ERK signaling pathway

Accepted Manuscript Title: Inhibition of microRNA-34a protects against propofol anesthesia-induced neurotoxicity and cognitive dysfunction via the MAPK/ERK signaling pathway Authors: Guang Feng Li, Su Jing Zhuang, Guang Cai Li PII: DOI: Reference:

S0304-3940(18)30229-5 https://doi.org/10.1016/j.neulet.2018.03.052 NSL 33511

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

27-11-2017 5-3-2018 21-3-2018

Please cite this article as: Guang Feng Li, Su Jing Zhuang, Guang Cai Li, Inhibition of microRNA-34a protects against propofol anesthesia-induced neurotoxicity and cognitive dysfunction via the MAPK/ERK signaling pathway, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.03.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibition of microRNA-34a protects against propofol anesthesia-induced neurotoxicity and cognitive dysfunction via the MAPK/ERK signaling pathway

Guang feng Li 1, Su jing Zhuang 1, Guang cai Li 2,* Department of neurology medicine, Linyi Central Hospital, Linyi, Shandong, China.

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Department of Pharmacy, Linyi Central Hospital, Linyi, Shandong, China.

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Corresponding Author: Guang cai Li, Department of Pharmacy, Linyi Central Hospital, jian kang

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Road #17 of Yi shui County, Linyi, Shandong, 276400, China, Email: [email protected], Tel:

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+86-0539-2251934

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Highlights

Protective effect of microRNA-34a (miR-34a) on propofol-induced neurotoxicity and cognitive dysfunction was studied.

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Propofol anesthesia had an adverse effect on cell survival due to the increased expression of apoptosis-related genes

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MicroRNA-34a could improve anesthesia-induced cognitive dysfunction

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Abstract

Aim: To investigate the protective effect of microRNA-34a (miR-34a) on propofol-induced neurotoxicity and cognitive dysfunction. Methods: After SH-SY5Y cells were treated with propofol to induce neurotoxicity, microRNA-34a mimics and inhibitors were transfected into the cells. The expression of apoptosis-related genes and the proteins were measured by quantitative

reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Sprague-Dawley (SD) rats received intraperitoneal injections of propofol, and were treated with microRNA-34a mimics and lentivirus-mediated microRNA-34a inhibitors. The Morris water maze (MWM) test was used to detect changes in motor function. Results: Propofol anesthesia had an adverse effect on cell survival due to the increased expression of apoptosis-related genes such as cleaved caspase-3/8 and

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Bax, which was accompanied by reduced expression of ERK1/2, pERK1/2, and phosphorylated NF-kappaB p65 both in vivo and in vitro. Unexpectedly, microRNA-34a was upregulated after

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propofol treatment, and the inhibitors protected the SH-SY5Y cells from propofol-induced apoptosis. The microRNA-34a inhibitor suppressed the apoptosis-induced effects of propofol. This protection may have been partly diminished by PD98059, a MAPK kinase inhibitor. MicroRNA-

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34a inhibited or reverted the reduced expression of ERK1/2 and upregulated the expression of p-

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CREB significantly and specifically. Additionally, the microRNA inhibitors improved the learning

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and memory functions of animals suffering from neurologic impairment due to propofol treatment

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and reduced cell apoptosis in the hippocampus. Conclusion: microRNA-34a could improve anesthesia-induced cognitive dysfunction by suppressing cell apoptosis and recovering the

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expression of genes associated with the MAPK/ERK signaling pathway.

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Keywords: MicroRNA-34a; Propofol; neurotoxicity; cognitive dysfunction; MAPK/ERK

Introduction

Postoperative cognitive dysfunction (POCD) is a novel form of cognitive impairment that occurs

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following surgical procedures. Impairment in memory and performance related to intellectual tasks is most common. The incidence of POCD increases significantly with age [1, 2]. Steinmetz et al. found that patients with POCD have a higher mortality rate and greater utilization of healthcare services [3]. Clinical and animal experiments have shown that anesthetics may lead to cognitive dysfunction and neurologic impairment, especially in aged individuals or those with nervous system

disease [4]. Although POCD has garnered recent attention from surgeons and anesthetists, its mechanisms following are still obscure. Micro ribonucleic acids (microRNAs) are small oligonucleotide non-coding RNAs that regulate and control many biological processes, including cell proliferation, cellular differentiation, and apoptosis. They also play an important role in disease [5]. MicroRNA-34a (miR-34a) plays a

