Fig 2: Effect of withanolide A on 7KC-induced increase in mRNA expression of pro-inflammatory genes, determined by real time RT-PCR. hCMEC/D3 cells were treated with vehicle only, 7KC only, withanolide A only, or pre-treated with 1 μM withanolide A for 1 h, followed by co-incubation with 30 μM 7KC for 24 h (A–F). Treatment of cells with 7KC resulted in significant induction of (A–F) IL-1β, IL-6, IL-8, NF-κB, TNF-α and COX-2 mRNA expression. These increases were significantly reduced by co-treatment with withanolide A. VC: Vehicle, W: 1 μM withanolide A, 7KC: 30 μM 7KC. Data are represented as mean ± SD (n = 4). * Significant difference compared to controls. # Significant difference compared to 7KC (all p < 0.01).

Fig 3: Schematic illustration of inflammation signaling pathway in AD. Inflammatory cascade which was triggered by NBM lesion primarily involved NF-κB which regulates cytokines and COX-2 expression. IκBα/P-IκBα ratio, which was regulated by ROS molecules, directly amplifies the cascade. EE fruit juice could remarkably attenuate NF-κB pathway so, inhibit inflammation and following events

Fig 4: EE moderated COX-2 enzyme activity. Enzymatic activity of COX-2 was increased in the NBML rats while treatment of AD rats by EE juice reduced COX-2 enzyme activity to near that of the control. Each bar indicates the mean±SEM. **p<0.01, one-way ANOVA, control vs. NBM lesion. #p<0.05 and ##p<0.01, one-way ANOVA, NBM lesion vs. NBM lesion-EE treated rats

Fig 5: Indomethacin treatment reduces cyclooxygenase-1 (COX) function and PGE2 release in irradiated primary parotid acinar cells. Primary parotid acinar cells were prepared from C57BL/6J mice as described in section “Materials and Methods” and were untreated (UT) or treated with 5 Gy radiation (IR) with vehicle (Veh, saline with 0.1% ethanol) or indomethacin (Indo, 25 μM) for 1 h prior to IR. (A) Untreated cells were collected after 24 h of indomethacin treatment, protein was extracted and COX activities were measured with a COX assay kit selective for COX-1 and COX-2. (B) Cell-free supernatants from vehicle or indomethacin treated cells were collected at indicated timepoints post-IR and PGE2 secretion was measured with a PGE2 enzyme-linked immunosorbent assay. (A,B) Graphs represent the mean ± SEM. Each symbol represents an independent sample. Significant differences from UT+ Veh treated cells were determined via (A) a student’s t-test or (B) a one-way ANOVA followed by Bonferroni’s post hoc comparisons (**p < 0.01, ****p < 0.0001; ns is not significant).