Propofol inhibited apoptosis of hippocampal neurons in status epilepticus through miR-15a-5p/NR2B/ERK1/2 pathway

ABSTRACT

Although a previous study reported that propofol had a therapeutic effect in status epilepticus (SE), the mechanisms underlying the effect of propofol in SE remain unclear. The aim of this study was to explore the regulatory mechanisms underlying propofol-induced inhibition of SE.

A rat SE model was established using the lithium–pilocarpine injection method. A qRT-PCR and Western blot were utilized to detect the expression of relative molecules. Cell apoptosis was evaluated by a flow cytometry assay. The interaction between miR-15a-5p and NR2B was assessed using a luciferase reporter assay.

Propofol inhibited cell apoptosis and increased miR-15a-5p expression both in hippocampal tissues of SE rats and low Mg²⁺-induced hippocampal neurons. Propofol-induced attenuation of apoptosis of low Mg²⁺-induced hippocampal neurons was mediated by miR-15a-5p. miR-15a-5p targeted NR2B and negatively regulated its expression. Propofol downregulated NR2B expression, mediated by miR-15a-5p. In terms of the mechanism of action, propofol suppressed the apoptosis of Mg²⁺-induced hippocampal neurons through the miR-15a-5p/NR2B/ERK1/2 pathway. In vivo experiment suggested that propofol inhibited the apoptosis of hippocampal neurons in SE rats by upregulating miR-15a-5p.

In terms of the molecular mechanism of propofol, it appears to inhibit apoptosis of hippocampal neurons in SE through the miR-15a-5p/NR2B/ERK1/2 pathway. The findings provide theoretical support for propofol treatment of SE.

1. Introduction

Status epilepticus (SE) is a common neurological condition characterized by prolonged seizures or intermittent seizures and unconsciousness [1]. SE poses a serious threat to life and health, with high morbidity and mortality [2]. Therefore, developing effective drugs to control SE is important.

Propofol, 2, 6-diisopropyl phenol, is a widely used short-acting intravenous anesthetic in the clinic. Previous studies demonstrated that propofol had therapeutic value in various diseases, including various types of cancer [3], and hypoxia/reoxygenation injury [4]. In the 1990s, propofol was reported to exert a therapeutic effect on SE [5]. Recent studies confirmed the therapeutic effect of propofol on SE in animals and humans [6,7]. Although the therapeutic role of propofol in controlling SE has been proven, the regulatory mechanisms by which propofol inhibits SE remain unclear.

MicroRNAs (miRNAs), a class of small noncoding RNAs (approximately 22 nucleotides), are involved in the regulation of multiple diseases, including SE [8]. In recent years, many studies provided evidence that several miRNAs, such as miR-54, miR-324-5p, miR-345-5p, and miR-344b-2-3p, were dysregulated in SE and implicated these miRNAs in epileptogenesis [9–11]. As reported, miR-199a-5p provided protection against SE by modulating the SIRT1-p53 cascade [12]. It also downregulated miR-146a and inhibited SE by regulating the nuclear factor-κB pathway [13]. Furthermore, miR-187-3p affected SE by influencing the KCNK10/TREK-2 pathway [14].

The miRNA miR-15a-5p plays a role in various diseases. According to a previous study, in osteoarthritis chondrocytes, miR-15a-5p regulated cell viability and matrix degradation by targeting VEGFA [15]. In chronic myeloid leukemia, miR-15a-5p negatively modulated cell viability and metastasis by targeting CXCL10 [16]. Based on miRNA sequencing and a qRT-PCR assay, miR-15a-5p was expressed at a low level in the serum of SE patients [17]. However, the potential role of miR-15a-5p in SE has not been studied.

In the current study, we focused on the molecular mechanism underlying propofol-induced inhibition of SE. Our results showed that propofol decreased cell apoptosis and increased miR-15a-5p expression both in hippocampal tissues of SE rats and in low Mg²⁺-induced hippocampal neurons. Propofol-induced inhibition of apoptosis of hippocampal neurons was mediated by miR-15a-5p, which targeted NR2B. Our results provide evidence that propofol suppresses apoptosis of hippocampal neurons through the miR-15a-5p/NR2B/ERK1/2 pathway.

