| Objective: To construct miRNA-92 a inhibitor and diabetes mellitus-related ED(DMED)rat models,inject miRNA-92 a inhibitor into the penile cavernous of DMED rats,and detect the erectile function of rats to evaluate the effects of miRNA-92 a inhibitor on erectile function in DMED rats.Methods: 8-week-old fasting male SD rats were intraperitoneally injected with streptozotocin(STZ,60mg/kg)to construct diabetic rat models.The blood glucose concentration was measured regularly,and the diabetic rats were further screened by Apomorphine(APO)test.Forty SD rats were randomly divided into 4 groups(10 in each group),normal control group(Control group),diabetes mellitus-related ED group(DMED group),and penile cavernosal injection of miRNA-92 a antagomir group(DMED + miRNA-92 a antagomir group),penile cavernosal injection of miRNA-92 a antagomir control group(DMED + miRNA-92 a antagomir control group).After 2 weeks,the cavernous nerves were electrically stimulated to measure the intracavernous pressure(ICP)and mean arterial pressure(MAP)of rats in each group,and calculate the ICP/MAP ratio.The area under the ICP curve(Area Under Curve,AUC)to assess erectile function in all rats.After the detection,the penile cavernous tissue is separated and divided into 4 parts,one of which is taken and tissue RNA was extracted.The expression level of miRNA-92 a in the penile cavernous tissue of each group of rats is detected,and the remaining tissue blocks are properly stored for subsequent experiments.Results:After 12 weeks of STZ administration,the blood glucose level of the rats remained more than 16.7 mmol/L.Electrical stimulation of cavernous nerves in evaluating the erectile function of rats in each group showed that compared with the Control group,the values of ICP/MAP and AUC in the DMED group reduced significantly,while the DMED + miRNA-92 a antagomir group was significantly higher than the DMED group,close to the level of rats in the Control group.There was no significant change in DMED + miRNA-92 a antagomir control group compared with DMED group.The expression level of miRNA-92 a in the penile cavernous tissue of rats in the DMED group increased significantly,which was 4-5 times as that in the Control group.miRNA-92 a antagomir could down-regulate the expression level of miRNA-92 a,while miRNA-92 a antagomir control has no such effect.Conclusion:The DMED rat models were successfully constructed.miRNA-92 a antagomir can significantly improve the erectile function and reduce the expression level of miRNA-92 a in diabetic ED rats.Objective: To study the mechanisms of miRNA-92 a inhibitor in improving erectile function in diabetic ED rats.Methods: Frozen corpus cavernosum tissue of rat penile was used to obtain the total protein or RNA,and paraffin-embedded sections or frozen sections of rat penile cavernosum tissue were used to perform the experiments of this part.Western Blot,ELISA and other methods were used to detect the expression level or concentrations of key proteins in e NOS/NO/c GMP signaling pathway in corpus cavernosum tissue of rat penile.Immunofluorescence and Western Blot were used to detect n NOS expression level;Western Blot was used to detect VE-Cadherin,Occludin,Claudin-5 expression levels;Immunofluorescence was administrated to detect the CD31 expression level on endothelial cell surface to evaluate the function of endothelial cell;Oxidative stress in penile cavernous tissue was detected,including fluorescent probes detecting reactive oxygen species(ROS)levels,TBA method detecting malondialdehyde(MDA)concentration,superoxide dismutase(SOD)activity,and Western Blot detecting the expression levels of NADPH oxidase subunits;Immunofluorescence and Western Blot were used to detect the expression of HO-1 protein in the corpus cavernosum tissue of rats in each group.Results:Compared with the Control group,the e NOS/NO/c GMP signaling pathway was significantly down-regulated in the corpus cavernosum of rats in the DMED group.The expression of e NOS and its phosphorylated protein p-e NOS(S1177)decreased significantly,and the contents of c GMP and NO reduced significantly.The expression level of n NOS was significantly down-regulated,reflecting the weakened function of cavernous nerve;The expression levels of VE-Cadherin,Occludin,Claudin-5 decreased,and the fluorescence intensity and fluorescence area of CD31 on the surface of endothelial cells were significantly down-regulated,reflecting endothelial dysfunction;ROS levels,NADPH oxidase subunit expression levels increased significantly,malondialdehyde(MDA)concentration increased,superoxide dismutase(SOD)activity decreased significantly,HO-1 protein levels were significantly down-regulated,reflecting that the level of oxidative stress in cavernous tissue of rats in DMED group increased significantly compared with the control group.The above parameters were improved in DMED rats treated with miRNA-92 a antagomir,and some of the parameters were similar to those in the Control group,while the miRNA-92 a antagomir control showed no such improvement.Conclusion : The miRNA-92 a antagomir can activate the e NOS/NO/c GMP signaling pathway in the corpus cavernosum tissue of DMED rats,protect the function of corpus cavernosum nerve,protect the endothelial function,inhibit the levels of overactivated oxidative stress,and thereby improve erectile function in DMED rats.Objective: To study the effects of and mechanisms of miRNA-92a/miRNA-92 a inhibitors on the endothelial cells of rats in vitro,to predict and validate the target genes of miRNA-92 a.Methods: Type I collagenase was used to digest the aorta tissue,and plating with matrigel was used for the primary culture of endothelial cells.Primary aortic endothelial cells were cultured.After the successful culture,3-5 generations of endothelial cells were used for in vitro experiments.The glucose concentration in the medium was 5 m M and 30 m M,which were used as normal glucose medium and high glucose medium.Aortic endothelial cells were cultured in 6-well plates,which were divided into the following groups: a.Normal Glucose group(NG group),b.High Glucose group(HG group),c.HG + miRNA-92 a inhibitor group,d.NG + miRNA-92 a mimic group,e.HG + miRNA-92 a inhibitor + Compound C group.Real-time quantitative PCR(q RT-PCR)was used to detect the expression level of miRNA-92 a and AMPK functional subunits Prkaa1 and Prkaa2 in endothelial cells of each group;Western Blot,immunofluorescence,q RT-PCR and other methods were used to detect the levels of AMPK/e NOS signaling pathway and AMPK/Nrf2/HO-1 signal pathway;Bioinformatics was used to predict and verify the target genes of miRNA-92 a,3’UTR region reporter gene vectors of the target genes were constructed,and dual luciferase reporter gene method was used to verify target genes of miRNA-92 a.Results:Compared with the NG group,the expression of miRNA-92 a in endothelial cells of the HG group was significantly up-regulated,the m RNA levels of the two functional subunits of AMPK—Prkaa1 and Prkaa2 reduced significantly,and the AMPK/e NOS and AMPK/Nrf2/HO-1 signaling pathways were significantly inhibited.After the administration of miRNA-92 a mimic in endothelial cells in NG group,it showed similar biological changes as in HG group,and after the administration miRNA-92 a inhibitor,the above parameters in endothelial cells were improved.However,Compound C,an AMPK inhibitor,can reverse this improvement.Bioinformatics predicted 148 potential target genes of miRNA-92 a.After KEGG pathway enrichment analysis,sequence alignment,and double luciferase reporter gene detection,it is observed that Prkaa2 is one of the target genes of miRNA-92 a,while Prkaa1 is not the target gene of miRNA-92 a.Conclusion : miRNA-92 a was up-regulated in high glucose conditions,inhibiting AMPK/e NOS and AMPK/Nrf2/HO-1 signaling pathways in rat aortic endothelial cells via targeting Prkaa2,causing endothelial dysfunction and overactive oxidative stress,miRNA-92 a inhibitor can improve the above parameters. |
PDF Full Text Request