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critical role in cortical development [6]. As the critical target of the P53 gene, microRNA-34a acts as a tumor suppressor by promoting apoptosis in tumor cells [7]. Cell apoptosis, a typical mode of

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programmed cell death, may be involved in the mechanism of anesthesia-induced POCD [2, 8]. It has been demonstrated that both mitochondrial- and receptor-dependent apoptosis pathways play a critical role in regulating apoptosis [9, 10] . The inhibitors of NAPDH oxidase may prevent the

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memory impairment that occurs following sevoflurane exposure in mice by negatively regulating

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apoptosis and reducing the generation of cytochrome C [10]. A previous study has found that

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exposure to propofol anesthesia accelerates the expression of the Fas receptor and its ligand, which

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mediate pro-apoptotic and pro-inflammatory signaling in the brain [9]. Furthermore, propofol

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anesthesia has been shown to upregulate the expression of Fas/FasL and downstream caspase-8, more remarkably in the thalamus than in the cortex [5], demonstrating that distinct regions of the

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brain have different tolerances to propofol-induced apoptosis.

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In this study, we demonstrate that the inhibition of microRNA-34a could have beneficial effects on anesthesia-induced cognitive dysfunction by regulating apoptosis via the MAPK/ERK signaling pathway. The inhibition of microRNA-34a attenuated apoptosis in neurocytes and impairment of

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cognitive function by transfection in vivo. MicroRNA-34a represents a potential therapeutic target for the regulation of propofol-induced apoptosis in the hippocampus of mammals. Materials and Methods Culture, treatment, and transfection of cells

Human neuronal SH-SY5Y neuroblastoma cells were purchased from ATCC (Manassas, VA) and cultured in Dulbecco’s Minimum Essential Medium (DMEM) (Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS) Sigma, St. Louis, MO) and 100 U/mL penicillin (Sigma, St. Louis, MO) at 37°C with 5% CO2 in 96-well plates (BD Biosciences, Rockville, MD). The SH-SY5Y cells were treated with 1, 5, 10, and 20 μg/ml propofol (2,6-diisopropylphenol, Rueil Malmaison, France)

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for 24 h for in vitro experiments. In addition, the cells were pretreated with the MAPK/ERK pathway inhibitor PD98059 (Sigma, St. Louis, MO) for 30 min as described previously [11]. The SH-SY5Y cells were transfected with 100 nM microRNA mimic or inhibitor (SCR; Genepharma,

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China) using the Lipofectamine 2000 (Invitrogen, CA), according to the manufacturer’s protocol. The cells transfected with scrambled sequences by the Lipofectamine 2000 were used as the

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negative controls.

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Animal models

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The Institutional Animal Care and Use Committee of Linyi Central Hospital approved the experimental protocols involving animals. Prior to the experiments, Sprague-Dawley (SD) rats

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(male, 250 ± 10 g, six weeks of age; Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) were housed under a controlled 12:12 h light/dark cycle for seven days. The rats were then

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randomly divided into two groups: a sevoflurane group that received propofol at a concentration of

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5 mg·kg-1 in 0.9% normal saline daily and consecutively for 10 days, and a control group that received an equal volume of 0.9% normal saline. The inhibition of microRNA-34a in the hippocampus was performed using a lentivirus-mediated miR-34a inhibitor and a lentiviral mock

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vehicle. The procedure for lentivirus production was performed as described previously [12]. For animal

transfection,

lentivirus-mediated

microRNA-34a

inhibitors/mimics/scrambles

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administered into the left lateral cerebral ventricles through a pre-drilled skull hole within 10 min following propofol treatment, as described previously [12]. After the rats were sacrificed, the hippocampus was rapidly extracted, frozen on dry ice, and homogenized using TRIzol Reagent (provided by Invitrogen, USA) to perform the subsequent RNA isolation.

