Crocin Improve Erectile Function By Reducing Oxidative Stress And Apoptosis In Diabetic Rats

Objective:The pathogenesis of diabetes erectile dysfunction(DMED)is complex.Current studies have confirmed that high glucose environment can cause the increase of reactive oxygen species(ROS)in the body,and the oxidative stress in the cavernous penis is aggravated,leading to the impairment of vascular endothelial function of the cavernous penis and the structure/dysfunction of cavernous smooth muscle.Saffron has the effect of improving oxidative stress in the body,and there is no research on its specific role and mechanism in diabetes induced erectile dysfunction.This study aims to explore the improvement effect and potential molecular mechanisms of saffron on erectile function in DMED rats at the animal and cellular levels,providing a theoretical basis for clinical diagnosis and treatment of DMED.Methods:Animal experiments:① Fifty healthy male SD rats were given intraperitoneal injection of 60 mg/kg streptozocin(STZ)in 40 randomly selected rats after 12 hours of fasting,while the remaining rats were injected intraperitoneally with equivalent amounts of normal saline.After 72 hours,the random blood glucose of rats was detected.A total of 37 rats had a continuous blood glucose concentration of more than 16.7 mmol/L.These rats were considered as diabetes rats.After 8 weeks,DM rats were given a subcutaneous injection of 100μ Apomorphine(APO),g/kg,was administered to each rat.After injection,penile erection was continuously observed for 30 minutes in each rat.If no erection occurred,DMED rats were selected.A total of 30 DMED rats were selected.② The rats were divided into the following four groups:control group(Sham),DMED group(DM),DMED+low dose crocin group(DM+L),and DMED+high dose crocin group(DM+H).Rats in the DM+L group were given 25mg/kg of saffron daily by gavage,while rats in the DM+H group were given 50mg/kg of saffron daily by gavage.Rats in the Sham and DM groups were given the same amount of physiological saline daily by gavage for 8 weeks.③After 8 weeks of continuous treatment,the erectile function was evaluated by electrical stimulation of the cavernous nerve to detect the penile cavernous pressure(ICP)and carotid artery pressure(MAP)in rats.Subsequently,the rats were killed,and the cavernous tissue of the penis was collected for Masson trichrome staining to assess the degree of penile fibrosis and immunofluorescence observation α-The expression levels of SMA,eNOS,and vWF were detected by TUNEL,the levels of SOD and MDA were detected by the kit to assess the oxidative stress status,and the expression levels of Nrf2,HO-1,Bcl-2,Bax,and Cleaved-caspase-3 proteins in the cavernous tissue of the penis were measured by Western Blot.Cell experiment:① Corpus cavernosum smooth muscle cells(CCSMCs)of SD rats were isolated and cultured by collagenase digestion method,and the smooth muscle cells were identified by immunofluorescence method.②CCSMCs was divided into three groups when cells were subcultured to P4 generation:control group(Sham),hydrogen peroxide group(H2O2),and crocin group(CRO).Sham group:cultured in normal culture medium;H2O2 group:H2O2 concentration in the culture medium is 200 μMol/L,cultured for 12h;CRO group:100 μ After pretreatment with mol/L crocin for 24 hours,H2O2 solution was added for treatment for 12 hours.③Cell viability was measured by CCK-8,apoptosis and ROS levels of CCSMCs were detected by flow cytometry,and the expression levels of Nrf2,HO-1,Bcl-2,Bax,and Cleaved-caspase-3 proteins were measured by Western blot.Results:① In animal experiments,compared with the control group,the ICP/MAP ratio of rats in the DMED group decreased significantly,indicating that erectile function was impaired;Masson trichrome staining showed a decrease in the ratio of smooth muscle to collagen and an aggravation of penile fibrosis;TUNEL staining showed an increase in apoptosis in the cavernous tissue;Immunofluorescence observation of penile cavernous tissue The expression of α-SMA,eNOS,and vWF decreased;The decrease of SOD level and the increase of MDA level indicate the aggravation of oxidative stress;Western Blot observed a decrease in the expression of Nrf2,HO-1,and Bcl-2 in the cavernous tissue of the penis of DM rats,and an increase in the expression of Bax,and Cleaved-caspase-3.After 8 weeks of crocin treatment,the above indicators were improved to varying degrees,and the improvement was more significant in the high-dose crocin group.②In the cell experiment,compared with the control group,CCK-8 detection after H2O2 treatment showed a significant decrease in the activity of CCSMCs;Flow cytometry showed that H2O2 treatment could increase ROS level and apoptosis of cells;Western blot showed that the expression of Nrf-2,HO-1,and Bcl-2 proteins decreased,while the expression of Bax,Cleaved-caspase-3 proteins increased;Saffron and treatment can improve the above changes.Conclusion:① Animal experiments have shown that saffron can improve erectile function by reducing oxidative stress and reducing apoptosis in penile cavernous tissue of DMED rats.②Cell experiments have shown that saffron pretreatment can improve the imaging of hydrogen peroxide stimulation on CCSMCs cell viability and reduce ROS production and apoptosis.③ The above studies indicate that crocin can exert antioxidant stress and anti apoptosis effects in vivo and in vitro,which may be achieved by upregulating the Nrf2/HO-1 signaling pathway.