrophoretic separation of the bands, the proteins were transferred onto the Polyvinylidene fluoride (PVDF) membrane.Subsequently, the PVDF membranes were blocked in 5% skim milk and was incubated with primary antibodies anti-cleaved-caspase-3 (1:1000, A19654, ABclonal, Wuhan, China), anti-Bax (1:1000, A19684, ABclonal), anti-SIRT1 (1:1000, A19667, ABclonal), anti-mTOR (1:1000, A2445, ABclonal), anti-p62 (1:1000, A19700, ABclonal), anti-microtubule-associated protein light chain 3Ⅱ (LC3II) (1:1000, R381544, ZEN-BIOSCIENCE, Chengdu, China) and GAPDH (1:1000, A19056, ABclonal) at 4 °C overnight. After washing with Tris Buffered Saline with Tween (TBST), the membranes were incubated with secondary antibody HRP Goat Anti-Rabbit Immunoglobulin G (IgG) (1:2000, AS014, ABclonal) at 37 °C for 1 h. An Enhanced Chemiluminescence (ECL) exposure solution (34580, Thermo, Waltham, MA, USA) was mixed at a 1:1 ratio of solution A to solution B, evenly spread over the entire membrane, and exposed for 1 min. The membranes were then placed in a nucleic acid and protein gel imaging system (Universal Hood II, Bio-Rad, Hercules, CA, USA) for exposure detection.

After fixation, cell coverslips were washed with PBS three times, 5 min per round. A suitable amount of 0.3% Triton X-100 (ST795, Beyotime, Beijing, China) permeabilization solution was added to coverslips and incubated. These coverslips were then rinsed three times. Goat serum (C0265, Beyotime, Beijing, China) was added for blocking and then removed without rinsing. Primary antibody against SIRT1 (1:200, A0230, ABclonal, Wuhan, China) was used for incubation overnight at 4 °C. After washing three times with PBS, the specimens were cultured with secondary antibodies Cy3 Goat Anti-Rabbit IgG (H+L) (1:50, AS008, ABclonal, Wuhan, China) and FITC Goat Anti-Rabbit IgG (H+L) (1:50, AS011, ABclonal, Wuhan, China) in the dark, and followed by another three rounds of phosphate buffered saline (PBS) washing. 4’,6-diamidino-2-phenylindole (DAPI) (C1005, Beyotime, Beijing, China) was provided and the cells were cultured again to stain nuclei. Excess DAPI was removed by washing with PBS three times. The coverslips were sealed with an anti-fluorescence quencher. Image acquisition was performed via an Mshot MF53 inverted microscope (MF53, Mshot, Guangzhou, China).

Annexin V Fluorescein Isothiocyanate/Propidium Iodide (FITC/PI)-apoptosis kit (40302ES50, YEASEN, Shanghai, China) was applied to detect the cell apoptosis by flow cytometry. Cells were seeded in the 6-well plate and incubated in the CO₂ incubator. When the cell density reached 70%, cells were added into FITC-dextran (1 mg/mL) and incubated for 2 h. Subsequently, cells were treated with the following procedures: PBS washing, trypsin digestion, and centrifugation after neutralizing with serum-containing medium. The supernatant was abandoned and cell pellets were resuspended in PBS. Finally, flow cytometry was performed using a CytoFLEX flow cytometer (CytoFLEX, Beckman, Brea, CA, USA).

Data analyses and plotting were performed using the GraphPad Prism 8.01 software (GraphPad Software Inc., San Diego, CA, USA). The data are presented as mean $\pm$ standard error. The t-test was used for the comparison between the two groups, while ANOVA was applied for comparisons among multiple groups. Post hoc tests were performed using Tukey’s method. Statistical significance was set at p 0.05.

Outcomes of TUNEL assay (Fig. 1A) showcased a notable increase in the rate of cell apoptosis within the hippocampal tissue of fetal rats exposed to 3% sevoflurane, when compared to the control. Results from qPCR and western blot (Fig. 1B,C) revealed that in 3% sevoflurane treatment group, the mRNA and protein expression of cleaved-caspase-3, Bax, and SIRT1 were substantially elevated (p 0.05, p 0.001), while the mRNA and protein expressions of mTOR were greatly reduced (p 0.01, p 0.001). All thoses findings implied that sevoflurane treatment facilitated an up-regulation of both apoptosis-related proteins, cleaved-caspase-3 and Bax, along with an increase in SIRT1 expression; concurrently, there was a down-regulation of mTOR expression. Immunofluorescence analysis further confirmed the up-regulation of SIRT1 expression (Fig. 1D). SIRT1 protein level was significantly increased in 3% sevoflurane treatment group, which was consistent with our qPCR and western blot results. These findings implied that sevoflurane treatment might participate in cell apoptosis in fetal rat hippocampal tissue by regulating the expression of these factors, potentially serving as a crucial factor contributing to cognitive dysfunction.

Fig. 1.

Inhalation of sevoflurane begets apoptosis of hippocampal tissue in fetal rats. (A) Fetal rat hippocampal tissuel apoptosis was assessed through Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis. Scale bars, 50 µm. (B) Real-time quantitative polymerase chain reaction (qPCR) analysis was conducted on fetal rat hippocampal tissue for cleaved-caspase-3, Bax, Sirtuin 1 (SIRT1), and mechanistic target of rapamycin (mTOR). (C) Western blot analyses of fetal rat hippocampal tissue for cleaved-caspase-3, Bax, SIRT1, and mTOR. (D) Immunofluorescence combined with 4’,6-diamidino-2-phenylindole (DAPI) staining was utilized to examine expression of SIRT1 in fetal rat hippocampal tissue. Scale bars, 50 µm. *p 0.05, **p 0.01, ***p 0.001 vs. control. n = 3.

