The Mechanisms Of Nebivolol Ameliorating The Endothelial Dysfunction In 2 Type Diabetic Rats

Objective:To observe the effects and possible mechanisms of nebivolol on aortic endothelial dysfunction in type 2 diabetic rats at the overall and cellular levels and provide new ideas for the treatment of diabetic macrovascular complications..Methods:Animal experiments:12-week-old male Zucker Diabetic Fatty（ZDF）and Lean Zucker rats（LZR）were divided into:ZDF group（n=7,ig,distilled water）;ZDF+nebivolol group（n=7,ig,10mg/kg/day）;ZDF+atenolol group（n=7,ig,100mg/kg/day）;ZDF+captopril group（n=7,ig,40mg/kg/day）;LZR group（n=7,ig,distilled water）.During the experiment,the weights of rats in each group were measured every week and the drugs were given according to the weights.Blood glucose and oral glucose tolerance test（OGTT）were measured at the begin and end of the study.At the end of study,the systolic blood pressure（SBP）,diastolic blood pressure（DBP）and mean arterial pressure（MAP）were measured.Blood collected from the abdominal aorta was used to measure plasma nitric oxide（NO）and plasma insulin.The thoracic aortas were quickly isolated and divided into different parts:（1）used for the testing of vascular reaction in vitro;（2）embedded in paraffin for HE and Masson staining to observe the structures of rat aortas;（3）fixed in OCT for immunofluorescence testing of vWF,α-SMA and for fluorescence testing of reactive oxygen species（ROS）in frozen sections;（4）preserved at-80℃for preparation of aorta homogenate（detecting NO）and western blot(eNOS,eNOS uncouple,p-eNOS,NOX4,p22^phox,AMPK and p-AMPK,Akt and p-Akt protein expression).Cell experiments:（1）The effect of nebivolol on the endothelial dysfunction in human arterial endothelial cells（HAEC）induced by high glucose（HG）.HAEC were divided into:control group,osmotic pressure group,HG（30mmol/L）,nebivolol（0.01,0.1,1,10μmol/L）groups.The expression of vWF was detected by immunofluorescence.ROS detected by fluorescence probe.NO measured by NO fluorescence probe kit.NOS was detected.The activity of cells was detected by MTT.（2）Nebivolol inhibits HG induced HAEC ED through AMPK-PKC-NOX-ROS pathway.DPI（1μmol/L,NOX inhibitor）,AICAR（1mmol/L,AMPK activator）,Go6983（5μmol/L,PKC inhibitor）,Compound C（10μmol/L,AMPK inhibitor）and PMA（10nmol/L,PKC activator）were used to observe the mechanisms of nebivolol（1μmol/L）improving ED in HAEC induced by HG.The expressions of vWF and the migrations of p47^phox and Rac-1 were measured by immunofluorescence.The expressions of NOX4,p22^phox,p47^phox,Rac-1,PKC,AMPK and p-AMPK were detected by Western blot.（3）Nebivolol inhibits HG induced HAEC ED through AMPK-AKT-NOS pathway.AICAR（1mmol/L,AMPKactivator）,Compound C（10μmol/L,AMPK inhibitor）,L-NAME（100μmol/L,NOS inhibitor）and Uprosertib（10μmol/L,AKT inhibitor）were used to observe the mechanisms of nebivolol（1μmol/L）improving ED in HAEC induced by HG.vWF and NO were detected by immunofluorescence.NOS was measured by test kit.The expressions of Akt,p-Akt,eNOS,p-eNOS and eNOS uncouple were detected by western blot.Results:Animal experiment results:Compared with LZR group,body weight in ZDF rats reduced,accompanied with increased blood glucose,impaired OGTT,decreased plasma insulin,increased blood pressure,decreased plasma and aortic NO.The antihypertensive effects of nebivolol,captopril and atenolol were significant.Nebivolol and captopril increased plasma and aortic NO without influencing blood glucose.Atenolol had no significant effect on NO but increased blood glucose.There was no significant difference in the aortic structure（shown by HE,Masson staining andα-SMA immunofluorescence）between ZDF rats and LZR group,but the expression of vWF decreased in ZDF rats.Compared with LZR group,the contraction to phenylephrine（PE）increased in ZDF rats,the endothelium-dependent relaxation to acetylcholine（ACh）decreased,but the endothelium-independent vasodilation to sodium nitroprusside（SNP）did not changed.Nebivolol and captopril increased vWF expression and the relaxation to ACh,and nebivolol also decreased contraction to PE.Furthermore,it was found that nebivolol could also reduce aortic ROS in ZDF rats.That may be related to the enhancement of AMP activated protein kinase（AMPK）phosphorylation and the decrease of NADPH oxidase（NOX4）and its active unit p22^phoxhox expression in aorta after the treatment of nebivolol.Nebivolol treatment also increased the expressions of eNOS,p-eNOS,Akt and p-Akt and reduced eNOS uncoupling.Captopril also improved arotic ROS and NO in ZDF rats,while atenolol had little effect on the indexes mentioned.Cell test results:（1）Compared with the control group and the osmotic group,the expression of vWF decreased,the level of ROS increased,NO and NOS activity decreased in HG group.Nebivolol（1μmol/L,10μmol/L）can ameliorate the injury induced by HG.Nebivolol had no significant effect on the survival rate of HAEC,but nebivolol（1μmol/L,10μmol/L）could improve the decreased cell survival induced by HG.（2）Compared with the HG,nebivolol（1μmol/L）and DPI（1μmol/L,NOX inhibitor）increased the expression of vWF,decreased ROS level,inhibited the transfer of p47^phox and Rac-1.enhanced the expression of AMPK and p-AMPK,decreased the expression of NOX4,p22^phox,PKC,p47^phox and Rac-1.Furthermore,both nebivolol and AICAR（1mmol/L,AMPK activator）can reduce the protein expressions of NOX4,p22^phox and PKC.Compound C（10μmol/L,AMPK inhibitor）can inhibit the effects of nebivolol.The elevated protein expressions of HAEC NOX4 and p22^phox induced by HG were decreased by nebivolol and Go 6983（5μmol/L,PKC inhibitor）,while PMA（10nmol/L,PKC activator）decreased the effects of nebivolol.（3）Compared with the HG,nebivolol increased vWF,NO and NOS activity,increased the protein expressions of Akt,p-Akt,eNOS and p-eNOSand inhibited the decoupling of eNOS.Uptosertib（10μmol/L,Akt inhibitor）can inhibit the effects of nebivolol.Except for Akt,p-Akt the effects of nebivolol were abolished by L-NAME（100μmol/L,NOS inhibitor）.Furthermore,the expressions of Akt,p-Akt,eNOS,p-eNOS increased and eNOS decoupling was inhibited after the treatment of nebivolol and AICAR（1mmol/L,AMPK activator）.Compound C（10μmol/L,AMPK inhibitor）can inhibit the effects of nebivolol.Conclusion:Nebivolol can improve the aorta ED of type 2 diabetic rats by regulating AMPK-PKC-NOX-ROS and AMPK-Akt-eNOS pathways.