Biological Response of Treatment with Saffron Petal Extract on Cytokine-Induced Oxidative Stress and Inflammation in the Caco-2/Human Leukemia Monocytic Co-Culture Model

Abstract

The pathogenesis of Inflammatory Bowel Disease (IBD) involves complex mechanisms, including immune dysregulation, gut microbiota imbalances, oxidative stress, and defects in the gastrointestinal mucosal barrier. Current treatments for IBD often have significant limitations and adverse side effects, prompting a search for alternative therapeutic strategies. Natural products with anti-inflammatory and antioxidant properties have demonstrated potential for IBD management. There is increasing interest in exploring food industry waste as a source of bioactive molecules with healthcare applications. In this study, a co-culture system of Caco-2 cells and PMA-differentiated THP-1 macrophages was used to simulate the human intestinal microenvironment. Inflammation was induced using TNF-α and IFN-γ, followed by treatment with Saffron Petal Extract (SPE). The results demonstrated that SPE significantly attenuated oxidative stress and inflammation by downregulating the expression of pro-inflammatory mediators such as iNOS, COX-2, IL-1β, and IL-6 via modulation of the NF-κB pathway. Given that NF-κB is a key regulator of macrophage-driven inflammation, our findings support further investigation of SPE as a potential complementary therapeutic agent for IBD treatment.

Keywords: inflammation; inflammatory bowel disease; oxidative stress; saffron; saffron petal extract.

Figure 1

Design of experiments (DoE) flowchart of optimizing the tepal extraction process.

Figure 2

(a) Chromatogram of a petals extract at 265 nm. (b) Chromatogram of a petals extract at 530 nm.

Figure 3

Cytotoxicity of SPE in differentiated and undifferentiated Caco-2 and THP-1 cells. (a) Undifferentiated and differentiated cultured (a) Caco-2 and (b) THP-1 cells were treated with SPE (range 50 ng/mL–1 mg/mL) (24 and 48 h), and cell viability was determined using an MTT assay. Cell survival (100%) was assigned to the control without treatment, and changes in cell viability relative were calculated. The data are presented as the mean ± SD (n = 3). SPE, saffron petal extract; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 4

Measurement of SPE’s antioxidant activity against oxidative stress through NBT testing in a co-culture system. NBT assay visualises formazan crystal formation in Caco-2 cells treated with INF-γ and TNF-α plus SPE (50 ng/mL–1mg/mL). Results are registered as stimulation index (SI). SI value of 1 was assigned to control cells. Each bar represents ± SEM (n = 3). * p < 0.005 vs. CTRL; # p < 0.05 TNF-α + INF-γ.

Figure 5

Analysis of intestinal barrier function (a) Alkaline phosphatase activity of apical/basolateral (AP/BL) in Caco-2 cell during 21-day incubation. Cells were analysed eight days after reaching the confluence, 14 days post-confluency (differentiated) and 21 days post-confluence. (b) Changes in TEER values of co-cultured Caco-2 and THP-1 cells incubated for 21 days on Transwell. Values obtained from four experiments, each performed in triplicate, are shown. * p < 0.05.

Figure 6

Effects of SPE on the ZO-1 and occludin of Caco-2 in the co-culture system. (a) Representative images and quantification of Western blot analysis for the proteins ZO-1 and occludin in each experimental group. Each value represents the mean ± SD (n = 3). SPE, Saffron Petal Extract. § p < 0.05, vs. control cells and * p < 0.05 vs. TNF-α + INF-γ stimulated cells. (b) Expression of junctional proteins, occludin and ZO-1, in the apical region of Caco-2 monolayers. Green Colour: immunostaining for ZO-1. Red colour: immunostaining for occludin. Blue colour: colouration of cell nuclei. Bar = 20 µm.

Figure 7

Effect of SPE on Fbw7/NFkB signalling. Western blotting measured the cells’ expression levels of p-p65/p65, IκBα, and Fbw7 proteins. The overexpression of Fbw7 leads to the degradation of IκB α, activating the nuclear factor κB signalling pathway in cytokine-stimulated co-culture. Additionally, treatment with SPE protects cells from proinflammatory stimuli. Each bar represents the mean ± standard deviation of 6 groups of cells. § p < 0.05 vs. the ctrl. * p < 0.05 vs. INF-γ + TNF-α treated cells. SPE, saffron petal extract; ctrl, control.

Figure 8

Effect of SPE on COX-2, iNOS and HO-1. (a) RNA expression and activity (PGE2 level) for COX-2, GADPH being used as loading control; (b) RNA expression and activity (NO level) for iNOS, GADPH being used as loading control; (c) RNA expression and Western blot for HO-1, GADPH and actin being used as loading control. Data shown are means ± SD, § p < 0.05 compared with control cells; * p < 0.05 compared with cytokines treated cells; data were from at least three independent experiments, each performed in triplicate.

Figure 9

SPE inhibited the production of inflammatory cytokines in THP-1 cells from the co-culture system with Caco-2 cells. (a) The expression of IL-1β and IL-6 were analysed using qRT-PCR. (b) Commercially available ELISA kits were used to test interleukin (IL)-1β and IL-6 levels in the culture medium. Data shown are means ± SD, § p < 0.05 compared with control cells; * p < 0.05 compared with cytokines treated cells.