| Objective:Type 2 diabetic erectile dysfunction(DMED)has gradually become a common type of sexual dysfunction in clinical diagnosis and treatment,showing a trend of younger onset.PDE5 inhibitors,as a first-line drug for clinical treatment of sexual dysfunction,have little effect on some patients with DMED.With the mutual development and penetration of interdisciplinary,new therapeutic approaches have been found.Drug therapy with high site specificity and low toxicity and side effects has become a new treatment method,is feasible in theory and technology,and has gradually entered into clinical trial.Targeted antioxidant SS-31 is an antioxidant that acts on target organ of mitochondria and has a strong inhibitory effect on inflammation and apoptosis.SS-31 has been used in clinical treatments such as heart disease,cerebrovascular and inflammation symptoms,which may also work on the sexual dysfunction.Currently,the effect of SS-31 on the biological function of erectile dysfunction in diabetic patients and its underling mechanism are still lacking.The present research has the following three purposes:1.Establish diabetic erectile dysfunction SD rat model,which has pathophysiological characteristics of human type 2 diabetes mellitus patients.This model will facilitate the study of the mechanism and intervention of erectile dysfunction.2.Establish a rat model of type 2 diabetic erectile dysfunction with poor therapeutic effect using valdenafil hydrochloride tablets.This study is aimed at preparing for the therapeutic effect and mechanism of targeted antioxidant SS-31 on DMED rat model.3.Explore whether targeted antioxidant SS-31 can improve erectile dysfunction in type2 diabetic SD rats and subsequently,research its related underling mechanism.In addition,it will be determined whether SS-31 has potential application value in treating type 2 diabetic erectile dysfunction.Method:The research method is divided into two parts.Part I: Establishment of SD rat model of diabetic erectile dysfunctionOne hundred eight-week-old male SD rats with SPF grade were randomly divided into two groups: 20 normal control rats were fed with normal diet and 80 diabetic rats were fed with high-sugar and high-fat diet for 8 weeks,respectively.The rat weight was recorded at the treatment diet of 0,4,8 and 10 weeks,respectively.12 hours after fasting in the next day of the eighth week,the rats were given streptozotocin(65mg/kg/d)for two weeks by intraperitoneal injection of citric acid.At the tenth week,the rats were given intragastric glucose tolerance test and synchronous insulin release test.Blood glucose and insulin were also determined,and then glucose tolerance test and simultaneous insulin release test wererepeated after stabilization.The serum insulin levels in SD rats were measured by Elisa method,and the levels of free fatty acids,high density lipoprotein cholesterol,low density lipoprotein cholesterol,triglyceride and HOMA-IR were measured by spectrophotometer.After 10 weeks,apomorphine(APO)screening test was performed on the surviving diabetic SD rats.APO(+)and APO(-)were used as markers indicating good and bad erectile functions,respectively.Afterwards,the therapeutic effects of vardenafil hydrochloride tablets on APO(+)and APO(-)diabetic rats were evaluated,and the statistical difference was also analyzed.Part II:The therapeutic effect and mechanism of targeted antioxidant SS-31 on DMED rat model.In the first part of the experiment,APO screened out 55 DMED rat,from which,32 rats were randomly selected and divided into 4 groups,8 rats in each group.They were SS-31low-dose group,SS-31 medium-dose group,SS-31 high-dose group and DMED-free group.The injection dose of low,medium and high SS-31 groups was 50 mg/kg/d,100 mg/kg/d,150mg/kg/d,respectively.Eight normal SD rats were injected with the same amount of normal saline in the control group.After 4 weeks treatment,the rats were sacrificed under pentobarbital anesthesia.Blood glucose,insulin,nitric oxide and malondialdehyde were measured by vena cava.The rat penile cavernous tissue was observed by HE staining to see the cells and tissues morphological changes.The expression of eNOS was detected by immunohistochemistry.The mRNA expression of eNOS,PI3 K and AKT genes was detected by real-time fluorescence quantitative PCR,and