高频重复经颅磁刺激（rTMS）通过 let-7b-5p/HMGA2/NF-κB 信号通路抑制 M1 小胶质细胞极化，从而起到对缺血性中风的保护作用。
High-frequency repetitive transcranial magnetic stimulation (rTMS) protects against ischemic stroke by inhibiting M1 microglia polarization through let-7b-5p/HMGA2/NF-κB signaling pathway.
机构信息
Department of Neurology, Nanjing First Hospital, Nanjing Medical University, 68# Changle Road, Nanjing, 210029, Jiangsu, China.
Department of Neurology, Jinling Hospital, Medical School of Nanjing University, Nanjing, 210002, Jiangsu, China.
出版信息
BMC Neurosci. 2022 Aug 4;23(1):49. doi: 10.1186/s12868-022-00735-7.
BACKGROUND
Microglia assume opposite phenotypes in response to ischemic brain injury, exerting neurotoxic and neuroprotective effects under different ischemic stages. Modulating M1/M2 polarization is a potential therapy for treating ischemic stroke. Repetitive transcranial magnetic stimulation (rTMS) held the capacity to regulate neuroinflammation and astrocytic polarization, but little is known about rTMS effects on microglia. Therefore, the present study aimed to examine the rTMS influence on microglia polarization and the underlying possible molecular mechanisms in ischemic stroke models.
METHODS
Previously reported 10 Hz rTMS protocol that regulated astrocytic polarization was used to stimulate transient middle cerebral artery occlusion (MCAO) rats and oxygen and glucose deprivation/reoxygenation (OGD/R) injured BV2 cells. Specific expression levels of M1 marker iNOS and M2 marker CD206 were measured by western blotting and immunofluorescence. MicroRNA expression changes detected by high-throughput second-generation sequencing were validated by RT-PCR and fluorescence in situ hybridization (FISH) analysis. Dual-luciferase report assay and miRNA knock-down were applied to verify the possible mechanisms regulated by rTMS. Microglia culture medium (MCM) from different groups were collected to measure the TNF-α and IL-10 concentrations, and detect the influence on neuronal survival. Finally, TTC staining and modified Neurological Severity Score (mNSS) were used to determine the effects of MCM on ischemic stroke volume and neurological functions.
RESULTS
The 10 Hz rTMS inhibited ischemia/reperfusion induced M1 microglia and significantly increased let-7b-5p level in microglia. HMGA2 was predicted and proved to be the target protein of let-7b-5p. HMGA2 and its downstream NF-κB signaling pathway were inhibited by rTMS. Microglia culture medium (MCM) collected from rTMS treated microglia contained lower TNF-α concentration but higher IL-10 concentration than no rTMS treated MCM, reducing ischemic volumes and neurological deficits of MCAO mice. However, knockdown of let-7b-5p by antagomir reversed rTMS effects on microglia phenotype and associated HMGA/NF-κB activation and neurological recovery.
CONCLUSION
High-frequency rTMS could alleviate ischemic stroke injury through inhibiting M1 microglia polarization via regulating let-7b-5p/HMGA2/NF-κB signaling pathway in MCAO models.
背景
小胶质细胞在缺血性脑损伤中呈现相反的表型，在不同的缺血阶段发挥神经毒性和神经保护作用。调节 M1/M2 极化是治疗缺血性中风的一种潜在疗法。重复经颅磁刺激（rTMS）具有调节神经炎症和星形胶质细胞极化的能力，但人们对 rTMS 对小胶质细胞的影响知之甚少。因此，本研究旨在探讨 rTMS 对缺血性中风模型中小胶质细胞极化的影响及其潜在的分子机制。
方法
采用已报道的调节星形胶质细胞极化的 10 Hz rTMS 方案刺激短暂性大脑中动脉闭塞（MCAO）大鼠和氧葡萄糖剥夺/复氧（OGD/R）损伤的 BV2 细胞。通过 Western blot 和免疫荧光法测定 M1 标志物诱导型一氧化氮合酶（iNOS）和 M2 标志物 CD206 的特异性表达水平。通过高通量第二代测序检测到的 microRNA 表达变化通过 RT-PCR 和荧光原位杂交（FISH）分析进行验证。双荧光素酶报告试验和 miRNA 敲低用于验证 rTMS 调节的可能机制。收集不同组的小胶质细胞培养基（MCM）测量 TNF-α和 IL-10 浓度，并检测对神经元存活的影响。最后，TTC 染色和改良神经功能缺损评分（mNSS）用于确定 MCM 对缺血性中风体积和神经功能的影响。
结果
10 Hz rTMS 抑制缺血再灌注诱导的 M1 小胶质细胞，显著增加小胶质细胞中 let-7b-5p 的水平。HMGA2 被预测并证明是 let-7b-5p 的靶蛋白。rTMS 抑制 HMGA2 及其下游 NF-κB 信号通路。与未接受 rTMS 处理的 MCM 相比，来自接受 rTMS 处理的小胶质细胞的小胶质细胞培养基（MCM）中 TNF-α浓度较低，但 IL-10 浓度较高，减少 MCAO 小鼠的缺血体积和神经功能缺损。然而，用反义寡核苷酸逆转 let-7b-5p 的敲低可逆转 rTMS 对小胶质细胞表型及相关 HMGA/NF-κB 激活和神经恢复的影响。
结论
在 MCAO 模型中，高频 rTMS 通过调节 let-7b-5p/HMGA2/NF-κB 信号通路抑制 M1 小胶质细胞极化，从而减轻缺血性中风损伤。
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