Notch抑制剂DAPT体外改善L02细胞脂肪变研究*

doi:10.3969/j.issn.1672-5069.2024.01.005

摘要/Abstract

摘要： 目的探讨Notch抑制剂(DAPT)体外对脂肪变肝细胞细胞炎症因子及脂质相关基因水平的影响。方法采用棕榈酸(PA)干预体外构建肝细胞脂肪变模型,采用不同浓度的DAPT干预。采用CCK-8检测细胞存活率,采用尼罗红染色了解细胞脂滴量,采用RT-qPCR法检测TNF-α、IL-1β和IL-6和脂肪相关因子【固醇调节元件结合蛋白1c(SREBP1c)、FASN和ACACA】mRNA水平,采用Western blot法检测细胞p65和SREBP1c蛋白表达。结果在无PA干预的L02细胞,经1μM、2μM、5μM和10 μM DAPT处理细胞24 h,细胞存活率均较对照组显著下降(分别为85.2±5.3%、84.6±2.9%、84.4±6.0%和84.5±3.2%对100.0%,P均<0.05);经2 μM和5 μM浓度的DAPT干预,细胞TNF-α mRNA水平分别为(0.6±0.01)和(0.5±0.1),显著低于对照组【(1.0±0.0),P<0. 01】,IL-1β水平分别为(0.7±0.2)和(0.4±0.0),显著低于对照组【(1.1±0.1),P<0. 01】,IL-6水平分别为(0.8±0.1)和(0.6±0.1),显著低于对照组【(1.0±0.0),P<0. 05】,细胞p65蛋白表达量也显著降低(P<0.05); 2 μM、5 μM和10 μM DAPT处理细胞脂滴荧光强度较对照组显著减弱【分别为(0.2±0.1)、(0.3±0.0)和(0.1±0.0)对(0.7±0.0),P均<0.001】,2 μM和5 μM DAPT处理细胞FASN mRNA水平分别为(0.7±0.0)和(0.4±0.1),显著低于对照组【(1.0±0.0),P<0. 001】、ACACA mRNA水平分别为(0.6±0.1)和(0.3±0.0),显著低于对照组【(1.0±0.0),P<0. 001】;5 μM DAPT处理细胞SREBP1c蛋白表达量显著低于对照组(P<0.01)。结论 DAPT能有效降低体外脂肪变细胞炎症因子表达量,缓解细胞内脂滴形成,其机制可能与调控脂肪相关因子的表达有关,提示抑制Notch信号通路有改善细胞脂肪变发生和进展的潜力。

关键词: L02细胞, 脂肪变, Notch抑制剂, 炎症因子, 脂质, 体外

Abstract: Objective The purpose of this experiment was to investigate the effects of Notch inhibitor (DAPT) on cellular inflammatory factors and lipid related genes in L02 cells in vitro. Methods The steatosis of L02 cells was established by palmitic acid (PA) incubation in vitro, and also intervened by DAPT at 0μM, 1μM, 2μM, 5μM and 10 μM concentration. The cell survival rate was detected by CCK-8,the cell lipid droplets was observed by nile red staining, and the cellular TNF-α, IL-1 β and IL-6 mRNA and fat related factors [sterol regulatory element-binding protein 1c (SREBP1c), FASN and ACACA] mRNA loads were detected by RT-qPCR. The cell p65 and SREBP1c expression was detected by Western blot. Results The cell survival rates of L02 cell without PA intervention at 1μM, 2μM, 5μM and 10 μM DAPT incubation for 24 h were all decreased compared to in the control(85.2±5.3%, 84.6±2.9%, 84.4±6.0% and 84.5±3.2% vs. 100.0%, respectively, P<0.05); in 2 μM and 5 μM DAPT-intervened cells, the TNF-α mRNA levels were (0.6±0.01) and (0.5±0.09),significantly lower than in the control [(1.0±0.0), P<0.01], the IL-1 β mRNA levels were (0.7±0.2) and (0.4±0.0), significantly lower than [(1.1±0.1), P<0.01] in the control, and the IL-6 mRNA levels were (0.8±0.1) and (0.6±0.1), significantly lower than [(1.0±0.0), P<0. 05] in the control group; the p65 expression showed also remarkably decreased (P<0.05); the lipid droplets in 2 μM, 5 μM and 10 μM DAPT-intervened cells were significantly weaker than in the control group [(0.2±0.1), (0.3 ±0.0), (0.1 ±0.0) vs. (0.73±0.0), P<0.001], the FASN mRNA loads in 2 μM and 5 μM DAPT-intervened cells were (0.7±0.0) and (0.4±0.1), much lower than [(1.0±0.0), P<0.001] in the control group, and the ACACA mRNA load in 2 μM and 5 μM DAPT-intervened cells were (0.6±0.1) and (0.3±0.0), much lower than [(1.0±0.0), P<0.001] in the control group; the expression of SREBP1c protein at 5 μM and 10 μM DAPT-intervened cells was significantly weaker than in the control group (P<0.01). Conclusion The DAPT could effectively inhibit the expression of inflammatory factors and ameliorate the formation of intracellular lipid droplets in L02 cells with PA-induced steatosis in vitro, hinting the mechanism might be related to the regulation of fat-related factors. Our findings suggest that the inhibition of Notch signal pathway might have a potential to alleviate the occurrence and progression of cell steatosis.

Key words: L02 cells, Steatosis, Notch inhibitors, Inflammatory cytokines, Lipids, In vitro

吴伟杰, 丁雯瑾, 杨蕊旭, 范建高. Notch抑制剂DAPT体外改善L02细胞脂肪变研究^*[J]. 实用肝脏病杂志, 2024, 27(1): 16-19.

Wu Weijie, Ding Wenjin, Yang Ruixu, et al. Notch inhibitor DAPT ameliorates steatosis of L02 cells in vitro[J]. Journal of Practical Hepatology, 2024, 27(1): 16-19.

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Notch抑制剂DAPT体外改善L02细胞脂肪变研究^*

Notch inhibitor DAPT ameliorates steatosis of L02 cells in vitro

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