Effect Of Liraglutide On The Secretion Of Atrial ANP And Its Mechanism

Objective:Atrial natriuretic peptide（ANP）has natriuretic and diuretic properties,participates in the regulation of water-salt balance,inhibits cell proliferation,reduces myocardial hypertrophy and myocardial fibrosis,and participates in immune regulation and blood pressure regulation.In addition,ANP also plays an important regulatory role in resisting myocardial protection such as ischemia-reperfusion injury and myocardial infarction.Glucagon-like peptide-1（GLP-1）,an insulin-stimulating hormone,has been widely used in clinical research as a new drug for the treatment of type 2 diabetes.Meanwhile,GLP-1 also has cardioprotective effects,including optimizing myocardial metabolism,directly protecting the heart by binding to cardiac receptors and signaling through subcellular ischemia regulatory pathways.However,the secretory regulatory role of GLP-1 and ANP,both of which have a protective effect on cardiovascular disease,remains unclear.In this experiment,the signaling pathway PI3K/Akt/m TOR activated by G protein-coupled receptors was selected to explore the mechanism of GLP-1 analog Liraglutide regulating the secretion of atrial ANP.Based on the effect of Liraglutide on atrial pulsatile pressure,calcium ion regulatory proteins Cathepsin K and Piezo1were selected for further mechanism research.Therefore,in this experiment,plasma samples from clinical patients with acute coronary syndrome,isolated beating rat atrial perfusion models,in vivo mouse models and DPP-4 knockout mouse models were used to observe the effect of Liraglutide on ANP secretion.At the same time,the mechanism of PI3K/Akt/m TOR,Cathepsin K and Piezo1 in the regulation of ANP secretion by Liraglutide was discussed.Methods:（1）Acute coronary syndrome includes acute myocardial infarction and unstable angina pectoris,myocardial infarction is divided into ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction.In this study,the plasma samples of clinical patients were collected,and the contents of GLP-1 and ANP in the plasma samples of the patients were determined.（2）In vitro rat atrial perfusion experiment:The experimental model is an improved isolated beating rat atrial perfusion model.The changes of atrial pulsatile pressure were observed throughout the experiment,and a peak-to-peak value of pulsatile pressure was taken every 10 min for calculation.Atrial perfusate was taken every 30min to measure the content of ANP in the atrium,and radioimmunoassay was used for the determination of ANP content.After the experiment,the atria of each experimental group were harvested for protein content determination.（3）In vivo mouse experiment:The experimental groups included the control group and the liraglutide group.Two weeks later,blood was drawn from the right heart,and the supernatant was collected after centrifugation for ANP content determination,and the heart was harvested for protein content determination by Western blotting.（4）DPP-4 knockout mouse experiment:The experimental groups included the control group and the Liraglutide group.Two weeks later,blood was drawn from the right heart,and the supernatant was collected after centrifugation for ANP content determination,and the heart was harvested for protein content determination by Western blotting.Results:（1）Plasma samples were collected from clinical patients with acute coronary syndrome.The results of GLP-1 content determination showed that the GLP-1concentration in STEMI group,NSTEMI group and UA group was significantly decreased;the determination of plasma ANP content showed that the plasma ANP content in UA group was slightly lower.The plasma ANP content in STEMI group and NSTEMI group increased significantly.（2）In the rat isolated beating atrial perfusion model,different concentrations of liraglutide were used for atrial perfusion experiments,the concentrations were 1.6nmol/L;2.4 nmol/L;3.2 nmol/L;4.8 nmol/L.The results of ANP assay showed that the secretion of atrial ANP did not change significantly when the concentration of Liraglutide was 1.6 nmol/L,while the concentration of liraglutide decreased the secretion of atrial ANP when the concentration of liraglutide was 2.4 nmol/L,3.2nmol/L and 4.8 nmol/L,and the concentration of liraglutide was 3.2 nmol/L./L than when its concentration was 4.8 nmol/L,the effect of reducing the secretion of atrial ANP was more significant.The results of pulsatile pressure showed that liraglutide concentrations of 1.6 nmol/L;2.4 nmol/L;3.2 nmol/L;4.8 nmol/L could significantly reduce isolated rat atrial pulsatile pressure.（3）In the in vivo mouse model and the DPP-4 knockout mouse model,the plasma ANP content of DPP-4-/-mice was significantly decreased after injection of liraglutide.（4）In the rat isolated beating atrial perfusion model,0.3 nmol/L Exendin9-39 was selected to further observe the changes of ANP secretion and beating pressure in isolated rat atria.The ANP assay results showed that the secretion of Exendin9-39increased significantly after 30 minutes of administration,and reached a peak at 90minutes of administration.The results of pulsatile pressure showed that although Exendin9-39 atrial pulsatile pressure showed a downward trend,Exendin9-39inhibited the effect of liraglutide on reducing atrial pulsatile pressure to a certain extent.（5）In the rat isolated beating atrial perfusion model,10μmol/L PI3K inhibitor LY294002 was selected to further observe the changes of ANP secretion and beating pressure in isolated rat atria.The ANP assay results showed that after LY294002pretreatment for 30 minutes,the combined use of LY294002 and liraglutide for 30 to60 minutes could significantly resist the effect of liraglutide on reducing ANP secretion.The results of pulsatile pressure showed that LY294002 could not resist the effect of liraglutide to reduce pulsatile pressure when LY294002 was used in combination with liraglutide after 30min of pretreatment with LY294002.（6）The results of Western blot in vitro showed that liraglutide significantly up-regulated the expression of GLP-1R,and Exendin9-39 could reduce the effect of liraglutide on the expression of GLP-1R.Liraglutide significantly upregulated the expression of PI3K/Akt/m TOR;Exendin9-39 significantly decreased the expression of PI3K/Akt/m TOR.Liraglutide significantly down-regulated the expression of Piezo1;Exendin9-39 slightly resisted the inhibitory effect of liraglutide on Piezo1.Cathepsin K expression was up-regulated after administration of Exendin9-39.（7）The results of Western blot in vivo showed that the expression of GLP-1R was significantly up-regulated after the tail vein injection of liraglutide in WT mice;the expression of GLP-1R in DPP-4^-/-mice was significantly up-regulated;the expression of GLP-1R was further up-regulated in DPP-4^-/-mice injected with liraglutide.The expression of PI3K/Akt/m TOR was up-regulated in WT mice injected with liraglutide by tail vein;the expression of PI3K/Akt/m TOR was up-regulated in DPP-4^-/-mice;PI3K/Akt/m TOR was up-regulated in DPP-4^-/-mice after administration of liraglutide effect is more pronounced.The expression of Piezo1 was down-regulated by tail vein injection of liraglutide in WT mice;Piezo1 protein expression was significantly down-regulated 14 days after injection of liraglutide in DPP-4^-/-mice.The expression of Cathepsin K was down-regulated in DPP-4^-/-mice;the expression of Cathepsin K was down-regulated after liraglutide was injected into the tail vein of WT mice;the expression of Cathepsin K was down-regulated more significantly in DPP-4^-/-mice after administration of liraglutide.Conclusions:Liraglutide reduces atrial ANP secretion by activating GLP-1R and reduces atrial pulsatile pressure.PI3K/Akt/m TOR signaling pathway,Piezo1 and Cathepsin K may be involved in the mechanism by which liraglutide regulates atrial ANP secretion and atrial pulsatile pressure.