The protective effects of miR-128-3p on sevoflurane-induced progressive neurotoxicity in rats by targeting NOVA1

Abstract

MiR-128 is highly expressed in the central nervous system and may regulate the directional differentiation of bone marrow stromal stem cells into nerve cells. However, its role and mechanism in sevoflurane-induced progressive neurotoxicity in rats are rarely reported. Therefore, this study aims to explore the protection of miR-128-3p on sevoflurane-induced neurotoxicity. Hippocampal neurons were isolated and sevoflurane was used to treat the cells. Cell counting kit-8 (CCK-8) was used to detect cell viability. Immunofluorescence was used to detect enrichment of GFAP or βIII tubulin to identify nerve cells. Dual luciferase assay was used to identify the targeted binding relationship between miR-128-3p and NOVA1. The effect of miR-128-3p and sevoflurane on cells regarding apoptosis was detected by flow cytometry. The expression of apoptosis-related protein and oxidative stress-related proteins were detected by western blot. Enzyme-linked immuno-sorbent assay (ELISA) was used to measure inflammatory cytokine levels. Hippocampal neurons’ cell viability was significantly decreased by treatment with sevoflurane. MiR-128-3p was down-regulated after sevoflurane treatment in cells. Overexpressed miR-128-3p partially reversed the role of sevoflurane treatment in triggering cell apoptosis, enhancing the expression of Bax and cleaved caspase-3 and inhibiting Bcl-2 expression obviously. Overexpressed miR-128-3p partially reversed the role of sevoflurane treatment in promoting the expression of NOX1and NOX4, and inflammatory cytokine levels by targeting with NOVA1. MiR-128-3p might be a potential therapeutic target for the prevention or treatment of sevoflurane-induced neurotoxicity by targeting with NOVA1.

INTRODUCTION

In recent years, with the rapid development of medical technology, the proportion of general anesthesia has gradually increased (Gale et al., 1982). Sevoflurane has been widely used in clinical practice due to its advantages such as rapid induction, quick recovery and little impact on liver and kidney function (Han et al., 2015). Studies have found that the injection of anesthetic drugs can induce the apoptosis of central nervous cells, which is a risk factor for impaired cognitive function (Dabbagh and Rajaei, 2013). At present, studies have described the toxic effects of sevoflurane general anesthesia on cerebral nerves and studies have found that sevoflurane anesthesia damages the cognitive function of the body by activating inflammation and promoting neuronal cell apoptosis (Cui et al., 2018). Therefore, it may be an important measure to reduce the neurocognitive impairment induced by sevoflurane by exploring the treatment methods to protect against neuronal apoptosis and reduce the inflammatory response of the body.

MicroRNAs (miRNAs) are non-coding small RNAs with a length of 19–23 nucleotides and they were found to modulate various genes or signaling pathways at their post-transcriptional levels (Fan et al., 2017). In recent years, a study found that microRNAs’ role in brain development and differentiation of neurons and synaptic connections and dendritic formation process play an important role in neurological diseases (Kawahara, 2008). Meanwhile, other studies found that microRNAs are involved in Alzheimer's disease, schizophrenia and other processes of cognitive function disorder, and the abnormal expression not only exists in the diseased brain regions but also exists in peripheral body fluids such as plasma and cerebrospinal fluid (Soreq, 2014; Sudesh et al., 2018). Therefore, these findings make it possible to use miRNAs as markers for early detection and evaluation of cognitive dysfunction. In addition, miRNAs can regulate gene transcription, protein translation and other life processes, which further reveals the pathogenesis of cognitive dysfunction and explores new therapeutic approaches (Xiang et al., 2017).

Studies have found that miR-128 is highly expressed in the central nervous system, which may affect the directional differentiation of bone marrow stromal stem cells into nerve cells by regulating Wnt3a (Wu et al., 2014). However, its role and mechanism in sevoflurane-induced progressive neurotoxicity are rarely reported. In this study, miR-128-3p was found to play a protective role in sevoflurane-induced progressive neurotoxicity in rats by upregulation.

