Tripartite motif 72 inhibits apoptosis and mitochondrial dysfunction in neural stem cells induced by anesthetic sevoflurane by activating PI3K/AKT pathway

Minmin Cai; Xiang Gao; Shenghui Yu

doi:10.4103/cjop.CJOP-D-22-00082

ORIGINAL ARTICLE

Year : 2023 | Volume : 66 | Issue : 1 | Page : 36-42

Tripartite motif 72 inhibits apoptosis and mitochondrial dysfunction in neural stem cells induced by anesthetic sevoflurane by activating PI3K/AKT pathway

Minmin Cai, Xiang Gao, Shenghui Yu
Department of Anesthesiology, The Affiliated People's Hospital of Ningbo University, Ningbo, Zhejiang, China

Date of Submission	22-Aug-2022
Date of Decision	24-Oct-2022
Date of Acceptance	27-Oct-2022
Date of Web Publication	20-Feb-2023

Correspondence Address:
Dr. Minmin Cai
Department of Anesthesiology, The Affiliated People's Hospital of Ningbo University, No. 251, Baizhang East Road, Ningbo
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-22-00082

Abstract

Anesthetics exposure induces neurocognitive deficits during brain development and impairs self-renewal and differentiation of neural stem cells (NSCs). Tripartite motif 72 (TRIM72, also known as mitsugumin 53, MG53) is involved in tissue repair and plasma membrane damage repair. The neuroprotective effect of TRIM72 against sevoflurane-induced neurotoxicity of NSCs was investigated in this study. First, human NSCs were exposed to different concentrations of sevoflurane. Results showed that TRIM72 was downregulated in sevoflurane-treated NSCs. Exposure to sevoflurane reduced cell viability in NSCs. Second, sevoflurane-treated NSCs were stimulated with recombinant human TRIM72 (rhTRIM72). Treatment with rhTRIM72 enhanced the cell viability in sevoflurane-treated NSCs. Moreover, treatment with a rhTRIM72-attenuated sevoflurane-induced increase in caspase-3 activity in NSCs. Third, JC-1 aggregates were deceased and JC-1 monomer was increased in sevoflurane-treated NSCs, which were reversed by rhTRIM72. Furthermore, rhTRIM72 also weakened sevoflurane-induced decrease in superoxide dismutase and glutathione peroxidase and increase in malondialdehyde and reactive oxygen species in NSCs. Finally, reduced phosphorylation levels of protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K) in sevoflurane-treated NSCs were upregulated by rhTRIM72. In conclusion, TRIM72 inhibited cell apoptosis and reduced the mitochondria membrane potential of sevoflurane-treated NSCs through activation of the PI3K/AKT pathway.

Keywords: Apoptosis, mitochondrial dysfunction, neural stem cells, neurotoxicity, PI3K/AKT pathway, sevoflurane, tripartite motif 72

How to cite this article:
Cai M, Gao X, Yu S. Tripartite motif 72 inhibits apoptosis and mitochondrial dysfunction in neural stem cells induced by anesthetic sevoflurane by activating PI3K/AKT pathway. Chin J Physiol 2023;66:36-42

How to cite this URL:
Cai M, Gao X, Yu S. Tripartite motif 72 inhibits apoptosis and mitochondrial dysfunction in neural stem cells induced by anesthetic sevoflurane by activating PI3K/AKT pathway. Chin J Physiol [serial online] 2023 [cited 2023 Sep 28];66:36-42. Available from: https://www.cjphysiology.org/text.asp?2023/66/1/36/370014

Introduction

Sevoflurane is widely used as pediatric sedation and anesthesia.^[1] However, substantial evidence from various animal models has shown the correlation between sevoflurane exposure and neurodevelopmental disorders.^{[2],[3],[4],[5]} Sevoflurane exposure induced neuronal apoptosis, synaptic dysfunction, and neuroinflammation,^[4] and stimulated neurodegeneration and neurocognitive deficits in neonatal rats.^[6] Therefore, developing of strategies to protect against anesthetic exposure-induced neurotoxicity might be important for pediatric sedation and anesthesia.

