Abrocitinib Attenuates Microglia-Mediated Neuroinflammation after Traumatic Brain Injury via Inhibiting the JAK1/STAT1/NF-κB Pathway

Abstract

Background and purpose: Neuroinflammation has been shown to play a critical role in secondary craniocerebral injury, leading to poor outcomes for TBI patients. Abrocitinib, a Janus kinase1 (JAK1) selective inhibitor approved to treat atopic dermatitis (AD) by the Food and Drug Administration (FDA), possesses a novel anti-inflammatory effect. In this study, we investigated whether abrocitinib could ameliorate neuroinflammation and exert a neuroprotective effect in traumatic brain injury (TBI) models.

Methods: First, next-generation sequencing (NGS) was used to select genes closely related to neuroinflammation after TBI. Then, magnetic resonance imaging (MRI) was used to dynamically observe the changes in traumatic focus on the 1st, 3rd, and 7th days after the induction of fluid percussion injury (FPI). Moreover, abrocitinib's effects on neurobehaviors were evaluated. A routine peripheral blood test was carried out and Evans blue dye extravasation, cerebral cortical blood flow, the levels of inflammatory cytokines, and changes in the numbers of inflammatory cells were evaluated to investigate the function of abrocitinib on the 1st day post-injury. Furthermore, the JAK1/signal transducer and activator of transcription1 (STAT1)/nuclear factor kappa (NF-κB) pathway was assessed.

Results: In vivo, abrocitinib treatment was found to shrink the trauma lesions. Compared to the TBI group, the abrocitinib treatment group showed better neurological function, less blood-brain barrier (BBB) leakage, improved intracranial blood flow, relieved inflammatory cell infiltration, and reduced levels of inflammatory cytokines. In vitro, abrocitinib treatment was shown to reduce the pro-inflammatory M1 microglia phenotype and shift microglial polarization toward the anti-inflammatory M2 phenotype. The WB and IHC results showed that abrocitinib played a neuroprotective role by restraining JAK1/STAT1/NF-κB levels after TBI.

Conclusions: Collectively, abrocitinib treatment after TBI is accompanied by improvements in neurological function consistent with radiological, histopathological, and biochemical changes. Therefore, abrocitinib can indeed reduce excessive neuroinflammation by restraining the JAK1/STAT1/NF-κB pathway.

Keywords: JAK1/STAT1/NF-κB; TBI; abrocitinib; neuroinflammation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Gene expression analysis for TBI and control mice. Volcano plot of differential analysis (A). Heatmap of differential analysis (B). Comparison of TNF-α, IL-1β, IL-6, NLRP3, and ASC expression between TBI group and control group (C) (n = 5, Kruskal-Wallis test). Bubble chart of BP, CC, and MF analysis (D). Bubble chart of KEGG analysis (E). Enrichment plots for GO and KEGG items from gene set enrichment analysis (GSEA) (F,G). Heatmap of immune infiltration in sham and TBI groups (H). Violin plot of immune infiltration in sham and TBI groups (I). All data are shown as mean ± SD. * p < 0.05, ** p < 0.01.

Figure 2

Effects of abrocitinib on hematoma absorption, the results of behavioral tests, cerebral edema, and cerebral cortical blood flow. Changes in trauma focus were dynamically observed with the help of MRI (A,B) (n = 6, repeated measures ANOVA with Tukey’s post-hoc test). In addition, neural function was evaluated using mNSS scores (C) (n = 6, repeated measures ANOVA with Tukey’s post-hoc test). FPI-induced vascular leakage was measured via Evans blue dye extravasation (D,E) (n = 4, Kruskal-Wallis test) and cerebral cortical blood flow (F,G) (n = 5–6, one-way ANOVA with Tukey’s post-hoc test) was also monitored on the 1st day after TBI. All data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Changes in pathological morphology. Morphological changes were observed using H&E staining (A). The number of surviving neurons (B,D) and the changes in apoptotic cells around the wound focus were observed via Nissl staining and TUNEL staining, respectively (DAPI—blue; TUNEL—red) (C,E) (n = 6, one-way ANOVA with Tukey’s post-hoc test). All data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Effects of abrocitinib on the JAK1/STAT1 pathway. On the 1st day after TBI, JAK1 and STAT1 showed a dramatic increase in activation. After abrocitinib treatment, the activated JAK1 and STAT1 were significantly decreased, as shown in the WB results above and can be displayed through WB (n = 5–9, one-way ANOVA with Tukey’s post-hoc test). All data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Changes in the infiltration of inflammatory cells and the activation of microglia. From the IHC staining, the increased neutrophils and microglia around the trauma focus caused by TBI were attenuated by abrocitinib (A–D) (n = 6 Kruskal-Wallis test). The effect of abrocitinib on LPS-mediated BV2 microglial polarization was also observed in vitro (E). M1-phenotype: iNOS + (red) and Iba-1 + (green); M2-phenotype: Arg-1 + (red) and Iba-1 + (green). Scale bar = 20 μm (n = 6 one-way ANOVA with Tukey’s post-hoc test). The changes in inflammatory cytokines (IL-6 and TNF-α) after brain injury and the effects of abrocitinib were determined with the help of ELISA (F–G) (n = 5–9 one-way ANOVA with Tukey’s post-hoc test). All data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Effects of abrocitinib on NFκB-related inflammation and pyroptosis pathways. On the 1st day after TBI, NFκB-related inflammation and the activation of pyroptosis pathways were dramatically increased. After abrocitinib treatment, the indicators of NFκB-related inflammation and pyroptosis pathways were significantly decreased, as can be seen from the WB (n = 5–9 one-way ANOVA with Tukey’s post-hoc test) and GSDMD IHC staining (A–C) (n = 6 Kruskal-Wallis test). From the ELISA results, the changes in inflammatory cytokines (IL-1β and IL-18) after brain injury and the effects of abrocitinib were precisely revealed (D,E) (n = 5–6 one-way ANOVA with Tukey’s post-hoc test). All data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.