丁香酚通过AMPK及SREBP1途径改善非酒精性脂肪肝的机制

Mechanism of eugenol improving nonalcoholic fatty liver disease through AMPK and SREBP1 pathways

目的:研究丁香酚对高脂饲料诱导小鼠非酒精性脂肪肝(NAFLD)的作用机制。方法:40只C57BL/6小鼠分为正常组,模型组,丁香酚高、低剂量(40、20mg/kg)组,每组10只,除正常组外,其余各组给予高脂饲料建立NAFLD模型的同时灌胃给丁香酚(1次/d),每周测2次体质量,持续15周。15周后,取小鼠肝脏组织,称肝重量;检测甘油三酯、胆固醇、非游离脂肪酸、谷草转氨酶、谷丙转氨酶水平;HE染色观察肝脏病理变化;油红O染色观察肝组织内脂肪滴情况;Western Blot法检测腺苷酸活化蛋白激酶(AMPK)、乙酰辅酶A羧化酶(ACC)、哺乳动物雷帕霉素靶蛋白(mTOR)、核酸S6蛋白激酶(p70S6k)的磷酸化表达,PCR法检测脂肪合成转录因子SREBP1及其靶基因(FAS、SCD1、GPAT)的基因表达。结果:与模型组比较,丁香酚高剂量组肝重与体质量的比值显著升高(P<0.05)。HE染色显示,与正常组比较,模型组的肝细胞出现增大并伴有大泡性脂肪变性,给药组明显改善肝脏病理状态和肝细胞脂肪变性;油红O结果显示,丁香酚高、低剂量组的脂滴较模型组显著减少。Western Blot结果表明,与模型组比较,丁香酚高、低剂量组的AMPK与ACC的磷酸化显著增加(P<0.01),mTOR和p70S6k的磷酸化表达则显著抑制(P<0.01,P<0.05);PCR结果表明,与正常组比较,模型组脂肪合成转录因子SREBP1及其靶基因表达显著增强(P<0.01,P<0.05);与模型组比较,丁香酚高、低剂量组中上述表达基因显著被抑制(P<0.01)。结论:丁香酚通过AMPK/ACC/mTOR/p70S6k信号通路和抑制脂肪合成转录因子SREBP1及其靶基因的表达来改善NAFLD。

Objective: To study the mechanism of eugenol on nonalcoholic fatty liver disease(NAFLD) induced by high-fat diet in mice. Methods: Forty C57BL/6 mice were divided into normal group, model group, eugenol high and low dose(40, 20 mg/kg) groups, with 10 mice in each group. They were given high-fat diet to establish NAFLD model. At the same time,eugenol was given by gavage(once a day). The body mass was measured twice a week for 15 weeks. After 15 weeks, the liver tissue of mice was taken and the liver weight was weighed;The levels of triglycerides, cholesterol, non free fatty acids, aspartate aminotransferase and alanine aminotransferase were detected;The pathological changes of liver were observed by HE staining;The fat droplets in liver tissue were observed by oil red O staining;The phosphorylation expression of AMPK, ACC, mTOR and P70S6K was detected by Western blot, and the gene expression of fat synthesis transcription factor SREBP1 and its target genes(FAS, SCD1 and GPAT) was detected by PCR. Results: Compared with the model group, the ratio of liver weight to body mass in eugenol high-dose group was significantly higher(P<0.05). HE staining showed that compared with the normal group, hepatocytes in the model group increased and accompanied with bullous steatosis. High and low dose eugenol groups significantly improved liver pathological state and hepatocyte steatosis;The results of oil red O showed that the lipid droplets of eugenol high and low dose groups were significantly lower than those of model group(P<0.01, P<0.05). The results of Western blot showed that compared with the model group, the phosphorylation of AMPK and ACC in eugenol high and low dose groups was significantly increased(P<0.01), while the phosphorylation expression of mTOR and P70S6K was significantly inhibited(P<0.01, P<0.05);PCR results showed that compared with the normal group, the expression of fat synthesis transcription factor SREBP1 and its target gene in the model group were significantly increased(P<0.01, P<0.05). Compared with the model group, the expression of these genes was significantly inhibited in eugenol high and low dose groups(P<0.01). Conclusion: Eugenol can improve NAFLD through AMPK/ACC/mTOR/P70S6K signaling pathway and inhibiting the expression of fat synthesis transcription factor SREBP1 and its target genes.