Mechanistic insights into the anti-aging effects of Crocus sativus in a D-Gal-induced in vitro neural senescence model

Abstract

Background: The increasing number of elderly individuals has made age-related disorders a significant health concern. Aging is a natural, progressive and gradual phenomenon that leads to irreversible modifications in all molecules, cells, tissues and organs of an organism. Brain senescence is associated with increased risk of developing various neurological diseases, such as Alzheimer's disease, Parkinson's disease, and stroke. Therefore, finding effective strategies to counteract or delay brain senescence is of great importance for improving the quality of life and health span of the elderly population. Previous studies demonstrated that D-galactose is an appropriate agent to induce aging effects in in vivo and in vitro models.

Purpose: In the present study, we evaluated anti-aging effects of a local Saffron extract (SE from Central Italy) on D-GAL-induced aging model in vitro. Based on promising preliminary results, future studies will focus on testing this specific Crocus sativus stigma preparation in animal models of aging.

Methods: The potential anti-aging effect was evaluated using assessment of cell proliferation, live-cell cytotoxicity, Beta-Galactosidase (β-GAL), lipid peroxidation, intracellular reactive oxygen species (ROS), advanced glycation end products (AGEs) and malondialdehyde (MDA) levels. Additionally, the effects of SE pretreatment were examined on cell cycle and endoplasmic reticulum stress. Additionally, we employed a novel approach to analyze deeper changes upon saffron extract treatment, which is label-free holotomography.

Results: Overall, our findings suggested that pretreatment with SE was protective against D-GAL-induced senescence, by counteracting oxidative and endoplasmic reticulum stress and proteins that regulate cell death.

Conclusion: We obtained interesting results since pre-treatment with SE ameliorated overall condition, and for the first time we observed the strong anti-aging effect of SE not only in term of morphology, but also dynamics and total dry mass of cells. Overall, our work introduces a novel and promising approach to counteract or delay brain senescence, potentially improving the quality of life and health span of the elderly population.

Fig 1. Spectrophotometric analysis UV-Vis.

Fig 2. A) MTS assay in human SH-SY5Y upon different treatments at 24h.

ANOVA one-way: **, P ≤  0.01 vs CTR; ##, P ≤  0.01, #, P ≤  0.05 vs D-Gal. N = 6; B) Cytotox Incucyte Assay graph 0-24h and C) representative images at 0h and 24h in human SH-SY5Y upon different treatments. ANOVA two-way: ***, P ≤  0.001 vs CTR; ##, P ≤  0.001, ##, P ≤  0.01, #, P ≤  0.05 vs D-Gal. N = 9. D) Senescence-associated β-GAL levels in human SH-SY5Y upon different treatments; ANOVA two-way: ***, P ≤  0.001 vs CTR; ###, P ≤  0.001, vs D-Gal. N = 4.

Fig 3. Oxidative stress analysis on D-Gal-induced senescence upon SE treatment.

A) ROS assay ANOVA one-way: ***, P ≤  0.001 vs CTR; ###, P ≤  0.001 vs D-Gal. N = 4; B) MDA levels C) SOD enzymatic activity assay, ANOVA one-way: **, P ≤  0.01 vs CTR; ##, P ≤  0.01, #, P ≤  0.05 vs D-Gal. N = 4. D) CAT enzymatic activity assay, ANOVA one-way: **, P ≤  0.01 vs CTR; ##, P ≤  0.01, #, P ≤  0.05 vs D-Gal. N = 4.

Fig 4. Live-cell label-free holotomographic microscopy.

Graph reports Total dry mass of cells upon different conditions. ANOVA one-way: ***, P ≤  0.001 vs CTR; ###, P ≤  0.001 vs D-Gal. N = 4.

Fig 5. Cell cycle pathway on D-Gal-induced senescence upon SE treatment.

A) p21 protein levels and representative image. B) Cyclin D1 protein levels and representative image. C) Survivin protein level and representative image. D) p-Akt protein levels and representative image. E) RT-PCR of Survivin. ANOVA one-way: ***, P ≤  0.001 vs CTR; ###, P ≤  0.001 vs D-Gal. N = 3.

Fig 6. RT-PCR of CHOP and Grp78 on D-Gal-induced senescence upon SE treatment.

ANOVA one-way: **, P ≤  0.005 vs CTR; #, P ≤  0.05 vs D-Gal. N = 3.