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Abstract

Objective

Ketamine is an anesthetic that induces neurotoxicity when administered at high doses. In this work, we explored the protective effects of lipoxin A₄ methyl ester (LXA₄ ME) against ketamine-induced neurotoxicity and the underlying protective mechanism in pheochromocytoma (PC12) cells.

Methods

PC12 cells were treated with 50 μM of ketamine and different LXA₄ ME concentrations of LXA4 ME (5–50 nM) for 24 h, and their viability, apoptosis, and oxidative status were assessed.

Results

Quantitative real-time polymerase chain reaction experiments showed that ketamine downregulated miR-22 expression and upregulated Bcl-2-associated athanogene 5 (BAG5) in PC12 cells in a concentration-dependent manner. LXA₄ ME induced the opposite effects, thus attenuating ketamine-induced neurotoxicity. Further in vitro assays showed that miR-22 directly targeted BAG5, thus promoting cell viability by suppressing cell apoptosis and oxidative stress. Under expression miR-22 or upregulation of BAG5 antagonized the effects of LXA₄ ME.

Conclusion

LXA₄ ME can protect PC12 cells from ketamine-induced neurotoxicity by activating the miR-22/BAG5 signaling pathway. Thus, LXA₄ ME can be used as a protective drug against ketamine-induced neural damage.

Keywords

ketamine neurotoxicity miR-22 BAG5

Introduction

Ketamine, a N-methyl-d-aspartate receptor blocker, is widely used in the clinic as an anesthetic and analgesic.¹ However, prolonged exposure to ketamine may lead to cortical neuronal death and cause permanent memory deficit.^2,3 Ketamine can also promote cortical neurotoxicity, leading to neuronal apoptosis and enhanced generation of reactive oxygen species (ROS).^4,5 The mechanisms of ketamine-stimulated neurotoxicity remain unclear, although it is known to involve multiple factors.⁶ Greater understanding of this neurotoxicity may help guide the development of therapies against it.

MicroRNAs (miRNAs) are small non-coding RNA molecules expressed in most eukaryotes, including humans,⁷ and their discovery has significantly contributed to the elucidation of mechanisms that regulate gene expression under various physiological and pathological conditions. Several studies have shown that miRNAs can silence the translation of target proteins by binding to the 3′-untranslated region (UTR) of the target mRNA.⁸ Among the miRNAs highly expressed in brain tissue that regulate several neurological functions,⁹ miR-22 is involved in various processes, such as the regulation of neuronal cell apoptosis and of the growth and apoptosis of pheochromocytoma (PC12).¹⁰ It is well known that PC12 cells exert typical neuron features and highly express glucocorticoid receptors, which have been widely used as an in vitro model to study the neuronal injury.¹¹ This miR-22 exerts its effects by targeting transient receptor potential melastatin 7.¹² Its potential role in ketamine-induced neurotoxicity has not yet been clearly investigated.

Lipoxin A₄ methyl ester (LXA₄ ME) is a relatively stable lipoxin analog that is more potent than endogenous lipoxins.¹³ It exerts neuroprotective effects against early brain injury,¹⁴ cognitive deficits,¹⁵ and vascular cognition impairment.¹⁶ Given that LXA₄ acts as an anti-inflammatory agent by regulating miRNAs, such as miR-21 or miR-126–5p,^17,18 we hypothesized that LXA₄ ME could reduce ketamine-induced neurotoxicity in neuronal cells.

Therefore, in the present study, we explored the role of miR-22 in ketamine-induced neurotoxicity, and the ability of LXA₄ ME to protect against this toxicity. We examined whether LXA₄ ME might exert its neuroprotective effects via miR-22 and Bcl-2-associated athanogene 5 (BAG5).

Materials and methods

Cell culture and treatment

PC12 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37°C in a humidified 5% CO₂ atmosphere. The medium was changed every 48 h. At the end of treatments, cells were trypsinized with 0.25% trypsin and collected.

Transfection with lentiviral vectors

Lentiviral vectors were obtained from Hanbio (Wuhan, China) encoding each of the following: an miR-22 mimics (lenti-miR-22-mimics), miR-22 control mimics (lenti-NC-mimics), miR-22 inhibitor (lenti-miR-22-inhibitor), miR-22 control inhibitor (lenti-NC-inhibitor), BAG5 mimic (lenti-BAG5), or empty control vector (lenti-vector). The lentiviral vectors were transfected into PC12 cells following the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of miR-22 and BAG5 in transfected PC12 cells.

