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Fig. 6 | Journal of Neuroinflammation

Fig. 6

From: Inhibition of lysophosphatidic acid receptor 1–3 deteriorates experimental autoimmune encephalomyelitis by inducing oxidative stress

Fig. 6

Effect of Ki16425 on proinflammatory cytokine milieu and oxidative stress in spinal cords after EAElow induction. AM Protein lysates and total RNA were isolated from the lumbar spinal cords (n = 3 per group) of the sham, EAElow, EAElow + Ki16425 (15 and 30 mg/kg), and Ki16425 (30 mg/kg) groups at day 19–20 after immunization. AC The lysates (n = 3 per group) were analyzed by Western blot using COX-2 (A), p-NF-κB (B), and p-p38 MAPK (C) antibodies to represent inflammatory signaling pathways and the results were quantified (AC). DF Total RNA (n = 3 per group) was analyzed by real-time PCR to measure mRNA expression of COX-2 (D), iNOS (E), and TNF-α (F) as representative cytokines. GI Lysates of lumbar spinal cords (n = 3 per group) from sham, EAElow, EAElow + Ki16425 (15 and 30 mg/kg), and Ki16425 (30 mg/kg) groups at day 19–20 after immunization were analyzed by Western blot using 4-HNE antibody followed by quantification (G). Cryosections (n = 3 per spinal cord) of lumbar spinal cords (n = 3 per group) from each group were subjected to MitoSOX™ assay to measure mitochondrial superoxide level (H) followed by quantification (I). JM Lumbar spinal cords (n = 3) from each group were analyzed by real-time PCR to investigate mRNA expression levels of NOX1 (J), NOX-2 (K), NOX3 (L), and NOX4 (M). Bar = 100 µm. Data are expressed as mean value, fold induction, or cell number ± SEM (ANOVA test; #p < 0.05 versus sham group; *p < 0.05 and **p < 0.01 versus EAElow group)

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