MATERIALS AND METHODS

Preparation of hippocampal neurons

A total of 120 Lewis rats (7 days old, ILAS-00) were purchased from the Institute of Experimental Animal Medicine, Chinese Academy of Medical Sciences (Beijing, China). The rats were housed at 25°C under a 12 hr light/12 hr dark cycle conditions and allowed to acclimate for 18 days before sacrifice. The rats were sacrificed through decapitation under 70 mg/kg sodium pentobarbital (P5178, Sigma-Aldrich, St. Louis, MO, USA) anesthesia. Then the bodies of the rats were soaked in 75% alcohol (diluted from absolute ethanol, A500737, Sangon, Shanghai, China) for 2 min. After that, the epicranium was cut, the skull was separated, both sides of the brain were exposed and both hemispheres were placed in an ice-cooled dish. D-Hank’s solution (QN3583, Baiaolaibo Technology Co., Ltd., Beijing, China) was added to the dish. After the separation of cortex, the hippocampus tissues were found and washed with D-Hank’s solution 5 times. Then the hippocampus tissues were cut into small pieces. The tissues were centrifuged and added with equal volume of trypsin (T2600000, Sigma-Aldrich) for tissue digestion for 5 times. Dulbecco’s modified eagle medium (DMEM, D0822, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, 10099, Gibco, Waltham, MA, USA) was added to terminate the digestion and then the solution was centrifuged and the single cell mass was resuspended and separated into 10 mmol/L poly-D-lysine (P6407, Sigma-Aldrich) coated cultural flasks (0030710118, Eppendorf, Hamburg, Germany). After overnight incubation in 37°C, 5% CO₂ incubator (3110, Thermo Fisher Scientific, Waltham, MA, USA), the medium was replaced by neurobasal medium (A3582901, Gibco) containing 2% B27 (A3582801, Gibco) and 1% N2 (17502001, Gibco).

Isolation and purification of hippocampal neurons

The culture flask was removed from the incubator and placed on a table concentrator (TS-8, Haimen Kylin-Bell Lab Instruments Co., Ltd., Jiangsu, China, http://www.ql-lab.com/products_detail/productId=38.html) to vibrate for 5 min at a speed of 60 rotations per minute. Most glial cells were eluted and floated in the culture medium. After washing with the culture medium, the culture was continued. The cellular morphology of hippocampus neurons at 0 days (d), 3 d and 7 d were observed at inverted microscope (CKX41, Olympus, Tokyo, Japan).

Immunofluorescence

Hippocampal neurons were washed three times with PBS (02-024-1A, Biological Industry, Cromwell, CT, USA). βIII tubulin conjugated with Alexa Fluor 488 antibody (ab195879, mouse monoclonal, 1:100, Abcam, Cambridge, UK) and GFAP conjugated with Alexa Fluor 488 (ab194324, rabbit monoclonal, 1:50, Abcam) antibody were added to the cells respectively, spend the night at 4°C heat preservation box incubation. After PBS washing for three times, DAPI solution (ab228549, Abcam) was added and the cells were sent to incubation for 3 min. Finally, fluorescent determination was carried out using fluorescence microscope (Delta Optical IB-100, Delta Optical, Warsaw, Poland).

Sevoflurane-treated hippocampal neurons

Hippocampal neurons were divided into four groups: 1% sevoflurane (CAS no: 28523-86-6, molecular formula: C₄H₃F₇O, Purity: 98%, Shanghai Acmec Biochemical Technology Co., Ltd., https://www.acmec-e.com/cas/28523-86-6/, Shanghai, China) treated group (the cells were treated with 1% sevoflurane for 6 hr at 37°C for neurotoxicity induction), 2% sevoflurane-treated group (the cells were treated with 2% sevoflurane for 6 hr at 37°C), 4% sevoflurane-treated group (the cells were treated with 4% sevoflurane for 6 hr at 37°C) and blank group (the cells were cultured under normal conditions for 6 hr).

Cell viability detection

The 1 × 10⁵/mL cells were inoculated into the 96-well plate (713011, Wuxi Nest Biotechnology Co., Ltd., Jiangsu, China, https://www.cell-nest.com/page94?product_id=162), 10 μL of cell counting kit-8 (CCK-8) (96992, Sigma-Aldrich) solution was added to each well, and the cells were incubated for 4 hr in CO₂ incubator. The absorbance at 450 nm was determined by enzyme microscopy (Multiskan SkyHigh, ThermoFisher, Waltham, MA, USA). The process was repeated in triplicate and the average value was calculated.