2. Materials and methods

2.1. Establishment of an SE model and propofol treatment

Sprague-Dawley (SD) rats (180–220 g) were purchased from Better Biotechnology Co., Ltd (Nanjing, China). The animals were maintained under standard conditions (20–25°C, 12 h light/dark cycle, and 50–60% humidity) and had access to food and water ad libitum. Before the experiments, the animals were allowed to adapt to the housing environment for 1 wk. The experiments were approved by the animal ethics committee of The First Affiliated Hospital of Zhengzhou University.

The rats were randomly divided into three groups: a sham group (n = 6), an SE group (n = 6), and an SE + propofol group (n = 6). A rat model of SE was created using the lithium–pilocarpine injection method, as described previously [13]. Briefly, the rats were intraperitoneally injected with lithium chloride (127 mg/kg) and atropine sulfate (1 mg/kg) 18 h later to prevent peripheral adverse reactions induced by pilocarpine. After 30 min, the rats were given an intraperitoneal injection of pilocarpine hydrochloride (30 mg/kg).

The Racine scale was used to assess the severity of seizures. Rats with stage IV or V seizure severity according to the Racine scale were considered a successful SE model. Thirty minutes after continuous seizures, propofol (50 mg/kg) was injected intraperitoneally. The rats in the sham group (control) were injected with normal saline of equal volume intraperitoneally. After the establishment of the SE model, the latency and frequency of the seizures were recorded. All the rats were sacrificed after 24 h, and hippocampal tissues were collected for further study.

2.2. Isolation and culture of primary rat hippocampal neurons

Primary rat hippocampal neurons were isolated from brains of embryonic d 18 SD rats as previously described [18]. In brief, the embryos were separated from maternal rats and then euthanized by decapitation in ice-cold dissection medium containing Hank’s buffered salt solution, 0.01 M HEPES, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Hippocampal tissues were isolated from the brains of embryos and digested with preheated trypsin, followed by trituration using a Pasteur pipette. The cells were then collected by centrifugation and cultured in Neurobasal culture medium, supplemented with 2% B27 serum-free supplement (Invitrogen, USA), 100 U/mL of penicillin (Invitrogen), 100 μg/mL of streptomycin (Invitrogen), 0.5 mM glutamine (Invitrogen, USA), and 10 μM glutamate (Invitrogen, USA) at 37°C, 5% CO₂.

2.3. Low Mg²⁺ induction

Low Mg²⁺ induction of hippocampal neurons was conducted as previously reported [19]. Dissected hippocampal neurons were pretreated with 10 μg/mL of propofol. Then, 2 h later, the cells were placed in an MgCl₂-free physiological bath recording solution (pBRS) to induce SE-like electrographic activity under low Mg²⁺ conditions. The hippocampal neurons in the control group were placed in pBRS containing 1 mM MgCl₂.

2.4. Whole-cell current-clamp recording

The hippocampal neurons were placed in an extracellular solution supplemented with NaCl (137 mM), KCl (5 mM), MgCl₂ (1 mM), CaCl₂ (2 mM), glucose (20 mM), and HEPES (10 mM). The osmolarity and pH of the extracellular solution were adjusted to 310–320 mOsM and 7.40, respectively. Glass pipettes were filled with internal medium containing CsCl (137 mM), MgCl₂ (2 mM), CaCl₂ (1 mM), EGTA (11 mM), ATP (3 mM), and HEPES (10 mM), with input resistance of 4–7 MΩ. The osmolarity and pH of the internal medium were adjusted to 290–300 mOsM and 7.30, respectively. Whole-cell current-clamp recordings were performed using a Patch Clamp PC-505 B (Warner Instruments, USA) amplifier in the current-clamp mode. The data were transferred to a computer and analyzed using relevant software [20].

