蒙花苷通过抑制TLR4/NF-κB通路抑制小鼠脊髓损伤后小胶质细胞活化介导的神经炎症和神经元凋亡

doi:10.12122/j.issn.1673-4254.2024.08.18

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (8): 1589-1598.doi: 10.12122/j.issn.1673-4254.2024.08.18

• • 上一篇

蒙花苷通过抑制TLR4/NF-κB通路抑制小鼠脊髓损伤后小胶质细胞活化介导的神经炎症和神经元凋亡

肖林雨¹(), 段婷², 夏勇生⁴, 陈悦¹, 孙洋¹, 许轶博⁶, 徐磊¹, 闫兴洲¹, 胡建国³^,⁵()

^1.蚌埠医科大学第一附属医院，康复科，安徽蚌埠 233004
^2.蚌埠医科大学第一附属医院，急诊内科，安徽蚌埠 233004
^3.蚌埠医科大学第一附属医院，炎症相关性疾病基础与转化研究安徽省重点实验室，安徽蚌埠 233004
^4.蚌埠医科大学第一附属医院，胃肠外科，安徽蚌埠 233004
^5.蚌埠医科大学第一附属医院，检验科，安徽蚌埠 233004
^6.蚌埠医科大学基础医学院组织与胚胎学教研室，安徽蚌埠 233030

收稿日期:2024-06-03 出版日期:2024-08-20 发布日期:2024-09-06
通讯作者: 胡建国 E-mail:xiaolinyu1123@163.com;jghu9200@bbmc.edu.cn
作者简介:肖林雨，在读硕士研究生，E-mail: xiaolinyu1123@163.com
基金资助:
国家自然科学基金(82071360);蚌埠医学院第一附属医院高水平科技创新团队(BYYFY2022TD002);安徽省新时代育人质量工程项目（研究生教育）(2023xscx122)

Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway

Linyu XIAO¹(), Ting DUAN², Yongsheng XIA⁴, Yue CHEN¹, Yang SUN¹, Yibo XU⁶, Lei XU¹, Xingzhou YAN¹, Jianguo HU³^,⁵()

^1.Department of Rehabilitation Medicine,
^2.Department of Emergency Medicine,
^3.Anhui Provincial Key Laboratory of Basic and Translational Research of Inflammation-related Diseases,
^4.Department of Gastrointestinal Surgery,
^5.Clinical Laboratory, First Affiliated Hospital of Bengbu Medical University, Bengbu 233004, China
^6.Department of Histology and Embryology, College of Basic Medical Sciences, Bengbu Medical University, Bengbu 233030, China

Received:2024-06-03 Online:2024-08-20 Published:2024-09-06
Contact: Jianguo HU E-mail:xiaolinyu1123@163.com;jghu9200@bbmc.edu.cn
Supported by:
National Natural Science Foundation of China(82071360)

摘要/Abstract

摘要：

目的探讨蒙花苷（LIN）在脊髓损伤（SCI）后对激活小胶质细胞介导的炎症和神经元凋亡的神经保护作用和潜在机制。方法将50只8~10周龄C57BL/6J小鼠随机分为假手术组（Sham组）、脊髓损伤模型组（SCI组）和LIN药物治疗组（12.5、25、50 mg/kg），10只/组。使用巴索小鼠评分量表（BMS）、斜板实验和脚印分析评估SCI小鼠的运动功能恢复情况；通过HE染色和LFB染色分别评估脊髓组织损伤面积和髓鞘化面积。采用Nissl染色、免疫荧光及Western blotting观察损伤脊髓组织前角运动神经元的保留情况。体外实验通过LPS诱导BV2细胞建立体外炎症模型，同时给予蒙花苷进行干预，将BV2细胞分为对照组（Con-B）、脂多糖组（LPS-B）及蒙花苷组（LIN-B）。通过免疫荧光、Western blotting、RT-qPCR及ELISA检测体内外小胶质细胞活化及炎症因子的释放情况，进一步采用Western blotting验证信号通路的改变。构建体外BV2细胞和HT22细胞共培养模型，分为Con-H组、LPS-H组以及LIN-H组。采用Western blotting检测激活小胶质细胞介导的HT22细胞凋亡情况。结果行为学检测发现，LIN组小鼠BMS评分显著提高，承受的斜板高度更高，后肢行走协调且足迹清晰（P<0.05）。组织学染色结果显示，LIN组小鼠脊髓组织损伤面积减少，髓鞘化面积增加，并且小鼠脊髓前角运动神经元的数量增加（P<0.05）。体内外实验证实，LIN显著抑制小胶质细胞的活化和炎症因子（iNOS、COX-2、TNF-α、IL-6、IL-1β）的释放，并减少了体内神经元的凋亡（P<0.05）。Western blotting显示LIN抑制小胶质细胞活化可能与抑制TLR4/NF-κB信号通路有关。体外共培养实验结果显示，LIN的干预能够减少炎症介导的神经元凋亡数量（P<0.05）。结论 LIN可能通过抑制TLR4/NF-κB信号通路抑制小胶质细胞的激活，减轻神经炎症，改善SCI后的神经元凋亡，提高SCI小鼠运动能力，发挥其神经保护作用。

