Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: RUX restores EAAT2 loss in astrocytes by inhibiting the activation of STAT3 in vitro. (A) Representative western blotting of p-JAK2, JAK2, p-STAT3 and STAT3 expression of astrocytes in each group pre-treated with RUX (0.2, 0.5 and 1 µM) ( n = 3 per group). (B) Quantitative analysis of p-JAK2/JAK2 and p-STAT3/STAT3 level in each group. (C) Immunocytochemistry of GFAP (green, Alexa Fluor 488) and p-STAT3 (red, Alexa Fluor 594) in primary mouse astrocytes. DAPI (blue) was used to stain nuclei. The A1IM group exhibited higher nuclear p-STAT3 expression compared with the Control group, whereas the RUX group displayed decreased nuclear p-STAT3 expression compared with the A1IM group. Scale bar: 40 µm. (D) Quantitative results of relative p-STAT3 intensity. (E) Representative western blotting of STAT3 expression of astrocytes pre-treated with S3I-201 or vehicle. (F) Quantitative results of relative STAT3 expression level. (G) Primary mouse astrocytes were pretreated with S3I-201 (50 μM) or RUX for 1 hour and then exposed to LPS (1 µg/mL) or PBS for 5 hours. Western blotting was conducted to assess EAAT2 expression level, followed by quantitative analysis of EAAT2 protein expression (H). (I) Glutamate (100 nM) was introduced into each culture medium, and the relative glutamate uptake of astrocytes in each group was measured. Data are normalized to the control group. Data are presented as the mean ± SD ( n = 3 per group). ## P < 0.01, vs. control group; * P < 0.05, ** P < 0.01, vs. A1IM group (one-way analysis of variance followed by Tukey’s post hoc test). A1IM: A1-like astrocyte induction medium; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; EAAT2: excitatory amino acid transporter 2; GFAP: glial fibrillary acidic protein; JAK2: Janus kinase 2; ns: not significant; p-JAK2: phosphorylated JAK2; p-STAT3: phosphorylated STAT3; RUX: ruxolitinib; S3I-201: a STAT3 inhibitor; STAT3: signal transducer and activator of transcription 3.

Article Snippet: In experiments, cells were starved in DMEM for 1 hour before being treated with RUX (0.2, 0.5, and 1 µM), S3I-201 (50 µm; Selleck, Houston, TX, USA), or solvent (dimethyl sulfoxide, DMSO).

Techniques: Activation Assay, In Vitro, Western Blot, Expressing, Immunocytochemistry, Staining