Abstract
BackgroundOligodendrocytes (OLs) death after spinal cord injury (SCI) contributes to demyelination, even leading to a permanent neurological deficit. Besides apoptosis, our previous study demonstrated that OLs underwent receptor-interacting serine-threonine kinase 3(RIP3)/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis. Considering that necroptosis is always accompanied with pro-inflammatory response and quercetin has long been used as anti-inflammatory agent, in the present study we investigated whether quercetin could inhibit necroptosis of OLs and suppress the M1 macrophages/microglia-mediated immune response after SCI as well as the possible mechanism.MethodsIn this study, we applied quercetin, an important flavonoid component of various herbs, to treat rats with SCI and rats injected with saline were employed as the control group. Locomotor functional recovery was evaluated using Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay. In vivo, the necroptosis, apoptosis, and regeneration of OLs were detected by immunohistochemistry, 5′-bromo-2′-deoxyuridine (BrdU) incorporation. The loss of myelin and axons after SCI were evaluated by Luxol fast blue (LFB) staining, immunohistochemistry, and electron microscopic study. The polarization of macrophages/microglia after SCI and the underlying mechanisms were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. In vitro, the ATP and reactive oxygen species (ROS) level examination, propidium iodide (PI) labeling, and Western blotting were used to analyze the necroptosis of cultured OLs, while the signaling pathways-mediated polarization of cultured macrophages/microglia was detected by qRT-PCR and Western blotting.ResultsWe demonstrated that quercetin treatment improved functional recovery in rats after SCI. We then found that quercetin significantly reduced necroptosis of OLs after SCI without influencing apoptosis and regeneration of OLs. Meanwhile, myelin loss and axon loss were also significantly reduced in quercetin-treated rats, as compared to SCI + saline control. Further, we revealed that quercetin could suppress macrophages/microglia polarized to M1 phenotype through inhibition of STAT1 and NF-κB pathway in vivo and in vitro, which contributes to the decreased necroptosis of OLs.ConclusionsQuercetin treatment alleviated necroptosis of OLs partially by inhibiting M1 macrophages/microglia polarization after SCI. Our findings suggest that necroptosis of OLs may be a potential therapeutic target for clinical SCI.
Highlights
Oligodendrocytes (OLs) death after spinal cord injury (SCI) contributes to demyelination, even leading to a permanent neurological deficit
Our previous study demonstrated that some OLs were receptor-interacting serine-threonine kinase 3(RIP3)/mixed lineage kinase domain-like protein (MLKL)-positive after SCI, indicating that necroptosis occurred in OLs [9]
Quercetin prevented necroptosis of OLs without affecting apoptosis and regeneration of OLs after SCI Given that the survival of OLs is crucial for functional recovery after SCI [4, 28], we focused on death and Quantification of cells In our study, the method of profile counts followed by calibration with the empirical method was adopted for unbiased data [23, 24].The 12-μm thickness of the section ensures that the plane of central canal is cut through on every slide, making random selection of the slide possible
Summary
Oligodendrocytes (OLs) death after spinal cord injury (SCI) contributes to demyelination, even leading to a permanent neurological deficit. Our previous study demonstrated that OLs underwent receptor-interacting serine-threonine kinase 3(RIP3)/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis. Given that the survival of OLs is crucial for functional recovery after SCI, in the present study we focused on death and regeneration of OLs. Apoptosis of OLs occurs mainly in the first several hours after SCI, and the injury is accompanied by chronic inflammatory demyelination [5,6,7], indicating other types of cell death accounted for the delayed loss of OLs. It has been reported that necroptosis of OLs mediates axonal degeneration in ALS [8]. Our previous study demonstrated that some OLs were receptor-interacting serine-threonine kinase 3(RIP3)/mixed lineage kinase domain-like protein (MLKL)-positive after SCI, indicating that necroptosis occurred in OLs [9]. There is an urgent need for new treatment strategies to prevent OLs death and to ameliorate the M1 macrophages/microglia-mediated immune response after SCI
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