关键词: 脊髓损伤, 蒙花苷, 神经炎症, 神凋亡, TLR4/NF-κB

Abstract:

Objective To investigate the mechanism underlying the neuroprotective effect of linarin (LIN) against microglia activation-mediated inflammation and neuronal apoptosis following spinal cord injury (SCI). Methods Fifty C57BL/6J mice (8-10 weeks old) were randomized to receive sham operation, SCI and linarin treatment at 12.5, 25, and 50 mg/kg following SCI (n=10). Locomotor function recovery of the SCI mice was assessed using the Basso Mouse Scale, inclined plane test, and footprint analysis, and spinal cord tissue damage and myelination were evaluated using HE and LFB staining. Nissl staining, immunofluorescence assay and Western blotting were used to observe surviving anterior horn motor neurons in injured spinal cord tissue. In cultured BV2 cells, the effects of linarin against lipopolysaccharide (LPS)‑induced microglia activation, inflammatory factor release and signaling pathway changes were assessed with immunofluorescence staining, Western blotting, RT-qPCR, and ELISA. In a BV2 and HT22 cell co-culture system, Western blotting was performed to examine the effect of linarin against HT22 cell apoptosis mediated by LPS-induced microglia activation. Results Linarin treatment significantly improved locomotor function (P<0.05), reduced spinal cord damage area, increased spinal cord myelination, and increased the number of motor neurons in the anterior horn of the SCI mice (P<0.05). In both SCI mice and cultured BV2 cells, linarin effectively inhibited glial cell activation and suppressed the release of iNOS, COX-2, TNF-α, IL-6, and IL-1β, resulting also in reduced neuronal apoptosis in SCI mice (P<0.05). Western blotting suggested that linarin-induced microglial activation inhibition was mediated by inhibition of the TLR4/NF-κB signaling pathway. In the cell co-culture experiments, linarin treatment significantly decreased inflammation-mediated apoptosis of HT22 cells (P<0.05). Conclusion The neuroprotective effect of linarin is medicated by inhibition of microglia activation via suppressing the TLR4/NF‑κB signaling pathway, which mitigates neural inflammation and reduce neuronal apoptosis to enhance motor function of the SCI mice.

Key words: spinal cord injury, linarin, neuroinflammation, apoptosis, TLR4/NF-κB

肖林雨, 段婷, 夏勇生, 陈悦, 孙洋, 许轶博, 徐磊, 闫兴洲, 胡建国. 蒙花苷通过抑制TLR4/NF-κB通路抑制小鼠脊髓损伤后小胶质细胞活化介导的神经炎症和神经元凋亡[J]. 南方医科大学学报, 2024, 44(8): 1589-1598.

Linyu XIAO, Ting DUAN, Yongsheng XIA, Yue CHEN, Yang SUN, Yibo XU, Lei XU, Xingzhou YAN, Jianguo HU. Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway[J]. Journal of Southern Medical University, 2024, 44(8): 1589-1598.

