Introduction

General anesthesia is the most common practice for surgery or procedural pain relief in modern anesthesia (Brown et al. 2018). Epidemiological studies showed that general anesthesia in neonates was associated with potential neurocognitive impairment later in life (Presečki et al. 2010; Sprung et al. 2017). Moreover, increasing experimental data have shown that early exposure to general anesthetics may induce widespread neuroapoptosis, inhibit neurogenesis, and cause long-term neurocognitive deficits (Sall et al. 2009; Ramage et al. 2013a; Lee et al. 2014). Therefore, it is imperative to uncover the mechanisms of anesthesia-induced neural impairment and develop potential protective strategies.

Sevoflurane is a commonly used anesthetic in adults as well as in infants and young children (Yasuda et al. 1991). It has been reported that prolonged exposure to sevoflurane may cause neurocognitive impairment in the developing brain (Ling et al. 2017a; Tian et al. 2015). During brain development, neural stem cells (NSCs) are widely distributed in brain areas. They are destined to divide, proliferate, and undergo multi-lineage differentiation into neurons, astrocytes and oligodendrocytes (Kim 2010). NSCs are responsible for continuous brain development and repairing central and peripheral nervous system injuries after birth (Wang et al. 2017b; Zhang et al. 2016). However, emerging evidences indicated that sevoflurane had detrimental effects on NSCs (Nie et al. 2013; Qiu et al. 2015). Therefore, one of the important ways to prevent the neurocognitive impairment after anesthesia is to reduce sevoflurane-induced NSC injury.

Tetramethylpyrazine (TMP), one of the important biological active components extracted from rhizoma Chuanxiong (Kang et al. 2009), possesses broad activities, such as anti-oxidant, anti-fibrosis, anti-inflammatory, and anti-neoplastic (Kim et al. 2014; Wang et al. 2016; Wu et al. 2015; Zhao et al. 2017). Additionally, TMP has been found to be a potent neuroprotective compound. TMP alleviates neuronal apoptosis in spinal cord injury via the down-regulation of microRNA-214-3p (Fan and Wu 2017). TMP protects neurons and inhibits glioma by down regulating chemokine receptor CXCR4 expression (Chen et al. 2013b). Importantly, TMP could promote neurogenesis in adult rat brain after focal ischemia (Xiao et al. 2010), probably through the enhanced proliferation and differentiation of NSCs after injury (Tian et al. 2010). However, whether TMP could attenuate NSC injury induced by sevoflurane exposure was still unclear. Thus, the objective of this study was to investigate the effect of TMP on sevoflurane-induced NSC injury and the possible involvement of Akt/GSK-3β pathway in TMP-induced protection of NSCs.

Materials and methods

NSC culture

Rat primary NSC cultures were derived from Sprague-Dawley rat embryos (14–15 days). The forebrain portion was quickly removed, dissected and placed in ice-cold Hank’s solution (Gibco, Carlsbad, CA, USA). The cells were dissociated using mechanical agitation method with a fire-polished Pasteur pipette. Briefly, the tissues were cut into several pieces, blown into cell suspension using a pipette, and then filtered into a centrifuge tube through a 300 mesh strainer (Harms and Tansey 2013). After centrifugation (Thermo Fisher Scientific, Waltham, MA, USA), the cells (2 × 105 cells/well) were resuspended in free-serum DMEM/F12 medium (Gibco, Carlsbad, CA, USA) supplemented with 2% B-27 (Gibco, Carlsbad, CA, USA), 20 ng/ml epidermal growth factor (EGF; Gibco, Carlsbad, CA, USA), 20 ng/ml basic fibroblast growth factor (bFGF; Gibco, Carlsbad, CA, USA), and 1% penicillin and streptomycin (Wang et al. 2018). The medium was changed every 2 days. All experiments were performed three times in duplicate.