2.5. Cell transfection

The hippocampal neurons were transfected with an miR-15a-5p inhibitor, miR-15a-5p mimic, si-NR2B, and corresponding controls (negative control [NC], pre-NC, and si-control) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

2.6. Flow cytometry assay

To evaluate the apoptosis of hippocampal neurons, a flow cytometry assay was conducted using Annexin VFITC staining kits (Bimake, China). The hippocampal neurons were seeded in an 48-well plate at a concentration of 1 × 10⁵ cells/well. Annexin V-FITC and PI were added to each well, and the cells were incubated for 15 min in the dark. Prior to analysis, binding buffer was added to every well. The apoptosis of hippocampal neurons was analyzed using Summit v4.3 software.

2.7. TUNEL staining

To assess cell apoptosis in rat hippocampal tissues, TUNEL staining was performed (One Step TUNEL Apoptosis Assay Kit; Beyotime, China). Briefly, the hippocampal tissue was cut into sections 5 µm thick after fixation, dehydration, transparency, and paraffin embedding. The sections were dewaxed with dimethylbenzene, followed by treatment with DNase-free protease K. After washing with PBS buffer, TUNEL detection solution (50 µL) was dripped onto each slice. The sections were then observed under a fluorescence microscope (Olympus, Japan).

2.8. CCK-8 assay

The viability of the hippocampal neurons was determined using a Cell Counting Kit-8 (Beyotime, China) according to the manufacturer’s instructions. In brief, the cells were seeded in a 48-well plate at a density of 2 × 10³ cells/well. Subsequently, CCK-8 solution (10 µl) was added to each well. After incubation at 37°C, 5% CO₂ for 24 h, the absorption at a wavelength of 450 nm was measured using a microplate reader (BioTek, USA).

2.9. Luciferase reporter assay

A rat NR2B 3′-UTR fragment containing a wild-type (WT) miR-15a-5p binding site or mutated (MUT) miR-15a-5p binding site was inserted into a vector encoding luciferase. The hippocampal neurons were then co-transfected with a luciferase reporter gene vector (NR2B 3′-UTR-WT or NR2B 3′-UTR-MUT) and miR-15a-5p inhibitor (or NC). Post-48 h of transfection, relative luciferase activity was measured using a Dual Luciferase (Promega, USA) kit.

2.10. RNA extraction, cDNA synthesis, and qRT-PCR

RNA was extracted from rat hippocampal tissues and hippocampal neurons using Trizol (Takara, Japan) and phenol-chloroform. To synthesize cDNA, 1000 ng of RNA and an RT reagent kit (Takara, Japan) were used. For the qRT-PCR, an SYBR Premix Ex Taq™ kit (Takara, Japan) and IQ5 Fast Realtime System (Bio-Rad, USA) were used. The relative expression of miRNAs and NR2B was normalized to U6 and β-actin, respectively. The 2^−ΔΔCt method was used to calculate relative expression.

2.11. Western blot

For protein isolation, the rat hippocampal tissues and hippocampal neurons were lysed using RIPA lysis buffer. The concentration of protein was determined using a BCA kit (CWBIO, China). After separation of the proteins by SDS-PAGE, the proteins were transferred on PVDF membranes and incubated with primary antibodies overnight. The PVDF membranes were then incubated with corresponding secondary antibodies for 1 h at room temperature. Subsequently, the immunoreactive bands were visualized using an ECL kit (Beyotime, China). The primary antibodies were as follows: anti-NR2B (Abcam, 1:500), anti-ERK1/2 (Abcam, 1:1000), anti-p-ERK1/2 (Abcam, 1:1000), anti-cleaved caspase-3 (Abcam, 1:1000), and anti-β-actin (anti-β-actin). β-Actin was used as a reference.

2.12. Intracerebroventricular injection

The miR-15a-5p inhibitor was injected into the lateral ventricle of the SD rats using the stereotactic method [21]. Briefly, the rats were anesthetized and then they were intubated and injected with 50 μL of the miR-15a-5p inhibitor or NC via the intracerebroventricular route. Six hours later, the rats were used to create an SE model using the lithium–pilocarpine method as described above. After 30 min of continuous seizures, the rats were injected with propofol (50 mg/kg) intraperitoneally. The rats in the sham group were injected with normal saline of equal volume intraperitoneally. After the establishment of the SE model, the latency and frequency of seizures were recorded. All the rats were sacrificed after 24 h, and hippocampal tissues were collected for further study.