图/表 7

图1 LIN治疗改善SCI小鼠运动功能

Fig.1 Linarin (LIN) treatment improves motor function in SCI mice. A: BMS score. B: Inclined plate experiment. C, D: Footprint score. *P<0.05 vs SCI group.

图2 LIN治疗改善SCI小鼠脊髓组织病理损伤

Fig.2 LIN treatment improves spinal cord histopathology in SCI mice. A: HE staining of a cross section at the center of spinal cord injury. B: Quantitative analysis of spinal cord injury area in LIN treatment group and SCI group. C: LFB staining of a cross section at the center of spinal cord injury. D: Quantitative analysis of myelination area in LIN treatment group and SCI group. *P<0.05 vs SCI group.

图3 LIN治疗抑制脊髓组织中小胶质细胞的活化和炎症因子的分泌

Fig3 LIN treatment inhibits microglia activation and inflammatory factor secretion in mouse spinal cord tissues. A: Microglia marker (CD11b) and activation marker (CD68) detected by immunofluorescence assay. B: Statistical graphs of the number of CD11b+CD68+ cells. C, D: Protein expression of iNOS and COX-2 detected by Western blotting and quantitative analyses. E-G: RT-qPCR for detecting relative expression levels of TNF-α, IL-6 and IL-1β mRNA. H-J: ELISA for detecting protein expression levels of TNF-α, IL-6 and IL-1β. #P<0.05 vs Sham group, *P<0.05 vs SCI group.

图4 LIN治疗减轻SCI后神经元凋亡

Fig.4 LIN treatment reduces neuronal apoptosis in mouse spinal cord after SCI. A: Nissl staining of the center of injury. B: Number of residual motor neurons in the anterior horn of the spinal cord in LIN-treated group and SCI group. C: Immunofluorescence detection of neuronal cell marker NeuN and apoptotic protein cleaved-caspase 3 (c-caspase 3). D: Statistical graph of the number of NeuN+c-caspase 3+ cells. E-H: Statistical graph of c-caspase 3, Bax and Bcl-2 protein expressions detected by Western blotting. #P<0.05 vs Sham group, *P<0.05 vs SCI group.

图5 LIN干预抑制小胶质细胞的活化和体外炎症因子的分泌

Fig.5 LIN inhibits microglia activation and secretion of inflammatory factors in BV2 cells. A: Cytotoxicity assay of LIN in BV2 cells. B: Optimal time determination of LPS-induced BV2 cell injury. C: Effect of LIN on LPS-induced BV2 cell viability. D, E: Statistical graphs of protein expression and quantitative analysis of iNOS and COX-2 detected by Western blotting. F: Immunofluorescence detection of microglia marker (CD11b) and activation marker (CD68) in BV2 cells. G: Statistical graphs of the number of CD11b+CD68+ cells. H-J: Relative mRNA expression levels of TNF-α, IL-6 and IL-1β detected by RT-qPCR. K-M: Protein expression levels of TNF-α, IL-6 and IL-1β detected by ELISA. #P<0.05 vs Con-B group, *P<0.05 vs LPS-B group.

图6 LIN抑制TLR4/NF-κB信号通路

Fig6 LIN inhibits the TLR4/NF‑κB signaling pathway. A-E: Statistical graphs of the expression and quantitative analysis of TLR4/NF-κB pathway proteins after LIN intervention detected by Western blotting. #P<0.05 vs Con-B group, *P<0.05 vs LPS-B group.

图7 LIN干预对炎症介导的神经元凋亡的保护作用

Fig.7 Protective effect of LIN agaisnt inflammation-mediated neuronal apoptosis. A: CCK8 detection of LIN toxicity on HT22 cells. B-E: Statistical graphs of cleaved caspase-3 (c-caspase 3), Bax and Bcl-2 protein expression and quantitative analysis in HT22 cells detected by Western blotting. #P<0.05 vs Con-H group, *P<0.05 vs LPS-H group.

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蒙花苷通过抑制TLR4/NF-κB通路抑制小鼠脊髓损伤后小胶质细胞活化介导的神经炎症和神经元凋亡

Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway

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