the contents of PI3 K,AKT and eNOS proteins were measured by Western Blot.Results:Part I: Establishment of SD rat model of diabetic erectile dysfunction results:After 8 weeks of high sugar and high fat feeding,the SD Rats body weight increased to1.32 times of that in the normal control group,with significant difference between the two groups(P < 0.05).There was no significant difference in blood glucose levels between the two groups of SD rats before injection of STZ(P > 0.05).After STZ injection,the blood glucose concentrations of both groups rose rapidly and stabilized at 14.06 ± 3.71mmol/L,which was significantly higher than that before injection 4.85 ± 0.57mmol/L(P < 0.05).The serum insulin in the high sugar and high fat group before injection of STZ was 10.17 ± 1.05mmol/L and decreased to 6.75 ± 1.43 mmol/L after injection,but still higher than the normal control group 4.09 ± 0.67 mmol/L(P < 0.05).The normal group of insulin resistance index was the lowest,which was 1.00 ± 0.05 followed by the diabetic group before the model built of 3.56 ± 0.45,and the Diabetes group of 4.27 + 1.25.And the two groups of insulin resistance index compared to the normal group have significant difference(P < 0.05).Free fatty acids,triglycerides and low-density lipoprotein cholesterol were shown to be significantly higher than those in the normal control group,with the opposite of high-density lipoprotein cholesterol in the diabetic model.After injection of STZ,3 SD rats were died,77 SD rats and 20 normal SD rats were screened for APO in tenth week post menstrual,and the erection rate of the two groups was separated,the erectile dysfunction was not obvious,and the erection rate was 100% in the normal group,22 of the 77 rats in the diabetic group showed an erection,namely APO(+),with an erection rate of 28.57%,significantly lower than the normal diet group(P < 0.01).Subsequently,the APO(+)and APO(-)Diabetic rats in the treatment of the non-tablet,the results showed that APO(+)Diabetic rats on the non-tablets of hydrochloric acid reactivity is good,basically can reach normal level,and APO(-)Diabetic rats on the non-tablets of the reaction of the poor,still below the normal level.Part II: The therapeutic effect and mechanism of targeted antioxidant SS-31 on DMED rat model results:After electric stimulation,the ICP/MPA value of the rat penile cavernous tissue increased as target antioxidant SS-31 treatment dose increased.The ICP/MPA value of the SS-31 medium dose group(0.79 ± 0.11),high dose group(0.83 ± 0.06)was close to that in the normal control group(0.86 ± 0.02)(P > 0.05),but the value was significantly higher than the DMED group(0.21 ± 0.09)(P < 0.05).After the target antioxidant SS-31 treatment,the SOD and GSH value of DMED group were 45.26 ± 3.62,19.78 ± 2.16,respectively,which was 68.36 ± 4.33,26.74 ± 3.79 in SS-31 low dose group,82.52 ± 3.26,33.68 ± 1.89 in SS-31 medium dose group,96.27 ±5.15,37.54 ± 4.21 in SS-31 high dose group.In compare with DMED group,the SOD and GSH values of all SS-31 groups were significantly increased(P < 0.01),and showed a dose dependent trend.The SOD and GSH values of SS-31 high dose group were very close to the normal control group(SOD and GSH value were 90.98±5.7,36.17±2.65,respectively),and the difference was not obvious(P > 0.05).The MDA content of SS-31 low,medium and high dose groups was 10.08 ± 0.86,6.13 ± 0.63,4.21 ± 0.49,respectively,which was decreased in compared with DMED group(MDA value of 11.35 ± 1.13)and showed the opposite tendency of SOD and GSH.However.Through measuring the values of NO and AGEs,the value of the normal control group was 6.35 ± 0.37 and 38.63 ± 2.15,respectively,which was 1.53 ± 0.12 and 61.25 ± 7.02 in the DMED group,2.98 ± 0.21 and 56.72 ± 5.39 in the SS-31 low dose group,4.67 ± 0.28 and 44.33 ± 5.25 in the SS-31 medium dose group,and 4.89 ± 0.54 and 42.11 ± 5.01 in the SS-31 high dose group.In compare with the DMED group,the content of NO in the medium and high dose groups of targeted antioxidant SS-31 significantly increased(P < 0.05),but the content of AGEs significantly decreased(P < 0.05).The HE staining results of the corpus cavernosum of rats in each group showed that there was no obvious corpus cavernosum space in the normal control group,the left and right corpus cavernosum spaces were not