Effect of Klotho on oxidized low density lipoprotein-induced endothelial cell senescence via activation of autophagy / 解放军医学杂志

ABSTRACT

Objective: To investigate the effect of Klotho on oxidized low density lipoprotein (ox-LDL)-induced human coronary artery endothelial cell (HCAEC) senescence via activation of autophagy. Methods: HCAECs were cultured in vitro, and divided into control group and ox-LDL group [treated with ox-LDL at different concentrations (0, 25, 50, 75, 100 μg/ml) or different durations (0, 24 h, 48 h, 72 h, 96 h) 2 h after pre-incubation with Klotho (400 pmol/L)]. Cell viability was tested by CCK-8, senescent cells were detected by SA-β-GAL staining, immunofluorescence was used to detect the intracellular expression of P53 and P16, and Western blotting was performed to quantitatively analyze the P53, P16, LC3 and P62 protein expression. HCAECs were pre-incubated separately with Klotho (400 pmol/L), rapamycin (5 μmol/L) or chloroquine (2 μmol/L) for 2 hours under normal culture condition, and then cultured under ox-LDL exposure (75 μg/ml) for 72 hours. Cell viability was tested by CCK-8, senescent cells were detected by SA-β-GAL staining, autophagic flux was assayed by adenovirus GFP-LC3, and Western blotting was performed to quantitatively analyze the P53, P16, LC3, and P62 protein expression. Results: The results of CCK-8 assay, SAKlotho β-GAL staining and Western blotting respectively showed that Klotho decreased ox-LDL-induced inhibition of HCAECs viability (P<0.05), reduced the proportion of senescent cells (P<0.05), and down-regulated the expression of aging-related proteins P53 and P16 (P<0.05). Western blotting revealed that ox-LDL decreased LC3-II/LC3-I ratio and increased P62 protein expression level in a time-dependent (P<0.05) or dose-dependent manner (P<0.05) to some extent. Furthermore, Klotho increased LC3-II/LC3-I ratio (P<0.01), decreased P62 protein expression level (P<0.01), and reduced the inhibition of autophagy by chloroquine under ox-LDL exposure (P<0.05). The results of CCK-8 assay, SA-β-GAL staining and Western blotting respectively indicated that the HCAECs viability (P<0.01), the proportion of senescent cells (P<0.01), and the protein expression levels of P53 (P<0.01) and P16 (P<0.01) were significantly alleviated in ox-LDL+Klotho group and ox-LDL+rapamycin group than those in ox-LDL group; while the HCAECs viability (P<0.01), the proportion of senescent cells (P<0.01), and the protein expression levels of P53 and P16 (P<0.05) were obviously worse in chloroquine+ox-LDL group than those in ox-LDL group. Conclusion: Klotho ameliorates ox- LDL-induced HCAEC senescence via activation of autophagy.