Sevoflurane exposure

For the exposure to sevoflurane, NSCs were exposed to 3% sevoflurane for 6 h in a gas mixture (21% O2, 5% CO2 and 74% N2) at 37℃ in a tightly sealed plastic chamber. For cell viability assay, NSCs were treated with 0, 50, 100, 150 or 250 µM TMP (Sigma-Aldrich Inc. St. Louis, MO, USA) for 2 h, followed by exposure to 3% sevoflurane. For cell proliferation and apoptosis assays, NSCs were treated with 150 µM TMP for 2 h (Chen et al. 2018, 2019), followed by exposure to 3% sevoflurane. Additionally, NSCs were pretreated with 10 µM LY294002 (Sigma-Aldrich Inc. St. Louis, MO, USA) concurrently with TMP for 2 h to inhibit the activation of Akt (Wang et al. 2018). Data were collected from 3 independent experiments.

Lactate dehydrogenase (LDH) release assay

After treated with 0, 50, 100, 150, 250 and 500 µM TMP or equivalent volume of phosphate buffer saline (PBS) for 24 h, NSCs were collected and centrifuged at 400 g for 5 min. The LDH levels in the supernatant were quantified using an LDH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance at 450 nm was measured using a microplate reader (Bio-Tek, Winooski, VT, USA).

Cell viability assay

NSCs were treated with different concentrations of TMP (0, 50, 100, 150 or 250 µM) or equivalent volume of PBS for 2 h prior to sevoflurane exposure. After that, cell viability of NSCs was determined by the cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay. In brief, the NSCs were added with 10 µl CCK-8 solutions and incubated for 4 h. The absorbance at 450 nm wavelength was measured using a microplate reader (Bio-Tek, Winooski, VT, USA).

5-bromo-2’-deoxyuridine (BrdU) labeling

The NSCs were seeded onto the cover glass coated with 100 µg/ml poly-L-lysine (PLL). The cultured cells were treated with 150 µM TMP or equivalent volume of PBS for 2 h, and then exposed to sevoflurane for 6 h. After that, 10 µM BrdU was added and the NSCs were incubated for another 4 h. Cells were then fixed with 4% paraformaldehyde for 50 min before treatment with 2 M HCl containing 1% Triton X-100. Cells were incubated with the rat anti-BrdU (1:200; Abcam, UK) antibody and FITC-conjugated goat anti-rat secondary antibody (1:300; Abcam, UK), followed by propidium iodide (PI) dye (Sigma-Aldrich Inc. St. Louis, MO, USA) staining for 8 min. The stained cells were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

After different treatments, the apoptosis of NSCs was determined by TUNEL assay using In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany). Briefly, the fixed cells were incubated with TUNEL reaction mixture for 1 h, and then stained with DAPI (Sigma-Aldrich Inc. St. Louis, MO, USA) for 8 min. The apoptotic cells were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

Immunofluorescence staining

For the identification of NSCs, cells were plated on PLL-coated coverslips, fixed in 4% paraformaldehyde for 20 min at room temperature, treated with 2% TritonX-100 for 30 min, and then incubated with the primary antibodies: mouse anti-nestin antibody (1: 200, Millipore, Germany), rat anti-BrdU antibody (1: 200, Abcam, UK), rabbit anti-β-tubulin III antibody (1: 200, Abcam, UK) and rabbit anti-glial fibrillary acidic protein antibody (GFAP; 1: 200, Cell Signaling Technology, USA). After washing for 3 times with 0.1 M PBS, the cells were incubated with Cy3-conjugated goat anti-mouse (1: 500, Abcam, UK), Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-rat secondary antibodies (1:1500, Cell Signaling Technology, USA) for 2 h at room temperature. Then the sections were stained with DAPI for 8 min. Cells were examined by fluorescence microscopy (Olympus, Tokyo, Japan).

Western blotting

Cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors (Sigma-Aldrich Inc. St. Louis, MO, USA). The lysates were centrifuged at 14,000 rpm for 15 min at 4 °C and then the determination of protein concentration was performed with a bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich Inc. St. Louis, MO, USA). Equal amounts of the proteins were used for western blotting analysis. Proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then the resolved bands were transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with blocking buffer (5% BSA) for 1 h at room temperature and then incubated with the following primary antibodies: rabbit anti-cleaved caspase-3 (1: 1500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-p-Akt [Ser473] (1: 1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Akt (1: 1500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-p-GSK-3β [Ser9] (1: 1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-GSK-3β (1: 2500, Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-β-actin (1: 1500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. After incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h, the signals were detected using enhanced chemiluminescence (ECL) substrates.