obvious,and a large number of irregular blood sinus spaces were visible.There are flat endothelial cells attached to the surface of the blood sinus space,and the blood sinus trabecula contains a large number of smooth muscle cells andcollagen fibers.In DMED group,the number of smooth muscle cells in the corpus cavernosum decreased,the density of collagen fibers increased,and the vascular wall of the interstitium of the penis thickened,showing fibrous changes and irregular lumen.The low dose group of targeted antioxidant SS-31 was similar to that of the DMED group.The number of smooth muscle cells in penile tissues of rats in the SS-31 medium and high dose group increased in different degree.Immunohistochemistry results of rat penile corpus cavernosum showed that the positive expression of eNOS protein was localized in the cytoplasm and nucleus of the intrinsic cells of rat penile corpus cavernosum,appearing as brownish yellow particles.In the control group,abundant expression was found in the cytoplasm and nucleus of glomerular cells.Compared with the control group,eNOS protein expression in the DMED group was less.Compared with the DMED group,as the SS-31 treatment dose increase,the expression of eNOS protein in the nucleus showed increased trend.Real-time fluorescence quantitative PCR results showed that compared with the normal control group,the mRNA expression of PI3 K,AKT and eNOS genes in DMED group was lower.Compared with DMED group,the mRNA expression of the above three genes in different dose groups of targeted antioxidant SS-31 increased and showed a dose dependent increase trend.At the protein level,the western blot experiment results showed a trend consistent with the gene expression level.Conclusion:1.High-sugar and high-fat feeding can induce rat body weight gain,dyslipidemia and insulin resistance in rats.Small dose of STZ injection after 8 weeks diet feeding can partially damage islet cells on this basis,similar to the pathogenesis of type 2 diabetes.At the tenth week,the APO screening test showed that the erection rate was only 28.57%,and the incidence of erectile dysfunction increased to 71.43%.This model can be used as a rat model of type 2 diabetic erectile dysfunction.After treatment with valdenafil hydrochloride tablets,Apo(-)diabetic rats showed poor responsiveness to valdenafil hydrochloride tablets,still lower than normal levels,and finally successfully established a diabetic rat model of erectile dysfunction with poor treatment effect with valdenafil hydrochloride tablets.2.Targeted antioxidant SS-31 may increase the production of SOD,GSH and NO by inhibiting the production of AGEs and MDA,and relax the corpus cavernosum of the penis,thus improving the erectile function of SD rats and a series of oxidative stress responses of diabetic rats.3.By observing the expression of PI3 K,AKT and eNOS genes and proteins in rat corpus cavernosum,we understand PI3K/AKT/eNOS signaling pathway and eNOS.By detecting the expression of SOD,GSH,MDA,NO and AGES,we prove that the PI3K/AKT/eNOS signaling pathway pairs in the induction of oxidative stress by targeted antioxidant SS-31 in the corpus cavernosum cells of DMED rats.It plays a regulatory role.Targeted antioxidant SS-31 has the effect of treating erectile dysfunction in SD rats.Itsmechanism may be through activating PI3K-Akt-eNOS signaling pathway,which can relieve the inhibition of eNOS synthesis and increase the amount of NO,so as to improve erectile function.PI3K-Akt-eNOS antioxidant system is the key endogenous pathway to regulate the transcriptional expression of various antioxidant enzymes,which provides a new way to achieve effective and moderate antioxidant therapy.4.This topic is the first time to apply targeted antioxidant SS-31 to the treatment of penile erectile dysfunction caused by diabetes mellitus.The whole process is still an exploratory study.There are inevitably some omissions and inaccuracies.Because of its relatively shallow mechanism of action,a series of proteins are involved in the signaling pathway.In the next study,RNA interference technology can be used to knock out the related genes in PI3K/AKT/eNOS signaling pathway in rats to verify whether its role is transmitted along PI3K/AKT/eNOS signaling pathway. |
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