FXR信号通路介导何首乌导致脂质沉积的作用及机理研究

目的 探索何首乌对肝细胞脂质沉积的影响及其机制。方法 利用永生化肝细胞系LO2,采用1 mmol/L游离脂肪酸（FFA）处理,建立脂肪肝沉积细胞模型。同时加入不同浓度的何首乌提取物处理（0、2.5、5、10 μg/mL）,采用油红O染色以观察何首乌对LO2细胞脂肪沉积的作用;采用实时荧光定量PCR进行实验组LO2细胞内法尼酯X受体（FXR）基因及其下游通路胆盐输出泵（BSEP）、小分子异源二聚体伴侣（SHP）、胆固醇7α-羟化酶1（CYP7A1）关键基因的检测;并采用蛋白免疫印迹法检测FXR及其下游通路关键蛋白的表达水平。结果 何首乌处理后的LO2细胞中,油红O的染色量会随着药物浓度增加而增加;FXR的mRNA表达下降,而FXR蛋白诱导促进的下游基因BSEP、SHP的表达相应下降,而其下游抑制基因CYP7A1的表达则上升（P均<0.05）。何首乌处理的LO2肝细胞中,FXR的蛋白水平明显降低,蛋白激酶C （PKC）蛋白磷酸化水平明显降低,而PKC总蛋白表达水平无明显改变,SHP的蛋白水平也明显下降。结论 何首乌可通过抑制FXR-PKC/SHP信号转导通路,从而促进LO2肝细胞内的脂质沉积。为何首乌导致胆汁淤积所引起的肝损伤的治疗提供新的理论依据。

The effect and mechanism of FXR signaling pathway on lipid accumulation induced by Polygonum multiflorum thumb

Objective To investigate the effect and mechanism of Polygonum multiflorum thumb （PMT） on lipid accumulation in hepatic cells. Methods Immortalized hepatocyte LO2 cell line was treated with non free fatty acids （FFA） to establish the lipid accumulation cell model. Following treatment with 0, 2.5, 5 and 10 μg/mL PMT for 24 h, the LO2 cells were assessed for lipid accumulation by Oil red O staining. The expression levels of FXR, BSEP, SHP and CYP7A1 were quantitatively measured by real-time quantitative fluorescence PCR. The expression levels of FXR and key proteins in the downstream signaling pathway were detected by Western Blot. Results In LO2 cells treated with PMT, the intensity of Oil red O staining was increased with the increasing concentration of PMT. The expression level of FXR mRNA was significantly down-regulated, those of BSEP and SHP mRNA in the downstream signaling pathway were down-regulated accordingly, whereas that of CYP7A1 mRNA in LO2 cells was significantly up-regulated （all P < 0.05）. In PMT-treated LO2 cells, the expression level of FXR protein was significantly down-regulated, the phosphorylated level of PKC was considerably decreased, the expression level of total PKC was not significantly changed and that of SHP protein was significantly down-regulated. Conclusions PMT may aggravate lipid accumulation via suppressing the FXR-PKC/SHP signaling pathway. These findings provide novel theoretical evidence for the treatment of liver injury caused by PMT induced-intrahepatic cholestasis.