Western blotting Protein isolation and immunoblotting were performed as described previously [13]. RIPA lysis and extraction buffer (Invitrogen, Thermo Scientific, USA) was employed for cell pellet lysis. The concentration of protein was detected using a BCA protein assay kit (Thermo Fisher Scientific, USA). Equal amounts of protein were transferred to PVDF membranes (EMD Millipore, Billerica,

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MA). Subsequently, the PVDF membranes underwent incubation with primary antibodies overnight at 4 °C, and were then washed with tris-buffered saline with Tween-20 (TBST) 3 times, followed

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by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Inc., MA; 1:5,000) at room temperature for 90 min. Chemiluminescence was employed to visualize the blotting bands. The following primary antibodies were used: anti-

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caspase-8 (cat. no. 9504; 1:1,000), anti-caspase-3 (cat. no. 6992; 1:1,000), anti-phosphorylated (p)

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extracellular signal-regulated kinases (cat. no. 9101; 1:1,000; ERK), anti-Bax (ADP-ribose)

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polymerase (cat. no. 9542; 1:1,000; PARP), anti-ERK (cat. no. 9272; 1:1,000), anti-nuclear factor

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(NF)-κB (cat. no. NB100-2176; 1:1,000; p65; Novus Biologicals LLC, America), anti-GAPDH (cat.

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no. sc-32233; 1:2,000), and anti-phosphorylated CREB (cat. no. 4060; 1:2,000).

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RNA extraction and qRT-PCR

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Total RNA was extracted from SH-SYSY cells using TRIzol or a microRNA Mini Kit (Qiagen) 6h following treated by propofol or the microRNA mimic/inhibitor/scramble. Total RNA (500 ng) was used for reverse transcription along with a TaqMan microRNA RT kit (provided by Applied

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Biosystems) and microRNA-specific stem loop primers. RT-PCR analysis for mRNA was performed as described previously [13]. The cDNA was employed to perform polymerase chain reaction of the tested genes in the ABI PRISM 7700 system (Applied Biosystems; Thermo Fisher Scientific). The relative expression of microRNA was determined by the △△Ct value after normalization with snRNA U6. GAPDH was used as the internal control for the normalization of

the relative quantification of expression of the tested genes. The 2–△△Ct method, in which △△Ct = (Ct Target gene – Ct housekeeping gene) group1 − (CtTarget gene – Ct housekeeping gene) group2, was used for calculation of the real-time qRT-PCR results. Morris water maze test The Morris water maze (MWM) test was carried out as previously described, with minor

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modifications [14]. The Morris water maze consisted of a circular pool (150 cm in diameter, 20 cm in depth) filled with warm (22–25 °C) water. Directed by distal cues attached to the wall, the rats

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were trained to swim through the water to a submerged platform (1.5 cm × 1.5 cm, 1.5 cm below the surface of the water). All animal movements were recorded using an automatic

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tracking/analyzing video system. In the acquisition phase, four training sessions per day were

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conducted on four consecutive days. Rats were allowed to swim freely to the platform within a 2-

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min time frame, followed by 20 s of rest on the platform. If the platform was not located within 2

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min, the rats were manually placed on the platform for 20 s at the end of the trial. On the fifth day, a probe trial was conducted in which both the tracking length and time to reach the platform were

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recorded. The time to reach the platform was used as a measure of spatial memory and learning ability.

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Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling

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TUNEL was performed on SH-SY5Y cells as described previously [15]. TUNEL was performed using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) according to the instructions provided by the manufacturer. In brief, tissue sections (three sections per rat) were

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incubated in xylol for 30 min at room temperature to permeabilize the sections after deparaffinization and dehydration. Subsequently the sections were incubated with 3% H2O2 in PBS for 10 min. After addition of the TUNEL reaction mixture, the sections were incubated for 60 min at 37°C, rinsed, and then visualized using a converter-POD for 30 min at 37°C. The sections were then subjected to PBS washing, followed by incubation with a chromogen (50-100 µL 0.05% 3,3′-

diaminobenzidine (DAB)) for 10 min. After PBS washing, the number of TUNEL-positive cells was counted in the CA1 area of the hippocampus under a light microscope. Statistical analysis Data are presented as mean ± standard deviation. All assays were repeated a minimum of three times. Student's t-tests were used for statistical analysis, and SPSS software (version 22.0) was