2.13. Statistical analysis

All data were analyzed using SPSS 22.0 software and expressed as the mean ± standard deviation. The Student’s t-test and a one-way analysis of variance were used to compare differences between the groups. A P value of <0.05 was considered statistically significant.

3. Results

3.1. Propofol attenuated apoptosis of nerve cells and upregulated miR-15a-5p in SE rats

To investigate the role of propofol in SE, we established a rat model of SE using the lithium–pilocarpine method and then treated the SE rats with propofol. We first verified the effects of propofol on the latency and frequency of seizures in rats. The results showed that propofol increased the latency and decreased the frequency of seizures in SE rats (Figure 1(a)). As a previous study demonstrated that nerve cell apoptosis was involved in the pathogenesis of SE [22], we then evaluated the impacts of propofol on cell apoptosis in hippocampal tissues. Flow cytometry showed that cell apoptosis was significantly inhibited in the SE + propofol group as compared with that in the SE group (Figure 1(b)). As shown by TUNEL staining, the number of TUNEL-positive cells in the SE + propofol group was lower than that in the SE group (Figure 1(b)). Thus, both the flow cytometry and TUNEL staining results revealed that propofol attenuated nerve cell apoptosis in SE rats. We then assessed the effect of propofol on five miRNAs previously shown to be dysregulated in SE [9–11,23]. Of these five abnormally expressed miRNAs, only the expression of miR-15a-5p was reversed inceased by propofol in SE rats, while the expressions of the other four miRNAs were not altered by propofol (Figure 1(c)). Those results suggested that propofol-induced inhibition of SE might be associated with miR-15a-5p.

Figure 1.

Effect of propofol on the apoptosis of hippocampal cells and miR-15a-5p in SE rats. An SE rat model was established by the lithium–pilocarpine intraperitoneal injection method. The SE rats were treated with propofol (50 mg/kg). (a) The effect of propofol on the latency and frequency of seizures of SE rats. (b) Cell apoptosis of hippocampal tissues isolated from rats, as detected by flow cytometry and a TUNEL staining assay. (c) The relative expression of miRNAs in hippocampal tissue was examined by a qRT-PCR. n = 6; ***P < 0.001 vs. the sham group, ^###P < 0.05 vs. the SE group.

3.2. Propofol inhibited apoptosis of low Mg²⁺-induced hippocampal neurons and increased miR-15a-5p expression

To explore the mechanism of propofol-induced inhibition of apoptosis of hippocampal neurons in vitro, primary hippocampal neurons were induced by low Mg²⁺ conditions to imitate an SE model. As shown by SE-like electrogram activity, low Mg²⁺ induction increased the spike frequency of hippocampal neurons, and propofol changed the effect of Mg²⁺ induction on the spike frequency (Figure 2(a)). Low Mg²⁺ induction suppressed miR-15a-5p expression in hippocampal neurons, whereas propofol-induced miR-15a-5p expression (Figure 2(b)). The flow cytometry results revealed that low Mg²⁺ induction contributed to apoptosis of hippocampal neurons and that propofol reversed this effect (Figure 2(d)). As demonstrated by the CCK-8 assay, low Mg²⁺ stimulation inhibited the vitality of hippocampal neurons, and propofol reversed this effect (Figure 2(c)). The Western blot analysis revealed that propofol reduced the protein levels of cleaved caspase-3 and Bax in low Mg²⁺-induced hippocampal neurons and that it increased the protein levels of procaspase-3 and Bcl-2 (Figure 2(e)).

Figure 2.