Cell viability assay

PC12 cells were cultured in 96-well plates and treated for 24 h with 50 μM of ketamine and different concentrations of LXA₄ ME (1–100 nM). Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 1 h. The optical density (OD) was measured at 450 nm by a microplate reader (Bio-Rad, Hercules, CA, USA), and the relative cell viability (%) was defined as OD_experiment/OD_control × 100.

To further assess cell viability after treatment with ketamine and LXA₄ ME, the amount of lactate dehydrogenase (LDH) in the cell medium was estimated using a commercial kit. The OD values at 450 nm were measured using a microplate reader.

Caspase-3 or -9 activity assay

Caspase-3 and caspase-9 assay kits (Sigma-Aldrich, St Louis, MO, USA) were used to evaluate the activity of caspases three and nine in PC12 cells. In brief, the cells were suspended in lysis buffer and incubated on ice for 15 min. The cell homogenates were then centrifuged at 13,000 r/min and 4°C for 12 min, and the collected supernatants were incubated at 37°C for 2 h with the caspase-3DEVD-p-NA substrate and reaction buffer containing DTT. The absorbance was measured at 405 nm.

Oxidative status assessment

After treatment, the PC12 cells were dissolved in radio-immunoprecipitation (RIPA) lysis buffer (Sigma-Aldrich) on ice for 15 min. The cell homogenates were then centrifuged at 13,000 r/min and 4°C for 12 min, and the supernatants were collected. The protein concentration in each sample was measured using the bicinchoninic acid (c) protein assay kit (Sigma-Aldrich). The levels of malondialdehyde (MDA) and ROS, as well as the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were evaluated using an enzyme-linked immunosorbent assay (Huamei Biotechnology, Wuhan, China) following the manufacturer’s instructions.

qRT-PCR

Total RNA was extracted from PC12 cells using TRIzol reagent (Invitrogen). A TaqMan reverse transcription kit (Sigma-Aldrich) was used to reverse transcribe RNA into cDNA. qRT-PCR was then performed using 2 μL of diluted cDNA (1:20, vol/vol) and a Mx3000 P system (Stratagene, USA). Levels of miR-22 were determined using the miRVana qRT-PCR miRNA detection kit (ABI company), while levels of BAG5 mRNA were measured using a SYBR Green qRT-PCR kit (ABI company). The following primers were used: miR-22 forward, 5′-AAGCUGCCAGUUGAAGAACUGU-3′; miR-22 reverse 5′-ACUACUGAGUGACAGUAGA-3′; BAG5 forward, 5′-CCCCGCTCAGACCTAGT-3′; BAG5 reverse, 5′-TTCTATCAAACGGCTCGCTCA-3′; caspase-3 forward, 5′-ACGCTAAGCTGGGCCCAGTGTTGTACG-3′; caspase-3 reverse, 5′-GTCAAGCCGGATTTGGCTGAAGCTGAG-3′; caspase-9 forward, 5′-CCTTGAGTGCATGTAGGCATAATC-3′; caspase-9 reverse, 5′-CTGGAATGCGTCCTGAAAGTCGATA-3′; β-actin forward, 5′-AACGGATTTGGTCGTATTGGG-3′; and β-actin reverse, 5′-TCGCTCCTGGAAGATGGTGA T-3′. The relative expression of miR-22 and BAG5 was normalized to that of U6 or β-actin and analyzed using the 2^−ΔΔCt method.

Dual-luciferase reporter assay

The wild-type (WT) BAG5 3′-UTR containing the miR-22 complementary pairing sequence or mutant BAG5 3′-UTR with substitutions in this sequence were cloned into pmirGLO (Invitrogen), giving rise to BAG5-WT-pmirGLO and BAG5-Mut-pmirGLO plasmids. HEK293 T cells were co-transfected with miR-22 or negative control (NC) mimics together with BAG5-WT or BAG5-Mut vectors for 48 h using Lipofectamine 2000 (Invitrogen). The luciferase activity was then measured using a dual-luciferase reporter system (Promega, Madison, WI), and the relative luciferase activity was defined as the ratio of firefly to Renilla luciferase activity.