Transfection

Hippocampal neurons treated with sevoflurane, since 2% sevoflurane and 4% sevoflurane have similar cytotoxic effects on cells, 2% sevoflurane was used for subsequent experimental studies. For transfection assay, firstly, cell culture: the cells were first digested and thoroughly mixed, and the cells (1 × 10⁶/mL) were seeded into the 96-well plate and then evenly distributed in an orifice plate. The next day, the cells were transfected after being 80–90% confluence. Secondly, transdye preparation: the 20 pmol of the miR-128-3p mimic (M, sense: 5’-UCACAGUGAACCGGUCUCUUU-3’) and mimic control (MC), and NOVA1 overexpression vector (NOVA1 group) were synthesized from Sangon Biotech Co., Ltd. (Shanghai, China) through inserting the whole PCR products of NOVA1 sequence into pcDNA3.1 vector. Then the PCR product and pcDNA3.1 plasmid were digested using BamH I and Xho I enzyme (1010A, 1094A, Takara Biotechnology, Dalian, China) and then the digested products were mixed and ligated. The empty vector was used as negative control (NC group). The miR-128-3p mimic or mimic control or NOVA1 overexpression plasmid were respectively dissolved in 50 μL of neurobasal medium containing B27 and N2 and mixed as the transfected group A. The 50 μL neurobasal medium containing B27 and N2 was used to dissolve the 1 μL Lipofectamine 2000 (11668027, Invitrogen, Carlsbad, CA, USA). Then the mixture of medium and Lipofectamine 2000 was set aside for 5 min at room temperature and then mixed with the transfected group A to be set as the transfection group B. Finally, the transfection group B was added into the corresponding hole of the 96-well plate, and then the cell culture plate was placed in a CO₂ incubator for further culture. Afterwards, the medium was changed 24 hr after transfection, and then the cells were collected after 72 hr of culturing.

Double luciferase assay

Dual Luciferase Reporter Assay System (E1910, Promega, Madison, WI, USA) was utilized to verify the targeting relation between NOVA1 and miR-128-3p. Sequences of NOVA1-Wild Type (WT) (5’-GTGAATGTAGATTTTACTGTGAA-3’) and NOVA1-Mutant type (MUT) (5’- GTGAATGTAGATTTTTGACTGAA-3’) were cloned onto pMirGLO luciferase vectors (50 ng, E1330, Promega). The cells were co-transfected with the pMirGLO-NOVA1-WT or pMirGLO-NOVA1-MUT and miR-128-3p mimic or miR-128-3p MC as needed by Lipofectamine 2000 transfection reagent for 24 hr. After co-transfection, the cells were lysed by diluted Lysis Buffer (50 μL, 16189, ThermoFisher) and added with Luciferase Assay Reagent II (100 μL). The reaction intensity of firefly luciferase, which was normalized to that of renilla luciferase was measured by a luminometer (GloMax 20/20, E5311, Promega).

Quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA of cells was extracted using Trizol reagent (15596018, Thermo Fisher Scientific), and the Nanodrop (Thermo Scientific, San Diego, CA, USA) was used to measure the concentration of RNA, and then the RNA concentration was diluted to 500 ng/μL. Total RNA (1 µg) was converted into cDNA using a RevertAid first-strand cDNA synthesis System (K1621, Invitrogen). The genome DNA was erased by genome DNA wiper kit in the procedure of cDNA synthesis. The mRNA expression levels were determined by SYBR-Green PCR Master Mix (4309155, Thermo Fisher Scientific) in the 7500 Real-Time PCR system (Thermo Fisher Scientific). The following components were combined in a 10 μL solution: 4 μL cDNA, 5 μL SYBR, 1 μL Primer. The PCR cycle was as follows: pretreatment at 95°C for 1 min for pre-denaturation, at 95°C for 30 sec, at 58°C for 20 sec, at 70°C for 20 sec (three steps all in 40 cycles) for amplification. The expression levels of RT-PCR products were determined by the 2^-ΔΔCT method (Livak and Schmittgen, 2001). All primer sequences are listed in Table 1.

Table 1. Primers used in real-time PCR analysis.

