Effect of CHI3L1 on the oxidative stress of human umbilical vein endothelial cells treated with palmitic acid / 解放军医学杂志

ABSTRACT

Objective To observe the effect of chitinase-3-like protein 1 (CHI3L1) on the oxidative stress of human umbilical vein endothelial cells (HUVECs) after treatment with palmitic acid, and preliminarily explore the molecular mechanism. Methods HUVECs were cultured in vitro, treated with different concentrations of CHI3L1 for 24 hours, and the MTT assay was used to determine the proliferation of HUVECs. The cells were divided into the control group (untreated group), palmitic acid group and CHI3L1+palmitic acid group. Cells in palmitic acid group were treated with 100 μmol/L palmitic acid for 24 hours, and in CHI3L1+palmitic acid group were pretreated with 100 ng/ml CHI3L1 for 2 hours, then 100 μmol/L palmitic acid were added and co-treating for 24 hours. Western blotting was used to detect the contents of nuclear and cytoplasmic p65, nuclear Lamin B, Actin, heme oxygenase (HMOX), reduced coenzyme /Ⅱ dependent quinone oxidoreductase (NQO1), phosphorylated protein kinase B (p-Akt), endoplasmic reticulum redox 1-like protein (Ero1-Lα), Calnexin, protein disulfide isomerase (PDI), chaperonin/ glucose regulatory protein (Bip1/Grp) 78, endoplasmic reticulum nuclear signal transduction protein (IRE)-1α and phosphorylated eukaryotic translation initiation factor (p-eIF) 2α. The contents of IL-6 and TNF-α were measured by ELISA, the activity of NF-κB was measured by DNA binding assay, and the nuclear translocation of Nrf2 was detected by immunofluorescence. Results MTT results showed that compared with the control, treatment with 100 ng/ml CHI3L1 for 24 hours did not decrease the activity of HUVECs (94.17±6.13 vs. 100.00±0.00). Western blotting results showed that the p65 level in nucleus of CHI3L1+palmitic acid group was lower than that in palmitic acid group (0.77±0.04 vs. 0.92±0.09, P<0.05), but in cytoplasm was higher than that in palmitic acid group (0.45±0.04 vs. 0.27±0.05, P<0.05). The DNA binding activity of NF-κB in CHI3L1+palmitic acid group was lower than that in palmitic acid group (0.26±0.04 vs. 0.43±0.07, P<0.05). ELISA results showed that the contents of TNF-α and IL-6 were lower in CHI3L1+palmitic acid group than those in palmitic acid group [(85.91±21.16) pg/ml vs. (221.12±18.71) pg/ml; (71.43±10.56) pg/ml vs. (95.03±11.2) pg/ml, P<0.05]. Western blotting results showed that the levels of HMOX and NQO1 were significantly higher in CHI3L1+palmitic acid group than those in palmitic acid group (0.58±0.07 vs. 0.32±0.09; 1.08±0.04 vs. 0.62±0.09, P<0.05). Immunofluorescence results showed that compared with palmitic acid group, the content of Nrf2 increased significantly in CHI3L1+palmitic acid group (10.26%±4.53% vs. 78.64%±3.16%, P<0.05). Western blotting results showed that the phosphorylation level of PI3K/Akt was significantly higher in CHI3L1+palmitic acid group than those in palmitic acid group (0.51±0.04 vs. 0.15±0.09, P<0.05), and the expression levels of Ero1-Lα, Calnexin, PDI, BIP1/GRP78, IRE-1α and p-eIF2α in endoplasmic reticulum were significantly reduced in CHI3L1+palmitic acid group than those in palmitic acid group (0.32±0.02 vs. 0.39±0.09; 0.42±0.04 vs. 0.54±0.07; 0.19±0.02 vs. 0.24±0.05; 0.11±0.01 vs. 0.17±0.03; 0.19±0.03 vs. 0.33±0.04; and 0.22±0.07 vs. 0.67±0.09. P<0.05). Conclusion CHI3L1 can inhibit the cytokine secretion of HUVECs treated with palmitic acid, and reduce the stress level of endoplasmic reticulum.