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employed for data processing. One-way analysis of variance (ANOVA) and Bonferroni post hoc tests were used for multiple group comparisons. P < 0.05 was used to indicate a significant

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difference. Results

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Propofol treatment induces the apoptosis of SH-SY5Y and upregulates the expression of

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apoptosis-related genes

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To investigate the mechanism of anesthesia-induced neurotoxicity, we detected the apoptosis of

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SH-SY5Y cells treated by propofol in vitro. The SH-SY5Y cells were treated with concentrations of

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1, 5, 10, or 20 μg/ml propofol or phosphate buffer. The expression levels of apoptosis-related genes were measured by qRT-PCR. The expression levels of cascade-3, caspase-8, and Bax increased at

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concentrations of 5, 10, and 20 μg/ml propofol (Pro) compared with the negative control group (Fig. 1A and B). In the 10 and 20 μg/ml propofol groups, the expression of cleaved caspase-3/8 and Bax

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increased, and were accompanied by a significant decrease in p-CREB (Fig. 1B and C). The result of TUNEL showed that the percentage and amount of positively stained SH-SY5Y cells in the

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groups treated with propofol at concentrations >5 μg/ml were significantly larger compared to the group treated with phosphate buffer (Fig. 1D). These results demonstrated that propofol treatment induced the apoptosis of SH-SY5Y and directly increased the expression of apoptosis genes. Propofol treatment upregulates microRNA-34a and inhibition of microRNA-34a prevents apoptosis in SH-SY5Y cells

We found that, in vitro, the transcriptional level of microRNA 34a in SH-SY5Y cells significantly increased in the groups treated with propofol at a concentration of 10 μg/ml (Fig. 2A). Therefore, we regarded microRNA-34a as a potential target in anesthesia-induced neurocyte apoptosis and cognitive dysfunction. In the groups treated with propofol at concentrations >10 μg/ml, the microRNA 34a inhibitor (100 nM) suppressed apoptosis, as shown by an increased percentage and

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amount of TUNEL-positive staining compared with the group treated by the scrambled microRNA (negative control) (Fig. 2B). In accompaniment with the higher percentage and amount of apoptotic

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cells, the activation of apoptosis-related proteins decreased simultaneously after treatment with the microRNA-34a inhibitor following propofol treatment (Fig. 2C and 2D). On the other hand, the phosphorylation of p65, a key subunit of NF-kappaB, significantly decreased after propofol

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treatment. Unexpectedly, the inhibitor of microRNA-34a also reversed the decreased

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phosphorylation of p65, which suggests that propofol might have an adverse effect on neurocyte

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survival via multiple mechanisms. The results from the in vitro experiments demonstrated that

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microRNA-34a may be involved in the mechanism of propofol-induced apoptosis and that the

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inhibition of microRNA-34a might protect neurocytes from anesthetic toxicity. MicroRNA-34a suppresses the apoptosis of neurocytes after treatment with anesthetic drugs

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via the MAPK/ERK signaling pathway

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The MAPK/ERK signaling pathway is crucial for cell proliferation, apoptosis, and differentiation. Previous studies using reporter gene assays have identified MEK1 as a direct target of microRNA34a. Therefore, we analyzed the expression levels of pERK1/2, ERK1/2, and p-CREB following

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treatment with microRNA-34a inhibitors in vitro. In the cells treated with propofol after treatment with the inhibitor, the expression of ERK1/2, pERK1/2, and p-CREB significantly increased (Fig. 3A and 3B) and had a higher ratio of pERK/ERK compared with the negative control (Fig. 3C). The application of microRNA inhibitor remarkably improved the expression levels of ERK1/2 and pERK1/2, which demonstrated that microRNA-34a not only regulates the transcription level of ERK in a targeted manner, but is also a switch of post-translational phosphorylation. To explore the

role of the MAPK/ERK signaling pathway in the presence of a microRNA inhibitor, we detected the apoptosis rate and levels of apoptosis-related proteins in SH-SY5Y cells after simultaneously treating with microRNA and PD98059, a MEK kinase inhibitor. Treatment with PD98059 eliminated the apoptosis resistance attributed to the microRNA inhibitor (Fig. 3D), which was illustrated by the increased proportion and amounts of TUNEL-positive cells (P<0.05). Further, the

