沙棘叶对HepG2细胞脂质沉积的影响及其机制研究

Study on the effect and mechanism of seabuckthorn leaves on lipid deposition in HepG2 cells

摘要:

目的 探讨沙棘叶含药血清对HepG2细胞脂质沉积的干预作用及机制。方法 制备油酸-棕榈酸诱导的HepG2细胞脂质沉积模型，设置空白组、模型组、阳性药组、沙棘叶低剂量组（SJYD组）、沙棘叶中剂量组（SJYZ组）、沙棘叶高剂量组（SJYG组）。采用CCK-8法检测沙棘叶含药血清最适给药浓度；通过油红O染色和细胞内总胆固醇（TC）、甘油三酯（TG）含量测定，评估细胞内脂质沉积情况；运用实时荧光定量聚合酶链反应检测各组细胞腺苷酸活化蛋白激酶（AMPK）、乙酰辅酶A羧化酶（ACC）、肉碱棕榈酰转移酶1（CPT1）基因的相对表达量；并利用Western blotting测定P-AMPK/AMPK、P-ACC/ACC和CPT1蛋白的相对表达量。结果 CCK-8实验结果显示，0%、5%、10%、15%、20%、25%浓度含药血清组细胞存活率分别为（100±0.00）%、（91.39±1.96）%、（93.41±5.60）%、（81.50±6.09）%、（65.20±3.93）%、（62.23±2.91）%。0%含药血清组与10%含药血清组细胞存活率比较，差异无统计学意义（P >0.05），随着含药血清浓度升高（>10%），细胞存活率逐渐降低（P <0.05）。油红O染色结果显示，与空白组相比，模型组细胞经油酸-棕榈酸混合溶液诱导后产生大量脂滴；与模型组相比，阳性药组、SJYD组、SJYZ组、SJYG组HepG2细胞内脂质沉积明显减少。与空白组相比，模型组细胞内TG、TC含量均升高（P <0.05）；与模型组相比，阳性药组、SJYZ组、SJYG组细胞内TC、TG含量均降低（P <0.05）。与空白组相比，模型组HepG2细胞中AMPK、CPT1基因相对表达量均降低（P <0.05）；与模型组相比，阳性药组、SJYZ组、SJYG组AMPK基因相对表达量均升高，ACC基因相对表达量均降低，阳性药组和SJYZ组CPT1基因相对表达量均升高（P <0.05）。与空白组相比，模型组HepG2细胞中P-AMPK/AMPK、P-ACC/ACC及CPT1蛋白相对表达量均降低（P <0.05）；与模型组相比，各给药组P-AMPK/AMPK蛋白和CPT1蛋白相对表达量均升高，阳性药组、SJYZ组、SJYG组P-ACC/ACC蛋白相对表达量均升高（P <0.05）。结论 沙棘叶改善HepG2细胞脂质沉积，作用机制与调节AMPK/ACC/CPT1信号通路有关。

Abstract:

Objective To explore the intervention effect and mechanism of seabuckthorn leaf-containing serum on lipid deposition in HepG2 cells.Methods An oleic acid-palmitic acid-induced HepG2 cell lipid deposition model was constructed, and groups were set as follows: blank group, model group, positive drug-containing serum group, seabuckthorn leaf low-dose-containing serum group (SJYD group), seabuckthorn leaf medium-dose-containing serum group (SJYZ group), and seabuckthorn leaf high-dose-containing serum group (SJYG group). The CCK-8 method was used to detect the optimal administration concentration of seabuckthorn leaf-containing serum. Oil red O staining and determination of intracellular total cholesterol (TC) and triglyceride (TG) contents were performed to evaluate intracellular lipid deposition. Real-time fluorescent quantitative polymerase chain reaction was used to detect the relative expression levels of AMPK, ACC, and CPT1 genes in cells of each group. Western blotting was used to determine the relative expression levels of P-AMPK/AMPK, P-ACC/ACC, and CPT1 proteins.Results The results of the CCK-8 experiment showed that the cell survival rates in the drug-containing serum groups with concentrations of 0%, 5%, 10%, 15%, 20%, and 25% were (100 ± 0.00)%, (91.39 ± 1.96)%, (93.41 ± 5.60)%, (81.50 ± 6.09)%, (65.20 ± 3.93)%, and (62.23 ± 2.91)% respectively. Compared with the 0% drug-containing serum group, there was no statistically significant difference in cell survival rate when the serum concentration was 10% (P > 0.05), while the cell survival rate decreased when the serum concentration was higher than 10% (P < 0.05). The results of oil red O staining showed that compared with the blank group, the model group cells produced a large number of lipid droplets after induction with the oleic acid-palmitic acid mixed solution; compared with the model group, the lipid deposition in HepG2 cells of the positive drug group, SJYD group, SJYZ group, and SJYG group was significantly reduced. Compared with the blank group, the contents of TG and TC in the model group cells were increased (P < 0.05); compared with the model group, the contents of TC and TG in the positive drug group, SJYZ group, and SJYG group were decreased (P < 0.05). Compared with the blank group, the relative expression levels of AMPK and CPT1 mRNA in HepG2 cells of the model group were decreased (P < 0.05); compared with the model group,the relative expression level of AMPK mRNA in the positive drug group, SJYZ group, and SJYG group was increased (P < 0.05), while the relative expression level of ACC mRNA in these groups was decreased (P < 0.05). The relative expression level of CPT1 mRNA in the positive drug group, SJYZ group was increased (P < 0.05). Compared with the blank group, the relative protein expression levels of P-AMPK/AMPK, P-ACC/ACC and CPT1 in HepG2 cells of the model group were decreased (P < 0.05); compared with the model group, the relative protein expression levels of P-AMPK/AMPK and CPT1 in all administration groups were increased, and the relative protein expression level of P-ACC/ACC in the positive drug group, SJYZ group and SJYG group was increased (P < 0.05).Conclusion Seabuckthorn leaves can improve lipid deposition in HepG2 cells, and the mechanism is related to the regulation of the AMPK/ACC/CPT1 signaling pathway.