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

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.

Keywords: TLR4/NF-κB; apoptosis; linarin; neuroinflammation; spinal cord injury.

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.