Protein expression assessment

The protein expression was evaluated by Western blot and 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). PC12 cells were dissolved in RIPA lysis buffer (Sigma-Aldrich) on ice for 15 min. The cell homogenates were then centrifuged at 13,000 r/min and 4°C for 12 min, and the supernatants were collected. The protein content in each sample was measured using the BCA protein assay (Sigma-Aldrich). The proteins (50 μg) of each sample were then fractionated by SDS–PAGE and transferred to nitrocellulose membranes. The non-specific membrane binding sites were blocked with 5% skim milk at room temperature for 1.5 h on a shaking table. Membranes were then incubated at 4°C overnight with rabbit anti-mouse monoclonal antibodies (all diluted 1:1000) against BAG5 (ab4435, Abcam, Cambridge, UK) and β-actin (ab6324, Abcam). After washing three times with phosphate-buffered saline (PBS) containing Tween-20, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:4000) at room temperature for 2 h. The proteins were detected using luminol reagent and peroxide solution (Millipore, Billerica, MA, USA), and densitometry images were obtained using Image J software.

Statistical analysis

Statistical analysis was performed using an independent-sample t-test (comparison between two groups) and one-way analysis of variance (multi-group comparison) using GraphPad statistical software 6.0 (GraphPad Prism, Chicago, IL). Data were expressed as mean ± standard deviation (SD). Differences associated with p < 0.05 were considered statistically significant.

Results

LXA₄ ME enhanced the viability of ketamine-treated PC12 cells

The viability of PC12 cells fell significantly with increasing ketamine concentration, and treatment with 50 μM of ketamine reduced cell viability by approximately 50% (Figure 1(a)). Thus, this concentration was selected for subsequent experiments. Treatment of PC12 cells only with LXA₄ ME (1–100 nM) for 24 h did not affect cell viability, indicating that LXA₄ ME was non-toxic (Figure 1(b)).

Figure 1.

Effect of LXA₄ ME on the viability of ketamine-treated PC12 cells. (a,b) Cell viability of PC12 cells after treatment with (a) 0.1–400 μM of ketamine and (b) 1–100 nM of LXA₄ ME for 24 h. (c) Protective effect of LXA₄ ME on ketamine-treated PC12 cells. (d) LDH activity in PC12 cells treated with 50 μM of ketamine and LXA₄ ME (1–100 nM) for 24 h. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to 0 μM ketamine or control group (CN); ^#p < 0.05, compared to 0 μM ketamine. PC12 cells: PC12 cells; LXA₄ ME: lipoxin A₄ methyl ester; LDH: lactate dehydrogenase. LXA₄ ME reduced apoptosis in ketamine-treated PC12 cells.

In order to explore the ability of LXA₄ ME to protect against ketamine-induced neurotoxicity, PC12 cells were simultaneously treated with 50 μM of ketamine and different LXA₄ ME concentrations (1–100 nM) for 24 h. LXA₄ ME significantly increased the viability of ketamine-treated cells (Figure 1(c)) while significantly reducing LDH activity (Figure 1(d)), both in a concentration-dependent manner.

Addition of only 50 μM of ketamine significantly increased apoptosis, an effect associated with upregulation of caspase-3 and caspase-9 expression and activity. LXA₄ ME antagonized these changes in caspase-3 and caspase-9 expression and activity, in a concentration-dependent manner (Figures 2(a)–(d)).

Figure 2.

LXA₄ ME reduced the apoptosis of PC12 cells treated with 50 μM of ketamine and different LXA₄ ME concentrations (5–50 nM) for 24 h. Levels of mRNAs encoding (a) caspase-3 and (b) caspase-9, as measured by quantitative real-time polymerase chain reaction. Enzymatic activity of (c) caspase-3 and (d) caspase-9, as estimated using assay kits. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to the control group (CN); ^#p < 0.05, compared to 0 μM ketamine. PC12 cells: pheochromocytoma cells; LXA₄ ME: lipoxin A₄ methyl ester. LXA₄ ME reduced oxidative stress in ketamine-treated PC12 cells.

Ketamine significantly induced oxidative stress in PC12 cells by increasing the production of ROS and MDA (Figures 3(a) and (b)) and inhibiting the activity of the antioxidant enzymes SOD and GPx (Figures 3(c) and (d)). LXA₄ ME reversed all these effects in a concentration-dependent manner. Thus, LXA₄ ME can reduce oxidative stress in ketamine-treated cells by maintaining the oxidant/antioxidant balance.

Figure 3.

