二甲双胍通过调节成纤维细胞生长因子21抑制肥胖小鼠脂肪肝及改善白色/棕色脂肪平衡

Metformin inhibits fatty liver and improves white/brown fat balance in obese mice by regulating FGF21

目的探讨二甲双胍抑制肥胖小鼠脂肪肝的效应及可能的机制。方法40只4周龄C57BL/6小鼠随机分为对照组(NC组,n=10)和高脂饮食组(n=30),喂养12周后,将高脂饮食组小鼠随机分为肥胖模型组(Mod组,n=10)、二甲双胍低剂量组(L-Met组,n=10)和二甲双胍高剂量组(H-Met组,n=10),L-Met组与H-Met组分别以10 mg/(Kg·d)、50 mg/(Kg·d)二甲双胍灌胃,治疗6周。稳定培养3T3L1细胞并传代,接种至24孔板,在24孔板中加入二甲双胍(5 mmol/L)或抗成纤维细胞生长因子21(FGF21)培养24 h。检测小鼠外周血生化指标,流式细胞法检测Th1细胞变化,HE染色检测肝脏组病理情况,RT-PCR法检测白色脂肪组织(WAT)和棕色脂肪组织(BAT)中相关基因的表达。结果(1)与Mod组相比,L-Met组及H-Met组小鼠体重均明显减轻,H-Met组体重减轻更为明显(P<0.05),但均明显高于NC组(P<0.05)。经二甲双胍治疗后,L-Met组及H-Met组肥胖小鼠均出现血糖不耐受和胰岛素抵抗水平的降低。(2)与Mod组相比,二甲双胍治疗组WAT相关基因,包括PPAR-r、C/EBP-a、aP2、Adipsin的表达水平显著降低(P<0.05),而BAT相关基因,包括UCP1、Elov13、Cida、Cox7a1、PGC1a的表达水平升高(P<0.05)。透射电子显微镜显示,与H-Met组相比,Mod组小鼠的脂滴数量显著增加。共聚焦染色分析显示,H-Met组肝脏中FGF21表达显著上调。(3)细胞实验显示,抗FGF21抗体的使用不影响3T3L1细胞的脂滴形成。经二甲双胍治疗后,肥胖小鼠Foxp3阳性的Treg细胞数量显著减少。(4)流式细胞检测显示,二甲双胍治疗的肥胖小鼠的Treg细胞数量明显低于未治疗的肥胖小鼠。结论二甲双胍可通过诱导肥胖小鼠和3T3L1细胞中FGF21的产生,进而抑制高脂饮食诱导的肥胖和相关的炎症反应。

Objective To investigate the effect of metformin on fatty liver in obese mice and its possible mechanism.Methods Forty 4-week-old C57BL/6 mice were randomly divided into NC group,Mod group,L-Met group and h-met group.3T3L1 cells were stably cultured and passaged,and then inoculated into 24 well plate.Metformin(5 mmol/L)or afgf21 was added to the 24 well plate for 24 h.Flow cytometry was used to detect Th1 cell changes,he staining was used to detect the pathological changes of liver,and RT-PCR was used to detect the expression of related genes in Wat and bat.Results(1)compared with Mod group,the weight of mice in L-Met group and H-Met group were significantly reduced,and the weight loss of H-Met group was more obvious(P<0.05),but it was significantly higher than that of NC group(P<0.05).After metformin treatment,both L-Met group and H-Met group had decreased levels of glucose intolerance and insulin resistance.(2)Compared with mod,the expression levels of wat related genes,including PPAR-r,C/EBP-a,aP2 and Adipsin,were significantly decreased in metformin treated group(P<0.05),while the expression levels of bat related genes,including UCP1,Elov13,Cida,COX7al and PGC1a,were significantly increased(P<0.05).Transmission electron microscopy showed that compared with H-Met group,the number of lipid droplets in mod group was significantly increased.Confocal staining analysis showed that FGF21 expression was significantly up-regulated in H-Met group.(3)Cell experiments showed that the use of anti FGF21 antibody did not affect the formation of lipid droplets in 3T3L1 cells.After metformin treatment,the number of Foxp3 positive Treg cells in obese mice decreased significantly.(4)Flow cytometry showed that the number of Treg cells in metformin treated obese mice was significantly lower than that in untreated obese mice.Conclusion metformin can inhibit obesity and related inflammation induced by high-fat diet by inducing the production of FGF21 in obese mice and 3T3L1 cells.