Effect of propofol on the apoptosis of hippocampal cells and miR-15a-5p in low Mg²⁺-induced hippocampal neurons. Primary rat hippocampal neurons were isolated from brains of embryonic d 18 SD rats. The cells were pretreated with 10 μg/mL of propofol for 2 h, followed by low Mg²⁺ induction for another 24 h. Control cells were exposed to pBRS containing 1 mM MgCl₂ and no propofol treatment. (a) The spike frequency of hippocampal neurons was detected. (b) The relative expression of miR-15a-5p in hippocampal neurons was detected by a qRT-PCR. (d) Apoptosis of hippocampal neurons was assessed by a flow cytometry assay. (c) The viability of hippocampal neurons was measured by a CCK-8 assay. (e) The protein levels of cleaved caspase-3, procaspase-3, Bax, and Bcl-2 were determined by a Western blot. NS: no significant difference. **P < 0.01, ***P < 0.001 vs. the control group, ^#P < 0.05, ^##P < 0.01 vs. the low Mg²⁺ group.

3.3. Propofol-induced suppressions of apoptosis of hippocampal neurons were mediated by miR-15a-5p

To investigate whether propofol-induced suppression of apoptosis of hippocampal neurons involved miR-15a-5p, hippocampal neurons were treated with propofol following transfection with an miR-15a-5p inhibitor. Cell apoptosis and cleaved caspase-3 protein level were then detected by a flow cytometry assay and a Western blot, respectively. Propofol abrogated the increase in cell apoptosis induced by low Mg²⁺, and this effect was reversed by miR-15a-5p silencing (Figure 3(a)). Propofol also abolished the upregulation of cleaved caspase-3 protein expression induced by low Mg²⁺, and this effect was reversed by miR-15a-5p inhibitor transfection (Figure 3(b-c)). These results indicated that propofol suppressed the apoptosis of hippocampal neurons, mediated by miR-15a-5p.

Figure 3.

Propofol-induced suppression of apoptosis of hippocampal neurons was mediated by miR-15a-5p. Hippocampal neurons were transfected with an miR-15a-5p inhibitor (or NC) and then treated with 10 μg/mL of propofol for 2 h, followed by low Mg²⁺ induction for another 24 h. Control cells were exposed to pBRS containing 1 mM MgCl₂ and no propofol treatment. (a) Apoptosis of hippocampal neurons was assessed by a flow cytometry assay. (b and c) The protein level of cleaved caspase-3 in hippocampal neurons was determined by a Western blot. NC: negative control of the miR-15a-5p inhibitor. **P < 0.01 vs. the control group, ^##P < 0.01 vs. the low Mg²⁺ group, ^&P < 0.05 vs. the low Mg²⁺ + propofol + NC group.

3.4. MiR-15a-5p targeted NR2B

A bioinformatics analysis using miRanda software showed that NR2B might be a downstream target of miR-15a-5p (Figure 4(a)). Thus, we performed a luciferase reporter assay to confirm whether miR-15a-5p targeted NR2B. The results showed that miR-15a-5p knockdown significantly increased the luciferase activity of the WT NR2B 3′-UTR, as well as the mRNA and protein levels of NR2B, whereas it had no apparent effect on the luciferase activity of the 3′-UTR of MUT NR2B (Figure 4(b)). In contrast, miR-15a-5p overexpression markedly reduced the luciferase activity of the WT NR2B 3′-UTR and the expression of NR2B, whereas it had no significant impact on the luciferase activity of the MUT NR2B 3′-UTR (Figure 4(c)). The results suggested that miR-15a-5p targeted NR2B and negatively regulated its expression.

Figure 4.

MiR-15a-5p targeted NR2B. (a) The predicted miR-15a-5p binding site on the NR2B 3′-UTR. A fragment of NR2B containing a wild type or mutated miR-15a-5p binding site was cloned into vectors carrying the luciferase gene. The recombinant vectors and miR-15a-5p inhibitor (or miR-15a-5p mimic) were co-transfected into hippocampal neurons. (b) The effect of the miR-15a-5p inhibitor on relative luciferase activity was detected using a luciferase reporter assay. The effect of the miR-15a-5p inhibitor on the expression of NR2B was determined by qRT-PCR and Western blot methods. (c) The effect of the miR-15a-5p mimic on relative luciferase activity was detected using a luciferase reporter assay. The effect of the miR-15a-5p mimic on the expression of NR2B was determined by qRT-PCR and Western blot methods. NC: negative control of the miR-15a-5p inhibitor; pre-NC: negative control of the miR-15a-5p mimic. **P < 0.05 vs. the NC group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the pre-NC group.