LXA₄ ME reduced oxidative stress in PC12 cells treated with 50 μM of ketamine and different LXA₄ ME concentrations (5–50 nM) for 24 h. Levels of (a) ROS and (b) MDA. Antioxidant activity of (c) SOD and (d) GPx. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to the control group (CN); ^#p < 0.05, compared to 0 μM ketamine. PC12 cells: pheochromocytoma cells; LXA₄ ME: lipoxin A₄ methyl ester; ROS: reactive oxygen species; MDA: malondialdehyde; SOD: superoxide dismutase; GPx: glutathione peroxidase. Ketamine reduced signaling via the miR-22/BAG5 pathway in PC12 cells.

Treatment with >10 μM of ketamine significantly downregulated miR-22 and upregulated BAG5 (Figure 4), suggesting that ketamine induces neuronal cell damage by suppressing signal transduction via the miR-22/BAG5 pathway.

Figure 4.

Ketamine downregulated miR-22 and upregulated BAG5 in PC12 cells. Relative levels of mRNAs encoding (a) miR-22 or (B) BAG5, as measured by Western blot after treatment with different ketamine concentrations (0.1–400 μM) for 24 h. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to 0 μM ketamine. PC12 cells: pheochromocytoma cells; BAG5: Bcl-2-associated athanogene 5. Upregulation of miR-22 attenuated ketamine-induced neurotoxicity in PC12 cells.

The expression of miR-22 was significantly higher in PC12 cells transfected with lenti-miR-22-mimics than in the control group (Figure 5(a)). Thus, PC12 cells transfected with lenti-NC-mimics or lenti-miR-22-mimics were further treated with 50 μM of ketamine for 24 h, and the neurotoxicity was assessed by the CCK-8 assay. The overexpression of miR-22 in the lenti-miR-22-mimics group led to higher cell viability (Figure 5(b)) and lower apoptosis than in the lenti-NC-mimics group, and these effects were associated with lower activity of caspases-3 and -9 (Figures 5(c) and (d)). At the same time, the upregulation of miR-22 in ketamine-treated PC12 cells inhibited ROS production and enhanced SOD activity, thus reducing oxidative stress (Figures 5(e) and (f)). These results clearly support that miR-22 plays an important role in alleviating ketamine-triggered neurotoxicity in PC12 cells.

Figure 5.

MiR-22 overexpression alleviated ketamine-induced neurotoxicity in PC12 cells. After transfection for 4 h, then cells were treated with 50 μM of ketamine for 24 h. (a) Relative miR-22 expression in PC12 cells transfected with lenti-NC-mimics or lenti-miR-22-mimics, as measured by quantitative real-time polymerase chain reaction. (b) Viability of ketamine-treated PC12 cells transfected with lenti-NC-mimics or lenti-miR-22-mimics, as measured by the CCK-8 assay. (c) Caspase-3 activity, (d) caspase-9 activity, (e) ROS levels, and (f) SOD activity in ketamine-treated PC12 cells transfected with lenti-NC-mimics or lenti-miR-22-mimics. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to the lenti-NC-mimics group. PC12 cells: pheochromocytoma cells; ROS: reactive oxygen species; SOD: superoxide dismutase; lenti-NC-mimics: lentiviral vector of miR-22 control mimics; lenti-miR-22-mimics: lentiviral vector of miR-22 mimics.

miR-22 directly targets BAG5

Potential binding sites of miR-22 and BAG5 were identified using the TargetScan database (Figure 6(a)), and the binding sequence of BAG5 was mutated to investigate the interaction between miR-22 and BAG5. The luciferase activity of BAG5-WT was lower in HEK293 T cells transfected with lenti-miR-22-mimics than in those transfected with lenti-NC-mimics, whereas the luciferase activity of BAG5-Mut remained unchanged (Figure 6(b)). In addition, lenti-miR-22-mimics downregulated BAG5 at the mRNA and protein levels in ketamine-treated PC12 cells (Figures 6(c)–(e)). Thus, miR-22 can attenuate ketamine-induced neurotoxicity in PC12 cells by directly targeting BAG5.

Figure 6.

MiR-22 directly targets BAG5. (a) Binding sites in miR-22, BAG5-WT, and BAG5-Mut, as determined using the TargetScan database. (b) Relative luciferase activity of BAG5-WT and BAG5-Mut in HEK293 T cells transfected with lenti-NC-mimics or lenti-miR-22-mimics. After transfection for 4 h, then cells were treated with 50 μM of ketamine for 24 h. (c) Relative BAG5 mRNA expression in PC12 cells transfected with lenti-NC-mimics or lenti-miR-22-mimics, as measured by quantitative real-time polymerase chain reaction. (d) Representative Western blots for BAG5. (e) Relative protein levels of BAG5 (normalized to levels of β-actin) in PC12 cells transfected with lenti-NC-mimics or lenti-miR-22-mimics. Data are shown as mean ± SD (n = 6). *p < 0.05, compared to the lenti-NC-mimics group. BAG5: Bcl-2-associated athanogene five; lenti-NC-mimics: lentiviral vector encoding miR-22 control mimics; lenti-miR-22-mimics: lentiviral vector encoding miR-22 mimics.

LXA₄ ME activated the miR-22/BAG5 signaling pathway in ketamine-treated PC12 cells

Since ketamine can downregulate miR-22 expression and induce BAG5 expression, we explored whether LXA₄ ME exerts its protective effects by altering the miR-22/BAG5 cascade in ketamine-treated PC12 cells. LXA₄ ME upregulated miR-22 (Figure 7(a)) and downregulated BAG5 (Figure 7(b)), both in a concentration-dependent manner. Thus, LXA₄ ME protects PC12 cells from ketamine-induced neurotoxicity by altering miR-22 and BAG5 expression in a way that activates the miR-22/BAG5 signaling pathway.

Figure 7.

Effect of LXA₄ ME on the activation of the miR-22/BAG5 signaling pathway in PC12 cells treated with 50 μM of ketamine and different LXA₄ ME concentrations (5–50 nM) for 24 h. Relative levels of mRNAs encoding (a) miR-22 or (b) BAG5, as determined by quantitative real-time polymerase chain reaction. (c) Representative Western blots for BAG5. (d) Relative protein levels of BAG5, normalized to levels of β-actin. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to the control group (CN); ^#p < 0.05, compared to 0 μM ketamine. PC12 cells: pheochromocytoma cells; BAG5: Bcl-2-associated athanogene five; LXA₄ ME: lipoxin A₄ methyl ester. MiR-22 under expression antagonized the ability of LXA₄ ME to protect against ketamine-induced neurotoxicity in PC12 cells.

Transfection with lenti-miR-22-inhibitor significantly reduced expression of miR-22 (Figure 8(a)). In cells treated with ketamine and LXA₄ ME, the levels of BAG5, caspase-3 activity, and ROS were significantly higher when the cells were expressing miR-22 inhibitor (Figure 8(c), (e), and F), whereas cell viability was significantly lower (Figure 8(d)). These results suggest that miR-22 under expression partially reverses the ability of LXA₄ ME to protect against ketamine-induced neurotoxicity. These results confirm that LXA₄ ME can reduce neurotoxicity in ketamine-treated PC12 cells by regulating the expression of miR-22.

Figure 8.

MiR-22 under expression weakened the protective effect of LXA₄ ME against ketamine-induced neurotoxicity in PC12 cells. After transfection for 4 h, then cells were treated with 50 μM of ketamine and 20 nM of LXA₄ ME for 24 h. (a) Relative miR-22 expression in PC12 cells transfected with lenti-NC-mimics or lenti-miR-22-inhibitor, as measured by quantitative real-time polymerase chain reaction. (b) Representative Western blots for BAG5. (c) Relative expression of BAG5, normalized to that of β-actin. (d) Viability of PC12 cells treated in different ways, as determined by CCK-8 assay. (e) Caspase-3 activity, as determined using a commercial kit. (F) Reactive oxygen species (ROS) levels, as determined by flow cytometry. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to the control group (CN); ^#p < 0.05, compared to the ketamine group; ⁺p < 0.05, compared to the ketamine + LXA₄ ME group. PC12 cells: pheochromocytoma cells; LXA₄ ME: lipoxin A₄ methyl ester; BAG5: Bcl-2-associated athanogene five; lenti-NC-mimics: lentiviral vector encoding miR-22 control mimics; lenti-miR-22-mimics: lentiviral vector encoding miR-22 mimics.

BAG5 overexpression antagonized the ability of LXA₄ ME to protect against ketamine-induced neurotoxicity in PC12 cells

Expression of BAG5 was significantly higher in cells transfected with lenti-BAG5 than in cells transfected with lenti-vector (Figure 9(a)). The viability of cells treated with ketamine and LXA₄ ME was significantly lower when the cells were overexpressing BAG5 (Figure 9(b)), while caspase-3 activity (Figure 9(c)) and ROS levels (Figure 9(d)) were significantly higher. These results suggest that BAG5 overexpression can partially reverse the protective effect of LXA₄ ME on neurotoxicity. These results confirm that LXA₄ ME can reduce ketamine-induced neurotoxicity in PC12 cells by downregulating BAG5.