Neural stem cells (NSCs) are important for cognitive formation during brain development^[7] due to their ability to differentiate into astrocytes, oligodendrocytes, and neurons,^[8] and self-renewal ability into neurospheres.^[9] Dysregulation of NSCs has been shown to induce megalencephaly or microcephaly, resulting in severe birth defects.^[10] Sevoflurane exposure inhibited the proliferation and differentiation of NSCs^[11],[12] and promoted the degeneration of NSCs, leading to cognitive dysfunction in brain development.^[13]

Tripartite motif 72 (TRIM72, also known as mitsugumin53) is widely expressed in the brain, skeletal muscle, and cardiac tissues, and belongs to the component of membrane repair machinery.^[14] TRIM72 discriminates between injured and intact membranes and is tethering into the injury membranes through membrane-delimited signals.^[15] Therefore, TRIM72 was involved in the plasma membrane repair of multiple tissues, including the brain, skeletal muscle, heart, skin, kidney, and lung.^[14] TRIM72 also exerted cardioprotection effects in sepsis or cardiac ischemic.^[14],[16] Moreover, TRIM72 reduced lipopolysaccharide-induced cell apoptosis and secretion of inflammatory cytokines in umbilical cord mesenchymal stem cells (UC-MSCs).^[17] TRIM72 was also involved in M1/M2 phenotype polarization of microglia and attenuated lipopolysaccharide-induced memory impairment in mice.^[17] TRIM72 alleviated anxiety and depressive-like behaviors, reduced neurological deficits, and mitigated brain edema in murine traumatic brain injury through promoting of UC-MSC proliferation and migration.^[18] However, the role of TRIM72 in anesthetic neurotoxicity remains unclear.

In this study, the effects of TRIM72 on cell apoptosis, oxidative stress, and mitochondrial dysfunction in sevoflurane-treated NSCs were investigated.

Materials and Methods

Cell culture and treatment

Human NSCs were purchased from Gibco (Thermo Fisher, Waltham, MA, USA), and grown in StemPro^™ NSC SFM (Thermo Fisher) at 37°C incubators with 5% CO₂. Cells were treated with 2%, 3%, or 4% sevoflurane (Sigma-Aldrich, St. Louis, MO, USA) for 2 h per day for 3 consecutive days. Cells under 4% sevoflurane exposure were incubated with 10 or 20 μg/mL recombinant human TRIM72 (rhTRIM72) (Abcam, Cambridge, UK) for another 2 h before subsequently experiments.

Cell viability and apoptosis

Human NSCs (1 × 10⁴ cells/well) were seeded in 96-well plates and subjected to the treatment. Cells were then cultured for another 24, 48, or 72 h. Cells were treated with 5 mg/mL MTT solution (Beyotime, Beijing, China) for 4 h and incubated with dimethyl sulfoxide after the discard of the solution. Absorbance at 490 nm was examined through Microplate Autoreader (Thermo Fisher). For detection of cell apoptosis, human NSCs (1 × 10⁶ cells) were lysed in RIPA buffer (Beyotime) and centrifuged at 12,000 g and 4°C to collect supernatants. The supernatants were subjected to Caspase-3 Assay Kit (Abcam) to detect the activity of caspase-3.

Mitochondria membrane potential assay

Human NSCs (1 × 10⁶ cells) were collected and incubated with 1 μg/ml JC-1 solution (JC-1-Mitochondrial Membrane Potential Assay Kit; Abcam) for 20 min. Cells were centrifuged at 1000 g at 4°C to discard the solution and washed with dilution buffer. Cells were stained with DAPI and analyzed under fluorescence microscopy (Olympus, Tokyo, Japan). The JC-1 fluorescent intensity was calculated using Microplate Autoreader.

Enzyme-Linked Immunosorbent Assay

Human NSCs (1 × 10⁶ cells) were lysed in adenosine triphosphate (ATP) lysis buffer of an enhanced ATP assay kit (Beyotime) and centrifuged at 15,000 g at 4°C to collect supernatants. The supernatants were incubated with 100 μL of ATP working solution. The luminescence was measured using a luminometer (Promega, Madison, WI, USA). Levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the supernatants were analyzed by commercial enzyme-linked immunosorbent assay kits (Abcam). Level of reactive oxygen species (ROS) was determined using OxiSelect in vitro ROS/RNS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA).

