The Study Of Dimethyl Fumarate Ameliorates Erectile Dysfunction In Cavernous Nerve Injury Rat

Objective: To investigate the therapeutic effect of dimethyl fumarate（DMF）on improving erectile function of cavernous nerve（CN）injury rats.Methods: Bilateral cavernous nerve injury（BCNI）rat model was established by clamping bilateral CN.DMF was given by gavage（Low-dose group: 20 mg/kg/d;High-dose group:40 mg/kg/d;Duration: 4 weeks）.After the intervention,intracavernous pressure was detected by electrical stimulation to CN.Penis and CN tissues were collected,and molecular biological methods such as Masson staining,immunohistochemistry,Western blot,and tissue immunofluorescence were used to detect the content of molecules and proteins in the penis and CN tissues.Transmission electron microscope was used to observe the morphologic changes of CN.The cell viability was detected by CCK-8.Nfe2l2 and Ho-1gene knockdown PC-12 cell lines（Commonly used nerve cell lines）were constructed by lentiviral transfection,respectively.H₂O₂ treated PC-12 cells to induce oxidative stress cell model.PC-12 cells without any treatment were taken as Control group;PC-12 cells treated with H₂O₂ were taken as H₂O₂ group;DMF treatment based on H₂O₂ group was taken as DMF group;DMF treatment based on H₂O₂ induced Nfe2l2 gene knockdown and Ho-1gene knockdown PC-12 cells was taken as sh-Nrf2 group and sh-HO-1 group,respectively.And then molecular biological methods such as cell immunofluorescence and Western blot were used to detect the molecules and proteins content in cells.Results: Erectile function was severely impaired and fibrosis occurred in penis corpus cavernosum of BCNI rats.The nerve fiber numbers of CN were decreased.The content of nNOS,NO and cGMP was decreased,and the content of Ca²⁺ was increased in penis corpus cavernosum.The level of oxidative stress and the activation of NLRP3 inflammasomes were increased in CN.After the intervention of DMF,the erectile function of BCNI rats was improved and the degree of penis corpus cavernosum fibrosis was reduced.The morphology of CN was improved and the nerve regeneration function was enhanced.New axons appeared and the numbers of nerve fibers were increased in CN.The content of nNOS,NO,cGMP and Ca²⁺ was reversed in penis corpus cavernosum.The results also showed oxidative stress level and NLRP3 inflammasomes activation were reduced,and the content of Nrf2 and HO-1 proteins was increased in CN.In vitro,DMF could activate Nrf2/HO-1pathway,increase cell viability,decrease oxidative stress level,and inhibit NLRP3inflammasomes-mediated pyroptosis in H₂O₂ induced PC-12 cells.Nfe2l2 knockdown and Ho-1 knockdown could significantly weaken the protective effect of DMF in H₂O₂-induced PC-12 cells,respectively.After the production of ROS was reduced by ROS inhibitor,Nacetylcysteine,in H₂O₂ induced PC-12 cells,NLRP3 inflammasomes activation was also reduced.Conclusions: DMF improved the erectile function of BCNI rats by promoting nerve repair and regeneration through inhibiting oxidative stress level and NLRP3 inflammasomemediated pyroptosis in CN via activation of Nrf2/HO-1 pathway.Part Ⅰ: Therapeutic effect of dimethyl fumarate on erectile dysfunction in cavernous nerve injury ratsObjective: To explore the therapeutic effect of DMF on erectile dysfunction in cavernous nerve injury rats.Methods: BCNI rat model was established by clamping bilateral CN.The rats were divided into 4 groups: sham group;BCNI group;low-dose DMF treatment group（20 mg/kg/d）and high-dose DMF treatment group（40 mg/kg/d）.After 4 weeks of gavage,intracavernous pressure was detected by electrical stimulation to CN.The penis and CN tissues were collected,then fibrosis of penis corpus cavernosum,the structure and numbers of nerve fibers,and the content of nNOS,NO,cGMP and Ca²⁺ of penis corpus cavernosum were detected by Masson staining,immunohistochemistry,tissue immunofluorescence,transmission electron microscopy,Western blot and kits.Results: After BCNI rat model was constructed,the treatment of DMF could improve erectile function,reduce the degree of penis corpus cavernosum fibrosis,increase the content of nNOS,NO and cGMP,and reduce the content of Ca²⁺ in penis corpus cavernosum.Compared to Sham group,the nerve fibers were reduced in the CN of BCNI rats.The normal nerve structure was destroyed and the numbers of normal nerve fibers were reduced observed by transmission electron microscopy in CN.After the treatment