Figure 9.

BAG5 overexpression weakened the protective effect of LXA₄ ME against ketamine-induced neurotoxicity in PC12 cells. After transfection for 4 h, then cells were treated with 50 μM of ketamine and 20 nM of LXA₄ ME for 24 h. (a) Relative BAG5 mRNA expression in PC12 cells transfected with lenti-BAG5, as measured by quantitative real-time polymerase chain reaction. (b) Cell viability, as determined by the CCK-8 assay. (c) Caspase-3 activity, as determined using a commercial kit. (d) Reactive oxygen species (ROS) levels, as determined by flow cytometry. Data are shown as mean ± SD (n = 6). ^*p < 0.05, compared to the control group (CN); ^#p < 0.05, compared to the ketamine group; ⁺p < 0.05, compared to the ketamine + LXA₄ ME group. PC12 cells: pheochromocytoma cells; LXA₄ ME: lipoxin A₄ methyl ester; BAG5: Bcl-2-associated athanogene 5.

Discussion

Ketamine is a commonly used anesthetic, but high doses can cause neurotoxicity in the brain.^19,20 Here we provide evidence that LXA₄ ME can protect PC12 cells from ketamine-induced neurotoxicity by activating the miR-22/BAG5 signaling pathway.

Several studies have shown that miRNAs might be involved in ketamine-induced neurotoxicity in neuronal cells. For example, miR-34c downregulation significantly alleviates ketamine-mediated memory impairment,²¹ while the under expression of miR-124 can improve memory performance in both humans and animals suffering from neurotoxicity due to overanesthesia.²² Moreover, miR-206 plays a key role in regulating ketamine-induced neural damage,²³ whereas miR-429 may suppress neuronal apoptosis.²⁴ Upregulation of miR-22 can suppress myoblast proliferation and promote myoblast differentiation into myotubes.²⁵ In addition, miR-22 regulates vascular smooth muscle cell function, which may be a new approach for treating vascular diseases.²⁶ Here we found that ketamine can significantly reduce miR-22 expression in PC12 cells in a concentration-dependent manner, and that upregulation of miR-22 attenuates ketamine-induced apoptosis and reduces oxidative stress. These results suggest that miR-22 can help protect neuronal cells from ketamine-induced neurotoxicity.

One of the targets of miR-22 is the mRNA encoding BAG5, a molecular chaperone that binds to the Bcl-2 protein and regulates cell survival.²⁷ BAG5 can also interact with the DJ-1 protein, inhibiting its protective role in neurons,²⁸ and BAG5 can promote Parkin-induced apoptosis and death of dopaminergic neurons by inhibiting Parkin-dependent mitophagy and by interacting with Hsp70.^29,30 On the other hand, other BAG family members have been shown to protect cell death from ketamine-induced.⁴ In our study, we found that miR-22 can directly bind to BAG5 mRNA and inhibit its translation, and that ketamine upregulates BAG5 in a concentration-dependent manner. This indicates that miR-22 mitigates ketamine-induced neurotoxicity in PC12 cells in part by downregulating BAG5. Thus, the miR-22/BAG5 pathway can be clinically exploited to develop targeted drugs against ketamine-induced neurotoxicity.

LXA₄ ME is known to improve neuronal functions and reduce cognitive impairment by attenuating oxidative injury and limiting neuronal apoptosis in the hippocampus.^14–16 Studies of chronic cerebral hypoperfusion have shown that LXA₄ ME exerts neuroprotective effects via anti-oxidative and anti-apoptotic mechanisms.¹⁵ Whether the compound can protect against ketamine-induced neurotoxicity and, if so, via what mechanisms are unclear. Given that ketamine can induce neuronal apoptosis during early retinal development in rats,⁴ we examined the effects of LXA₄ ME on viability, apoptosis and oxidative stress in ketamine-treated PC12 cells.¹⁵ We found that inhibition of BAG5 through overexpression of miR-22 reduced ketamine-induced neuronal damage and apoptosis. Since miR-22 suppresses neuronal apoptosis induced by ischemia/reperfusion in PC12 cells,⁴ we further observed the effects of LXA₄ ME on the activity of the miR-22/BAG5 signaling pathway. The results indicated that LXA₄ ME can upregulate miR-22 and downregulate BAG5 to alleviate ketamine toxicity against PC12 cells, and that miR-22 under expression or BAG5 overexpression antagonizes these protective effects. We conclude that LXA₄ ME protects PC12 cells from ketamine-induced neurotoxicity by activating the miR-22/BAG5 signaling pathway through its influence on expression of miR-22 and BGA5. These findings may help guide the development of effective pharmacological or genetic agents targeting the miR-22/BAG5 axis.

