灵芝三萜酸经Pnpla3基因的降脂效果体外试验——基于长爪沙鼠肝原代细胞的脂变模型

Lipid-lowering effect of Ganoderma lucidum triterpene acid(GLTA)via Pnpla3: an in vitro steatosis model in liver parenchyma cells of the Mongolian gerbil

目的建立长爪沙鼠Pnpla3基因敲减的肝原代细胞的脂变模型,并用于评价灵芝三萜酸的降脂效果。方法分离培养长爪沙鼠肝原代实质细胞,以棕榈酸(PA)诱导成肝脂变模型,根据Pnpla3三个外显子序列设计、合成三对Pnpla3基因的干扰RNA载体,并通过q PCR及Western blot法检测Pnpla3的表达量及干扰效率筛选出一对稳转肝实质的干扰载体。以高(1μmol/L)、中(0.1μmol/L)、低(0.01μmol/L)三个剂量的灵芝三萜酸培养细胞,并以阳性药物蛋氨酸(7μmol/L)为对照,检测干扰Pnpla3基因后的细胞甘油三酯(TG)的含量以进行降脂效果观察。结果诱导24 h内,长爪沙鼠肝脂变细胞脂滴增加的较多且大多数细胞形状正常的PA 400μmol/L为造模的合适浓度,以Pnpla3的第二外显子为靶点设计的si RNA2组敲低效果最好,干扰效率高于70%。干扰Pnpla3基因可以起到降低细胞内TG的作用,单独加入中高剂量的灵芝三萜酸有明显的降脂效果(P <0.01),干扰Pnpla3基因与加入灵芝三萜酸的降脂效应具有累积作用(P <0.01)。结论建立了长爪沙鼠肝实质原代细胞的脂变模型,灵芝三萜酸对干扰Pnpla3基因后的长爪沙鼠肝实质细胞中TG的含量有明显的抑制或减轻作用,灵芝三萜酸降脂效应与磷脂代谢相关2个通路(ko00561,ko00564)中Pnpla3基因的表达相关。

Objective To establish a steatosis model in Mongolian gerbil primary liver cells and to evaluate the effect of Pnpla3 knockdown and Ganoderma lucidum triterpenic acid(GLTA)on lowering lipid content.Methods Primary gerbil liver parenchyma cells were isolated and cultured,and a liver steatosis model was induced by palmitic acid(PA4;400μmol/L).Three interfering RNA vectors targeting three Pnpla3 exons were constructed and stably transfected into parenchyma liver cells.The efficiency of Pnpla3 interference was determined.Cells were cultured with high(1μmol/L),medium(0.1μmol/L)and low(0.01μmol/L)doses of GLTA and with the positive control drug,methionine(7μmol/L),and triglyceride(TG)content was determined with and without Pnpla3 knockdown.Results Within 24 hours of induction,PA increased the number of lipid droplets and most cells were of normal shape.si RNA2,designed against the second exon of Pnpla3,had the best knockdown effect and interference efficiency was higher than 70%.Pnpla3 knockdown reduced intracellular TG content.Medium and high dose GLTA had obvious lipid-lowering effects(P<0.01),and Pnpla3 knockdown and GLTA had cumulative effects(P<0.01).Conclusions A lipid transformation model in primary gerbil hepatocytes was established.GLTA inhibited or reduced the TG content in cells after Pnpla3 knockdown,and the lipid lowering effect of GLTA was associated with the expression of Pnpla3 in two KEGG pathways(ko00561 and ko00564),which are related to phospholipid metabolism.