Gong Fanyong, Wei Yiting
Department of Neurosurgery, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China.
Int J Neurosci. 2025 May;135(5):573-587. doi: 10.1080/00207454.2024.2312998. Epub 2024 Mar 9.
Intracerebral hemorrhage (ICH) has significant morbidity and mortality. TXNIP and the competing endogenous RNA (ceRNA) regulatory mechanism involved in long non-coding RNA (lncRNA) play roles in ICH. We probed the upstream microRNAs (miRNAs)/lncRNAs that regulated TXNIP expression in the ceRNA mechanism.
ICH mouse model was established, and ICH secondary injury was simulated in BV2 microglia by hemin treatment. TXNIP was silenced 48 h before ICH modeling. The ICH mouse brain water content (BWC) and brain lesion volume after ICH were recorded. Neuronal apoptosis and neurological deficits were evaluated by double staining of NeuN and TUNEL/modified Garcia/corner turn/forelimb placement tests. Iba1 + microglia number and tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)/IL-10/TXNIP/PVT1/miR-128-3p levels were assessed by immunohistochemistry, Western blot, ELISA, and RT-qPCR. Cell viability/death of BV2 cells conditioned medium-treated neuron HT22 cells were assessed by CCK-8/LDH assays. miRNA that had a targeted binding relationship with TXNIP was screened. The targeted bindings of miR-128-3p to TXNIP/PVT1 to miR-128-3p were verified by dual-luciferase reporter gene assay.
TXNIP knockdown reduced post-ICH microglial activation/release of pro-inflammatory factors/brain edema/brain lesion volume/neurological deficits in mice and increased releases of anti-inflammatory factors. TXNIP/PVT1 knockdown inhibited hemin-induced inflammatory responses in BV2 cells and protected co-cultured HT22 cells. PVT1 was a sponge of miR-128-3p to repress TXNIP expression. miR-128-3p knockdown diminished PVT1 knockdown-inhibited hemin-induced BV2 cell inflammatory responses/neurotoxicity.
PVT1 silencing reduced hemin-induced neuroinflammation and had a protective effect on neurons by increasing the targeted inhibition of TXNIP by miR-128-3p.
脑出血（ICH）具有显著的发病率和死亡率。硫氧还蛋白相互作用蛋白（TXNIP）以及长链非编码RNA（lncRNA）参与的竞争性内源性RNA（ceRNA）调控机制在脑出血中发挥作用。我们探究了ceRNA机制中调控TXNIP表达的上游微小RNA（miRNA）/lncRNA。
建立脑出血小鼠模型，并通过血红素处理在BV2小胶质细胞中模拟脑出血继发性损伤。在脑出血建模前48小时沉默TXNIP。记录脑出血后小鼠的脑含水量（BWC）和脑损伤体积。通过NeuN和TUNEL双重染色/改良的加西亚测试/转角试验/前肢放置试验评估神经元凋亡和神经功能缺损。通过免疫组织化学、蛋白质免疫印迹法、酶联免疫吸附测定（ELISA）和逆转录定量聚合酶链反应（RT-qPCR）评估离子钙结合衔接分子1（Iba1）阳性小胶质细胞数量以及肿瘤坏死因子-α（TNF-α）/白细胞介素-1β（IL-1β）/白细胞介素-10（IL-10）/TXNIP/ Pvt1基因（PVT1）/miR-128-3p水平。通过细胞计数试剂盒-8（CCK-8）/乳酸脱氢酶（LDH）检测评估经BV2细胞条件培养基处理的神经元HT22细胞的细胞活力/死亡情况。筛选与TXNIP具有靶向结合关系的miRNA。通过双荧光素酶报告基因检测验证miR-128-3p与TXNIP/PVT1与miR-128-3p之间的靶向结合。
TXNIP基因敲低减少了脑出血后小鼠小胶质细胞的激活/促炎因子的释放/脑水肿/脑损伤体积/神经功能缺损，并增加了抗炎因子的释放。TXNIP/PVT1基因敲低抑制了血红素诱导的BV2细胞炎症反应，并保护了共培养的HT22细胞。PVT1是miR-128-3p的海绵，可抑制TXNIP表达。miR-128-3p基因敲低减弱了PVT1基因敲低对血红素诱导的BV2细胞炎症反应/神经毒性的抑制作用。
PVT1基因沉默通过增加miR-128-3p对TXNIP的靶向抑制作用，减少了血红素诱导的神经炎症，并对神经元具有保护作用。