Conclusions

In this study we showed that ketamine downregulates miR-22 and upregulates BAG5 in PC12 cells, which LXA₄ ME partially reversed, thereby alleviating ketamine-induced toxicity. However, under expression of miR-22 or overexpression of BAG5 antagonized these protective effects of LXA₄ ME, confirming that miR-22, through its influence on BAG5 expression, is significantly involved in alleviating ketamine-induced neurotoxicity. LXA₄ ME appears to act, at least in part, by regulating the expression of miR-22 and BAG5 in a way that activates the miR-22/BAG5 signaling pathway. Additional studies are needed to verify and explore the role of LXA₄ ME in regulating the miR-22/BAG5 axis in PC12 cells.

Footnotes

Acknowledgments

The authors thank the Department of Anesthesiology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Jin Gao

References

Nowacka

Borczyk

. Ketamine applications beyond anesthesia - a literature review. Eur J Pharmacol 2019; 860: 172547.

Yang

J-J

Cao

, et al. Iron overload contributes to general anaesthesia-induced neurotoxicity and cognitive deficits. J Neuroinflammation 2020; 17(1): 110.

Hladik

Buratovic

Toerne

, et al. Combined treatment with low-dose ionizing radiation and ketamine induces adverse changes in CA1 neuronal structure in male murine hippocampi. Int J Mol Sci 2019; 20(23): 6103.

Zhang

Sun

, et al. Laminin degradation by matrix metalloproteinase 9 promotes ketamine‐induced neuronal apoptosis in the early developing rat retina. CNS Neurosci Ther 2020; 26(10): 1058–1068.

Cao

Zhang

, et al. Small interfering LncRNA-TUG1 (siTUG1) decreases ketamine-induced neurotoxicity in rat hippocampal neurons. Int J Neurosci 2019; 129(10): 937–944.

Molero

Ramos-Quiroga

Martin-Santos

, et al. Antidepressant efficacy and tolerability of ketamine and esketamine: a critical review. CNS Drugs 2018; 32(5): 411–420.

Saliminejad

Khorshid

HRK

Soleymani Fard

, et al. An overview of microRNAs: biology, functions, therapeutics, and analysis methods. J Cell Physiol 2019; 234(5): 5451–5465.

Bhaskaran

Mohan

. MicroRNAs. Vet Pathol 2014; 51(4): 759–774.

Lopez-Ramirez

Reijerkerk

de Vries

, et al. Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation. FASEB J 2016; 30(8): 2662–2672.

10.

Shui

Han

, et al. MicroRNA-22 attenuates neuronal cell apoptosis in a cell model of traumatic brain injury. Am Journal Translational Research 2016; 8(4): 1895–1902.

11.

Z-Y

Guo

Liu

Y-M

, et al. Neuroprotective effects of total saikosaponins of bupleurum yinchowense on corticosterone-induced apoptosis in PC12 cells. J Ethnopharmacology 2013; 148: 794–803.

12.

Yang

Zhang

, et al. Neuroprotective role of microRNA-22 in a 6-hydroxydopamine-induced cell model of parkinson’s disease via regulation of its target gene TRPM7. J Mol Neurosci 2016; 60(4): 445–452.

13.

X-H

Guo

P-P

, et al. Neuroprotective effect of lipoxin A4 methyl ester in a rat model of permanent focal cerebral ischemia. J Mol Neurosci 2010; 42(2): 226–234.

14.

Song

Yang

Cui

, et al Lipoxin A4 Methyl ester reduces early brain injury by inhibition of the nuclear factor kappa B (NF-κB)-Dependent matrix metallopeptidase 9 (MMP-9) pathway in a rat model of intracerebral hemorrhage. Med Sci Monitor 2019; 25: 1838–1847.

15.