Statistical analysis

Data were expressed as the mean ± SEM of the results from three replicates. Differences among the multiple groups were analysed by GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) with one-way analysis of variance (ANOVA) followed by the post-hoc Duncan’s test. Statistical significance was set at P ˂ 0.05.

Results

Identification of NSCs

We determined the characteristics of cultured cell. As indicated in Fig. 1 A, the majority of cells expressed the NSC marker nestin in adherent culture 3 days after seeding. To determine the differentiating potential, NSCs were cultured in differentiation medium supplemented with 1% FBS and lacking bFGF and EGF for 7 days. The staining results showed that the cultured cells expressed the specific maker for neuron β-tubulin Ш (Fig. 1B) and the astrocytic marker GFAP (Fig. 1 C). These findings indicated that the primary cultured cells were NSCs, and this culture system was used to conduct subsequent experiments.

Fig. 1
figure 1

Identification of NSCs. (A) Representative immunofluorescence images of nestin expression in adherent NSCs after 3 d culture. Nestin was stained red in adherent NSCs. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, blue). (B and C) Representative immunofluorescence images of NSCs differentiated into neurons and astrocytes. NSCs were cultured in differentiation medium for 7 days. β-tubulin III (green), GFPA (green), DAPI (blue). Scale bars = 100 μm

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TMP treatment increased the proliferation of NSCs after sevoflurane exposure

Firstly, NSCs were treated with 0, 50, 100, 150, 250 or 500 µM TMP for 24 h to examine the cytotoxicity of TMP. Data showed that the LDH release from NSCs treated with 500 µM TMP were markedly higher than that in control NSCs, indicating that the concentration of 500 µM TMP might have potential cytotoxicity on NSCs (P < 0.05, Fig. 2 A).

Fig. 2
figure 2

TMP increased the cell viability and proliferation of NSCs exposed to sevoflurane. (A) Cytotoxicity effect of TMP on NSCs measured by LDH release assay after TMP treatment for 24 h. (B) Cell viability of NSCs treated with TMP for 2 h following exposure to 3% sevoflurane. (C) Proliferation of NSCs by BrdU labeling after TMP treatment and sevoflurane exposure. PI, propidium iodide. (D) Quantification analysis of cell proliferation. *P < 0.05 vs. control group; #P < 0.05 vs. sevoflurane group. n = 5 for each group

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Next, we explored the protective effect of TMP on NSCs exposed to sevoflurane. CCK-8 assay demonstrated that cell viability of NSCs was markedly decreased after exposure to 3% sevoflurane, while pretreatment with TMP improved the cell viability of sevoflurane-exposed NSCs (P < 0.05, Fig. 2B). The concentration of 150 µM TMP was selected for further experiments, because it showed the most prominent protection of NSCs’ viability. BrdU labeling was performed to evaluate the cell proliferation of NSCs. The results showed that sevoflurane significantly decreased the proliferation of NSCs, however, pretreatment with 150 µM TMP reversed the inhibitory effect of sevoflurane on NSCs proliferation (P < 0.05, Fig. 2 C-2D).

TMP suppressed NSCs apoptosis induced by sevoflurane

In order to investigate the effect of TMP on NSC apoptosis, the expression level of cleaved caspase-3 was detected by western blotting. Compared to the control, cleaved caspase-3 expression level was markedly increased in NSCs exposed to sevoflurane (P < 0.05). However, pretreatment with 150 µM TMP prevented the increase of cleaved caspase-3 (P < 0.05, Fig. 3 A and 3B). Besides, cellular apoptosis was also measured by TUNEL assay. As indicated in Fig. 3 C-3D, the number of TUNEL-positive cells was obviously increased after sevoflurane exposure (P < 0.05), while pretreatment with TMP significantly reduced the number of TUNEL-positive cells (P < 0.05).

