CAJ | 학술논문

目的探讨利拉鲁肽改善肝脏脂肪沉积的潜在分子机制.方法 8周龄雄性C57BL/6J小鼠标准饲料喂养作为野生对照组(WT组,n=10);ApoE KO小鼠56只,随机分为标准饲料喂养组(ApoE KO组,n=10)、高脂喂养组(HF组,n=10)、高脂喂养+空载腺病毒组(Ad-shGFP组,n=6)、高脂喂养+脂联素RNM腺病毒组(Ad-shAcrp30组,n=10)、高脂喂养+脂联素RNAi腺病毒+利拉鲁肽组(Liraglutide组,n=10)、高脂喂养+脂联素RNAi腺病毒+等渗盐水组(Saline组,n=10).共喂养16周,于14和15周末给予Ad-hGFP组和Ad-hAcrp30组尾静脉分别注射Ad-shGFP和Ad-shAcrp30重组腺病毒(1×1010/ml).Liraglutide组第9周开始腹腔注射利拉鲁肽(1mg/kg,2次/d).16周末测血浆葡萄糖、血脂、脂联素及胰岛素等.取肝脏组织做HE染色及油红O染色.荧光实时定量PCR检测基因mRNA表达、蛋白免疫印迹Western blot检测相关蛋白表达.非正态分布数据采取自然对数转换.组间比较采用方差分析,组内比较采用SNK法.结果与Ad-shGFP组相比,Ad-shAcrp30组脂肪组织脂联素mRNA、蛋白表达及血浆脂联素水平均降低(均P＜0.01);HF组的空腹血糖(FBG)、胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)较WT组及ApoE KO组均显著升高(均P＜0.01),给予Ad-shAcrp30后,FBG、TC、TG、FFA进一步升高.Ad-hAcrp30组肝脏TC及TG含量较HF组升高(P值均＜0.05).油红O染色见Ad-shAcrp30组肝内大量脂滴沉积,HE染色示Ad-shAcrp30组肝小叶结构紊乱.与Saline组相比,Liraglutide组体质量、血糖、血脂及ALT均显著降低(均P＜ 0.01);脂肪组织脂联素蛋白表达及血浆脂联素水平均显著升高(均P＜ 0.01);肝脏TG、TC均减少(P值均＜0.05);油红O染色提示肝脏脂滴明显变小,且数量减少;肝脏ACC、FAS表达下调,而磷酸化AMPK蛋白表达增加(P＜ 0.01).结论利拉鲁肽可以显著改善高脂喂养联合低脂联素血症诱导的代谢紊乱并减轻肝脏脂质沉积.
목적 탐토리랍로태개선간장지방침적적잠재분자궤제.방법 8주령웅성C57BL/6J소서표준사료위양작위야생대조조(WT조,n=10);ApoE KO소서56지,수궤분위표준사료위양조(ApoE KO조,n=10)、고지위양조(HF조,n=10)、고지위양+공재선병독조(Ad-shGFP조,n=6)、고지위양+지련소RNM선병독조(Ad-shAcrp30조,n=10)、고지위양+지련소RNAi선병독+리랍로태조(Liraglutide조,n=10)、고지위양+지련소RNAi선병독+등삼염수조(Saline조,n=10).공위양16주,우14화15주말급여Ad-hGFP조화Ad-hAcrp30조미정맥분별주사Ad-shGFP화Ad-shAcrp30중조선병독(1×1010/ml).Liraglutide조제9주개시복강주사리랍로태(1mg/kg,2차/d).16주말측혈장포도당、혈지、지련소급이도소등.취간장조직주HE염색급유홍O염색.형광실시정량PCR검측기인mRNA표체、단백면역인적Western blot검측상관단백표체.비정태분포수거채취자연대수전환.조간비교채용방차분석,조내비교채용SNK법.결과 여Ad-shGFP조상비,Ad-shAcrp30조지방조직지련소mRNA、단백표체급혈장지련소수평균강저(균P＜0.01);HF조적공복혈당(FBG)、담고순(TC)、감유삼지(TG)、유리지방산(FFA)교WT조급ApoE KO조균현저승고(균P＜0.01),급여Ad-shAcrp30후,FBG、TC、TG、FFA진일보승고.Ad-hAcrp30조간장TC급TG함량교HF조승고(P치균＜0.05).유홍O염색견Ad-shAcrp30조간내대량지적침적,HE염색시Ad-shAcrp30조간소협결구문란.여Saline조상비,Liraglutide조체질량、혈당、혈지급ALT균현저강저(균P＜ 0.01);지방조직지련소단백표체급혈장지련소수평균현저승고(균P＜ 0.01);간장TG、TC균감소(P치균＜0.05);유홍O염색제시간장지적명현변소,차수량감소;간장ACC、FAS표체하조,이린산화AMPK단백표체증가(P＜ 0.01).결론 리랍로태가이현저개선고지위양연합저지련소혈증유도적대사문란병감경간장지질침적.
