| Aim:1.We established NAFLD model in vivo in T2 DM model mice and in vitro in PA-induced Hep G2 cells to investigate the effects of liraglutide on hepatocyte steatosis,gluconeogenesis and inflammation as well as the regulation of ACE/Ang II/AT1 R axis and ACE2/A1-7/MAS axis expression of local RAAS system in the liver by liraglutide,to explore the role and mechanism of RAAS system in the improvement of NAFLD by liraglutide.2.We used ACE2 knockout mice and exogenous A1-7 to treat with T2 DM C57BL/6J mice to down-regulate and up-regulate the ACE2/Ang-(1-7)/MAS axis,respectively,to further establish the participation of ACE2/Ang-(1-7)/MAS axis in NAFLD;Method: 1.Fifteen 6-week-old male C57BL/6J mice were selected to induce T2DM-nonalcoholic fatty liver disease model by long-term high-fat diet plus intraperitoneal low-dose streptozotocin(STZ)injection.After successful modeling,they were randomly divided into T2 DM model group(T2DM group)(n=5),liraglutide intervention group(liraglutide group)(n=5)and Ang-(1-7)intervention group(A1-7 group)(n=5).Another five normal male C57BL/6J mice were fed with normal diet as control group(C group)(n=5).The drug intervention groups continued to intervene for 4 weeks.At the end of experiments,Hepatic steatosis was detected by HE staining and oil red O staining in the livers.Triglyceride(TG)content in the livers were detected by TG detection kit. CPT-1a?ACOX-1?G6P?PEPCK?NF?B as well as IL-1?m RNA expression were detected by RT-PCR.2.the expression of ACE?AT1R?ACE2 and MAS m RNA of RAAS system in the livers were detected by RT-PCR in the normal control group?T2DM model group and liraglutide group.3.Three ACE2 knockout(ACE2 KO)mice and three wild type(WT)mice were selected respectively.All mice were fed with normal diet.Hepatic steatosis was detected by HE staining and oil red O staining in the livers.Triglyceride(TG)content in the livers were detected by TG assay kit.The expression of CPT-1a?ACOX-1?G6P?PEPCK?NF?B as well as IL-1?m RNA were detected by RT-PCR.4.Treatment of Hep G2 cells to 0.25 m M palmitic acid(PA)induced hepatocyte steatosis to establish NAFLD model in vitro.To determine the effect of liraglutide on the regulation of RAAS components expression in Hep G2 cells and the involvement of PI3K/AKT signaling in NAFLD condition in vitro,the cells were divided into the following groups: 1)normal control group;2)PA group(0.25 m M PA);3)PA + liraglutide(10 nmol/L)group(liraglutide group);4)PA + liraglutide(10 nmol/L)+ PI3 K inhibitor LY294002(20 umol/L)group(LY294002 group).After treatment for 24 h,the expression of AKT and P-AKT proteins in each group were detected by Western blot.The m RNA expression of ACE, AT1R,ACE2 and MAS of RAAS system were detected by RT-PCR.5.To determine whether upregulation of ACE2/Ang-(1-7)/MAS axis by liraglutide and liraglutide-mediated benefical effects on NAFLD is of functional relevance,we used MAS receptor-specific inhibitor A779 to inhibit ACE2/A1-7/MAS axis and detected the influence of blockade of ACE2/A1-7/MAS axis on liraglutide-mediated effects.Treatment of Hep G2 cells to 0.25 m M palmitic acid(PA)induced hepatocyte steatosis to establish NAFLD model in vitro.The cells were divided into the following groups in this part of the experiments : 1)normal control group;2)PA(0.25 m M PA)group;3)PA + liraglutide(10 nmol/L)group(liraglutide group);4)PA + Ang-(1-7)(10-7mol/L)group(A1-7 group);5)PA+ liraglutide(10 nmol/L)+ Ang-(1-7)(10-7mol/L)group(liraglutide + A1-7 combination group);6)PA + liraglutide(10 nmol / L)+ A779(10-7 mol / L)group(liraglutide + A779 group).After treatment for 24 h,the triglyceride(TG)content in cells of each group were detected by TG assay kit.The expression of CPT-1a?ACOX-1?G6P?PEPCK?NF?B as well as IL-1?m RNA were detected by RT-PCR.Nuclear translocation of NF ? Bp65 in Hep G2 cells was detected by immunofluorescence staining.The expression of AMPK and P-AMPK proteins in each group were detected by Western blot.6.The SPSS 11.5 software were used to analyze the datas.All values were represented