In 3% sevoflurane treatment group, apoptotic levels of fetal rat hippocampal neurons were markedly higher than the control, as indicated by the flow cytometry results (Fig. 2A). This suggested that sevoflurane treatment could lead to an increase in neuronal apoptosis, potentially exerting adverse effects on cognitive functions. Immunofluorescence detection results (Fig. 2B) revealed an apparent elevation in SIRT1 protein expression in 3% sevoflurane treatment group. SIRT1 functions as an essential regulator of cellular autophagy, and its increased expression during sevoflurane therapy likely signifies the activation of autophagic processes. Further qPCR and WB analysis results (Fig. 2C,D) demonstrated a substantial increase (p 0.01, p 0.001) in the mRNA and protein expression of cleaved-caspase-3, Bax, and SIRT1 in hippocampal neurons of fetal rats exposed to sevoflurane. Conversely, mRNA and protein expressions of mTOR were found greatly reduced (p 0.05, p 0.001). Simultaneously, alterations in the expression of autophagy-related genes and proteins were observed, specifically the up-regulation of LC3 expression and down-regulation of p62 expression.

Fig. 2.

Promoting effect of sevoflurane on apoptosis and autophagy of fetal rat hippocampal neurons. (A) Changes in apoptosis of fetal rat hippocampal neuron cells after 3% sevoflurane treatment. (B) The expression of SIRT1 was detected by immunofluorescence. Scale bars, 50 µm. (C) qPCR analysis of cleaved-caspase-3, Bax, SIRT1, mTOR, p62, and LC3II in fetal rat hippocampal neurons. (D) Western blot analyses of cleaved-caspase-3, Bax, SIRT1, mTOR, p62, and microtubule-associated protein light chain 3Ⅱ (LC3II) in fetal rat hippocampal neurons. *p 0.05, **p 0.01, ***p 0.001 vs. control. n = 3. UL: Upper Left; UR: Upper Right; LL: Lower Left; LR: Lower Right.

In this section, we employed strategies of SIRT1 disruption and mTOR suppression to evaluate effects of autophagy on sevoflurane-induced apoptosis in fetal rat hippocampal neurons. Flow cytometric analysis revealed (Fig. 3A) that hippocampal neuron cell apoptosis decreased in 3% sevoflurane + SIRT1 inhibitor group compared to 3% sevoflurane group, suggesting that SIRT1 interference is likely to produce a protective effect against sevoflurane-induced neuronal apoptosis. In our mTOR inhibition experiment, rapamycin was employed to suppress mTOR activity; this approach resulted in a higher incidence of hippocampal neuron apoptosis in 3% sevoflurane + SIRT1 inhibitor + rapamycin group compared to 3% sevoflurane + SIRT1 inhibitor group (Fig. 3A). This observed increase implies that mTOR suppression potentially negates the neuroprotective impact of SIRT1 knockdown, thereby intensifying sevoflurane-induced neuronal apoptosis. Immunofluorescence detection (Fig. 3B) implied an apparent decrease in the expression of SIRT1 in both 3% sevoflurane + SIRT1 inhibitor and 3% sevoflurane + SIRT1 inhibitor + rapamycin groups. Moreover, qPCR and WB results (Fig. 4A,B) further supported these findings. In 3% sevoflurane + SIRT1 inhibitor group, expression of SIRT1 significantly decreased, along with a significant decrease in cleaved-caspase-3, Bax, and LC3II expression (p 0.01, p 0.001), while that of mTOR and p62 substantially increased (p 0.001). In 3% sevoflurane + SIRT1 inhibitor + rapamycin group, expression of cleaved-caspase-3, Bax, and LC3II significantly increased (p 0.01, p 0.001), while mTOR and p62 expression was significantly decreased (p 0.01, p 0.001). Our experimental outcomes have furnished compelling evidence that substantiates SIRT1’s integral function in modulating sevoflurane-induced apoptosis in hippocampal neurons, concurrently highlighting mTOR’s antagonistic regulatory influence within this biological cascade.

Fig. 3.

Autophagy regulates apoptosis of hippocampal neurons in fetal rats induced by sevoflurane inhalation anesthesia. (A) Changes in apoptosis of fetal rat hippocampal neuron cells after different treatment. (B) The expression of SIRT1 was detected by immunofluorescence after different treatment. Scale bars, 50 µm. Statistical significance compared to the control group: **p 0.01, ***p 0.001; compared to 3% sevoflurane group: #p 0.05, ###p 0.001; compared to 3% sevoflurane + SIRT1 inhibitor group: &&p 0.01. n = 3.

Fig. 4.

Neuroprotective effect of autophagy on fetal rat nerve cell apoptosis induced by sevoflurane inhalation anesthesia. (A) qPCR analysis of cleaved-caspase-3, Bax, SIRT1, mTOR, p62, and LC3II in fetal rat hippocampal neurons. (B) Western blot analysis of cleaved-caspase-3, Bax, SIRT1, mTOR, p62, and LC3II in fetal rat hippocampal neurons. Statistical significance compared to the control group: *p 0.05, **p 0.01, ***p 0.001; compared to 3% sevoflurane group: #p 0.05, ##p 0.01, ###p 0.001; compared to 3% sevoflurane + SIRT1 inhibitor group: &&p 0.01, &&&p 0.001. n = 3.