Fig. 3
figure 3

TMP suppressed sevoflurane-induced NSCs apoptosis. (A, B) The protein expression of cleaved caspase-3 by western blotting assay. (C, D) Cellular apoptosis by TUNEL assay. *P < 0.05 vs. control group; #P < 0.05 vs. sevoflurane group. n = 5 for each group

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TMP increased the phosphorylation of Akt and GSK-3β in sevoflurane-exposed NSCs

It has been demonstrated that Akt/GSK-3β pathway was involved in the proliferation and apoptosis of NSCs (Sabelstrm et al. 2014). We thus tested whether it also played an important role in sevoflurane-injured NSCs and the effect of TMP on it. Firstly, we observed that the expressions of p-Akt and p-GSK-3β were markedly decreased in NSCs exposed to sevoflurane as indicated in Fig. 4 (Control vs. Sevoflurane, P < 0.05). However, the decreased levels of p-Akt and p-GSK-3β were partly reversed by TMP pretreatment (Sevoflurane vs. Sevoflurane + TMP, P < 0.05). Then, we added LY294002, an inhibitor of PI3K, to block the Akt/GSK-3β pathway in NSCs treated with sevoflurane plus TMP. The results showed that the effect of TMP on Akt/GSK-3β pathway in NSCs exposed to sevoflurane was abolished by LY294002 (Sevoflurane + TMP vs. Sevoflurane + TMP + LY294002, P < 0.05). Furthermore, we found that the promotion of TMP on the viability of sevoflurane-exposed NSCs was markedly hampered by LY294002 (P < 0.05, Fig. 5).

Fig. 4
figure 4

TMP regulated Akt/GSK-3β pathway in sevoflurane-exposed NSCs. (A, C) The expressions of p-Akt and Akt by western blot. (B, D) The expressions of p-GSK-3β and GSK-3β by western blot. *P < 0.05 vs. control group; #P < 0.05 vs. sevoflurane group, &P < 0.05 vs. sevoflurane + TMP group. n = 4 for each group

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Fig. 5
figure 5

LY294002 reversed the protective effect of TMP on cell viability in sevoflurane-exposed NSCs. NSCs were pretreated with 10 µM LY294002 and 150 µM TMP for 2 h and then exposed to 3% sevoflurane. Cell viability was detected by CCK-8 assay. *P < 0.05 vs. control group; #P < 0.05 vs. sevoflurane group, &P < 0.05 vs. sevoflurane + TMP group. n = 4 for each group

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Discussion

The present study illustrated that sevoflurane exposure decreased the viability, reduced the proliferation, and increased the apoptosis of NSCs in vitro, while TMP treatment attenuated these toxic effects of sevoflurane. In addition, TMP partly reversed the reduced phosphorylation of both Akt and GSK-3β after sevoflurane exposure. However, the increased phosphorylation levels of Akt and GSK-3β and the improved NSCs viability induced by TMP after sevoflurane exposure were abolished by the PI3K inhibitor LY294002. These results suggested that TMP could protect NSCs from sevoflurane-induced injury via the Akt/GSK-3β pathway.

NSCs remain in a quiescent state under normal physiological conditions in the central nervous system, while under injury conditions, NSCs can be stimulated to proliferate and differentiate into neurons, astrocytes and oligodendrocytes (Dulken et al. 2017). It has been demonstrated that NSCs are crucial for the brain development and brain tissue repair (Stenudd et al. 2015). Previous studies have shown that sevoflurane exposure decreases the viability, increases the apoptosis and causes cell cycle arrest of NSCs, ultimately leading to NSC degeneration and neurogenesis disturbances (Xie et al. 2013). Sevoflurane also elevated mRNA levels of antioxidant enzymes and cleaved caspase-3 expression in cultured NSCs (Zhou et al. 2017). Moreover, prolonged sevoflurane exposure could also decrease the self-renewal and differentiation capacities of hippocampal NSCs, resulting in cognitive deficits (Nie et al. 2013). These findings suggested that the detrimental effects of sevoflurane on neurogenesis and cognitive dysfunction could be the causation to its neurodevelopmental toxicity. Therefore, preventing the sevoflurane-induced NSC injury is important for the prevention of the neurocognitive impairment following sevoflurane anesthesia in children. In the present study, we showed that sevoflurane decreased the viability, inhibited the proliferation, and increased the apoptosis of NSCs in vitro.