of DMF,the nerve regeneration function was enhanced and new axons appeared,and the numbers of nerve fibers were increased.Conclusions: DMF improved erectile function in BCNI rats by reducing CN injury,promoting nerve regeneration and repair,improving nerve function recovery,and reducing fibrosis of penis corpus cavernosum.Part Ⅱ: Dimethyl fumarate alleviates oxidative stress level of nerves in cavernous nerve injury rats via activating Nrf2/HO-1 pathwayObjective: To explore whether the neuroprotective function of DMF was related to the antioxidant effect via activating Nrf2/HO-1 pathway in vivo and in vitro.Methods: DHE staining and immunohistochemistry were used to detect the content of oxidative stress-related proteins and molecules in tissues,and the content of Nrf2 and HO-1 was detected by tissue immunofluorescence.The cell viability was detected by CCK-8.Nfe2l2 and Ho-1 gene knockdown PC-12 cell lines were constructed by lentiviral transfection,respectively.H₂O₂ treated PC-12 cells to induce oxidative stress cell model.PC-12 cells without any treatment were taken as Control group;PC-12 cells treated with H₂O₂ were taken as H₂O₂ group;DMF treatment based on H₂O₂ group was taken as DMF group;DMF treatment based on H₂O₂ induced Nfe2l2 knockdown and Ho-1 knockdown PC-12 cells was taken as sh-Nrf2 group and sh-HO-1 group,respectively.Nrf2 and HO-1 were detected by Western blot and cellular immunofluorescence.Intracellular oxidative stress level was detected by H2DCFH-DA staining,cellular immunofluorescence,Western blot,SOD and MDA kits.Results: DMF reduced the content of ROS,gp91^phox and 3-NT in the CN of BCNI rats. DMF increased the content of Nrf2 and HO-1 in CN.In vitro,the results showed that DMF could increase the content of total Nrf2 and transfer Nrf2 to nucleus,then it could also increase the content of HO-1.The treatment of DMF increased cell viability and reduced the content of ROS,gp91^phox,3-NT,MDA and DNA damage markers in H₂O₂ induced PC-12 cells.DMF also increased the content of SOD.The protein levels of Nrf2 and HO-1 were both reduced in sh-Nrf2 group.The content of HO-1 was reduced and there was no effect on the content of Nrf2 in sh-HO-1 group.The results showed that both sh-Nrf2 and sh-HO-1 groups could reverse the protective effect of DMF on H₂O₂ induced PC-12 cells.Conclusions: DMF reduced nerve injury by inhibiting the level of oxidative stress in CN via activating Nrf2/HO-1 pathway.Part ⅡI: Dimethyl fumarate alleviates NLRP3 inflammasome-mediated pyroptosis of nerves in cavernous nerve injury rats via activating Nrf2/HO-1 pathwayObjective: To explore the role of NLRP3 inflammasome-mediated pyroptosis in neurological dysfunction of cavernous nerve injury rats and the effect and mechanism of DMF on NLRP3 inflammasome-mediated pyroptosis in vivo and in vitro.Methods: The content of GSDMD was detected by tissue immunofluorescence,and the content of NLRP3,Caspase-1 and IL-1β was detected by immunohistochemistry.The morphology of cells was detected by transmission electron microscopy.The LDH assay kit was used to detect LDH content in cell culture supernatant.PI/Hoechst33342 fluorescent staining was used to detect the level of pyroptosis in cells.Western blot was used to detect intracellular GSDMD-T,GSDMD-N,NLRP3,pro-Caspase-1,cle-Caspase-1,IL-1β,IL-18 content.The ELISA method was used to detect the content of IL-1β and IL-18 in cell culture supernatant.H2DCFH-DA staining was used to detect intracellular ROS content.Results: DMF could reduce the content of GSDMD,NLRP3,Caspase-1 and IL-1β in the CN of BCNI rats.In vitro,DMF could improve morphological changes of cells,decrease LDH release,inhibit the increase of ratio of pyroptosis cells,and decrease intracellular content of GSDMD-T,GSDMD-N,NLRP3,pro-Caspase-1,cle-Caspase-1,IL-1β and IL-18 caused by H₂O₂ treatment in PC-12 cells.After Nfe2l2 knockdown and Ho-1 knockdown,the protective effect of DMF on cells was significantly weakened.After the production of ROS was reduced by ROS inhibitor,N-acetylcysteine,in H₂O₂ induced PC-12 cells,the content of intracellular GSDMD-T,GSDMD-N,NLRP3,pro-Caspase-1,and cle-Caspase-1 was also reduced.Conclusions: NLRP3 inflammasome-mediated pyroptosis was involved in the pathological process of neurological dysfunction in BCNI rats.DMF could inhibit NLRP3 inflammasome-mediated pyroptosis in CN via activating Nrf2/HO-1 pathway,and this effect might be achieved through antioxidant effect of DMF.