1、J Intern Med Concepts Pract 2023,Vol.18,No.1 论著 生酮饮食诱导 db/db 小鼠肝脏脂肪沉积张梦潇1,孙烁烁2,韦晓2,张少红2,陈国芳2,刘超2(1.南京中医药大学附属盐城市中医院老年科,江苏盐城224000;2.南京中医药大学附属中西医结合医院内分泌科江苏省中医药研究院国家中医药管理局瘿病证治重点研究室,江苏南京210028)摘要目的:研究生酮饮食(ketogenic diet,KD)对db/db小鼠肝脏脂质沉积的影响及其机制,探讨KD治疗db/db小鼠的安全性。方法:选用8周龄db/db雄性小鼠20只作为肥胖2型糖尿病(type 2 diab
2、etes mellitus,T2DM)动物模型,适应性喂养3周后,最终18只纳入研究,随机数字表法分为正常喂养(ND)组、KD组、75%热量限制(calorierestriction,CR)组,每组6只。另将8周龄C57BL/6雄性小鼠6只作为正常对照(C)组,以标准饲料喂养。C组、ND组自由进食标准饲料,KD组自由进食生酮饲料,CR组作为阳性对照组,每日摄入ND组75%的标准饲料。干预4周后,由于实验过程中KD组及CR组分别有2只及1只小鼠不明原因死亡,按随机数字表法每组纳入3只小鼠进行统计分析。检测各组小鼠空腹甘油三酯(triglyceride,TG)、总胆固醇(total cholest
3、erol,TC)及低密度脂蛋白胆固醇(lowdensity lipoprotein cholesterol,LDL-C)水平;观察小鼠肝脏形态和结构及肝脏组织中脂滴大小和数量;定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)法检测肝脏组织固醇调节元件结合蛋白1C(sterol regulatory element-binding protein 1C,SREBP1C)、硬脂酰辅酶A去饱和酶-1(stearoyl-CoA desaturase 1,SCD1)、酰基辅酶A氧化酶1(acyl-CoA oxidase 1,ACOX1)、过氧化物
4、酶体增殖物激活受体(peroxisome proliferator-activated receptor,PPAR)、白细胞分化抗原36(cluster of differentiation 36,CD36)、肿瘤坏死因子-(tumor necrosis factor-,TNF-)、白介素-1(in-terleukin-1,IL-1)、基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)、组织金属蛋白酶抑制物1(tissue in-hibitor of metalloproteinase 1,TIMP1)、型胶原a1(type collagen a1,Col
5、3a1)及型胶原a1(type collagen a1,Col1a1)等相关因子的表达;免疫组化及蛋白质印迹法检测肝脏组织中CD36的表达水平。结果:与ND组相比,KD组小鼠TG、TC及LDL-C水平无明显改善,CR组小鼠TG水平显著降低(P0.05)。KD组较ND及CR组肝脏空泡变性增加,脂质沉积增多。qPCR结果显示,与ND组相比,KD组PPAR、ACOX1等脂质分解代谢基因差异无统计学意义;CD36表达明显升高(P0.05);IL-1、TNF-等炎症因子表达明显升高(P0.05);MMP13表达显著下降(P0.05),其余肝纤维化相关基因表达无明显变化。与ND组相比,KD组CD36蛋白表
6、达水平显著增加(P0.05)。结论:KD诱导db/db小鼠肝脏脂质沉积,加重肝脏炎症水平。因此,在使用KD治疗肥胖及肥胖相关疾病时,要密切关注肝功能的变化,优化KD方案,预防其不良反应。关键词:2型糖尿病;肥胖;生酮饮食;肝脏;脂肪沉积中图分类号:R587.1文献标志码:A文章编号:1673-6087(2023)01-0056-08DOI:10.16138/j.1673-6087.2023.01.018Ketogenic diet promotes hepatic lipid accumulation in db/db mice ZHANG Mengxiao1,SUN Shuoshuo2,WE
7、I Xiao2,ZHANG Shaohong2,CHEN Guofang2,LIU Chao2.1.Department of Geriatrics,Yancheng Hospital Affiliated to NanjingUniversity of Traditional Chinese Medicine,Yancheng 224000;2.Department of Endocrinology,Affiliated Hospital of In-tegrated Traditional Chinese and Western Medicine,Nanjing University of
8、 Traditional Chinese Medicine,Jiangsu Provin-cial Academy of Traditional Chinese Medicine;Key Laboratory of Physic Diseases and Treatment of State Administrationof Traditional Chinese Medicine,Nanjing 210028,ChinaAbstract Objectives To study the effect of ketogenic diet(KD)on liver lipid deposition
