Effect Of Liraglutide On Autophagy Activity Of Endothelial Cells In High Glucose Environment By AMPK/mTOR Signaling Pathway

Objective:（1）To observe the changes of autophagy in human umbilical vein endothelial cells（HUVECs）in high glucose environment,and to explore its possible mechanism.（2）To study the effect of glocalization peptide-1（Glocalization Peptide-1,GLP-1）receptor agonist Liraglutide（LIRA）on autophagy induced by high glucose in HUVECs and its possible mechanism.To determine whether it can be a possible therapeutic target for diabetic macroangiopathy.Methods:（1）HUVECs were cultured in vitro and divided into control group（group A）and high glucose group（group B）.Group A and group B were cultured respectively in 5.5mmol/L and 25mmol/L glucose medium for 12,24 and 48 hours.The expressions of autophagy-related genes BECN1/Beclin-1,LC3 and porpoin SQSTM1/p62 were determined by real-time fluorescent quantitative polymerize chain reaction（RT-PCR）,and the optimal culture time of high glucose condition on autophagy was determined.（2）The high glucose group at the optimal culture time point according to the first part of the experiment was treated with low,medium and high dose gradients（10nmol/L,50nmol/L,100nmol/L）of LIRA（C₁,C₂,C₃ groups）.The same method was used to detect the above indicators,and the optimal intervention concentration was obtained.（3）In addition to the treatment of the optimal dose of LIRA under high gluco se condition（group C）and high glucose culture alone（group B）,The amp-activate d protein kinsey（AMPK）inhibitor CompoundC（ComC,10μmol/L）was introduced（gr oup D and group E）.The fluorescence spots of green fluorescent protein（GFP）co njugated LC3（GFP-LC3）were observed by high-resolution laser confocal fluorescen cemicroscopy to monitor the formation of autophagosomes.The expression of cell a utophagy marker BECN1/Beclin-1,SQSTM1/p62 protein and LC3-II/LC3-I,the the ra tios of phosphorylation AMPK（p-AMPK）/AMPK,phosphorylation the mammalian tar get of rapamycin（p-mTOR）/mTOR were measured by western blotting.Results:（1）Compared with group A,the number of GFP-LC3 fluorescent spots in group B decreased,and the expression of BECN1/Beclin-1 and LC3 mRNA decreased at different times（all P<0.05）,while SQSTM1/p62 mRNA expression increased（P<0.05）,The above change was most significant at 24h.Thus 24h was chosen as the optimal culture time.Its protein expression is consistent with gene expression results.At the same time,the ratio of p-AMPK/AMPK in group B was lower than that in group A,and the ratio of p-mTOR/mTOR was higher than that in group A.（2）Compared with group B cultured in 24h,the expression of BECN1/Beclin-1 and LC3 mRNA was increased and the expression of SQSTM1/p62 mRNA was decreased in C₃ group（all P<0.05）.And the expression of LC3 mRNA in C₁,C₂ and C₃ groups all increased（all P<0.05）in a dose-dependent pattern.The optimal intervention concentration of LIRA was 100nmol/L.（3）Compared with group B,the fluorescence aggregation signal of GFP-LC3 w as significantly increased in group C.the expression of BECN1/Beclin-1 protein,the ratio of LC3-II/LC3-I and p-AMPK/AMPK increased significantly（all P<0.05）,SQ STM1/p62 protein,the ratio of p-mTOR/mTOR expression decreased（all P<0.05）.The ratio of LC3-II/LC3-Iand p-AMPK/AMPK in group D was lower than that in group C（all P<0.05）,and the ratio of p-mTOR/mTOR was higher than group C（P<0.05）.There was no significant difference in the expression level ofprotein BECN1/Beclin-1and SQSTM1/p62 between group C and D（all P>0.05）.（4）Compared with group D,LC3-II/LC3-I ratio decreased（P<0.05）and the expression of SQSTM1/p62 protein increased in group E（P<0.05）.The trend in changes of autophagy fluorescent spots GFP-LC3 was consistent with that of autophagy related protein.Conclusion:（1）The autophagy level of HUVECs cultured in high glucose was significantly inhibited,and the expression of BECN1/Beclin-1,LC3 protein decreased,while the expression of SQSTM1/p62 protein increased,and the number of fluorescent spots of GFP-LC3 decreased.The ratio of AMPK/AMPK protein was down-regulated and the ratio of p-mTOR/mTOR protein was up-regulated,suggesting that the AMPK/mTOR pathway is inhibited in the high glucose state.（2）LIRA can up-regulate BECN1/Beclin-1 protein,the ratio of LC3-II/LC3-I a nd p-AMPK/AMPK,and down-regulate SQSTM1/p62 protein and the ratio of p-mT OR/mTOR.That is,LIRA can activate the autophagy,which may activate AMPK/m TOR rmediated signaling pathway through phosphorylation,reduce the damage of H UVECs in hyperglycemia state,and have a protective effect on damaged HUVECs.