Gene	Primer sequence
miR-128-3p	Forward: 5'- GGTC ACAGTGAACCGGTC-3' Reverse: 5'- GTGCAGGGTCC GAGGT-3'
NOVA1	Forward: 5'- GGGTTCCCATAGACCTGGAC-3' Reverse: 5'- CGCTCAGTAGTACCTGGGTAA-3'
β-actin	Forward: 5′-GAGCCTCGCCTTTGCCGATCC-3′ Reverse: 5′-CGATGCCGTGCTCGATGGGG-3′
U6	Forward: 5′-TGACTTCCAAGTACCATCGCCA-3′ Reverse: 5′-TTGTAGAGGTAGGTGTGCAGCAT-3′

Abbreviations: miR, microRNA

Cell apoptosis

After 24 hr of transfection, the cells (1 × 10⁶/mL) were resuspended in a 1 × Annexin binding buffer, 5 μL of fluorescein isothiocyanate (FITC) Annexin V (C1062S, Beyotime Biotechnology, China) and 1 μL of 100 μg/mL Propidium Iodide solution and 300 μL 1 × Annexin Binding Buffer were added to the cell suspension for 15 min after conducting the reaction at room temperature. Finally, the stained cells were analyzed by flow cytometry (version 10.0, FlowJo, FACS CaliburTM, BD, Franklin Lakes, NJ, USA).

Western blotting

The total protein of cells was lysed by a RIPA buffer (P0013B, Beyotime, Shanghai, China). Next, the protein was boiled for 5 min at 100°C for protein denaturation, which was separated on the 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, P0012A, Beyotime) then moved to polyvinylidene fluoride (PVDF) membranes (IPSN07852, Millipore, Burlington, MD, USA). Following transfer, the membranes were sealed with 5% non-fat milk at room temperature for 1 hr. The membranes were then incubated with the primary anti-NOVA1 (rabbit, 1:2000, ab183024, 52 kDa, Abcam), anti-Cleaved-cysteinyl aspartate specific proteinase-3 (anti-C caspase-3, rabbit, 1:100, ab2302, 17 kDa, Abcam), anti-B-cell lymphoma-2 (anti-Bcl-2, rabbit, 1:1000, ab194583, 26 kDa, Abcam), anti-Bcl-2 associated x protein (anti-Bax, rabbit, 1:2000, ab32503, 21 kDa, Abcam), NADPH Oxidase (NOX) 1 (rabbit, 1:500, ab131088, 65 kDa, Abcam), NOX4 (rabbit, 1:1000, ab133303, 67 kDa, Abcam), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, mouse, 1:2000, ab8245, 36 kDa, Abcam) at 4°C overnight. After the primary antibody was added for incubation, the membrane was washed with TBST three times, and then incubated with secondary antibody (SA00001-1, SA00001-2, 1:5000, Proteintech Group, Inc., Rosemont, IL, USA) for 2 hr. Protein bands were detected using ECL Western blotting kit (70-P1421, MULTI SCIENCES, Hangzhou, China) and scanned by super sensitive multifunctional imager (Image J, version 4.7, National Institutes of Health, Bethesda, MD, USA).

Detection of levels of inflammatory factors

After 24 hr of transfection, interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α concentration in the concentrated hippocampal neurons supernatants was measured by IL-6 enzyme linked immuno-sorbent assay (ELISA) kit (RAB0311, Sigma-Aldrich) and IL-1β ELISA kit (SEKR-0002, Solarbio, Beijing, China), and TNF-α ELISA kit (RAB0480, Sigma-Aldrich) respectively. Briefly, the biotin antibody drops (100 μL) were added to each reaction hole, the reaction hole was sealed by plate sealing tape and incubated in a 37°C incubator for 60 min, and then washed with 2 mL PBS for five times. The enzyme binding working fluid (100 μL) was added to each reaction hole, the reaction hole was sealed by plate sealing tape and incubated in a 37°C incubator for 30 min, and then washed with 2 mL PBS for five times. Next 100 μL substrate chromogenic reagent was added to the reaction hole and incubated in a 37°C incubator for 30 min. Finally, 100 μL the termination solution was added into the reaction hole. After gently shaking the culture suspension, the absorbance values of each hole were measured and recorded at 450 nm by SpectraMax 190 Microplate Reader (Molecular Devices, Shanghai, China) immediately. The standard curve was drawn according to the absorbance values measured by the standard substances of different concentrations and the corresponding IL-6, IL-1β and TNF-α concentration was detected on the standard curve.

Statistical analysis

Prism 6 (version 6.01, GraphPad Software, Inc., San Diego, CA, USA) was used for data analysis. The results in our study are shown as mean ± standard deviation (SD) in triplet, and t test was used to compare the differences in the mean between the continuous variables. Differences between multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc. P < 0.05 was considered to be statistically significant.