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protein expression of Bax, cleaved caspase-3, and caspase-8 were consistent with the change in the apoptosis rate (Fig. 4A-D). These findings demonstrate that the protective effect of microRNA-34a

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inhibitors might occur via activation of the MAPK/ERK pathway.

Anesthetizing animals causes impairment of motor function, cell apoptosis, and upregulation

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of microRNA in the hippocampus

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To ensure the validation of propofol-induced apoptosis and neurotoxicity in animal models, we

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performed experiments in vivo. After SD rats received intraperitoneal anesthesia with propofol for

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10 consecutive days, they were tested for their learning and memory ability using the Morris water maze. The expression of miR-34a increased significantly in the propofol group (5 mg/kg of body

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weight) compared with the control group (P<0.05) (Fig. 4E). The rats treated with propofol showed a longer latency than the control group (Fig. 4F). Neurocyte apoptosis in the hippocampus (the

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percentage and amount of TUNEL-positive cells) was in line with the results of the in vitro

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experiments (Fig. 4G). The propofol-induced neurotoxicity and memory impairment identified in vivo resulted from the promotion of apoptosis in hippocampal CA1 neurons and upregulation of microRNA-34a.

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Inhibitors of microRNA-34a improve the motor function of anesthetized animals and inhibit apoptosis in the hippocampus A lentivirus-mediated microRNA-34a inhibitor or a lentivirus mock vehicle was transfected into the left lateral ventricle of anesthetized rats by intracerebroventricular injection after 10 days of continuous anesthesia. The animals transfected by the lentivirus mock vehicle were classified as

the negative group. The animals that received transfection of microRNA inhibitors showed a shorter latency than those without transfection (P<0.05) (Fig. 5A). There was no statistical discrepancy between the rats without transfection and the negative group. No difference was observed in swimming speed among groups (data not shown). The neurocyte apoptosis was reduced in rats that had received transfection with microRNA inhibitors compared with the

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negative control groups. There was no difference in the apoptosis between the animals transfected by the lentivirus mock vehicle and those without transfection (Fig. 5B). Meanwhile,

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the protein expression of apoptosis-related genes significantly decreased after transfection by the lentivirus-mediated microRNA inhibitor compared with the negative control (Fig. 5C and D).

These results provide strong evidence that the inhibitor of microRNA-34a improves the memory

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and cognitive function of anesthetized rats via suppression of neurocyte apoptosis in the

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hippocampus.

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Discussion

Aged patients frequently suffer from post-operative cognitive dysfunction (POCD), which is

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characterized by memory and cognitive impairment that typically develops from post-operative delirium [16]. It has been found that patients who experience post-operative cognitive dysfunction

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have a higher 1-year mortality rate after discharge from the hospital than those who do not [17].

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Recent clinical research has identified many risk factors for POCD, including extensive surgical procedures under general anesthesia, organ ischemia, alcohol abuse, and emotional disorders [18, 19]. Anesthetic neurotoxicity might be the major cause of age-related POCD [20].

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It has been reported that surgical procedures in mice activate the TNFα/NF-κB signaling

pathway, which mediates the pro-inflammatory response and leads to the release of cytokines [21]. Cytokines destroy the integrity of the blood-brain barriers and cooperate with the chemotactic facilitation of macrophages entering the hippocampus, which leads to neuronal apoptosis and observed memory impairment [21]. Additionally, numerous neurological studies in mammals have shown that apoptosis occurs in the hippocampus after propofol and sevoflurane anesthesia [22-24],

which might be associated with the cognitive dysfunction induced by anesthetics. The pro-apoptotic and pro-inflammatory effects of anesthesia have been confirmed by previous studies [25, 26]. Zhong, Y et al. found that propofol treatment decreased nuclear factor kappaB (NF-κB) p65 expression in primary hippocampus neuronal cells, and was accompanied by decreased expression of B-cell lymphoma 2 (Bcl-2), activation of caspase-3 protein, and increased expression of caspase-

