Table_1_Photobiomodulation Attenuates Neurotoxic Polarization of Macrophages by Inhibiting the Notch1-HIF-1α/NF-κB Signalling Pathway in Mice With Spinal Cord Injury.xlsx

Spinal cord injury (SCI) is a catastrophic disease with a complex pathogenesis that includes inflammation, oxidative stress, and glial scar formation. Macrophages are the main mediators of the inflammatory response and are distributed in the epicentre of the SCI. Macrophages have neurotoxic and neuroprotective phenotypes (also known as classically and alternatively activated macrophages or M1 and M2 macrophages) that are associated with pro- or anti- inflammatory gene expression. Our previous study demonstrated that photobiomodulation (PBM) alters the polarization state of macrophages in the SCI region towards the M2 phenotype and promotes the recovery of motor function in rats with SCI. However, the mechanism by which PBM promotes SCI repair remains largely undefined. This study is based on the replacement of conventional percutaneous irradiation with implantable biofibre optic in vivo irradiation. The aim was to further investigate the effects of PBM on SCI in mice under new irradiation patterns and its potential mechanisms of action. PBM was administered to male mice with clamped SCI for four consecutive weeks and significantly promoted the recovery of motor function in mice. Analysis of the macrophage phenotypes in the epicentre of the SCI in mice showed that PBM mainly inhibited the neurotoxic activation of macrophages in the SCI area and reduced the secretion of inflammatory factors such as IL-1α and IL-6; PBM had no effect on M2 macrophages. Immediately afterwards, we constructed in vitro models of the inflammatory polarization of macrophages and PBM intervention. We found that PBM attenuated the neurotoxicity of M1 macrophages on VSC 4.1 motor neurons and dorsal root ganglion (DRG) neurons. The effects of PBM on neurotoxic macrophages and the possible mechanisms of action were analysed using RNA sequencing (RNA-seq), which confirmed that the main role of PBM was to modulate the inflammatory response and immune system processes. Analysis of the differentially expressed genes (DEGs) associated with the inflammatory response showed that PBM had the most significant regulatory effects on genes such as interleukin (IL)-1α, IL-6, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) and had obvious inhibitory effects on inflammation-related Notch1 and hypoxia-inducible factor-1α (HIF-1α) pathway genes. RNA-seq analysis of the effect of PBM on gene expression in resting-state macrophages and M2 macrophages did not show significant differences (data not shown). In conclusion, PBM promoted better motor recovery after SCI in mice by inhibiting the neurotoxic polarization of macrophages and the release of inflammatory mediators by acting on the Notch1-HIF-1α/NF-κB Signalling Pathway.

脊髓损伤（Spinal cord injury, SCI）是一类发病机制复杂的灾难性疾病，涉及炎症反应、氧化应激及胶质瘢痕形成等过程。巨噬细胞（Macrophages）是炎症反应的主要介导因子，分布于脊髓损伤的损伤中心区域。巨噬细胞可分为神经毒性与神经保护两种表型，即经典活化与替代活化巨噬细胞，或称M1、M2型巨噬细胞，分别与促炎、抗炎基因表达相关。本团队既往研究表明，光生物调节作用（photobiomodulation, PBM）可使脊髓损伤区域的巨噬细胞极化为M2表型，并促进脊髓损伤大鼠的运动功能恢复。然而，光生物调节作用促进脊髓损伤修复的具体机制仍未完全阐明。本研究采用植入式生物光纤体内照射替代传统经皮照射，旨在进一步探究新型照射模式下光生物调节作用对小鼠脊髓损伤的影响及其潜在作用机制。本研究对接受脊髓钳夹损伤的雄性小鼠连续4周施加光生物调节干预，结果显示其可显著促进小鼠的运动功能恢复。对小鼠脊髓损伤中心区域的巨噬细胞表型分析显示，光生物调节作用主要抑制脊髓损伤区域巨噬细胞的神经毒性活化，并降低IL-1α、IL-6等炎症因子的分泌；而对M2型巨噬细胞无显著影响。随后，本研究构建了巨噬细胞炎性极化及光生物调节干预的体外模型，结果发现光生物调节作用可减轻M1型巨噬细胞对VSC 4.1运动神经元及背根神经节（dorsal root ganglion, DRG）神经元的神经毒性。本研究通过RNA测序（RNA sequencing, RNA-seq）分析了光生物调节作用对神经毒性巨噬细胞的影响及其潜在作用机制，证实其核心作用为调控炎症反应与免疫系统进程。对炎症相关差异表达基因（differentially expressed genes, DEGs）的分析显示，光生物调节作用对白介素（interleukin, IL）-1α、IL-6、环氧合酶-2（cyclooxygenase-2, COX-2）及诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）等基因具有最显著的调控作用，并对炎症相关Notch1及缺氧诱导因子-1α（hypoxia-inducible factor-1α, HIF-1α）通路基因具有明确的抑制效果。针对静息态巨噬细胞及M2型巨噬细胞的基因表达，RNA测序分析未发现显著差异（数据未展示）。综上，光生物调节作用可通过调控Notch1-HIF-1α/NF-κB信号通路，抑制巨噬细胞的神经毒性极化及炎症介质释放，从而促进小鼠脊髓损伤后运动功能的更好恢复。