RESULTS

Morphological observation and identification of hippocampal neurons

The cultured primary hippocampal neurons were observed by inverted microscope. At the beginning of inoculation, the cells were round, small in size, with clear cytoplasm, and dispersed evenly in suspension state. After 3 days of culture, the cells were showed typical morphological features of neurons, which were plump and spindle shaped, a few showed irregular shape, and the cytoplasm was abundant. Neurons were clustered into smaller clumps, synapses were more pronounced, and synapses were connected to each other in a network. After 7 days of culture, the volume of neuron cells were further increased, which could be seen that the cells were close to each other and migrated, some regions formed small clusters of neurons with interwoven synapses, and the neural fiber network was denser (Fig. 1A). Immunofluorescence experiments showed that isolated cultured cells expressed immunofluorescence specific neuronal protein βIII tubulin, but didn’t express glial-specific protein GFAP, indicating that the isolated cells were identified as hippocampal neurons (Fig. 1B).

Fig. 1

Morphological observation and identification of hippocampal neurons (A) The morphological characteristics of hippocampal neurons were observed by inverted microscope. (B) Immunofluorescence staining of hippocampal neurons was observed by fluorescence microscopy.

Toxicity of sevoflurane on hippocampal neurons and the role of miR-128-3p in sevoflurane-induced hippocampal neuron injury

The cells were treated with 1%, 2% and 4% sevoflurane for 6 hr at 37°C with 5% CO₂. In order to explore the toxicity of sevoflurane on cells, we used CCK-8 to detect the activity of cells, and the results showed that the cell viability treated with 1% sevoflurane was lower than that of the Blank group (Fig. 2A, P < 0.05), while the cell viability treated with 2% and 4% sevoflurane was more significantly lower than that of the Blank group (Fig. 2A, P < 0.001). We further investigated the expression of miR-128-3p in cells treated with 2% sevoflurane, and the results showed that the expression of miR-128-3p in cells treated with sevoflurane was significantly lower than that in the Blank group (Fig. 2B, P < 0.001), but obviously increased by treatment with miR-128-3p mimic compared with control group (Fig. 2C, P < 0.001).

Fig. 2

Toxicity of sevoflurane on hippocampal neurons and the role of miR-128-3p in sevoflurane-induced hippocampal neuron injury. (A) The viability of hippocampal neurons treated with sevoflurane (1%, 2% and 4%) was detected by CCK-8 (n = 3, P** < 0.05, P** < 0.001, vs. Blank). (B) QRT-PCR was used to detect the expression of miR-128-3p in hippocampal neurons (n = 3, P** < 0.001, vs. Blank). (C) After mimic was transfected into cells, qRT-PCR was used to detect the expression of miR-128-3p in hippocampal neurons (n = 3, P** < 0.001, vs. control and mimic control). (D–E) Cell apoptosis was detected by flow cytometry (n = 3, P** < 0.001, vs. Blank; P^{^^} < 0.001, vs. control and mimic control). (F–G) Western blotting was used to detect the expression of cleaved-caspase-3, Bcl-2, Bax, NOX1 and NOX4 (n = 3, P** < 0.001, vs. Blank; P^{^^} < 0.001, vs. control and mimic control). (H) ELISA was used to detect the expression levels of IL-6, IL-1β and TNF-α (n = 3, P** < 0.001, vs. Blank; P^{^^} < 0.001, vs. control and mimic control). The U6 was the internal reference. Blank group: hippocampus neurons did not receive sevoflurane treatment. Other groups except additional explanation: hippocampus neurons received 2% sevoflurane treatment.

We found the cell apoptosis was significantly increased in control and mimic control groups, but the cell apoptosis was obviously decreased by treatment with miR-128-3p mimic than control and mimic control groups (Fig. 2D and 2E, P < 0.001). In addition, the expression of Bax and cleaved caspase-3 were significantly increased and down-regulated Bcl-2 expression when the hippocampal neurons were treated with 2% sevoflurane or mimic control, respectively. Moreover, overexpression of miR-128-3p significantly attenuated the expression of Bax and cleaved caspase-3 and up-regulated Bcl-2 expression (Fig. 2F and 2G, P < 0.001). The expression of NOX1 and NOX4 were significantly increased when the hippocampal neurons were treated with 2% sevoflurane or mimic control, respectively, but were significantly decreased by treating mimic (Fig. 2F and 2G, P < 0.001). Finally, the expression levels of IL-6, IL-1β and TNF-α were detected by ELISA, and result showed that the expression levels were significantly up-regulated in cells by treatment with sevoflurane, and this effect was obviously attenuated treated with mimic (Fig. 2H, P < 0.001).

