Conclusion
Crocin protects renal epithelial cells against injury induced by high glucose, and the mechanism is associated with partial activation of the SIRT1-Nrf2 pathway.
Methods
Renal tubular epithelial HK-2 cells were cultured with D-glucose to establish an in vitro DN model. Cell viability was evaluated by CCK-8 assay. Apoptosis was detected by Annexin V-FITC kit. Oxidative stress was evaluated by colorimetry. RT-qPCR was carried out to determine the mRNA expressions of NF-E2-related factor 2 (Nrf2) and its pathway genes. Western blot was applied to determine the protein expressions of Nrf2 and related proteins.
Results
High glucose (5.5, 30, and 50 mM D-glucose) decreased cell viability at 72 hr, which was attenuated by crocin (25 and 50 μM). Crocin also attenuated the high glucose (30 mM D-glucose) induced apoptosis of HK-2 cells, decreased MDA content, and increased SOD activity in culture media. Crocin increased mRNA levels of Nrf2, HO-1, and NQO1. Moreover, crocin increased protein expressions of Nrf2, Sirtuin 1 (SIRT1), and p-Akt (Ser473). Inhibition of Nrf2 using siRNA, and inhibitors of SIRT1 (nicotinamide, NAM, 20 μM) and PI3K/Akt (LY294002, 50 μM) all attenuated the protective effect of crocin. Nrf2 siRNA and NAM also partially attenuated the inhibitory effect on oxidative stress and increase in the Nrf2 protein by crocin treatment.