3.5. Propofol-induced repression of NR2B expression was mediated by miR-15a-5p

A previous study reported that NR2B was involved in the pathogenesis of SE and that propofol inhibited its expression [7]. To further clarify whether propofol regulation of NR2B expression was mediated by miR-15a-5p, hippocampal neurons were treated with propofol after transfection with an miR-15a-5p inhibitor. We then examined the expression of miR-15a-5p and NR2B. As shown in Figure 5(a), propofol eliminated the downregulation of miR-15a-5p caused by low Mg²⁺ induction, and miR-15a-5p inhibitor transfection reversed this effect. In addition, propofol suppressed the expression of NR2B in low Mg²⁺-stimulated hippocampal neurons, and miR-15a-5p knockdown altered this trend (Figure 5(b-c)). Thus, the results demonstrated that propofol-induced inhibition of NR2B expression was mediated by miR-15a-5p.

Figure 5.

Propofol-induced repression of NR2B expression was mediated by miR-15a-5p. Hippocampal neurons were transfected with an miR-15a-5p inhibitor (or NC) and then treated with 10 μg/mL of propofol for 2 h, followed by low Mg²⁺ induction for another 24 h. Control cells were exposed to pBRS containing 1 mM MgCl₂ and no propofol treatment. (a) The expression of miR-15a-5p in hippocampal neurons was detected by a qRT-PCR. (b and c) The protein level of NR2B in hippocampal neurons was determined by a Western blot. NC: negative control of the miR-15a-5p inhibitor. ***P < 0.001 vs. the control group, ^##P < 0.01 vs. the low Mg²⁺ group, ^&&P < 0.05 vs. the low Mg²⁺ propofol + NC group.

3.6. Propofol inhibited hippocampal neuron apoptosis via the miR-15a-5p/NR2B/ERK1/2 pathway

Previous research demonstrated that the ERK1/2 pathway was involved in the development of SE and that NR2B activated the ERK1/2 pathway [24]. To determine whether propofol regulated ERK1/2 activation via the miR-15a-5p/NR2B pathway, hippocampal neurons were treated with propofol after transfection with silencing vectors of miR-15a-5p and NR2B. Silencing of miR-15a-5p canceled the effect of propofol on the activation of ERK1/2 in low Mg²⁺-stimulated hippocampal neurons, and NR2B knockdown reversed this trend. In addition, miR-15a-5p overexpression inhibited the activation of ERK1/2 in low Mg²⁺-stimulated hippocampal neurons, and NR2B overexpression altered this effect (Figure 6(a)). These results indicated that propofol suppressed ERK1/2 activation through the miR-15a-5p/NR2B pathway. To confirm the role of ERK1/2 in cell apoptosis regulated by propofol, hippocampal neurons were treated with propofol and ERK1/2 agonists (NGF). Propofol attenuated cell apoptosis of low Mg²⁺-induced hippocampal neurons. In contrast, NGF increased cell apoptosis (Figure 6(b)). These results revealed that propofol-induced attenuation of cell apoptosis was associated with inhibition of ERK1/2 activation.

Figure 6.

Propofol inhibited hippocampal neurons apoptosis via the miR-15a-5p/NR2B/ERK1/2 pathway. (a, c, and d) Hippocampal neurons were transfected with an miR-15a-5p inhibitor (or mimic) or si-NR2B and treated with 10 μg/mL of propofol for 2 h, followed by low Mg²⁺ induction for another 24 h. Control cells were exposed to pBRS containing 1 mM MgCl2 and no propofol treatment. (a) The protein levels of ERK1/2 and p-ERK1/2 were detected by a Western blot. (b) Hippocampal neurons were treated with 100 ng/mL of an ERK1/2 agonist (NGF) and 10 μg/mL of propofol 2 h before low Mg²⁺ stimulation. The apoptosis of hippocampal neurons was assessed using a flow cytometry assay. (c) The apoptosis of hippocampal neurons was assessed using a flow cytometry assay. (d) The protein levels of cleaved caspase-3, procaspase-3, Bax, and Bcl-2 were determined by a Western blot. NC: negative control of the miR-15a-5p inhibitor; pre-NC: negative control of the miR-15a-5p mimic; Si-control: negative control of si-NR2B; pcDNA: negative control of pcDNA-NR2B.