Western blot

Protein samples in the isolated supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membrane. Membranes were blocked with 5% skim milk and probed with primary antibodies at 4°C for overnight: anti-TRIM72 (ab170540) and anti-β-actin (ab179467) (1:2000), anti-BCL (ab32124) and anti-BAX (ab32503) (1:3000), anti-p-PI3K (ab278545) and anti-PI3K (ab32089) (1:4000), anti-p-AKT (ab38449) and anti-AKT (ab8805) (1:5000), and anti-cleaved caspase-3 (ab32042) (1:6000). Membranes were incubated with horseradish peroxidase-labeled secondary antibody (ab6721) (1:5000) and subjected to enhanced chemiluminescence (KeyGen Biotech, Jiangsu, China) to detect immunoreactivities. All the antibodies were purchased from Abcam.

Statistical analysis

All the data were expressed as mean ± standard error of the mean and analyzed through Student's t-test or one-way analysis of variance in GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered statistically significant.

Results

Tripartite motif 72 enhanced the proliferation of sevoflurane-treated neural stem cells

To determine the role of TRIM72 in sevoflurane-induced neurotoxicity, the expression of TRIM72 in sevoflurane-treated NSCs was investigated. Results showed that protein expression of TRIM72 was reduced in sevoflurane-treated NSCs in a dosage-dependent way [Figure 1]a. Sevoflurane-treated NSCs were then incubated with rhTRIM72 to assess the neuroprotective role of TRIM72 [Figure 1]b. Incubation with rhTRIM72 increased TRIM72 expression in sevoflurane-treated NSCs [Figure 1]c. Sevoflurane exposure decreased cell viability [Figure 1]d of NSCs. However, rhTRIM72 treatment enhanced cell viability [Figure 1]d of sevoflurane-treated NSCs. These results suggested that TRIM72 enhanced the proliferation of sevoflurane-treated NSCs.

Figure 1: TRIM72 stimulated proliferation of sevoflurane-treated NSCs. (a) Protein expression of TRIM72 was reduced in sevoflurane-treated NSCs using a dosage-dependent way. (b) Flow chart of cell treatment was shown. (c) Incubation with rhTRIM72 increased TRIM72 expression in sevoflurane-treated NSCs. (d) Incubation with rhTRIM72 enhanced cell viability of sevoflurane-treated NSCs. *P < 0.05, **P < 0.01, ***P < 0.001. TRIM72: Tripartite motif 72, NSC: Neural stem cell, rhTRIM72: Recombinant human TRIM72.

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Tripartite motif 72 suppressed cell apoptosis of sevoflurane-treated neural stem cells

The effect of TRIM72 on apoptosis of sevoflurane-treated NSCs was then evaluated. The activity of caspase-3 was increased in NSCs treated with sevoflurane [Figure 2]a. Treatment with rhTRIM72 decreased caspase-3 activity in sevoflurane-treated NSCs [Figure 2]a. Moreover, rhTRIM72 attenuated sevoflurane treatment-induced decrease in BCL2 and an increase in BAX and cleaved caspase-3 in NSCs [Figure 2]b. These results revealed the anti-apoptotic effect of TRIM72 on sevoflurane-treated NSCs.

Figure 2: TRIM72 suppressed cell apoptosis of sevoflurane-treated NSCs. (a) Treatment with rhTRIM72 decreased caspase-3 activity in sevoflurane-treated NSCs. (b) Treatment with rhTRIM72 attenuated sevoflurane-induced decrease of BCL2, increase of BAX, and cleaved caspase-3 in NSCs. **P < 0.01, ***P < 0.001. TRIM72: Tripartite motif 72, NSC: Neural stem cell, rhTRIM72: Recombinant human TRIM72.

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Tripartite motif 72 attenuated mitochondrial dysfunction of sevoflurane-treated neural stem cells

Sevoflurane exposure triggered mitochondrial dysfunction in NSCs through decreasing JC-1 aggregates (red) and increasing JC-1 monomer (green) [Figure 3]a. The red/green JC-1 fluorescence was decreased in NSCs treated with sevoflurane [Figure 3]b. Incubation with rhTRIM72 increased red/green JC-1 fluorescence to alleviate the mitochondrial dysfunction in sevoflurane-treated NSCs [Figure 3]a and [Figure 3]b. rhTRIM72 also weakened sevoflurane exposure-induced decreased ATP in NSCs [Figure 3]c, indicating that TRIM72 ameliorated mitochondrial dysfunction of sevoflurane-treated NSCs.

