Fig 2: Injury-induced CISD2 downregulation, enhanced GFAP mRNA and protein expression, and proinflammation in LPS-challenged ALT astrocytes. WBM can prevent the abovementioned detrimental effects. Results of mRNA expression of GFAP (a), GFAP protein (b), mRNA expression of IL-1ß (c), IL-6 (d), IL-4 (e), and CISD2 (f) in ALT cells with or without administration of WBM and a-ESA. Vertical bars indicate the mean ± standard error of the mean (SEM) of mRNA expression (n = 3). *P < 0.05vs. control,**P < 0.01vs. control,***P < 0.001vs. control, #P < 0.05, ##P < 0.01, and ###P < 0.001indicate a significant difference. Pair-wise multiple comparisons between groups were performed using the Newman-Keuls method.

Fig 3: Curcumin enhanced cell viability and attenuated LPS-challenge-driven cell death in non-stressed and LPS-challenged neural cells. The knockdown of CISD2 expression was shown to reduce the protective effects of curcumin. The eight groups of cells included the following: (i) scrambled RNA-transfected control cells, untreated with LPS and curcumin; (ii) scrambled RNA-transfected cells treated with curcumin; (iii) scramble RNA-transfected, LPS-challenged cells, without curcumin treatment; (iv) scramble RNA-transfected, LPS-challenged cells treated with curcumin; (v) siCID2-transfected control cells without LPS or curcumin treatment; (vi) siCID2-transfected cells treated with curcumin; (vii) siCID2-transfected, LPS-challenged cells without curcumin treatment; (viii) siCID2-transfected, LPS-challenged cells, treated with curcumin. Cell viability was measured using alamarBlue® assay. Vertical bars indicate the mean ± SEM (n = 3). #,&,* p < 0.05, &&,** p < 0.01, and ###,*** p < 0.001 indicate differences of statistical significance.

Fig 4: Knockdown of CISD2 expression in neural cells produced inflammatory responses, promoted apoptosis, and decreased cell viability. Results of mRNA expression of CISD2 (A), iNOS (B), RANTES (C), and BCL2 (D) in neural cells with or without siCISD2 transfection. (E) Cell viability was measured using alamarBlue® assay. Vertical bars indicate the mean ± (SEM) of mRNA expression (n = 3). * p < 0.05, and *** p < 0.001 indicate differences of statistical significance.

Fig 5: WBM abolished injury-triggered GFAP and attenuated injury-downregulated CISD2, PPAR-ß, and I?B protein expression in mice 24 h following spinal cord hemisection in vivo. (a–d) In vivo mouse model of SCI. Results of protein expression of CISD2 (a), PPAR-ß (b), I?B (c), and GFAP (d) for 3 conditions 8 h after SCI. (e–h) Results of protein expression of CISD2 (e), PPAR-ß (f), I?B (g), and GFAP (h) for 3 conditions 24 h after SCI. The lower panel indicates representative immunoblot of the proposed protein expression, and ß-actin (42 kDa) serves as an internal control. The results shown were from one of the experiments that were repeated for at least 3 times. The upper panel indicates the mean ± SEM of the proposed protein/ß-actin band intensity in ratio to the control group. Anti-inflammatory effects of WBM on SCI can be postulated as CISD2 preservation, and the potential upstream regulation of CISD2 on PPAR-ß, subsequent I?B stabilization, and therefore, NF-?B inhibition. Vertical bars indicate the mean ± standard error of the mean (SEM) of protein expression (n = 3). *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control, #P < 0.05, ##P < 0.01, and ###P < 0.001 indicate a significant difference. Pair-wise multiple comparisons between groups were performed using the Newman-Keuls method.