The Study On The Effect Of GLP-1 Inhibits Endothelial-mesenchymal Transition-induced Myocardial Fibrosis

Objective:1.Explore the complete mechanism of GLP-1 inhibiting myocardial remodeling and complete the basic mechanism research of GLP-1 reversing myocardial remodelin.2.Provide theoretical basis for intervention of myocardial fibrosis and prevention of chronic heart failure.Methods:1.Cell culture and groupingRat heart microvascular endothelial cells(RHMECs)were cultured in DMEM high glucose medium containing 10% fetal bovine serum(FBS)and placed in a 37 ° C incubator.Change the medium every 2-3 days and take cells in the logarithmic growth phase for testing.Grouping:blank control group,TGF-?1 group,GLP-1 group,inhibitor group A,inhibitor group B.All groups except the control group were given 10ng/ml TGF-?1 intervention for 48 h.GLP-1 group were given 5nmol/ml,10nmol/ml,20nmol/ml GLP-1 for 24 h.Inhibitor group A was given20nmol/ml GLP-1 and LY194002 for 24 h.Inhibitor group B was given 20nmol/ml GLP-1 and GSK690693 for 24 h.2.Test objects and methodsRHMECs skeleton protein,endothelial cell-specific marker VE-cadherin,interstitial cell-specific marker ?-SMA,SM22?,and key signaling pathway proteins PI3 k and Akt were selected as the detection targets.Immunofluorescence was used to detect the changes of RHMECs skeleton protein and cell morphology,and q-PCR was used to detect the expression of VE-cadherin,?-SMA,SM22?,PI3 k and Akt.Results:1.GLP-1 inhibits TGF-?1 induced morphological changes of RHMECs.The following results were observed by immunofluorescence: Compared with the control group,the morphology of RHMECs intervened by TGF-?1 changed from the appearance of closely arranged paving stones to scattered spindles.Scaffold protein proliferation and reconstruction(P<0.05).Cells present as interstitial cell-like changes.Compared with the TGF-?1group,GLP-1 intervention can attenuate the above changes,which is related to the drug concentration.The effect of the 20nmol/LGLP-1 group is most obvious.It is suggested that GLP-1 inhibits the morphology and interstitial cell-like changes of RHMECs induced by TGF-?1.2.GLP-1 inhibits TGF-?1-induced changes in the expression of RHMECs-specific markers.The q-PCR test results are shown below: Compared with the control group,TGF-?1 induced a decrease in the expression level of RHMECs endothelial-specific markers(VE-cadherin)(P<0.05),while the expression of interstitial-specific markers(?-SMA,SM22?)was up-regulated(P<0.05).GLP-1 can attenuate the above changes(P<0.05),which is related to the drug concentration.The effect of the 20nmol/LGLP-1 group is most obvious.It suggests that GLP-1inhibits the expression changes of RHMECs specific markers induced by TGF-?1.3.GLP-1 inhibits the activation of PI3 k and Akt in RHMECs induced by TGF-?1.The results of q-PCR detection are as follows: Compared with the control group,TGF-?1can induce the increase of PI3 k and Akt expression(P<0.05).GLP-1 can attenuate the above changes(P<0.05),and it is drug concentration-dependent.The intervention effect of the 20 nmol /LGLP-1 group is the most obvious.It is suggested that GLP-1 inhibits PI3 k and Akt activation induced by TGF-?1.4.GLP-1 inhibits TGF-?1 induced interstitial transdifferentiation of RHMECs via PI3k/Akt pathway.The above experimental results suggest that GLP-1 has an inhibitory effect on TGF-?1-induced interstitial transdifferentiation of RHMECs,and this effect is also applicable to PI3 k and Akt.To clarify the specific mechanism,we added two specific inhibitors(LY194002,GSK690693)to intervene and observed the q-PCR detection results of three specific markers(VE-cadherin,?-SMA,SM22?).The results showed that the expression levels of ?-SMA and SM22? were higher than those of GLP-1 group after adding inhibitors,but still lower than those of TGF-?1 group(P<0.05).The expression level of VE-cadherin was lower than that of GLP-1group,but still higher than that of TGF-?1 group(P<0.05).It is suggested that GLP-1 inhibits TGF-?1 induced interstitial transdifferentiation of RHMECs through the PI3 k / Akt pathway.Conclusion:GLP-1 inhibits TGF-?1-induced interstitial transdifferentiation of RHMECs through the PI3 k / Akt pathway,thereby inhibiting cardiac fibrosis.