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3 mRNA [27]. Given that apoptosis has been shown to be a possible mechanism of POCD in animal models,

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the abnormal regulation of apoptosis following anesthesia might also play an important role in POCD. Zhang, S et al. found that repeated injection of propofol intraperitoneally resulted in a significant downregulation of miR-132 levels and a decrease in the amount of dendritic spines in

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the hippocampus and was associated with learning and memory dysfunction in rats [28]. Twaroski

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et al. revealed that miR-21 was involved in apoptosis induced by propofol, which acted via the

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STAT3/Sprouty-2 pathway [15]. Through in vivo and in vitro models, the expression levels of miR-

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124, miR-137, and miR-34a are known to be modified during neurotoxicity induced by ketamine

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[29, 30]. Interestingly, microRNA-34a was downregulated in myocardial tissues, opposite to the upregulation found in the hippocampus [31]. Therefore, to elucidate the contradiction, we

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performed experiments involving propofol-induced apoptosis models to analyze the transcriptional level of microRNA-34a. Our results were consistent with previous research that suggested that the

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upstream regulation of microRNA-34a varies between tissue types. MicroRNA-34a is characterized by facilitating apoptosis in tumor cells and is the downstream

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target of P53 [32]. Ectopic expression of miR-34a induces apoptosis in neuroblastoma cells [33]. Suppression of miR-34a expression protects wild-type cells expressing P53 from apoptosis to some extent, demonstrating that miR-34a is at least partly necessary for the apoptosis induced by P53 [32]. Moreover, CREB and BIRC5 (Survivin), which are typical proteins that possess anti-apoptotic properties, have been recognized as targets of miR-34a and have degeneration that is involved in the pro-apoptotic activity of miR-34a [34]. In our study, apoptosis was detected using TUNEL and

behavioral experiments in vivo and in vitro. These results demonstrate that the inhibitor alleviated apoptosis both in vivo and in vitro, and subsequently improved cognitive function, which verified our hypothesis. A previous study has shown that intravenous administration of propofol markedly decreases phosphorylation of ERK1/2 in the hippocampus of adult mice via either NMDA receptors or PLC-

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and PKC-dependent pathways, suggesting that ERK1/2 represents a target for anesthetics in the brain [35]. In our study, it was found that the expression of ERK1/2 and pERK1/2 decreased after

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propofol treatment, which is consistent with other studies. Moreover, Ichimura et al. found that MEK1 is a direct target of microRNA-34a and that overexpression of microRNA-34a significantly suppresses the proliferation of K562 cells. This previous study provided us with a novel hypothesis

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that microRNA-34a promotes apoptosis after propofol anesthesia by targeting the degeneration of

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the mRNA of MEK1/2 and downregulating the MEK/ERK signaling pathway, a critical mitogen-

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activated protein kinase signal pathway [36]. Hence we demonstrated that the inhibition of

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microRNA-34a reduced apoptosis via activation of the MEK/ERK pathway. We did this by

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blocking the MAPK/ERK signaling pathway using the specific inhibitor PD98059. We found that PD98059 partly blocked the anti-apoptosis effect of the miRNA34a inhibitor. The results

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demonstrate that the microRNA-34a inhibitor attenuates apoptosis induced by propofol via

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activation of the MEK/ERK signaling pathway. In conclusion, our study highlights the importance of microRNA-34a in the regulation of apoptosis in hippocampal neurons and the impact of a microRNA inhibitor on protection from

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neurotoxicity induced by anesthetic drugs. MicroRNA-34a and its target mRNA were identified as key switches in terms of regulating neuronal apoptosis via the MEK/ERK signaling pathway. MicroRNA-34a might be a potential therapeutic target for POCD.

Authors' contributions Guang feng Li and Guang cai Li conceived the study and designed the experiments. Guang feng Li and Su jing Zhuang contributed to the data extraction, performed the analysis and interpreted the results. Guang feng Li wrote the first draft; Guang cai Li contributed to the critical revision of

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article. All authors read and approved the final manuscript.

