The First Affiliated Hospital, Department of Neurology, Hengyang Medical School, University of South China, Hengyang, China.
The First Affiliated Hospital, Department of Hepatopancreatobiliary Surgery, Hengyang Medical School, University of South China, Hengyang, China.
Brain Behav. 2023 May;13(5):e2935. doi: 10.1002/brb3.2935. Epub 2023 Mar 24.
Neuroinflammation caused by microglia cells activation and the apoptosis of neuron cells are associated with the occurrence of depression. Circ-Bnc2 has been shown to be significantly downregulated in depression mice, but its role in the progression of depression remains unclear.
Lipopolysaccharide (LPS) was used to treat BV2 microglial cells to induce neuroinflammation. The expression of circ-Bnc2, microRNA (miR)-497a-5p, and HECT domain E3 ubiquitin protein ligase 1 (HECTD1) was measured by quantitative real-time PCR. The protein levels of neuroinflammation markers, apoptosis markers, and HECTD1 were determined by western blot analysis. ELISA assay was used to examine the concentrations of inflammatory factors. After HT22 cells were cultured with the conditioned medium of LPS-induced BV2 cells, the proliferation and apoptosis of HT22 cells were assessed by cell counting kit 8 assay, EdU assay, and flow cytometry. In addition, the interaction between miR-497a-5p and circ-Bnc2 or HECTD1 was confirmed by dual-luciferase reporter assay, RIP assay, and RNA pull-down assay.
Our data showed that circ-Bnc2 was lowly expressed in LPS-induced BV2 cells. Function experiments suggested that circ-Bnc2 could inhibit LPS-induced neuroinflammation in BV2 cells to repress HT22 cell apoptosis and promote proliferation. Circ-Bnc2 could sponge miR-497a-5p, and the neuroprotective function of circ-Bnc2 could be reversed by miR-497a-5p overexpression. Additionally, miR-497a-5p could target HECTD1. miR-497a-5p inhibitor could alleviate LPS-induced neuroinflammation in BV2 cells and reduce HT22 cell apoptosis, which also could be reversed by HECTD1 knockdown. Moreover, circ-Bnc2 had a positive regulation on HECTD1 expression by sponging miR-497a-5p.
In summary, our results confirmed that circ-Bnc2 could inhibit neuroinflammation and neuron cell apoptosis by regulating miR-497a-5p/HECTD1 axis, suggesting that circ-Bnc2 might be a potential target for depression treatment.
小胶质细胞激活引起的神经炎症和神经元细胞凋亡与抑郁症的发生有关。Circ-Bnc2 在抑郁小鼠中表达明显下调，但它在抑郁症进展中的作用尚不清楚。
用脂多糖（LPS）处理 BV2 小胶质细胞诱导神经炎症。用实时定量 PCR 测定 circ-Bnc2、微小 RNA（miR）-497a-5p 和 HECT 结构域 E3 泛素蛋白连接酶 1（HECTD1）的表达。用 Western blot 分析测定神经炎症标志物、凋亡标志物和 HECTD1 的蛋白水平。用 ELISA 检测炎症因子的浓度。用 LPS 诱导的 BV2 细胞的条件培养基培养 HT22 细胞后，用细胞计数试剂盒 8 测定 HT22 细胞的增殖和凋亡，用 EdU 测定和流式细胞术。此外，通过双荧光素酶报告基因检测、RIP 检测和 RNA 下拉实验证实了 miR-497a-5p 与 circ-Bnc2 或 HECTD1 的相互作用。
我们的数据表明，circ-Bnc2 在 LPS 诱导的 BV2 细胞中低表达。功能实验表明，circ-Bnc2 可抑制 LPS 诱导的 BV2 细胞神经炎症，抑制 HT22 细胞凋亡，促进增殖。Circ-Bnc2 可以海绵 miR-497a-5p，circ-Bnc2 的神经保护功能可以被 miR-497a-5p 的过表达逆转。此外，miR-497a-5p 可以靶向 HECTD1。miR-497a-5p 抑制剂可减轻 LPS 诱导的 BV2 细胞神经炎症，减少 HT22 细胞凋亡，HECTD1 敲低也可逆转这一现象。此外，circ-Bnc2 通过海绵 miR-497a-5p 对 HECTD1 表达有正调节作用。
综上所述，我们的研究结果证实，circ-Bnc2 通过调节 miR-497a-5p/HECTD1 轴抑制神经炎症和神经元细胞凋亡，提示 circ-Bnc2 可能是治疗抑郁症的潜在靶点。