MiR-128-3p could target NOVA1 and inhibit its expression

TargetScan was used to predict the target gene of miR-128-3p; the results showed that miR-128-3p targeted NOVA1 (Fig. 3A). Next, the dual luciferase gene assay report was used to verify the relationship between miR-128-3p and NOVA1, and the results confirmed that NOVA1 was indeed the target gene of miR-128-3p (Fig. 3B, P < 0.001). Moreover, qRT-PCR was used to detect the expression of NOVA1 in the sevoflurane-treated cells that transfected with miR-128-3p mimic, the result showed that sevoflurane treatment increased NOVA1 expression in the cells, but overexpression of miR-128-3p significantly inhibited the NOVA1 expression in the sevoflurane-treated cells (Fig. 3C, P < 0.001). Meanwhile, Western blotting also confirmed the results of qRT-PCR (Fig. 3D and E, P < 0.001).

Fig. 3

MiR-128-3p could target NOVA1 and inhibit its expression. (A) TargetScan was used to predict the target gene of miR-128-3p. (B) The dual luciferase gene assay report was used to verify the relationship between miR-128-3p and NOVA1. (C) QRT-PCR was used to detect the expression of NOVA1 in the sevoflurane-treated cells that transfected with miR-128-3p mimic. (D–E) Western blotting also confirmed the results of qRT-PCR. (n = 3, P** < 0.001, vs. Blank; P^{^^} < 0.001, vs. mimic control). Blank group: hippocampus neurons did not receive sevoflurane treatment. Other groups: hippocampus neurons received 2% sevoflurane treatment.

Effects of NOVA1 overexpression on the improvement of sevoflurane nerve injury by miR-128-3p mimic

QRT-PCR and Western blotting were used to detect the transfection efficiency of NOVA1, the results showed that after mimic and NOVA1 was transfected to cells, the NOVA1 expression was significantly inhibited compared with that in the mimic control+NOVA1 group (Fig. 4A, B and C, P < 0.001). Flow cytometry was used to detect cell apoptosis. The results showed that NOVA1 overexpression significantly reversed mimic to inhibit cell apoptosis induced by sevoflurane (Fig. 4D, P < 0.001). Western blotting was used to detect apoptosis and oxidative stress-related proteins, and the results showed that the over-expression of NOVA1 significantly reversed the effect of mimic on the down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3 and NOX1/4 in 2% sevoflurane treatment (Fig. 4E and F, P < 0.001). The content of inflammatory factors was detected by ELISA, and the results showed that NOVA1 overexpression significantly reversed the effects of 2% sevoflurane treatment to increase the content of inflammatory factors (Fig. 4G, P < 0.001).

Fig. 4

Effects of NOVA1 overexpression on the improvement of sevoflurane nerve injury by miR-128-3p mimic. (A) QRT-PCR and (B–C) Western blotting were used to detect the transfection efficiency of NOVA1. (D) Flow cytometry was used to detect cell apoptosis. (E–F) Western blotting was used to detect apoptosis and oxidative stress-related proteins. (G) The content of inflammatory factors was detected by ELISA. (n = 3, P** < 0.001, vs. Control; P^{^^} < 0.001, vs. mimic+NC; P^&& < 0.001, vs. mimic control+NOVA1).

DISCUSSION

Sevoflurane is a commonly used inhalation anesthetic gas (Woll et al., 2017). At present, there is no definite result on whether sevoflurane has toxic effects on the nervous system. Many studies have confirmed that sevoflurane not only has toxic effects on the nervous system of normal people, but also causes certain nerve damage to patients with Alzheimer's disease (Brosnan and Bickler, 2013; Xiong et al., 2013). Research results show that 2.5% sevoflurane inhalation for 2 hr exhibits neurotoxicity in newborn rats (Shen et al., 2018; Zheng et al., 2013). In order to further explore the effects of sevoflurane on neurons of rats, in this study, we used 1%, 2% and 4% respectively of sevoflurane processing hippocampal neurons, action after 6 hr cell activity, the results show that after 1% sevoflurane treatment, the activity of cells did not deal with lower, and by 2% and 4% sevoflurane, significantly lower the activity of cells; therefore, sevoflurane have definite toxicity effects on hippocampal neurons. The concentration dependence forced medical researchers to further explore the protective mechanism to improve this toxic effect.