Objeetive To investigate the mechanism ofliraglutide-mediated protection against nonalcoholic fatty liver disease (NAFLD) using aApoE knockout (KO) mouse with high-fat diet (HFD) and Acrp30 knockdown.Methods Fifty-six male ApoE KO mice were divided into the following six modeling and experimental groups:regular chow fed (ApoE KO,n =10),HFD fed (HF,n =10),HFD+Adenovirus (Ad)-small hairpin (sh) Acrp30 (Ad-shAcrp30,n =10),HFD+Ad-shGreen Fluorescent Protein (GFP) (Ad-shGFP,n =6),HFD+Ad-shAcrp30+liraglutide (liraglutide,n =10),and HFD+Ad-shAcrp30+saline (saline,n =10).Weight-matched C57BL/6 mice on the regular chow diet were used as the control group (WT control,n =10).All mice were fed their assigned diet for 16 weeks.The Ad-shGFP or Ad-shAcrp30 was injected by tail vein at the end of 14 and 15 weeks.Mice in the liraglutide group received 1 mg/kg of the drug,twice daily,intraperitoneally for a total of 8 weeks (from the 9th to 16th week).Fasting blood samples were collected for testing levels of fasting plasma glucose (FPG),triglycerides (TGs),total cholesterol (TC),free fatty acid (FFA),alanine aminotransferase (ALT),Acrp30 and insulin.Liver tissue was procured for histological examination.Expression of mRNA was detected by real-time RT-PC and of protein was detected by western blot analysis.Results The Ad-shAcrp30 treated mice had reduced expression of Acrp30 at both the mRNA and protein levels in adipose tissues and plasma,as compared with the AdshGFP treated mice (all P ＜ 0.01).Compared to the WT and ApoE KO groups,the HF group showed higher levels of FPG,FFA,TGs and TC (all P ＜ 0.01); furthermore,the Ad-shAcrp30 treatment compounded these changes.The Ad-shAcrp30 treated group had markedly higher hepatic TC and TGs than the HF group (P ＜ 0.01 andP ＜0.05).Oil Red O staining showed that there was more lipid droplets in the liver tissue of the Ad-shAcrp30 treated group than in that of the HF group (P ＜ 0.01),and hematoxylin-eosin staining confirmed these results.Liraglutide treatment prevented the increase in body weight,FPG,FFA,TGs,TC and ALT levels,as compared to the saline controls (all P ＜ 0.01),but the plasma Acrp30 levels and the Acrp30 mRNA and protein expression in adipose tissues were elevated (allP ＜ 0.01).Oil-Red O staining indicated that the liraglutide group had a significantly lower hepatic lipid content than the saline group,and total hepatic TG and TC were reduced in the former group (P ＜ 0.01 andP ＜ 0.05).The liraglutide treatment significantly attenuated the mRNA expression of ACC and FAS (both P ＜ 0.01) but increased AMPK phosphorylation (P ＜ 0.01).Conclusion Administration of liraglutide prevented the development of HFD-and hypoadiponectinemia-induced metabolic disturbance and accumulation of hepatic lipids in this mouse model system of NAFLD.