Angiotensin (1-7) Improves Diabetes Mellitus-Induced Erectile Dysfunction In Rats By Downregulating Inducible Nitric Oxide Synthase Levels

Diabetic Mellitus-induced Erectile Dysfunction（DMED）is the most common chronic complication of hyperglycemia.75 percent of male diabetic patients have Erectile Dysfunction with varying degrees,and the erectile function declines as the disease progresses.The influences on the patients and their familys are laent and complicated.Few doctors would pay close attention to DMED in clinical practice.Current medical techniques and drug therapies produce insufficient potency to DMED.This study uses the plasma of DMED patients,type 2 diabetes mellitus（DM）rat model and cultured rat penile cavernous smooth muscle cells（CCSMCs）as the study objects.We aims to explore the effect of angiotensin1-7（Ang[1-7]）on DMED and the role of inducible nitric oxide synthase（iNOS）in the pathogenesis of DMED,so as to provide theoretical basis for clinical treatment of DMED.Part One Correlation between serum Ang（1-7）level and diabetic erect-ile dysfunctionObjective:To explore the serum Ang（1-7）and NO levels and their clinical significance in patients with diabetic erectile dysfunction.Methods:Male hospital inpatients of the Second Hospital of Hebei Medical University were selected as experimental subjects.Serum Ang（1-7）,SOD,MDA and NO content in 55 diabetic patients without erectile dysfunction,56 DMED patients and 60 healthy men with normal erectile function were compared.The correlation between Ang（1-7）and erectile function was compared.Results:1.The age of patients in DMED group was significantly higher than that in control group,with longer course of diabetes and more patients complicated with cardiovascular diseases（P<0.05）.The levels of Hb A1c,TG,CHOL,LDL,uric acid and homocysteine in DMED group were significantly higher than those in control group（P<0.05）.2.The serum Ang（1-7）concentration in DM group was significantly lower than that in control group,while the contents of NO and MDA were increased and SOD content was decreased（P<0.05）.The serum Ang（1-7）concentration and SOD content in DMED group were further lower than those in control group,while the serum NO and MDA content were higher（P<0.05）.3.Serum Ang（1-7）level was positively correlated with erectile function.Summary:1.DMED patients have poor control of biochemical indicators,increased cardiovascular risk factors and lower serum Ang（1-7）levels.2.Erectile dysfunction in men with type 2 diabetes may be associated with decreased serum Ang（1-7）levels.Part Two Ang（1-7）regulates nitric oxide synthase levels in the corpus cavernosum and improves erectile function of the DMED ratsObjective:This study aimed to observe the effects of Ang（1-7）on NOS levels and the improvement of Ang（1-7）on erectile function in DMED rats.Methods:Male Sprague-Dawley rats（6-9 weeks old）were used to establish a type 2 diabetes mellitus（DM）model by the intraperitoneal injection of streptozotocin（25 mg/kg）.Eight weeks after diabetes induction,erectile function was assessed in the DM group using the apomorphine（APO）test.The animals with ED were randomly divided into two groups according to treatment:treated group,intraperitoneally injected with Ang（1-7）（576μg/kg/day）for 4 weeks and a vehicle group,intraperitoneally injected with saline.Erectile function was assessed by measuring intracavernous pressure（ICP）and mean arterial pressure（MAP）after electrical stimulation.The expression of inducible nitric oxide synthase（iNOS）,endothelial nitric oxide synthase（e NOS）,e NOS phosphorylated at Ser 1177（p-e NOS[Ser 1177]）,Akt and the level of phosphorylated Akt（p-Akt）in corpus cavernosum was measured by Western blotting and immunofluorescence.The plasma levels of NO,SOD,malondialdehyde（MDA）,and ONOO-were determined.Results:1.Ang（1-7）regulated NOS level and increased p-Akt/Akt expression in the corpus cavernosum1)iNOS expression was considerably higher in DMED rats than in the control group,and Ang（1-7）treatment reduced this effect.p-e NOS expression was markedly lower in the DMED group than in the control group（P<0.05）,and Ang（1-7）attenuated this effect（P<0.05）,demonstrating that this hexapeptide restored local NO levels in DMED rats by decreasing iNOS while increasing p-e NOS expression（P<0.05）.Akt expression remained unchanged in all of the groups.The level of phosphorylated Akt was significantly lower in the penile tissues of DMED rats than in the control and DM groups,and Ang（1-7）abrogated this effect（P<0.05）.2)IF showed that the expression of iNOS increased and the p-e NOS expression decreased in DMED group compared with control and DM rats.Ang（1-7）treatment reversed this effect（P<0.05）.2.Ang（1-7）suppresses oxidative stress in DMED ratsThe serum level of NO was significantly lower and ONOO-level was significantly higher in DMED rats vs.the control,and Ang（1-7）treatment attenuated