Jin

Jia

Huang

, et al. Lipoxin A4 methyl ester ameliorates cognitive deficits induced by chronic cerebral hypoperfusion through activating ERK/Nrf2 signaling pathway in rats. Pharmacol Biochem Behav 2014; 124: 145–152.

16.

Jia

Jin

Xiao

, et al. Lipoxin A4 methyl ester alleviates vascular cognition impairment by regulating the expression of proteins related to autophagy and ER stress in the rat hippocampus. Cell Molecular Biology Letters 2015; 20(3): 475–487.

17.

Cai

X-H

, et al. Lipoxin A4 activates alveolar epithelial sodium channel gamma via the microRNA-21/PTEN/AKT pathway in lipopolysaccharide-induced inflammatory lung injury. Lab Invest 2015; 95(11): 1258–1268.

18.

Codagnone

Recchiuti

Lanuti

, et al. Lipoxin A 4 stimulates endothelial miR‐126-5p expression and its transfer via microvesicles. FASEB J 2017; 31(5): 1856–1866.

19.

Fan

Bian

Liu

, et al. MiRNA ‐429 alleviates ketamine‐induced neurotoxicity through targeting BAG5. Environ Toxicol 2021; 36(4): 620–627.

20.

Zhu

Ding

Zhang

, et al. Risks associated with misuse of ketamine as a rapid-acting antidepressant. Neurosci Bull 2016; 32(6): 557–564.

21.

Cao

S-E

Tian

Chen

, et al. Role of miR-34c in ketamine-induced neurotoxicity in neonatal mice hippocampus. Cel Biol Int 2015; 39(2): 164–168.

22.

Zhang

Zhou

, et al. The role of miR-124 in modulating hippocampal neurotoxicity induced by ketamine anesthesia. Int J Neurosci 2015; 125(3): 213–220.

23.

Yao

Wang

Gao

. LncRNA KCNQ1OT1 sponges miR-206 to ameliorate neural injury induced by anesthesia via up-regulating BDNF. Drug Des Dev Ther 2020; Volume 14: 4789–4800.

24.

Yang

Zhang

Yin

, et al. MiR-429/200a/200b negatively regulate notch1 signaling pathway to suppress CoCl2-induced apoptosis in PC12 cells. Toxicol Vitro 2020; 65: 104787.

25.

Wang

Zhang

Wang

, et al. miR-22 regulates C2C12 myoblast proliferation and differentiation by targeting TGFBR1. Eur J Cel Biol 2018; 97(4): 257–268.

26.

Yang

Chen

, et al. miR-22 is a novel mediator of vascular smooth muscle cell phenotypic modulation and neointima formation. Circulation 2018; 137(17): 1824–1841.

27.

Zou

Zheng

Cai

, et al. Identification of BAG5 from orange-spotted grouper (Epinephelus coioides) involved in viral infection. Dev Comp Immunol 2021; 116: 103916.

28.

Qin

Tan

Zhang

, et al. BAG5 interacts with DJ-1 and inhibits the neuroprotective effects of DJ-1 to combat mitochondrial oxidative damage. Oxid Med Cell Longev 2017; 2017: 5094934.

29.

De Snoo

Friesen

Zhang

, et al. Bcl-2-associated athanogene 5 (BAG5) regulates parkin-dependent mitophagy and cell death. Cel Death Dis 2019; 10(12): 907.

30.

Kalia

Lee

Smith

, et al. BAG5 inhibits parkin and enhances dopaminergic neuron degeneration. Neuron 2004; 44(6): 931–945.

Lipoxin A 4 methyl ester protects PC12 cells from ketamine-induced neurotoxicity via the miR-22/BAG5 pathway

Abstract

Objective

Methods

Results

Conclusion

Keywords

Introduction

Materials and methods

Cell culture and treatment

Transfection with lentiviral vectors

Cell viability assay

Caspase-3 or -9 activity assay

Oxidative status assessment

qRT-PCR

Dual-luciferase reporter assay

Protein expression assessment

Statistical analysis

Results

LXA4 ME enhanced the viability of ketamine-treated PC12 cells

miR-22 directly targets BAG5

LXA4 ME activated the miR-22/BAG5 signaling pathway in ketamine-treated PC12 cells

Discussion

Conclusions

Footnotes

Acknowledgments

Declaration of conflicting interests

Funding

ORCID iD

References

LXA₄ ME enhanced the viability of ketamine-treated PC12 cells

LXA₄ ME activated the miR-22/BAG5 signaling pathway in ketamine-treated PC12 cells