Figure 3: TRIM72 attenuated mitochondrial dysfunction of sevoflurane-treated NSCs. (a) Incubation with rhTRIM72 increased the fluorescence of JC-1 aggregates (red) and decreased the fluorescence of JC-1 monomer (green) in sevoflurane-treated NSCs. (b) Incubation with rhTRIM72 increased red/green JC-1 fluorescence in sevoflurane-treated NSCs. (c) Incubation with rhTRIM72 weakened sevoflurane-induced decrease of ATP in NSCs. *P < 0.05, ***P < 0.001. TRIM72: Tripartite motif 72, NSC: Neural stem cell, rhTRIM72: Recombinant human TRIM72.

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Tripartite motif 72 inhibited oxidative stress of sevoflurane-treated neural stem cells

The effect of TRIM72 on oxidative stress of sevoflurane-treated NSCs was also evaluated. Sevoflurane exposure triggered an increase in MDA and a decrease in SOD and GSH-Px in NSCs [Figure 4]a. Treatment with rhTRIM72 reduced MDA, enhanced levels of SOD, and GSH-Px in sevoflurane-treated NSCs [Figure 4]a. rhTRIM72 also decreased the level of ROS in sevoflurane-treated NSCs [Figure 4]b, demonstrating the antioxidant effect of TRIM72 on sevoflurane-treated NSCs.

Figure 4: TRIM72 inhibited oxidative stress of sevoflurane-treated NSCs. (a) Treatment with rhTRIM72 reduced MDA, enhanced levels of SOD, and GSH-Px in sevoflurane-triggered NSCs. (b) Treatment with rhTRIM72 decreased the level of ROS in sevoflurane-triggered NSCs. *P < 0.05, **P < 0.01, ***P < 0.001. TRIM72: Tripartite motif 72, NSC: Neural stem cell, rhTRIM72: Recombinant human TRIM72, MDA: Malondialdehyde, SOD: Superoxide dismutase, GSH-Px: Glutathione peroxidase, ROS: Reactive oxygen species.

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Tripartite motif 72 stimulated activation of PI3K/AKT in sevoflurane-treated neural stem cells

The underlying mechanism of the neuroprotective effect of TRIM72 on sevoflurane-treated NSCs was determined. Protein expressions of p-PI3K and p-AKT in NSCs were decreased by sevoflurane treatment [Figure 5]. Incubation with rhTRIM72 increased p-PI3K and p-AKT expression in sevoflurane-treated NSCs [Figure 5], suggesting that TRIM72 attenuated sevoflurane treatment-induced effects on NSCs through activation of PI3K/AKT pathway.

Figure 5: TRIM72 stimulated activation of PI3K/AKT in sevoflurane-treated NSCs. Incubation with rhTRIM72 increased p-PI3K and p-AKT expression in sevoflurane-treated NSCs. **P < 0.01, ***P < 0.001. TRIM72: Tripartite motif 72, NSC: Neural stem cell, rhTRIM72: Recombinant human TRIM72.

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Discussion

TRIM72 has been shown to protect against lipopolysaccharide-induced neuronal cell death, neuroinflammation, and memory impairment in mice, and it is involved in the pathogenesis and development of neurodegenerative disorders.^[19] This study found that TRIM72 protected NSCs against anesthetic sevoflurane-induced cell apoptosis and mitochondrial dysfunction.