as the meanS.D(standard deviation),t-test was used to compare the mean of the two samples,and one-way ANOVA was used to compare the differences among the groups,P < 0.05 was used as the significant difference threshold.Results: At the end of experiments,1.The HE staining and oil red O staining of hepatic tissue showed that the histological structure of the livers was disordered,the hepatocytes showed obvious fat deposition,and the red lipid droplets in the hepatocytes increased significantly in the T2 DM model group compared to C group.Liraglutide group showed significant improvements in liver histological structure and hepatic steatosis,lipid deposition and red lipid droplets decreased significantly.The TG content detection showed that the liver TG content in the T2 DM model group was significantly higher than that in the C group(P<0.01),and the liver TG content in the liraglutide group was significantly lower than that in the T2 DM group(P<0.01).RT-PCR results showed that the m RNA expression of CPT-1a and ACOX-1 genes related to fatty acid ?-oxidation in livers of T2 DM model group were lower than that of C group(P<0.01?P<0.05 respectively),while the m RNA expression of G6 P and PEPCK,the key enzymes of gluconeogenesis,were higher than that of C group(both P<0.01),and the m RNA expression of inflammatory signal NF?B and IL-1?were higher than that of C group(both P<0.01).The m RNA expression of CPT-1a ? ACOX-1 were significantly increased(P<0.01?P<0.05 respectively),the m RNA expression of G6P?PEPCK(both P<0.01)as well as NF?B and IL-1?(both P<0.01)were all significantly decreased in liraglutide group compared with the T2 DM model group.2.RT-PCR results showed that the m RNA expression of ACE?AT1R?ACE2 and MAS in livers of T2 DM model group were remarkedly higher than those of C group(P<0.01?P<0.05?P<0.01?P<0.05 respectively),and the m RNA expression of ACE and AT1 R were significantly decreased(both P<0.01),while the m RNA expression of ACE2 and MAS were further increased in livers of liraglutide group compared to T2 DM model group(both P<0.05).4.The HE staining and oil red O staining of hepatic tissue showed that the histological structure of the livers was disordered,the hepatocytes showed obvious fat deposition,and the red lipid droplets in the hepatocytes increased significantly in the ACE2 KO group compared to WT group.The TG content detection showed that the liver TG content in the ACE2 KO group was significantly higher than that in the WT group(P<0.01).ACE2 KO group showed decreased m RNA expression of CPT-1a and ACOX-1(P<0.01?P<0.05 respectively),increased m RNA expression of G6 P and PEPCK(both P<0.01)as well as NF?B and IL-1?(both P<0.01)in the livers relative to WT group.After 4 weeks of intervention T2 DM C57BL/6J mice with exogenous A1-7,the HE staining and oil red O staining also showed that hepatic steatosis was significantly improved.The TG assay showed that the TG content in the livers was decreased significantly(P<0.05).RT-PCR results showed that the m RNA expression of CPT-1a?ACOX-1 in the liver of A1-7 group were increased(P<0.01 ? P<0.05 respectively),while the m RNA expression of G6P?PEPCK(both P<0.05)and NF?B?IL-1?(both P<0.01)were decreased compared to T2 DM group.4.In vitro experiments showed that the level of P-AKT protein was decreased,and the m RNA expression of ACE?AT1R?ACE2 and MAS were all increased in Hep G2 cells of PA group compared with the C group(P<0.01?P<0.01?P<0.05?P<0.01 respectively).Compared with the PA group,the level of P-AKT protein was increased,the m RNA expression of ACE and AT1 R were decreased(P<0.05 ? P<0.01 respectively)meanwhile the m RNA expression of ACE2 and MAS were further increased(P<0.05?P<0.01 respectively)in the liraglutide group.Compared with the liraglutide group,the level of P-AKT protein was decreased,the m RNA expression of ACE and AT1 R were increased(P<0.05?P<0.01 respectively)while the m RNA expression of ACE2 and MAS were decreased(P<0.05?P<0.01 respectively)in LY294002 group.There was no significant difference in the expression of total AKT protein in the 4 groups.5.In vitro experiments showed that compared with the C group,the intracelluar TG content was increased(P<0.01);the m RNA expression of CPT-1a and ACOX-1 were decreased(both P<0.01),the m RNA expression of G6 P and PEPCK(both P<0.01)as well as NF?B and IL-1?