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Conflict of interests

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None.

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Acknowledgments

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None.

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19. Shoair OA, Grasso Ii MP, Lahaye LA, Daniel R, Biddle CJ, and Slattum PW. Incidence and risk factors for postoperative cognitive dysfunction in older adults undergoing major noncardiac surgery: A prospective study. J Anaesthesiol Clin Pharmacol, 2015;31(1):30-6. 20. Vlisides P and Xie Z. Neurotoxicity of general anesthetics: an update. Curr Pharm Des, 2012;18(38):6232-40. 21. Terrando N, Eriksson LI, Ryu JK, Yang T, Monaco C, Feldmann M, et al. Resolving postoperative neuroinflammation and cognitive decline. Ann Neurol, 2011;70(6):986-995. 22. Creeley C, Dikranian K, Dissen G, Martin L, Olney J, and Brambrink A. Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus macaque brain. Br J Anaesth, 2013;110 Suppl 1:i29-38. 23. Jia Z, Geng L, Xie G, Chu Q, and Zhang W. Sevoflurane impairs acquisition learning and memory function in transgenic mice model of Alzheimer's disease by induction of hippocampal neuron apoptosis. Int J Clin Exp Med, 2015;8(9):15490-7. 24. Zheng SQ, An LX, Cheng X, and Wang YJ. Sevoflurane causes neuronal apoptosis and adaptability changes of neonatal rats. Acta Anaesthesiol Scand, 2013;57(9):1167-74. 25. Cheng Y, He L, Prasad V, Wang S, and Levy RJ. Anesthesia-Induced Neuronal Apoptosis in the Developing Retina: A Window of Opportunity. Anesth Analg, 2015;121(5):1325-35. 26. He Y, Li Z, and Zuo YX. Nerve Blockage Attenuates Postoperative Inflammation in Hippocampus of Young Rat Model with Surgical Trauma. Mediators Inflamm, 2015;2015:460125. 27. Zhong Y, Liang Y, Chen J, Li L, Qin Y, Guan E, et al. Propofol inhibits proliferation and induces neuroapoptosis of hippocampal neurons in vitro via downregulation of NF-kappaB p65 and Bcl-2 and upregulation of caspase-3. Cell Biochem Funct, 2014;32(8):720-9. 28. Zhang S, Liang Z, Sun W, and Pei L. Repeated propofol anesthesia induced downregulation of hippocampal miR-132 and learning and memory impairment of rats. Brain Res, 2017;1670:156-164. 29. Cao SE, Tian J, Chen S, Zhang X, and Zhang Y. Role of miR-34c in ketamine-induced neurotoxicity in neonatal mice hippocampus. Cell Biol Int, 2015;39(2):164-8. 30. Xu HY, Zhang JJ, Zhou W, Feng YZ, Teng SY, and Song XS. The role of miR-124 in modulating hippocampal neurotoxicity induced by ketamine anesthesia. International Journal of Neuroscience, 2015;125(3):213-220. 31. Lucchinetti E, Hofer C, Bestmann L, Hersberger M, Feng JH, Zhu M, et al. Gene regulatory control of myocardial energy metabolism predicts postoperative cardiac function in patients undergoing off-pump coronary artery bypass graft surgery - Inhalational versus intravenous anesthetics. Anesthesiology, 2007;106(3):444-457. 32. Kofman AV, Letson C, Dupart E, Bao YD, Newcomb WW, Schiff D, et al. The p53microRNA-34a axis regulates cellular entry receptors for tumor-associated human herpes viruses. Medical Hypotheses, 2013;81(1):62-67. 33. Tivnan A, Tracey L, Buckley PG, Alcock LC, Davidoff AM, and Stallings RL. MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma. Bmc Cancer, 2011;11. 34. Sarkar S, Jun S, Rellick S, Quintana DD, Cavendish JZ, and Simpkins JW. Expression of microRNA-34a in Alzheimer's disease brain targets genes linked to synaptic plasticity, energy metabolism, and resting state network activity. Brain Research, 2016;1646:139-151. 35. Gao HY, Han MJ, Zhang LY, Wang QX, Wang HS, and Zhang BX. Anesthetics inhibit extracellular signal-regulated Kinase1/2 phosphorylation via NMDA receptor, phospholipase C and protein kinase C in mouse hippocampal slices. Neurochemistry International, 2017;103:36-44. 36. Ichimura A, Ruike Y, Terasawa K, Shimizu K, and Tsujimoto G. MicroRNA-34a Inhibits Cell Proliferation by Repressing Mitogen-Activated Protein Kinase Kinase 1 during Megakaryocytic Differentiation of K562 Cells. Molecular Pharmacology, 2010;77(6):10161024.

