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.

背景：体温过低会减弱小胶质细胞的活化，并对脑缺血再灌注（I / R）损伤发挥潜在的神经保护作用。但是，尚需阐明其基本机制。在这个体外 这项研究采用了氧葡萄糖剥夺然后恢复（OGD / R）的模型，以研究体温过低是否发挥抗炎和抗凋亡作用 通过 增强了泛素化和电压依赖性阴离子通道3（VDAC3）表达的下调。

方法：在有或没有体温过低的情况下，在OGD下将BV2小胶质细胞培养4小时，2、4或8小时。使用免疫荧光检测M1和M2小胶质细胞标志物[诱导型一氧化氮合酶（iNOS）和精氨酸酶（Arg）1]。使用酶联免疫吸附测定（ELISA）测定促炎细胞因子[肿瘤坏死因子（TNF）α，白介素（IL）-1β]和抗炎因子（IL-10）的水平。使用流式细胞仪通过JC-1染色法测定线粒体膜电位（ΔΨm）。使用蛋白质印迹分析评估caspase-3，切割的caspase-3和VDAC3的表达。使用双重免疫荧光染色和免疫沉淀（IP）分析鉴定了泛素和VDAC3的细胞位置和相互作用。也，

结果：体温过低抑制了OGD / R诱导的小胶质细胞活化并分化为M1型，具有促炎作用，而促进分化为M2型，具有抗炎作用。体温过低会降低OGD / R引起的Δψm损失以及凋亡相关蛋白的表达。与正常体温相比，低温在再灌注2小时和8小时时会增加BV2小胶质细胞中泛素化VDAC3的水平。此外，体温过低不会减弱OGD / R诱导的小胶质细胞中VDAC3 mRNA的表达。

结论：再灌注过程中的体温过低治疗，OGD / R引起的炎症减弱和BV2小胶质细胞凋亡。这可能是由于VDAC3泛素化的促进，将VDAC3确定为低温治疗的新靶点。