Effect Of NRG-1 On The Function Of Human Cardiac Micro Vascular Endothelial Cells And Its Mechanism

Objective:To investigate the effects of neuregulin-1(NRG-1)on the function of human cardiac microvascular endothelial cells and its effect on the activity of HCMECs under hypoxic conditions,and to explore its mechanism.Methods:(1)The expression levels of NRG-1 and ErbBs in HCMECs were detected by qPCR.(2)Transfection of HCMECs with siRNA lentivirus carrying shRNA interference sequences to inhibit the expression of NRG-1 in HCMECs.HCMECs were divided into control group,NRG-1 siRNA group(siRNA group),negative control virus group(con-siRNA group)and exogenously added rhNRG-1 group(rhNRG-1 group)for transfection.After successful transfection,qPCR and Western blot was used to detect the expression of NRG-1 in each group of HCMECs.CCK8 kits,scratch test,Transwell cell migration assay and Matrigel Matrigel tubular formation assay were used to detect the proliferation,migration and microvascular tubular formation of HCMECs.The cck8 kit was used to detect the cell viability of each group of HCMECs in hypoxia and low serum(1%FBS)environment.(3)HCMECs were divided into control group,rhNRG-1 group and inhibitor group(rhNRG-1+GW572016 group,rhNRG-1+LY294002 group and rhNRG-1+L-NEMA group).HCMECs were incubated with rhNRG-1 in the rhNRG-1 group.In each inhibitor group,HCMECs were incubated with rhNRG-1 with a pre-incubation of the inhibitor for 30 minutes.The proliferation of HCMECs in each group was detected by CCK8 kits.The expression of VEGF in HCMECs was detected by qPCR.The expression levels of T-eNOS,p-eNOS,T-Akt,p-Akt and VEGF in HCMECs were detected by Western Blot.(4)HCMECs were divided into normal group,control group,rhNRG-1 group and inhibitor group(rhNRG-1+GW572016 group,rhNRG-1+LY294002 group and rhNRG-1+L-NEMA group).HCMECs in the control group,rhNRG-1 group and each inhibitor group were cultured under hypoxia and low serum(1%FBS)conditions.In the rhNRG-1 group,HCMECs were incubated with rhNRG-1.In each inhibitor group,HCMECs were incubated with GW572016(1uM)(ErbB inhibitor),LY294002(1uM)(PI3K inhibitor)and L-NAME(1mM)(eNOS inhibitor)for 30 minutes,and then co-incubated with rhNRG-1.The cell viability of each group of HCMECs was detected by cck8 kits,and the expression levels of T-eNOS,p-eNOS,T-Akt,p-Akt and VEGF of HCMECs were detected by Western Blot.Results:(1)HCMECs highly expressed NRG-1,ErbB2,ErbB3,and low expression of ErbB4.(2)shRNA lentiviral transfection significantly inhibited the expression of NRG-1 in HCMECs(P<0.001).(3)Compared with the control group,the proliferation and migration ability of HCMECs in rhNRG-1 group was significantly enhanced(P<0.001),and the proliferation and migration ability of HCMECs in siRNA group were significantly inhibited(P<0.001).Compared with the control group,the number of vascular meshes formed by HCMECs in the siRNA group was significantly decreased(P<0.001),and there was no significant difference in the number of vascular meshes formed by HCMECs in the rhNRG-1 group(P>0.05).(4)HCMECs were incubated with inhibitor and rhNRG-1.Compared with rhNRG-1 group,the results of CCK8 kits showed that the proliferative effect of NRG-1 on HCMECs was significantly weakened(P<0.01).qPCR results showed that in each inhibitor group,inhibitors attenuated NRG-1-induced VEGF expression in HCMECs(P<0.05);Western Blot results showed that in each inhibitor group,inhibitors could attenuate the effect of NRG-1 on the expression of p-Akt,p-eNOS and VEGF in HCMECs(P<0.05).(5)In the hypoxia and low serum(1%FBS)environment,compared with the control group,HCMECs activity was significantly increased in the rhNRG-1 group(P<0.05),while HCMECs activity was significantly inhibited in the siRNA group(P<0.001).The protective effect of rhNRG-1 on HCMECs was weakened after the addition of corresponding inhibitors.Compared with the control group,the expression levels of p-Akt,p-eNOS and VEGF in the rhNRG-1 group were increased in the context of ischemia and low serum(1%FBS),while the expression levels of p-Akt,p-eNOS and VEGF in HCMECs were down-regulated after co-incubation with inhibitors.However,compared with the normal group,HCMECs in the control group showed decreased p-Akt expression and up-regulated p-eNOS expression in the ischemic and low-serum(1%FBS)environment,and no significant changes in VEGF.Conclusion:(1)NRG-1 can significantly promote the proliferation and migration of HCMECs.(2)NRG-1 may promote the proliferation of HCMECs through the ErbB-PI3K/Akt-eNOS-VEGF signaling pathway.(3)NRG-1 alleviates HCMECs injury in hypoxia and low serum environment,which is related to NRG-1 up-regulating the expression of p-Akt,p-eNOS and VEGF in cells.