TMP is a natural bioactive component from Ligusticum wallichii Franchat, which is called Chuanxiong in Chinese (Guo et al. 2016). It has been used in clinical treatment of cardiovascular diseases and neurovascular disorders (Zhao et al. 2016). In recent years, its neuroprotective effects have been widely studied and remarkable progress has been made, which suggest a promising therapeutic potential of TMP in the treatment of neural damage and neurodegenerative diseases (Michel et al. 2016; Yang et al. 2017). As for the underlying mechanisms, it was discovered that TMP could promote the proliferation of NSCs and also the differentiation of NSCs into neurons under hypoxic condition (Tian et al. 2010). In the present study, we investigated the role of TMP in sevoflurane-induced NSC injury in vitro. The results showed that TMP elevated the viability and proliferation, and suppressed cellular apoptosis in sevoflurane-exposed NSCs, indicating that TMP might have a potential protective effect on sevoflurane-damaged NSCs. This finding may support the use of TMP as a new neuroprotective drug in clinical anesthesia with sevoflurane.

It has been established that multiple abilities are responsible for the protective effects of TMP against neural damage including anti-oxidant, anti-apoptosis and Ca2+ antagonist effects (Li et al. 2010; Liang et al. 2011; Pang et al. 1996). PI3K/Akt/GSK-3β signaling pathway plays as an important role in neural survival, proliferation and differentiation, which has been found to be a therapeutic target for neurodegenerative disease (Xie et al. 2014; Yy et al. 2019; Zheng et al. 2017). PI3K/Akt is an important cell signaling pathway which regulates cell proliferation, metabolism, differentiation and survival. Under conditions like insulin, the pathway is stimulated and responsible for cell survival (Jafari et al. 2019). GSK-3β is a major direct target molecule that performs most functions of Akt by acting on a variety of signaling proteins and transcription factors to regulate cell proliferation, differentiation, and apoptosis (Jope et al. 2007). Phosphorylation of Akt at Ser473 leads to its activation. Activated Akt then inhibits GSK-3β by its Ser9 phosphorylation, which is vital to cell survival pathway (Lei et al. 2008). Likewise, PI3K/Akt/GSK-3β signaling pathway also participated in NSC proliferation and neuronal differentiation (Keishi et al. 2018; Tiwari et al. 2015). Notably, some recent studies proved that TMP played a protective role against acute myocardial ischemia injury by regulating the PI3K/Akt/GSK-3β signaling pathway (Qing et al., 2019; Yang et al. 2019). Our results showed that TMP pretreatment increased the levels of p-Akt and p-GSK3β in NSCs after exposure to sevoflurane, same as the findings in myocardial ischemia, suggesting that TMP may has a universal effect on Akt/GSK-3β pathway in different cell types. However, when treated with the PI3K inhibitor LY294002, the protective effect of TMP on sevoflurane-injured NSCs was reversed, indicating that TMP could protect NSCs from sevoflurane-induced injury via the Akt/GSK-3β pathway. In the study, we observed that the Ser9 phosphorylation of GSK-3β, namely its inhibition form could be responsible for the potent proliferation and differentiation abilities of NSCs rendered by TMP. Actually, inhibition of GSK-3β is a key switcher for NSC survival and proliferation. It was reported that inhibition of GSK-3β enhanced the proliferation and differentiation of NSCs in the subventricular zone in experimental Parkinson’s disease (Singh et al. 2018). Application with GSK-3β inhibitor lithium chloride (LiCl) reversed N,N,N’,N’-tetrakis-(2-pyridylmethy) ethylenediamine (TPEN)-induced downregulation of β-catenin and impairment of NSC proliferation (Zhao et al. 2015). These indicated that inhibition of GSK-3β might be an important mechanism for TMP-preserved neurogenesis ability of NSCs after sevoflurane exposure, which warrant further verification by manipulation of GSK-3β activity. Besides, the exact molecular mechanisms of TMP on the Akt/GSK-3β pathway need to be further studied.

Conclusions

Our study proved that TMP protected NSCs from sevoflurane exposure-induced injury through Akt/GSK-3β pathway. These findings supported the use of TMP as a potential protective agent for the prevention of the neurotoxicity induced by sevoflurane anesthesia. Nevertheless, there are still limitations in this study. First, we did not perform the in vivo study to observe the effect of TMP pretreatment on the developing brain. Second, as for the mechanisms of TMP’s neuroprotection, more experiments should be conducted in future. Third, the clinical concentration of TMP will require further experiments.