郝小惠, 邵京, 吴慧, 靳宜璇, 郭灵丽, 刘和亮, 杨方. 二氧化硅对MLE-12细胞脂质沉积及PI3K/AKT/mTOR通路的影响[J]. 环境与职业医学, 2022, 39(5): 506-511. DOI: 10.11836/JEOM21388
引用本文: 郝小惠, 邵京, 吴慧, 靳宜璇, 郭灵丽, 刘和亮, 杨方. 二氧化硅对MLE-12细胞脂质沉积及PI3K/AKT/mTOR通路的影响[J]. 环境与职业医学, 2022, 39(5): 506-511. DOI: 10.11836/JEOM21388
HAO Xiaohui, SHAO Jing, WU Hui, JIN Yixuan, GUO Lingli, LIU Heliang, YANG Fang. Effects of silicon dioxide exposure on lipid deposition and PI3K/AKT/mTOR signaling pathway in MLE-12 cells[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 506-511. DOI: 10.11836/JEOM21388
Citation: HAO Xiaohui, SHAO Jing, WU Hui, JIN Yixuan, GUO Lingli, LIU Heliang, YANG Fang. Effects of silicon dioxide exposure on lipid deposition and PI3K/AKT/mTOR signaling pathway in MLE-12 cells[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 506-511. DOI: 10.11836/JEOM21388

二氧化硅对MLE-12细胞脂质沉积及PI3K/AKT/mTOR通路的影响

Effects of silicon dioxide exposure on lipid deposition and PI3K/AKT/mTOR signaling pathway in MLE-12 cells

  • 摘要: 背景 脂质代谢失衡与多种疾病的发生发展密切相关,磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶点(PI3K/AKT/mTOR)信号通路是脂质代谢的重要调控通路,矽肺与脂质代谢异常是否有关尚待探索。

    目的 观察二氧化硅(SiO2)染毒后,肺泡Ⅱ型上皮细胞MLE-12细胞内脂质沉积情况,胆固醇及PI3K、AKT、mTOR磷酸化蛋白表达的变化,探讨SiO2是否通过该通路调控脂质变化。

    方法 (1)采用50 mg·L−1 SiO2混悬液染毒MLE-12细胞,分为对照组和12、24、48 h SiO2染毒组。(2)根据PI3K抑制剂LY294002对细胞增殖影响的实验结果,选择5 μmol·L−1进行后续实验。将细胞分为对照、50 mg·L−1 SiO2、50 mg·L−1 SiO2+5 μmol·L−1 LY294002、5 μmol·L−1 LY294002四组,均染毒细胞48 h。采用酶法试剂盒检测各组细胞内总胆固醇、游离胆固醇、胆固醇酯(总胆固醇与游离胆固醇的差值)、甘油三酯含量,采用油红“O”染色法观察细胞内脂质沉积状况,采用Western blotting检测PI3K、AKT、mTOR磷酸化蛋白的表达水平。

    结果 (1)50 mg·L−1的SiO2染毒后,与对照组相比,随着染毒时间的延长:细胞内总胆固醇、游离胆固醇和胆固醇酯的含量呈现增加的趋势,24、48 h组均明显升高,在48 h组,总胆固醇由对照组的(2.242±0.181)mg·g−1升高到(5.148±0.544)mg·g−1,游离胆固醇从(1.923±0.158)mg·g−1升高至(4.168±0.433)mg·g−1,胆固醇酯也从(0.318±0.067)mg·g−1升至(0.978±0.134)mg·g−1(均P<0.01);与对照组相比,甘油三酯含量无明显变化(P>0.05);细胞中的橙红色染色颗粒沉积增加,48 h橙红色颗粒细胞内沉积增加最明显(P<0.01);p-PI3K、p-AKT、p-mTOR蛋白表达呈现增高的趋势,在染毒48 h时达到高峰(均P<0.01)。(2)与SiO2染毒组相比,SiO2+LY294002组细胞内总胆固醇、游离胆固醇和胆固醇酯均降低(均P<0.01),细胞内橙红色脂质染色颗粒沉积减少,细胞p-PI3K、p-AKT、p-mTOR蛋白表达降低(均P<0.01)。

    结论 SiO2可能通过激活PI3K/AKT/mTOR信号通路诱导MLE-12细胞内胆固醇成分增加,促进细胞内脂质沉积。

     

    Abstract: Background Lipid metabolism imbalance is tightly linked to the development and progression of multiple diseases. The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is important for the regulation of lipid metabolism. However, whether silicosis is associated with lipid metabolic abnormalities has yet to be explored.

    Objective To observe the changes of lipid deposition, cholesterol, and phosphorylated proteins of PI3K/AKT/mTOR pathway in silicon dioxide (SiO2)-induced MLE-12 cells and to explore potential mechanism of lipid composition regulated though the pathway.

    Methods (1) MLE-12 cells were stimulated with 50 mg·L−1 SiO2 suspension, and divided into fourgroups: a control group and three SiO2 groups (12, 24, and 48 h of stimulation). (2) Cellproliferation was detected to determine an optimal dose of LY294002, an inhibitor of PI3K protein. LY294002 at 5 μmol·L−1 was used for further study, in which MLE-12 cells cultured for 48 h were divided into four groups: a control group; a 50 mg·L−1 SiO2 suspension stimulation group; a 50 mg·L−1 SiO2 suspension and 5 μmol·L−1 LY294002 treatment group; a 5 μmol·L−1 LY294002 treatment group. Total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE; total cholesterol minus free cholesterol), and triglycerides (TG) were measured with enzyme assay kits. Lipid deposition was observed using Oil Red O staining. The expressions of p-PI3K, p-AKT, and p-mTOR proteins were detected by Western blotting.

    Results (1) The contents of TC, FC, and CE in the 50 mg·L−1 SiO2-induced MLE-12 cells were increased compared to those of the control group in a time-dependent manner by trend analysis, and the increment at 24 and 48 h were significant. By 48 h, the contents of cholesterol indicators were all elevated: TC from (2.242±0.181) mg·g−1 to (5.148±0.544) mg·g−1, FC from (1.923±0.158) mg·g−1 to (4.168±0.433) mg·g−1, and CE from (0.318±0.067) mg·g−1 to (0.978±0.134) mg·g−1, compared with the control group (P<0.01). The changes of TG were not significant (P>0.05). The SiO2 suspension induced orange-red particle deposition in the MLE-12 cells, especially at 48 h (P<0.01). The protein expression levels of p-PI3K, p-AKT, and p-mTOR in SiO2-stimulated MLE-12 cells were higher than those of the control groups with the prolongation of stimulation time, which peaked at 48 h (P<0.01). (2) The contents of TC, FC, and CE in MLE-12 cells of the SiO2 + LY294002 group were decreased, comparing to those of the SiO2 stimulation only group (P<0.01), companied with less orange-red lipid deposition, and suppressed protein expression levels of p-PI3K, p-AKT, and p-mTOR (P<0.01).

    Conclusion SiO2 could induce increases of cholesterol and lipid deposition through activation of PI3K/AKT/mTOR signaling pathway in MLE-12 cells.

     

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