肝豆汤通过肝激酶B1/AMP活化蛋白激酶途径改善高脂摄入引起的小鼠肝脏脂肪变性

Gandou decoction improves fatty degeneration of mice liver caused by high fat intake through LKB1/AMPK pathway

目的研究肝豆汤对高脂摄入引起的小鼠肝脏脂肪变性的影响及可能的机制。方法采用60只C57BL/6雄性小鼠,按随机数字表法分为六组:正常饮食组,模型组,东宝肝泰组,肝豆汤低、中、高剂量组,每组10只。除正常饮食组外,其余各组均给予高脂饲料造模,同时分别灌胃给予生理盐水,东宝肝泰冲剂0.225 g/kg,肝豆汤(22,44,66 g/kg)各2 mL·kg^(-1)·d^(-1),药物干预6周后,禁食24 h,采用苏木精-伊红(HE)和油红O染色观察各组小鼠肝组织病理变化及肝脏脂滴数量,采用试剂盒测定各组小鼠肝组织总胆固醇、三酰甘油的含量及血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)的活性,采用蛋白质印迹法(Western blotting)检测小鼠肝脏3-羟基-3-甲基戊二酰辅酶A还原酶(HMGCR)、尼曼匹克C1型类似蛋白(NPC1L1)、固醇调节元件结合蛋白1(SREBP-1)及磷酸化肝激酶B1(p-LKB1)、磷酸化乙酰辅酶A羧化酶(p-ACC)、磷酸化AMP活化蛋白激酶(p-AMPK)相对表达情况。结果与正常组[(12.78±1.17)IU/L、(6.08±0.71)IU/L、(1.90±0.10)mmol/L、(1.83±0.09)mmol/L]相比,模型组小鼠肝组织出现脂肪变性,肝细胞肿大,脂肪滴增多等病变,小鼠血清中AST(18.81±1.14)IU/L、ALT活性(9.65±0.90)IU/L、肝组织中总胆固醇(2.48±0.08)mmol/L、三酰甘油含量(3.12±0.10)mmol/L及HMGCR、NPC1L1、SREBP-1蛋白相对表达量均升高,p-AMPK、p-LKB1、p-ACC蛋白相对表达量均降低,差异有统计学意义(P<0.05);与模型组相比,肝豆汤低、中、高剂量组小鼠肝组织病变依次减轻,血清AST[(16.30±1.18)IU/L、(14.020±1.15)IU/L、(12.20±1.12)IU/L]、ALT活性[(8.58±0.86)IU/L、(7.18±0.83)IU/L、(6.18±0.65)IU/L]、肝组织中总胆固醇[(2.36±0.11)mmol/L、(2.03±0.09)mmol/L、(1.91±0.10)mmol/L]、三酰甘油含量[(2.52±0.08)mmol/L、(1.96±0.09)mmol/L、(1.84±0.10)mmol/L]及HMGCR、NPC1L1、SREBP-1蛋白相对表达量依次降低,p-ACC、p-AMPK、p-LKB1蛋白相对表达量依次升高(P<0.05),东宝肝泰组小鼠血清中AST(14.52±1.16)IU/L、ALT活性(7.16±0.90)IU/L、肝组织中总胆固醇(2.01±0.09)mmol/L、三酰甘油含量(1.95±0.09)mmol/L及HMGCR、NPC1L1、SREBP-1蛋白相对表达量均降低,p-ACC、p-AMPK、p-LKB1蛋白相对表达量均升高(P<0.05),其中肝豆汤中剂量组与东宝肝泰组比较,差异无统计学意义(P>0.05)。结论肝豆汤可改善高脂摄入引起的小鼠肝脏脂肪变性,其机制可能与激活LKB1/AMPK信号通路有关。

Objective To study the effect of Gandou decoction on fatty degeneration of mice liver caused by high fat intake and its possible mechanism.Methods Sixty C57 BL/6 male mice were assigned into 6 groups by random number table:normal diet group,model group,Dongbao Gantai granule group,low-,medium-and high-dose Gandou decoction groups,with 10 mice in each group.In addition to the normal diet group,the other groups were given high-fat feed to make model,and at the same time were given normal saline,Dongbao Gantai granule 0.225 g/kg,Gandou decoction(22,44,66 g/kg)2 mL·kg^(-1)·d^(-1) respectively.After 6 weeks of drug intervention,the mice were fasted for 24 hours.Hematoxylin eosin(HE)and oil red O staining were used to observe the pathological changes of liver tissue and the number of lipid droplets,the contents of total cholesterol(TC),triglyceride(TG)and the activities of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by kit,and Western blotting was used to detect the relative expressions of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR),Niemann-Pick C1-like 1(NPC1 L1),sterol regulatory element binding protein 1(SREBP-1),phosphorylated liver kinase B1(p-LKB1),phosphorylated acetyl coenzyme A carboxylase(p-ACC)and phosphorylated AMP-activated protein kinase(p-AMPK)in mice liver.Results Compared with the normal group[(12.78±1.17)IU/L,(6.08±0.71)IU/L,(1.90±0.10)mmol/L,(1.83±0.09)mmol/L],the liver tissue of the model group showed fatty degeneration,hepatomegaly,increased fat droplets and other pathological changes.The activities of AST[(18.81±1.14)IU/L]and ALT[(9.65±0.90)IU/L]in serum,the contents of TC[(2.48±0.08)mmol/L]and TG[(3.12±0.10)mmol/L]in liver tissue and the relative expressions of HMGCR,NPC1 L1 and SREBP-1 increased,while the relative expressions of p-AMPK,p-LKB1 and p-ACC decreased,and the differences were statistically significant(P<0.05).Compared with the model group,the pathological changes of liver tissue in the low-,middle-and highdose Gandou decoction groups were alleviated in turn.The activities of AST[(16.30±1.18)IU/L,(14.020±1.15)IU/L,(12.20±1.12)IU/L]and ALT[(8.58±0.86)IU/L,(7.18±0.83)IU/L,(6.18±0.65)IU/L]in serum,the contents of TC[(2.36±0.11)mmol/L,(2.03±0.09)mmol/L,(1.91±0.10)mmol/L]and TG[(2.52±0.08)mmol/L,(1.96±0.09)mmol/L,(1.84±0.10)mmol/L]in liver tissue and the relative expressions of HMGCR,NPC1 L1 and SREBP-1 decreased in turn,while the relative expressions of p-AMPK,p-LKBl and p-ACC increased in turn(P<0.05).In Dongbao Gantai granule group,the activities of AST[(14.52±1.16)IU/L]and ALT[(7.16±0.90)IU/L]in serum,the contents of TC[(2.01±0.09)mmol/L]and TG[(1.95±0.09)mmol/L]in liver tissue and the relative expressions of HMGCR,NPC1 L1 and SREBP-1 decreased,while the relative expressions of p-AMPK,p-LKB1 and p-ACC increased(P<0.05),and there was no significant difference between the medium-dose Gandou decoction group and Dongbao Gantai Granule group(P>0.05).Conclusion Gandou decoction can improve the fatty degeneration of mice liver caused by high fat intake,and its mechanism may be related to the activation of LKB1/AMPK signal pathway.