CHI3L1对软脂酸处理后人脐静脉内皮细胞氧化应激的影响

Effect of CHI3L1 on the oxidative stress of human umbilical vein endothelial cells treated with palmitic acid

目的观察壳多糖酶3样蛋白1(CHI3L1)对软脂酸处理后人脐静脉内皮细胞(HUVECs)氧化应激水平的影响,并初步探讨其分子机制。方法体外培养HUVECs,采用不同浓度CHI3L1处理HUVECs 24 h,MTT法检测细胞增殖情况。按照处理因素分为对照组(未处理),软脂酸组及CHI3L1+软脂酸组,软脂酸组用100μmol/L软脂酸处理24 h,CHI3L1+软脂酸组先用100 ng/ml CHI3L1预孵育2 h,随后加入100μmol/L软脂酸共同处理24 h。Western blotting检测细胞核及细胞质p65、核纤层蛋白(Lamin)B、肌动蛋白(Actin)、血红素氧合酶(HMOX)、还原型辅酶Ⅰ/Ⅱ依赖醌氧化还原酶(NQO1)、磷酸化蛋白激酶B(p-Akt)、内质网氧化还原1样蛋白(Ero1-Lα)、钙连蛋白(Calnexin)、蛋白二硫键异构酶(PDI)、伴侣蛋白/葡萄糖调节蛋白(BIP1/GRP)78、内质网核信号转导蛋白(IRE)-1α及磷酸化真核细胞翻译启动因子(p-eIF)2α含量。ELISA法测定白细胞介素(IL)-6及肿瘤坏死因子(TNF)-α的分泌水平,DNA结合实验测定核因子(NF)-κB活性的变化。免疫荧光检测Nrf2的核转位。结果MTT结果显示,与对照组相比,100 ng/ml CHI3L1作用24 h并不能降低HUVECs活性(94.17±6.13 vs.100.00±0.00)。Western blotting检测结果显示,CHI3L1+软脂酸组细胞核中p65水平低于软脂酸组(0.77±0.04 vs.0.92±0.09,P<0.05),细胞质中p65含量高于软脂酸组(0.45±0.04 vs.0.27±0.05,P<0.05);CHI3L1+软脂酸组p65 DNA结合活性低于软脂酸组(0.26±0.04 vs.0.43±0.07,P<0.05)。ELISA法检测结果显示,CHI3L1+软脂酸组TNF-α(85.91±21.16)pg/ml及IL-6(71.43±10.56)pg/ml的含量明显低于软脂酸组[分别为(221.12±18.71)pg/ml及(95.03±11.2)pg/ml,P<0.05]。Western blotting检测结果显示,CHI3L1+软脂酸组中的HMOX及NQO1水平(分别为0.58±0.07、1.08±0.04)明显高于软脂酸组(分别为0.32±0.09、0.62±0.09,P<0.05)。免疫荧光结果显示,与软脂酸组比较,CHI3L1+软脂酸组细胞核中的Nrf2含量增加(10.26%±4.53%vs.78.64%±3.16%)。Western blotting结果显示,CHI3L1+软脂酸组PI3K/Akt的磷酸化水平明显高于软脂酸组(0.51±0.04 vs.0.15±0.09,P<0.05),CHI3L1+软脂酸组Ero1-Lα(0.32±0.02 vs.0.39±0.09)、Calnexin(0.42±0.04 vs.0.54±0.07)、PDI(0.19±0.02 vs.0.24±0.05)、BIP1/GRP78(0.11±0.01 vs.0.17±0.03)、IRE-1α(0.19±0.03 vs.0.33±0.04)及p-eIF2α(0.22±0.07 vs.0.67±0.09)等分子的表达量明显低于软脂酸组(P<0.05)。结论CHI3L1能抑制软脂酸处理后HUVECs中的细胞因子分泌,并减轻内质网应激水平。

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 100μmol/L palmitic acid for 24 hours,and in CHI3L1+palmitic acid group were pretreated with 100 ng/ml CHI3L1 for 2 hours,then 100μmol/L palmitic acid were addedand 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 Ⅰ/Ⅱ dependent quinone oxidoreductase (NQO1), phosphorylated proteinkinase 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 phosphorylatedeukaryotic translation initiation factor (p-eIF) 2α. The contents of IL-6 and TNF-α were measured by ELISA, the activity of NF-κBwas measured by DNA binding assay, and the nuclear translocation of Nrf2 was detected by immunofluorescence. Results MTTresults showed that compared with the control, treatment with 100 ng/ml CHI3L1 for 24 hours did not decrease the activity ofHUVECs (94.17±6.13 vs. 100.00±0.00). Western blotting results showed that the p65 level in nucleus of CHI3L1+palmitic acidgroup 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 inpalmitic 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 waslower 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 andIL-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 weresignificantly 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 increasedsignificantly in CHI3L1+palmitic acid group (10.26%±4.53% vs. 78.64%±3.16%, P<0.05). Western blotting results showed thatthe 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 inendoplasmic reticulum were significantly reduced in CHI3L1+palmitic acid group than those in palmitic acid group (0.32±0.02vs. 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;and0.22±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.