To determine whether propofol affected apoptosis via the miR-15a-5p/NR2B axis, hippocampal neurons were treated with propofol after transfection with an miR-15a-5p inhibitor and si-NR2B. As expected, silencing miR-15a-5p altered the influence of propofol on apoptosis of low Mg²⁺-stimulated hippocampal neurons, whereas NR2B knockdown abolished this effect (Figure 6(c)). Likewise, miR-15a-5p knockdown abrogated the effect of propofol on the protein level of cleaved caspase-3 in low Mg²⁺-stimulated hippocampal neurons, and si-NR2B transfection reversed this effect (Figure 6(d)). Collectively, these results demonstrated that propofol inhibited hippocampal neurons apoptosis through the miR-15a-5p/NR2B/ERK1/2 pathway.

3.7. Propofol-induced inhibition of apoptosis of hippocampal cells was mediated by miR-15a-5p in SE rats

To demonstrate that propofol-induced inhibition of cell apoptosis involved miR-15a-5p in vivo, an miR-15a-5p inhibitor was injected into the lateral ventricle of rats, and an SE model was created 6 h later by a lithium–pilocarpine intraperitoneal injection. The results showed that propofol increased the latency and decreased the frequency of seizures in SE rats, whereas miR-15a-5p silencing altered these effects (Figure 7(a)). Propofol treatment inhibited cell apoptosis of hippocampal tissues in SE rats, whereas miR-15a-5p knockdown reversed this trend (Figure 7(b)). In addition, propofol upregulated miR-15a-5p expression and downregulated the NR2B protein level in hippocampal tissues of SE rats, and these effects were reversed by silencing miR-15a-5p (Figure 7(c)).

Figure 7.

Propofol-induced inhibition of neuronal apoptosis was mediated by miR-15a-5p in SE rats. An miR-15a-5p inhibitor was injected into the lateral ventricle of SD rats using the stereotactic method. Then, 6 h later, an SE rat model was created by a lithium–pilocarpine intraperitoneal injection. After 30 min of continuous seizures, the rats were injected with propofol (50 mg/kg) intraperitoneally. (a) The latency and frequency of seizures in the rats. (b) The apoptosis of hippocampal tissues isolated from rats was detected by a flow cytometry assay. (c) The expression of miR-15a-5p and NR2B in hippocampal tissues. n = 6; ***P < 0.001 vs. the sham group, ^###P < 0.001 vs. the SE group, ^&&&P < 0.05 vs. the SE + propofol +NC group.

4. Discussion

According to the literature, propofol regulates miRNA expression in many diseases. In gastric cancer, propofol inhibited cell proliferation and invasion by downregulating miR-221 expression [3]. In hypoxia/reoxygenation injury, propofol exerted neuroprotective effects by reducing the expression of miR-134 [4]. Furthermore, propofol suppressed anthracycline-induced cardiomyocyte apoptosis by targeting miR-181a [25]. In this study, in hippocampal tissues of SE rats, propofol resulted in downregulation of miR-15a-5p, miR-324-5p, and miR-345-5p and upregulation of miR-344b-2-3p and miR-54. Among these five abnormally expressed miRNAs, propofol treatment increased the expression of only miR-15a-5p in SE rats, while had no significant effect on the expressions of the other four miRNAs. An in vitro cell experiment confirmed that propofol downregulated miR-15a-5p expression in low Mg²⁺-stimulated hippocampal neurons. In addition, the results showed that propofol suppressed apoptosis of low Mg²⁺-stimulated hippocampal neurons and reduced the protein level of apoptosis-related proteins (cleaved caspase-3), whereas miR-15a-5p silencing abrogated these effects. Together, these results suggested that propofol inhibited apoptosis of hippocampal neurons by upregulating miR-15a-5p expression in SE.