9、in db/db mice,and to in-基金项目:国家自然科学基金项目(项目编号:81800756);江苏省第十六批“六大人才高峰”高层次人才选拔培养项目(项目编号:WSN-035);江苏省研究生科研创新计划项目(项目编号:KYCX20_1557)通信作者:陈国芳E-mail:刘超E-mail:56内科理论与实践2023年第18卷第1期肥胖及2型糖尿病(type 2 diabetes mellitus,T2DM)等慢性代谢性疾病已成为严重影响国际公共卫生事业发展的重大医学问题,引起全球广泛关注。研究发现1,肥胖、高血糖是脂肪肝、肝纤维化等严重肝脏疾病的重要危险因素。生酮饮食(keto-
10、genic diet,KD)是一种高脂低碳及适当蛋白质的饮食结构,主要通过模拟禁食状态,以脂肪作为机体的主要能量来源,通过肝脏分解脂肪酸,诱导酮体生成,替代葡萄糖的氧化供能模式2。几个世纪以来,KD一直用于癫痫治疗,疗效显著。近年来,KD被证实对肥胖、T2DM等代谢性疾病有效。然而,随着KD的普及,关于其不良反应的报道也越来越多。有研究发现,长期KD会导致T2DM小鼠血脂水平异常、炎症水平增加及肝脏脂肪变性3,故KD治疗T2DM等代谢性疾病的安全性引起广泛争议。本研究通过KD干预肥胖T2DM模型db/db小鼠,评估KD对db/db小鼠肝脏形态和脂质代谢的影响,为临床应用KD减重、治疗肥胖的T2
11、DM提供安全性保障。材料与方法一、实验动物分组及处理8周龄雄性C57BL/6J小鼠6只和雄性db/db小鼠20只购自南通大学,生产许可证号:SCXK(苏)2014-0001,按清洁级饲养标准分笼。适应性喂养3周,自由摄食及饮水,保持相同温、湿度,12 h交替照明。环境温度(233),湿度55%5%。生酮饲料及标准饲料购自江苏协同医药生物工程有限公司。本实验已通过动物伦理审核,并严格按照国家实验动物福利伦理的相关规定执行。6只C57BL/6J小鼠和20只db/db小鼠适应性喂养3周后,将C57BL/6J小鼠设为正常对照组(C组)。db/db小鼠中,1只在适应性喂养过程中因体重骤降死亡,1只空腹血
12、糖值与其余db/db小鼠相差较大,予剔除。通过随机数字表法将db/db小鼠分为肥胖T2DM模型(ND)组、KD组、75%热量限制(calorie restriction,CR)组,每组6只。C组及vestigate the safety of KD treatment and molecular mechanism in db/db mice.Methods Twenty eight-week-old db/db malemice of obesity type 2 diabetes mellitus(T2DM)were selected.After 3 weeks of adaptive f
13、eeding,18 mice were enrolledand randomly divided into 3 groups:normal feeding(ND)group(n=6),KD group(n=6)and 75%caloric restriction(CR)group(n=6).In addition,6 eight-week-old C57BL/6 male mice were fed with standard control diet and considered as nor-mal(C)group.C and ND groups were free to get the
14、standard feed,KD group was free to get the ketogenic feed,and CRgroup was the positive control group,which consumed 75%of standard feed calories of ND group each day.After 4 weeksof diet treatment,2 mice in KD group and 1 mouse in CR group died.Three mice in each group were randomly includedfor stat
15、istical analysis,and the levels of fasting triglyceride(TG),total cholesterol(TC)and low-density lipoprotein choles-terol(LDL-C)in each group were detected and compared.The morphology and structure of liver were observed by hema-toxylin and eosin(HE)staining.The size and number of lipid droplets in
16、liver tissue of mice were observed by oil red Ostaining.The quantitative polymerase chain reaction(qPCR)was used to detect the expression of sterol regulatory element-binding protein 1C(SREBP1C),stearoyl-CoA desaturase 1(SCD1),acyl-CoA oxidase 1(ACOX1),peroxisome proliferator-activated receptor (PPAR),cluster of differentiation 36(CD36),tumor necrosis factor-(TNF-),interleukin-1(IL-1),matrix metalloproteinase-13(MMP13),matrix metalloproteinase-inhibitor 1(TIMP1),collagen type alpha 1(COL3a1)andc