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Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery.

ABSTRACT: Background: Hypothermia attenuates microglial activation and exerts a potential neuroprotective effect against cerebral ischemic-reperfusion (I/R) injury. However, the underlying mechanism remains to be elucidated. In this in vitro study, a model of oxygen-glucose deprivation, followed by recovery (OGD/R), was used to investigate whether hypothermia exerts anti-inflammatory and anti-apoptosis properties via enhanced ubiquitination and down-regulation of voltage-dependent anion channel 3 (VDAC3) expression. Methods: BV2 microglia were cultured under OGD for 4 h following reperfusion with or without hypothermia for 2, 4, or 8 h. M1 and M2 microglia markers [inducible nitric oxide synthase (iNOS) and arginase (Arg)1] were detected using immunofluorescence. The levels of pro-inflammatory cytokines [tumor necrosis factor (TNF) α, interleukin (IL)-1β], and anti-inflammatory factor (IL-10) were determined using enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was assayed by JC-1 staining using a flow cytometer. Expression of caspase-3, cleaved caspase-3, and VDAC3 were assessed using western blot analysis. The cellular locations and interactions of ubiquitin and VDAC3 were identified using double immunofluorescence staining and immunoprecipitation (IP) assay. Also, the level of the VDAC3 mRNA was determined using a quantitative polymerase chain reaction (qPCR). Results: Hypothermia inhibited the OGD/R-induced microglia activation and differentiation into the M1 type with pro-inflammatory effect, whereas it promoted differentiation to the M2 type with anti-inflammatory effect. Hypothermia attenuated OGD/R-induced loss of Δψm, as well as the expression of apoptosis-associated proteins. Compared to normothermia, hypothermia increased the level of ubiquitinated VDAC3 in the BV2 microglia at both 2 and 8 h of reperfusion. Furthermore, hypothermia did not attenuate VDAC3 mRNA expression in OGD/R-induced microglia. Conclusions: Hypothermia treatment during reperfusion, attenuated OGD/R-induced inflammation, and apoptosis in BV2 microglia. This might be due to the promotion of VDAC3 ubiquitination, identifying VDAC3 as a new target of hypothermia.

SUBMITTER: Zhao S

PROVIDER: S-EPMC7289978 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionThe extracellular deposition of β-amyloid (Aβ) is a pathological hallmark in Alzheimer's disease (AD), which induces microglial activation in the pathology of AD. The expression of serine/threonine-protein kinase 2 (SRPK2) is increased in the brain tissues of patients with AD. In this study, we examined the effect of SRPK2 in the activation of microglia.MethodsMicroglia (BV2) cells were cultured and the expression of SRPK2 was enhanced by transfection of SRPK2 recombinant vectors or knockdown by SRPK2 small interfering RNA (siRNA). The cells were stimulated by lipopolysaccharide (LPS) + interferon-γ (IFN-γ) or Aβ in vitro, generating inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-10, and IL-6], which were investigated by real-time quantitative PCR (qPCR) and ELISA. The proliferation ability of the BV2 cells with/without SRPK2 expression was evaluated by WST-1 under pressure in the presence of Aβ. The effects of SRPK2 on microglia polarization were evaluated by investigating the expression of CD16/32 and CD206 by western blot and the expression of ionized calcium-binding adapter molecule-1 (IBA-1) and arginase-1 (Arg-1) by immunofluorescence. Hippocampal cells HT-22 were cultured with a BV2 cell (with/without SRPK2 expression)-derived medium stimulated by Aβ or LPS + IFN-γ, prior to the evaluation of HT-22 cytotoxicity by assessment of cell viability. Possible relationships between Akt and SRPK2 in the BV2 cells were investigated by western blot.ResultsThe expression of SRPK2 was related to the phenotype polarization changes of microglia with increased expression of CD16/32 and IBA-1. The expression of proinflammatory cytokines IL-6 and TNF-α was increased, whereas the expression of anti-inflammatory cytokine IL-10 was decreased in the BV2 cells with SRPK2 overexpression. Moreover, with the expression enhancement of SRPK2, the BV2 cells had a higher proliferation rate. Aβ treatment can promote SRPK2 expression in BV2 cells. Aβ or LPS + IFN-γ promoted the production of cytokines IL-6 and TNF-α but decreased cytokine IL-10 in the BV2 cells. SRPK2 deficiency alleviated the cytotoxic effects of Aβ or LPS + IFN-γ exposed microglia on HT22 cells. In addition, the activated Akt pathway promoted the expression of SRPK2 in the BV2 cells.ConclusionOur data have found that enhanced SRPK2 expression contributed to the proinflammatory activation of microglia. Thus, SRPK2 may be a key modulating pathway of inflammatory mediators in AD pathology.

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Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery.

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