Study On The Mechanism Of Action Of Activating Blood And Resolving Stasis To Improve Diabetes Mellitus Erectile Dysfunction

Objective: Erectile dysfunction(ED)is a complex and common complication of diabetes mellitus for which there is a lack of effective treatment.In this study,we firstly used Mendelian randomization(MR)analysis to clarify the causal relationship between blood stasis and diabetes mellitus erectile dysfunction(DMED)and to provide a clinical basis for the use of the method of activating blood stasis to treat DMED.Secondly,to explore the main active components of Shaofu Zhuyu decoction(SFZYD),and clarify the underlying mechanism.Finally,in vivo and in vitro experiments were used to explore the mechanism of action of isorhamnetin,the main active ingredient in SFZYD,in improving DMED.Methods: Genome wide association study(GWAS)data were selected from internationally available databases and screened for instrumental variables,and two-sample Mendelian randomization analyses were conducted to verify whether there was a significant causal relationship between the exposure to blood stasis and the outcome of DMED and the direction of the relationship,and sensitivity analyses were conducted to verify whether the results were robust.A diabetic rat model was induced with streptozotocin(STZ).After intragastric administration,erectile function was assessed by the maximum intracavernous pressure(ICPmax)/mean arterial pressure(MAP).Corpus cavernosum fibrosis was evaluated by Masson staining,and ELISA methods were used to determine the serum levels of IL-6,TNF-α,IL-10,IL-4 and IL-1β to evaluate inflammation.Then,the main active components of SFZYD were identified by UPLC-MS/MS.Finally,the target and biological mechanism of SFZYD in improving DMED were predicted by combined network pharmacology and transcriptomics,which was also validated by molecular docking and cellular thermal shift assay(CETSA)experiments.Thirty male SD rats were randomly grouped and 24 rats were injected intraperitoneally with streptozotocin to establish a diabetes model.Erectile function of rats was assessed by ICPmax/MAP,and penile cavernous fibrosis was assessed by Masson staining and immunohistochemical detection of α-SMA expression.Serum levels of IL-6,TNF-α,IL-10,IL-4 and IL-1β were measured by ELISA to assess the level of inflammation,and commercial kits were used to determine the activities of superoxide dismutase SOD,MDA,GPx,and CAT as well as NO in the penile tissues of rats in each group.Immunofluorescence was used to detect the expression of CD31,and RT-q PCR and Western blot were used to detect the expression levels of key proteins and m RNAs in the PI3K/AKT/eNOS signaling pathway.TUNEL staining was used to assess apoptosis in vivo.High glucose-induced corpus cavernosum endothelial cells(CCECs)were used to further validate the anti-apoptotic effects of isorhamnetin in vitro and the modulation of the PI3K/AKT/eNOS signaling pathway.Results: Two of the 10 blood stasis-related markers demonstrated a causal relationship with DMED,with MPV being a protective factor for DMED and LDL being a risk factor for the development of DMED.SFZYD significantly improved erectile dysfunction and inhibited inflammatory responses and local tissue fibrosis in diabetic rats.A total of 1846 active components were identified by UPLC-MS/MS,and isorhamnetin was the main active component.The transcriptomic results were used to identify differentially expressed genes among the control,DM and SFZYD groups,and 1264 differentially expressed genes were obtained from the intersection.The network pharmacology results showed that SFZYD acts on core targets such as AKT1,ALB,HSP90AA1 and ESR1 through core components such as isorhamnetin,quercetin and chrysophanic acid.Further combined analysis revealed that multiple targets,such as CYP1B1,DPP4,NOS2 and LCN2,as well as the regulation of the PI3K-AKT signaling pathway,may be important mechanisms by which SFZYD improves DMED.Molecular docking verification showed that isorhamnetin,the key component of SFZYD,has good binding ability with several core targets,and its binding ability with CYP1B1 was the strongest.The CETSA results showed that isorhamnetin binds to CYP1B1 in CCECs.Isorhamnetin improves erectile function in diabetic rats,decreases serum levels of proinflammatory factors IL-6,TNF-α,and IL-1β,increases levels of anti-inflammatory factors IL-10 and IL-4,increases SOD,GPx,and CAT activities as well as NO levels in penile tissues,and decreases MDA levels.Isorhamnetin also increased the content of CD31 in penile tissues of diabetic rats,activated the expression of PI3K/AKT/eNOS signaling pathway,and inhibited apoptosis.Conclusion: There is a certain causal correlation between blood stasis and DMED,so it is feasible to use the method of activating blood and resolving stasis to treat DMED.SFZYD improves DMED,inhibits the inflammatory response and alleviates local tissue fibrosis.The combined application of transcriptomic,network pharmacology,molecular docking and CETSA approaches was helpful for revealing the mechanism by which SFZYD improves DMED,which may be related to the regulation of CYP1B1 and the PI3K-Akt signaling pathway.Isorhamnetin exerts a protective effect on erectile function in diabetic rats by inhibiting the inflammatory response,attenuating the level of oxidative stress and cavernous fibrosis,improving the endothelial function,and inhibiting apoptosis,the mechanism of which may be related to the activation of the PI3K/AKT/eNOS signaling pathway.