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    Ruxolitinib attenuates secondary injury after traumatic spinal cord injury
  • Figure 1|RUX mitigates IFN-γ-induced pro-inflammatory cytokines in microglia by targeting the JAK/STAT1 axis. 

    The viability of microglia treated with different doses of RUX was determined by cell counting kit-8 assay at 24 hours post-SCI. The results indicated that concentrations ranging from 0.1 to 1 mM seldom decreased cell viability, while doses at 2 and 5 mM significantly reduced microglial viability (Figure 1A). Next, we determined the effect of RUX at 0.1 to 1 mM on microglia pretreated with IFN-γ for 6 hours. The western blot results indicated that the p-JAK1/JAK1 (P < 0.0001), p-JAK2/JAK2 (P = 0.0003), and p-STAT1/STAT1 (P < 0.0001) ratios were significantly reduced after treatment with 1 mM RUX compared with the IFN-γ group (Figure 1B–E). We also measured the levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 and the anti-inflammatory cytokines IL-10 and IL-27. TNF-α (P < 0.0001), IL-1β (P < 0.0001), and IL-6 (P < 0.0001) mRNA levels were markedly decreased in an RUX concentration–dependent manner (Figure 1F–H); however, IL-10 (P = 0.5665) and IL-27 (P = 0.0574) mRNA levels showed no significant changes compared with the IFN-γ group (Figure 1I and J). Taken together, these data suggest that RUX mitigates IFN-γ-induced pro-inflammatory effects by inhibiting the JAK/STAT1 axis in microglia.

    Figure 2|RUX reduces microglial activation and proliferation in vitro. 

    To further ascertain the anti-inflammatory role of RUX in IFN-γ-stimulated microglia, we selected 1 mM RUX to examine its effect on polarization. Specifically, the inflammatory marker iNOS was detected in microglia by immunofluorescence, and the results indicated that IFN-γ increased iNOS expression in microglia, but that subsequent treatment with RUX decreased the IFN-γ-stimulated high expression level of iNOS (Figure 2A). IL-1β and IL-6 production also increased after IFN-γ stimulation, whereas RUX attenuated their expression in microglia (Figure 2B). Moreover, western blotting showed that iNOS (P = 0.0002) and COX-2 (P < 0.0001) expressions were significantly decreased by treatment with RUX following IFN-γ stimulation (Figure 2C–E). Cell proliferation, as measured by the 5-ethynyl-2′-deoxyuridine assay, was clearly decreased after RUX treatment (Figure 2F). Consistent with this, expression of Ki67, a cell proliferation marker (Scholzen and Gerdes, 2000), was also inhibited by RUX (Figure 2G). Taken together, these results indicate that RUX inhibits IFN-γ–induced microglial inflammation and proliferation.

    Figure 3|RUX alleviates inflammation in the spinal cords of SCI mice.  

    In vivo, we used the activated microglial marker iNOS to assess microglial-induced neuroinflammation. The distribution of activated p-STAT1+ microglia was then assessed. Immunofluorescence staining showed that the number of IBA1+/iNOS+ microglia was increased post-SCI; however, administration of RUX reduced this amount (Figure 3A). In addition, the increase in the number of p-STAT1+ microglia was mitigated by treatment with RUX by day 3 post-SCI (Figure 3A). We also found that the increased expression of IL-1β, IL-6, TNF-α, and COX-2 post-SCI was significantly reduced by treatment with RUX by day 3 post-SCI (Figure 3B). Furthermore, enzyme-linked immunosorbent assays were used to measure the levels of TNF-α, IL-1β, and IL-6 at 1 and 3 days post-SCI. The results showed that high levels of TNF-α, IL-1β, and IL-6 were present at 1 day post-SCI, and that the levels slightly dropped at 3 days post-SCI. Strikingly, mice treated with RUX exhibited a marked decrease in expression of the three cytokines compared with the untreated SCI mice on days 1 and 3 post-SCI (P < 0.0001; Figure 3C and D). H&E staining indicated a smaller area of pathological damage in SCI mice after RUX treatment on day 3 post-SCI (P < 0.0001; Figure 3E and F). Collectively, these results indicate that RUX reduces the inflammatory response post-SCI.

    Figure 4|RUX ameliorates inflammation-induced neuropathology in SCI mice.   

    The glial scar, which is composed of microglia and astrocytes, was detected by immunofluorescence staining. At 7 days post-SCI, the initial glial scar had formed; however, the number of microglia and astrocytes was reduced markedly after RUX treatment in SCI mice (P < 0.0001; Figure 4A–C). Notably, SCI mice exhibited dispersed distribution of activated microglia; however, we found that activated microglia (indicated with white arrows) gathered near the injured foci, and that other microglia (indicated with yellow arrows) remained distal to the foci (Figure 4A). SCI mice treated with RUX consistently showed a less dispersed and smaller glial scar at 28 days post-SCI compared with SCI mice (P < 0.0001; Figure 4D–F). Interestingly, RUX administration promoted a reduction in the area of the glial scar, to a point where it encompassed only the injured epicenter, potentially providing more space for axonal regeneration (Figure 4D). As expected, severe demyelination and axonal loss were evident at 7 days post-SCI and were significantly ameliorated after RUX treatment (Figure 4G–I). Therefore, RUX may protect against secondary neurological injury due to its potent anti-inflammatory properties.

    Figure 5|RUX reduces the degree of histological damage and functional disorder following SCI. 

    Given that formation of the glial scar was nearly complete at 28 days post-SCI, we compared histological staining of the injured spinal cord at 7 and 28 days post-SCI. H&E and Nissl staining at 7 days post-SCI showed that treatment with RUX resulted in a larger number of viable neurons compared with untreated mice (P < 0.0001; Figure 5A–C) and a reduction in SCI-induced tissue loss. In contrast, the number of neurons was unchanged both in RUX-treated and untreated mice at 28 days post-SCI (P < 0.0001; Figure 5D–F). In the swimming test, RUX-treated mice exhibited a smaller angle between their trunk and the water surface, as well as a higher frequency of hindlimb movements compared with SCI mice at 28 days post-SCI (Figure 5G). The LSS scores in the SCI + RUX group were higher than those in the SCI group beginning at 14 days post-SCI (P = 0.0109; Figure 5H). The Basso mouse scale tests also showed that RUX administration significantly improved locomotor scores beginning at 7 days post-SCI (P < 0.0001; Figure 5I). Taken together, these results indicate that treatment with RUX improves tissue protection and functional recovery post-SCI.


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  • 发布日期: 2022-03-10  浏览: 327
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