Many studies reported that miRNAs exerted functions in SE by regulating the expression of target genes. For example, downregulating miR-146a alleviated lithium-pilocarpine-induced SE by regulating the nuclear factor-κB pathway [13], and miR-187-3p affected SE by mediating the regulation of the KCNK10/TREK-2 potassium channel [14]. In addition, miRNAs regulated N-methyl-D-aspartate (NMDA) receptor (NMDAR) signaling. Kocerha et al. [26] indicated that miR-219 modulated neurobehavioral dysfunction by regulating NMDAR signaling. They confirmed that miR-139-5p regulated NR2A expression negatively in SE rats and in patients with temporal lobe epilepsy [27]. In the present study, we predicted a miR-15a-5p binding site on the 3′-UTR of NR2B, suggesting that miR-15a-5p might exert functions by targeting NR2B. To test this prediction, we performed a luciferase reporter assay. The results showed that the relative luciferase activity of NR2B 3ʹ-UTR increased following miR-15a-5p silencing and decreased following miR-15a-5p overexpression when the miR-15a-5p -binding site was not mutated. These data suggested that miR-15a-5p targeted NR2B. Our results also revealed that silencing miR-15a-5p caused upregulation of NR2B, whereas overexpressing miR-15a-5p resulted in downregulation of NR2B. Knockdown of miR-15a-5p abolished the effect of propofol on the NR2B protein level. Thus, the present results demonstrated that miR-15a-5p targeted NR2B and that propofol regulation of NR2B expression was mediated by miR-15a-5p.

It has been confirmed that NR2B, an NMDAR subunit, participated in SE. A previous study indicated that upregulation of NR2B in the cortex contributed to epileptogenesis in humans [28]. In an in vitro model of SE, overexpression of NR2B in hippocampal neurons triggered neuronal hyper-excitability [29]. In pilocarpine-induced SE, activation of NR2B promoted SE-induced neuronal cell apoptosis due to phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) [24,30]. In the present study, an increase in the NR2B protein level was accompanied by activation of ERK1/2 in low Mg²⁺-stimulated hippocampal neurons. This result implied that ERK1/2 activation was mediated by high expression NR2B in SE. Previous reports also indicated that propofol affected the NR2B/ERK1/2 pathway. For instance, propofol effectively controlled SE by downregulating NR2B expression [7], and it exerted an analgesic effect by regulating the NMDAR/ERK1/2 signaling pathway in inflammation-induced pain [31]. Studies also reported that propofol inhibited NMDAR-mediated ERK1/2 activation in hippocampal neurons [32,33]. Similarly, in the present study, propofol reduced the protein levels of NR2B and p-ERK1/2 in low Mg²⁺-stimulated hippocampal neurons. In addition, miR-15a-5p knockdown reversed the effect of propofol on ERK1/2 activation and cell apoptosis, as well as the cleaved caspase-3 protein level, and NR2B silencing reversed these trends. Therefore, these results demonstrated that propofol inhibited apoptosis of hippocampal neurons and controlled SE via the miR-15a-5p/NR2B/ERK1/2 pathway.

In the current study, we conducted only a preliminarily analysis of the mechanisms by which propofol regulates the apoptosis of nerve cells via the miRNA-15a/NR2B/ERK1/2 pathway in SE. Various potential mechanisms underlying the actions of propofol in SE need to be further studied. Recently, inflammasomes have attracted widespread attention due to their important role in the occurrence and development of nervous system diseases. Some studies reported that the inflammasome NLRP1 directly bound to the caspase-1 precursor and led to pyroptosis of nerve cells, thereby causing neuropathy [34,35]. In a future study, we intend to investigate the role of neuronal pyroptosis in the development of SE and to explore whether propofol regulates neuronal pyroptosis in SE.

In summary, this study sheds light on one molecular mechanism, regulation of the miR-15a-5p/NR2B/ERK1/2 pathway, by which propofol suppresses apoptosis of hippocampal neurons in SE. Our findings provide theoretical support for propofol treatment of SE.

Funding Statement

This study was supported by grants from the National Natural Science Foundation Youth Project of China [No.81601201].

Disclosure statement

No potential conflict of interest was reported by the authors.