Studies have found that the content and species of miRNA in the brain of primates are more abundant (Zhao et al., 2014a), and there are more miRNAs in the synapses of brain neurons, which can target and regulate the synthesis of over 90% of synaptic proteins (Hu and Li, 2017). Therefore, miRNA is involved in the physiological activities of the nervous system. Studies have found that miR-128 is a brain-enriched miRNA, which is usually involved in the development of the nervous system and the maintenance of neurophysiological functions (Huang et al., 2015), but its protective effect on sevoflurane-induced neurotoxicity has been rarely reported. Therefore, this study further explored its protective effect on sevoflurane-induced neurotoxicity.

Many scholars believe that sevoflurane anesthesia can lead to changes in the miRNA expression profile of the body, for example, Ishikawa et al. found that 2.5% sevoflurane can lead to differential expression of miRNA in rat liver (Ishikawa et al., 2012). Sevoflurane and propofol anesthesia induced changes in miRNA expression profiles in the brain of rats (Lu et al., 2015). Sevoflurane anesthesia induced changes in miRNA expression in the hippocampus of newborn rats (Ye et al., 2016). In this study, we found that the expression of miR-128-3p in hippocampal neurons was significantly down-regulated after sevoflurane treatment. Consistent with the results of this study, the expression of miR-206 was significantly down-regulated when sevoflurane induced hippocampal astrocytes in aged rats (Liu et al., 2018). These results preliminarily revealed the effect of sevoflurane on miR-128-3p in hippocampal neurons of rats.

Multiple clinical studies have shown that inhalation of sevoflurane induces extensive neuronal apoptosis in the brain and can cause long-term cognitive and memory impairment in patients (Chen et al., 2016; Zhang et al., 2017). According to the results of detecting apoptosis related proteins expression, sevoflurane treatment is to promote neurons apoptosis, and miR-128-3p mimic transfection after sevoflurane treatment is to reverse the aggravated neuron apoptosis. In addition, the results of this study is different with reports of Xiang LV et al. that inhibition of miR-27a-3p reduced the hippocampus neuron cell apoptosis induced by sevoflurane (Lv et al., 2017). Thus, overexpression of miR-128-3p can inhibit sevoflurane-induced apoptosis.

As we all know, miRNA and mRNA can play a regulatory role in the life process of cells through their targeting relationship (Fabian et al., 2010). NOVA1 is a new type of RBP that plays an important role in the occurrence and development of tumors (Yu et al., 2018). Studies have shown that NOVA1 is down-regulated by miR-181b-5p and plays a common regulatory role in inhibiting the proliferation, migration and invasion of astrocytoma and promoting cell apoptosis (Zhi et al., 2014). There is no report on the interaction between NOVA1 and miR-128-3p in disease research, so in this study we explored the mechanism of NOVA1 and miR-128-3p in nerve cells induced by sevoflurane.

The effects of sevoflurane on cognitive function may be caused by stimulating oxidative stress reaction in the body, causing inflammation in the central nervous system, and leading to intracellular calcium imbalance (Tian et al., 2015). NOX1 and NOX4 are specific marker proteins of oxidative stress response, and the changes in their levels are positively correlated with the degree of stress response (Hou et al., 2018). In this study, overexpression of miR-128-3p inhibited the effect of sevoflurane-induced increased intracellular levels of NOX1 and NOX4, while overexpression of NOVA1 had an opposite effect. IL-6, IL-1β and TNF-α are considered to be the key factors to induce inflammation (Gao et al., 2017; Zhao et al., 2014b). Therefore, in this study, the levels of these inflammatory factors were detected, and we found that overexpression of miR-128-3p could inhibit the effect of sevoflurane-induced increased levels of intracellular inflammatory factors, while overexpression of NOVA1 reversed the effect of miR-128-3p mimic.

In summary, our results provide some insights into the mechanism of sevoflurane-mediated neurotoxicity, and may provide a new therapeutic method for reducing the damage of anesthesia on brain nerves.

ACKNOWLEDGMENTS

This study was supported by the Science &Technology Development Fund of Tianjin Municipal Education Commission [2020KJ174].

Conflict of interest

The authors declare that there is no conflict of interest.

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