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(SiO_(2))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SiO_(2)是否通过该通路调控脂质变化。[方法](1)采用50 mg·LSiO_(2)混悬液染毒MLE-12细胞,分为对照组和12、24、48 h SiO_(2)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5μmol·L^(-1)进行后续实验。将细胞分为对照、50 mg·L-1 SiO_(2)、50 mg·LSiO_(2)+5μmol·L^(-1)LY294002、5μmol·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的SiO_(2)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升高到(5.148±0.544)mg·g,游离胆固醇从(1.923±0.158)mg·g升高至(4.168±0.433)mg·g,胆固醇酯也从(0.318±0.067)mg·g升至(0.978±0.134)mg·g(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SiO_(2)染毒组相比,SiO_(2)+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]SiO_(2)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。
[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(PI3 K/AKT/m TOR) 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 PI3 K/AKT/m TOR pathway in silicon dioxide(SiO_(2))-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·LSiO_(2)suspension, and divided into four groups: a control group and three SiO_(2)groups(12, 24, and 48 h of stimulation).(2) Cellproliferation was detected to determine an optimal dose of LY294002, an inhibitor of PI3 K 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·LSiO_(2)suspenSiO_(2)n stimulation group;a 50 mg·LSiO_(2)suspenSiO_(2)n 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 expresSiO_(2)ns of p-PI3 K, p-AKT, and p-m TOR proteins were detected by Western blotting.[Results](1) The contents of TC, FC, and CE in the 50 mg·LSiO_(2)-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 SiO_(2)suspenSiO_(2)n induced orange-red particle deposition in the MLE-12 cells, especially at 48 h(P < 0.01). The protein expresSiO_(2)n levels of p-PI3 K, p-AKT, and p-m TOR in SiO_(2)-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 SiO_(2)+ LY294002 group were decreased, comparing to those of the SiO_(2)stimulation only group(P < 0.01), companied with less orange-red lipid deposition, and suppressed protein expresSiO_(2)n levels of p-PI3 K, p-AKT, and p-m TOR(P < 0.01).[ConcluSiO_(2)n] SiO_(2)could induce increases of cholesterol and lipid deposition through activation of PI3 K/AKT/m TOR signaling pathway in MLE-12 cells.
作者
郝小惠
邵京
吴慧
靳宜璇
郭灵丽
刘和亮
杨方
HAO Xiaohui;SHAO Jing;WU Hui;JIN Yixuan;GUO Lingli;LIU Heliang;YANG Fang(School of Public Health,North China University of Science and Technology,Tangshan,Hebei 063210,China;Hebei Key Laboratory of Organ Fibrosis,North China University of Science and Technology,Tangshan,Hebei 063210,China)
出处
《环境与职业医学》
CAS
CSCD
北大核心
2022年第5期506-511,共6页
Journal of Environmental and Occupational Medicine
基金
河北省自然科学基金项目(H2018209332)。