Emerging evidence has shown that sevoflurane exposure induced developmental neurotoxicity through neuroendocrine effects, tau phosphorylation, plasticity and assembly of neural circuits, neural cell damage, and neural cell death.^[20] Sevoflurane exposure upregulated levels of pro-apoptotic factors (BAX/BAK) and downregulated anti-apoptotic factors (BCL-2, BCL-XL, and MCL1) to induce permeabilization of the mitochondrial outer membrane, release of mitochondrial cytochrome C, and promote activation of caspase 3, resulting in neuroapoptosis.^[20] Suppression of neuroapoptosis alleviated sevoflurane-induced neurotoxicity in NSCs.^[21] TRIM72 reduced lipopolysaccharide-induced cell apoptosis of HT22 and promoted newborn cell survival in lipopolysaccharide-induced mice.^[19] This study showed that the expression of TRIM72 was reduced in sevoflurane-treated NSCs, and treatment with rhTRIM72 enhanced the cell viability of sevoflurane-treated NSCs. Moreover, rhTRIM72 also reduced the activity of caspase-3 in sevoflurane-treated NSCs. Protein expression of BCL2 in sevoflurane-treated NSCs was increased, whereas BAX and cleaved caspase3 were decreased by incubation with rhTRIM72. Therefore, TRIM72 enhanced the proliferation and inhibited apoptosis inhibited sevoflurane-induced neurotoxicity in NSCs by promoting the proliferation and inhibition of apoptosis of NSC.

Mitochondrial dysfunction is regarded as a pathological hallmark of neurodegenerative diseases.^[20] Sevoflurane exposure induced calcium homeostasis deregulation in mitochondria, and stimulated ATP reduction, ROS activation, and mitochondrial respiration impairment, leading to neural cell damage and neuroapoptosis.^[20] Mitochondrial membrane potential, the crucial factor for cell viability and ATP synthesis of mitochondria, was reduced by sevoflurane exposure.^[22] TRIM72 initiated the assembly of membrane repair machinery through the regulation of calcium homeostasis,^[23] and increased JC-1 aggregates (red)/JC-1 monomer (green) fluorescent to attenuate mitochondrial membrane potential dysfunction in UC-MSCs.^[17] Our results also showed that incubation with rhTRIM72 increased JC-1 aggregates (red)/JC-1 monomer (green) fluorescent and ATP production in sevoflurane-treated NSCs, suggesting that TRIM72 might protect against sevoflurane-induced neurotoxicity through mitigation of mitochondrial dysfunction.

Oxidative stress has been implicated in the pathogenesis of sevoflurane-induced neurotoxicity.^[24] Sevoflurane exposure induced ROS activation and promoted oxidative stress in NSCs, leading to neural cell damage and neuroapoptosis.^[24] Suppression of sevoflurane anesthesia-mediated oxidative stress contributed to the amelioration of cognitive dysfunction and neurotoxicity.^[25] TRIM72 reduced ischemia reperfusion-induced oxidative stress and preserved the mitochondrial integrity in cardiomyocytes.^[26] This study demonstrated that treatment with rhTRIM72 reduced MDA and ROS, and enhanced the levels of SOD and GSH-Px in sevoflurane-treated NSCs, indicating the anti-oxidant effect of TRIM72 on sevoflurane-induced neurotoxicity. Moreover, sevoflurane anesthesia-induced systemic inflammation triggered neuronal damage and stimulated developmental neurotoxicity in neonatal rats.^[27] Considering that TRIM72 protected UC-MSCs against lipopolysaccharide-induced neuroinflammation,^[17] TRIM72 might exhibit an anti-inflammatory effect on sevoflurane-induced neurotoxicity.

The PI3K/Akt/mTOR pathway is involved in sevoflurane anesthesia-induced developmental neurotoxicity.^[28] Sevoflurane anesthesia suppressed p-AKT/AKT and promoted activation of autophagy to induce mitochondrial dysfunction and neurodegeneration.^[20] Sevoflurane exposure also promoted neuroapoptosis in the developing brain through the regulation of PI3K/AKT.^[20] Activation of PI3K/AKT pathway protected against sevoflurane-induced neurotoxicity in neonatal rats.^[29] TRIM72 interacted with the p85 subunit of PI3K and promoted activation of PI3K/AKT signaling.^[30],[31] Our results also revealed that incubation with rhTRIM72 increased protein expression of p-PI3K and p-AKT in sevoflurane-treated NSCs, indicating that TRIM72 might protect against sevoflurane-induced neurotoxicity through activation of PI3K/AKT signaling.

Conclusion

TRIM72 reduced neuroapoptosis and oxidative stress and ameliorated mitochondrial dysfunction through activation of PI3K/AKT signaling in sevoflurane-treated NSCs. Therefore, TRIM72 might be a novel target for the treatment of sevoflurane-induced neurotoxicity. However, the effect of TRIM72 on sevoflurane-induced neurotoxicity in in vivo animal models should be investigated in further research.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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