(both P<0.01)were increased(both P<0.01),P-AMPK protein expression was significantly reduced in the PA group.Significant nuclear translocation of NF?Bp65 was observed in the PA group.compared with the PA group,liraglutide and/or A1-7 treatment significant decreased the intracelluar TG content(both P<0.01),increased m RNA expression of CPT-1a(P<0.05?P<0.01 respectively)and ACOX-1(both P<0.01),decreased m RNA expression of G6P(both P<0.01)?PEPCK(both P<0.01)as well as NF ? B(P<0.01 ? P<0.05 respectively)?IL-1?(both P<0.01),increased P-AMPK protein expression,and significantly inhibited nuclear translocation of NF ? Bp65.The intracelluar TG content was further decreased(P<0.05 ? P<0.01 respectively),the m RNA expression of CPT-1a(both P<0.05), ACOX-1(P<0.05?P<0.01 respectively)were further increased,while the m RNA expression of G6P(both P<0.05),PEPCK(P<0.01 ? P<0.05 respectively)as well as NF?B(P<0.01?P<0.05 respectively),IL-1?(P<0.05?P<0.01 respectively)were further decreased in the combination group of liraglutide and A1-7 compared with their respective monotreatment.However,there was no significant difference in P-AMPK protein levels between the combination group and the two monotherapeutic groups.Compared with the liraglutide group,the intracelluar TG content was increased(P<0.01),the m RNA expression of CPT-1a,ACOX-1 were decreased(both P<0.05),while the m RNA expression of G6 P,PEPCK(both P<0.01)as well as NF?B,IL-1?(both P<0.05)were increased,P-AMPK protein expression was reduced in liraglutide+A779 group.NF ? Bp65 showed significant nuclear translocation again in liraglutide+A779 group.There was no significant difference in the expression of total AMPK protein in the 6 groups.Conclusions: 1.Liraglutide can improve NAFLD effectively in T2 DM model mice in vivo and in PA-induced Hep G2 cells in vitro,including the improvement of gene expression related to fatty acid ?-oxidation,the inhibition of gene expression related to hepatic gluconeogenesis and inflammatory signals in liver.2.In vivo and in vitro NAFLD model,the local RAAS system in the liver is over-activated,as evidenced by marked up-regulation of the m RNA expression of ACE,AT1 R,ACE2 and MAS,and liraglutide can obviously down-regulate the m RNA expression of ACE and AT1 R while further up-regulate the m RNA expression of ACE2 and MAS,indicating the dual regulatory effects of liraglutide on the two axes of the local RAAS system in the liver.Furthermore,blockade of the ACE2/A1-7/MAS axis with MAS-specific inhibitor A779 in vitro can significantly attenuate or even block liraglutide-mediated improvement of NAFLD,including the enhancement of fatty acid ?-oxidation,inhibition of gluconeogenesis and inflammation in the liver,suggesting that the improvement of NAFLD by liraglutide depends at least in part on its up-regulation of ACE2/A1-7/MAS axis in the liver.3.The regulatory effects of liraglutide on the m RNA expression of ACE/Ang?/AT1 R axis and ACE2/A1-7/MAS aixs of RAAS system is at least partially mediated by the activation of PI3K/AKT signal.4.ACE2 knockout mice exhibited a severe NAFLD phenotype,characterized by impaired fatty acid oxidation and increased gluconeogenesis as well as activation of inflammatory signals in the liver.While exogenous A1-7 treatment of T2 DM C57BL/6J mice can significantly improve NAFLD,including enhancement of fatty acid ?-oxidation,inhibition of hepatic gluconeogenesis and inflammation,showing that inhibition of ACE2/A1-7/MAS axis activity can promote the development of NAFLD,while activation of ACE2/A1-7/MAS axis can prevent it.These results reveal that the ACE2/A1-7/MAS axis may be a potential target for the treatment of NAFLD. |
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