this effect（P<0.05）.Compared with the control,the serum level of SOD was significantly lower and MDA was consider ably higher in the DM and DMED groups,and Ang（1-7）treatment partially reversed this effect in the latter group（P<0.05）.3.Ang（1-7）improved erectile function in DMED ratsCompared with the controls,DM rats could maintain a normal erection with a slightly lower ICP/MAP ratiop.Compared with the control and DM groups,the DMED group showed a slower response to electrical stimulation,and a shorter duration of erection（P<0.05）.The ICP/MAP ratio was significantly lower in the DMED group than in the DM and control group.Ang-（1-7）treatment markedly restored the ICP/MAP ratio and improved the erectile function（P<0.05）.Summary:Ang（1-7）treatment regulates NOS levels,alleviates oxidative damage,restores erectile fuction,and reversed the decrease of phosphorylated Akt in DMED rats.Part Three Ang（1-7）regulates NOS expression in high-glucose cultured CCSMCs through the Mas/Akt pathway.Objective:To observe Ang（1-7）regulates NO synthesis through the Akt/NOS pathway in penile cavernous smooth muscle cells cultured with high glucose,and explore its possible mechanism of inhibiting oxidative stress injury.Methods:A primary culture of corpus cavernosum smooth muscle cells（CCSMCs）was obtained from i Cell Bioscience Inc.Cells from passages 2 to 4were used.1.Rat penile cavernous smooth muscle cells were cultured with high glucose.Ang（1-7）was used to intervene CCSMCs in high glucose culture.The concentration gradient of Ang（1-7）was set.The expression of iNOS,p-e NOS/e NOS was detected by Western Blot,and the content of NO,SOD,MDA and ONOO-in the supernatant was detected by ELISA.2.The effect of iNOS knockdown and iNOS overexpression on CCSMCs.iNOS si RNA inhibited the protein expression of iNOS.The CCSMCs were divided into blank group,high glucose group,high glucose+Ang（1-7）group,high glucose+iNOS si RNA group,and high glucose+NC group（NC is the transfection reference）.CCSMCs overexpressing iNOS were transfected with lentivirus and divided into blank group,iNOS overexpression group,high glucose group,high glucose+iNOS overexpression group,and high glucose+NC group.Cells were collected in order to detect the content of calcium ion using flow cytometry..3.Pathway inhibitors were used to intervene the cultured cells.Cells were divided into blank group,high glucose group,high glucose+Ang（1-7）group,high glucose+Ang（1-7）+A779 group,high glucose+Ang（1-7）+LY294002 group,and high glucose+Vehicle（DMSO）group.The detection purpose and method were the same as above.At the same time,the expression of P-Akt/Akt protein was detected,and the intracellular calcium ion content was detected by flow cytometry.Results:1.High glucose stimulated the expression of iNOS,down-regulated the level of p-e NOS/e NOS,and increased oxidative stress injury in a time-dependent manner,with a significant change at 48 hours（P<0.05）.Ang（1-7）antagonized the effect of high glucose culture on CCSMCs,down-regulated the expression of iNOS,up-regulated the level of p-e NOS/e NOS,and reduced oxidative stress injury at the degree of 10^-6mol/L in a concentration-dependent manner（P<0.05）.2.iNOS si RNA transfection significantly down-regulated the expression of iNOS compared with the high glucose group（P<0.05）,but had NO significant effect on p-e NOS/e NOS and NO synthesis（P>0.05）;SOD synthesis increased,ONOO-,MDA content decreased significantly（P<0.05）.iNOS overexpression significantly increased iNOS expression compared with blank group,（P<0.05）,the level of p-e NOS/e NOS was slightly decreased,and NO content was not significantly changed（P>0.05）,SOD synthesis decreased,ONOO-and MDA contents increased significantly（P<0.05）.Ang（1-7）antagonized cell damage caused by iNOS overexpression,increased NO synthesis and reduced oxidative stress（P<0.05）.High glucose and overexpression of iNOS significantly increased intracellular calcium ion content,while high glucose and lentivirus overexpression of iNOS further increased intracellular calcium ion content.Ang（1-7）and iNOS si RNA could down-regulate the calcium content in CCSMCs cultured with high glucose（P<0.05）.3.Mas receptor antagonist A779（1 mol/L）and Akt antagonist LY294002（100 pmol/L）eliminated the protective effect of Ang（1-7）.High glucose,A779 and LY294002 significantly increased intracellular calcium content（P<0.05）.Summary:iNOS increased oxidative stress but had a minor impact on NO synthesis.Ang（1-7）reverses the inhibitory effect of HG on the Akt pathway and exerts a protective effect through the MAS/Akt pathway.Conclusions:1.Erectile dysfunction in men with type 2 diabetes may be associated with decreased serum Ang（1-7）levels.2.iNOS increased oxidative stress but had a minor impact on NO synthesis.Ang（1-7）reverses the inhibitory effect of HG on the Akt pathway and exerts a protective effect through the MAS/Akt pathway.