Luteolin inhibits H2O2-induced cellular senescence via modulation of SIRT1 and p53

Abstract

Luteolin, a sort of flavonoid, has been reported to be involved in neuroprotective function via suppression of neuroinflammation. In this study, we investigated the protective effect of luteolin against oxidative stress-induced cellular senescence and its molecular mechanism using hydrogen peroxide (H₂O₂)-induced cellular senescence model in House Ear Institute-Organ of Corti 1 cells (HEI-OC1). Our results showed that luteolin attenuated senescent phenotypes including alterations of morphology, cell proliferation, senescence-associated β-galactosidase expression, DNA damage, as well as related molecules expression such as p53 and p21 in the oxidant challenged model. Interestingly, we found that luteolin induces expression of sirtuin 1 in dose- and time-dependent manners and it has protective role against H₂O₂-induced cellular senescence by upregulation of sirtuin 1 (SIRT1). In contrast, the inhibitory effect of luteolin on cellular senescence under oxidative stress was abolished by silencing of SIRT1. This study indicates that luteolin effectively protects against oxidative stress-induced cellular senescence through p53 and SIRT1. These results suggest that luteolin possesses therapeutic potentials against age-related hearing loss that are induced by oxidative stress.

Keywords: Cellular senescence; Hydrogen peroxide; Luteolin; Sirtuin 1; Tumor suppressor protein p53.

Fig. 1. Luteolin reduces hydrogen peroxide (H₂O₂)-induced cellular senescence in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells.

(A–C) Cells were pretreated with luteolin for 12 h, then incubated in 30 μM H₂O₂ for 3 days. Cell viability was determined by MTT assay (A). The percentage of SA-β-gal positive cells out of total cells was counted and the average data was obtained from three independent experiments (B). The senescent phenotype of HEI-OC1 cells was detected by the SA-β-gal assay (×100) (C). (D) Cell growth was evaluated by MTT assay at various time points indicated in the figure after addition of H₂O₂. Data represents means values of triple experiments. *p < 0.05 vs. control, ^#p < 0.05 vs. H₂O₂. Piperine was used as a positive control.

Fig. 2. Protective effect of luteolin against hydrogen peroxide (H₂O₂)-induced cellular senescence is dependent on p53.

(A) Cells were pretreated with 2 μM luteolin for 12 h, then incubated in 30 μM H₂O₂ for 3 days. (B) Cells were transfected with various concentrations siRNAs of control or p53, then incubated with 30 μM H₂O₂ for 3 days. The protein levels were detected by Western blotting and quantified by densitometry based on immunoblot images (A, B). (C) Representative images of SA-β-gal staining of HEI-OC1 (×100). (D) The percentage of senescent cells were calculated in (D). Data represents means values of triple experiments. *p < 0.05 vs. control; ^#p < 0.05 vs. H₂O₂-treated cells.

Fig. 3. Effects of luteolin on sirtuin 1 (SIRT1) expression in House Ear Institute-Organ of Corti 1 cells (HEI-OC1).

(A, C) HEI-OC1 cells were treated with luteolin (0–4 μM, 12 h). (B, D) Cells were treated with luteolin (2 μM, 0–12 h). The proteins level (A, B) and SIRT1 activity (C, D) were performed with Western blotting and SIRT1 fluorometric kit, respectively. The proteins were quantified by densitometry based on immunoblot images. Data represents means values of triple experiments. Significance vs. control: *p < 0.05.

Fig. 4. Luteolin protects against hydrogen peroxide (H₂O₂)-induced p53 phosphorylation via sirtuin 1 (SIRT1) protein expression.

House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were transfected with 80 nM siRNAs of control or SIRT1, then incubated in 2 μM luteolin for 12 h, followed by 30 μM H₂O₂ treatment for additional 3 days. (A, B) The protein levels were detected by Western blotting and quantified by densitometry based on immunoblot images. (C) Representative images of SA-β-gal staining of HEI-OC1 (×100). (D) The percentage of senescent cells were calculated in (D). Data represents means values of triple experiments. *p < 0.05 vs. control; ^#p < 0.05 vs. H₂O₂-treated cells.

Fig. 5. Luteolin prevents DNA from hydrogen peroxide (H₂O₂)-induced damage.

(A, B) House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were transfected with 80 nM siRNAs of sirtuin 1 (SIRT1) or p53, then incubated in 2 μM luteolin for 12 h, followed by 30 μM H₂O₂ treatment for additional 3 days. Comet assay was performed as described in Methods. Data show the results of one of three independent experiments. *p < 0.05 vs. H₂O₂, ^#p < 0.05 vs. luteolin/H₂O₂/control siRNA.

Fig. 6. A schematic illustration summarizing the protective effect of Luteolin against hydrogen peroxide (H₂O₂)-induced premature senescence in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells.