Crocin I delays cellular senescence potentially via modulating SIRT1/PKM2 expression to improve lactate homeostasis

Abstract

Crocin I has a protective effect against D-galactose-induced aging in rat models; however, its specific mechanism on delaying cellular senescence remains unclear. The aim of the present study was to investigate the interventional effect and related molecular mechanism of Crocin Ⅰ on replicative senescence and premature senescence of human embryonic lung diploid fibroblasts (WI-38) cells. Replicative senescence model was established through long-term culture and passaging to >55 PD. The premature senescence model was established using WI-38 cells treated with 2,2'-azobis-2-methy-propanimidamide dihydrochloride. The cell viability was detected by using a cell counting kit-8 assay, and the effects of Crocin Ⅰ on senescent phenotype of WI-38 cells were detected by senescence-associated β-galactosidase (SA-β-Gal) staining. In addition, the intracellular reactive oxygen species (ROS) level was detected by flow cytometry, and gene expression levels were detected by quantitative PCR. RNA sequencing analysis was performed to explore the alteration genes in senescent cells and the Crocin Ⅰ intervention group. The proteins PKM2, SIRT1, p53 and p21 in WI-38 cells were detected by western blotting, whereas cellular lactate level was detected by L-lactic acid content detection kit and pyruvate kinase (PK) enzyme activity was measured using PK activity assay kit. Crocin Ⅰ promoted cell proliferation within the range of 10-40 µM. In addition, Crocin Ⅰ decreased the SA-β-Gal staining positive rate on both replicative and premature senescent WI-38 cells. Furthermore, the intracellular ROS level was decreased by Crocin Ⅰ intervention. The differentially expressed genes were mainly enriched in phosphorylation, oxidation-reduction process, glucose metabolic process, cellular senescence pathway, glycolysis, hippo signaling pathway and pyruvate metabolism. RNA sequencing analysis and molecular docking revealed that PKM2 was one of the potential targets in Crocin Ⅰ against senescence activity. Furthermore, Crocin Ⅰ could enhance the expression of SIRT1 following with decreasing the level of PKM2, p21 and p53. Meanwhile, Crocin Ⅰ significantly decreased the cellular lactate level in WI-38. In conclusion, Crocin Ⅰ alleviated the replicative and premature senescence of WI-38 cells partially through regulating SIRT1 and PKM2 expression. Furthermore, this function may involve in glycolysis pathway.