Figure legends Fig. 1. Propofol induces the apoptosis of SH-SY5Y cells and upregulates the apoptosis genes. A. The mRNA transcriptional level of the apoptosis gene was determined by quantitative real-time RT-PCR. B. Representative results of western blot demonstrating that propofol (Pro) treatment induces the upregulation of apoptosis gene expression and the downregulation of p-CREB

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expression. GAPDH was used as the control. C. Quantification of signal intensities from Western blots. D. The application of propofol increases the number and percentage of TUNEL-positive cells

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in a dose-dependent manner. Statistical analysis results of TUNEL-positive cells are shown in the bar graphs. Student’s t-tests were used for single comparisons, *P < 0.05 versus control group. All

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experiments were replicated at least three times.

Fig. 2. Inhibition of microRNA-34a attenuated the apoptosis induced by propofol treatment. A. The transcriptional level of microRNA-34a significantly increased after propofol (Pro) treatment at a concentration of 10 μg/ml.* P<0.05 versus the control group, **P < 0.01 versus control group . B. The percentage of TUNEL-positive SH-SY5Y cells in each group, demonstrating the apoptosis rate. C. Representative results of western blot showing that the microRNA-34a mimic (miR-mimic)

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decreased the expression of apoptosis genes and increased the expression of the NF-kappaB p65 subunit after propofol treatment. GAPDH was used as the control. D. Quantification of signal

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control. All experiments were replicated at least three times.

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intensities from Western blots. * P<0.05 versus the negative control, **P<0.01 versus negative

Fig. 3. The effect of microRNA-34a on apoptosis via the MAPK/ERK signaling pathway. A. Representative results of western blot demonstrating that the microRNA-34a mimic decreased the expression of apoptosis genes and increased the expression of the NF-kappaB p65 subunit after propofol treatment. GAPDH was used as the control. B. Semi-quantitative analysis of the protein expression levels. *P<0.05 versus the negative control, **P<0.01 versus negative control. C. The

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ratio of pERK/ERK. D. The apoptosis rate of SH-SY5Y cells treated by inhibitor and PD98059 significantly increased compared with the group treated only by inhibitor. * P<0.05 versus the

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control group, **P < 0.01 versus control group. All experiments were replicated at least three times.

Fig. 4. Increased apoptosis in the hippocampus and upregulation of the microRNA-34a level after propofol treatment. A. The protein expression levels of Bax, cleaved caspase-3, and caspase8 were determined by western blot. B-D. Quantification of signal intensities from Western blots. E. After propofol treatment the hippocampal microRNA-34a level significantly increased compared with the control group (n=5 per group). F. Propofol treatment significantly prolonged the latency

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time (n=5 per group). There was no significant difference in swimming speed between the two groups (data not shown). G. The apoptosis rate (i.e., the percentage of TUNEL-positive cells in the

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hippocampus) in the propofol treatment group increased significantly compared with the control

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group, * P<0.05 versus the control group, **P < 0.01 versus control group (n=5 per group).

Fig. 5. The microRNA-34a inhibitor improved cognitive function by suppressing apoptosis. A. The application of microRNA inhibitor significantly improved latency time (n=5 per group). B. The apoptosis rate was analyzed by the percentage of TUNEL-positive cells. C. The protein expression levels of cleaved caspase-3, caspase-8 and Bax were determined by western blot. D. Quantification of signal intensities from Western blots. * P<0.05 versus the control group, **P < 0.01 versus

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control group (n=5 per group).