| ObjectiveDiabetes mellitus is an independent risk factor for ED.Diabetic men are three times more likely to suffer from ED than non-diabetic men.The incidence of diabetic ED reached 32%-90%.The development of diabetic ED is a multifactorial and multistep process involving multiple factors,such as neurotransmitters,blood vessels and vasoactive substances,endocrine and metabolic factors.The apoptosis of smooth muscle cells mediated by oxidative stress is an important pathogenesis of diabetic ED.The smooth muscle,the final organization of various factors,was recognized very important in the process of penile erection.In the condition of diabetes,a large number of reactive oxygen species(ROS)accumulates in the corpus cavernosum tissue,resulting in the apoptosis of the smooth muscle cells in corpus cavernosum and diabetic ED.In view of this,we believe that reducing apoptosis mediated by oxidative stress is an effective way to improve diabetic erectile dysfunction.Although its exact mechanism is not very clear,ADSCs have previously been used for treatment of ED and proved to significantly improve erectile function of experimental animal.Now it is commonly thought the biological functions of ADSCs on restoring erectile function of diabetes-related ED rats not mainly exert through directly differentiating into endothelial cell,smooth muscle cell or neuron-like cell to replace damaged ones but via secreting abundant bioactive substances,so-called paracrine effects,to repair the damaged penile tissues.Recently,increasing attention has been paid to the EXOs,produced by almost all cell types including MSCs,which are defined as phospholipid bilayer vesicles with a diameter of 40-100 nm.EXOs contain numerous proteins and lipids,as well as nucleic acid material.EXOs can deliver their cargo to adjacent or distant cells and locations,and can control of critical processes.EXOs contain bioactive substances such as proteins,lipids,and RNAs,which can facilitate the communication between cells and was recognized as the key messenger for intercellular communication.Whether EXOs are the key component of ADSCs to improve the erectile dysfunction in diabetic rats,and undering molecular mechanism for improving diabetic ED?Nrf2 as the key transcription factor of anti-oxidative stress response in cell,it can bind to ARE and increase the expression of antioxidants proteins and detoxifying enzymes ?,then enhance the ability of anti-oxidation stress,therefore Nrf2 signaling pathway plays an important role in the process of cell defense to oxidative stress.In recent years,the studys found that Nrf2 signaling pathway has a protective effect in variety diseases of oxidative stress as the main pathogenesis.However,whether the Nrf2 pathway is one of the molecular mechanisms of ADSCs-EXOs in improve erectile dysfunction in diabetic rats?The purpose of this study was to study the effects and molecular mechanisms of ADSCs-EXOs to improve diabetic ED by animal and cell experiments.This will provide a theoretical basis for ADSCs-EXOs as a new method of cell therapy without cell therapy.Materials and methods1.ADSCs were isolated from the inguinal adipose tissue using a modified version of a previously published protocol.Osteosynthesis and adipogenic differentiation induction and flow cytometry were used to identify ADSCs.EXOs were isolated from culture medium of ADSCs via ultracentrifugation.Nanoparticle Tracking Analysis,transmission electron microscopy and western blot were adopted to identify EXOs.2.The model of type 2 diabetes mellitus rat generated by a high-fat diet combined with multiple injections of low-dose streptozotocin.Cultured ADSCs and ADSCs-EXOs were resuspended in PBS for implantation.4 weeks after intracavernous injection,erectile function,the number of smooth muscle cells and endothelial cells,cell apoptosis,the level of oxidative stress and Nrf2-ARE pathway in penile cavernous tissue were evaluated.3.The corpus cavernosum smooth muscle cell was cultured with the tissue explants adherent method.Treatment with different concentrations of hydrogen peroxide to induced CCSM cells injury as the model of apoptosis mediated by oxidative stress.CCSM cells were treated with different concentrations of ADSCs-EXOs for 24-48h and then treated with H2O2,cell viability,cell apoptosis,the level of oxidation stress,Nrf2-ARE pathway and nuclear-plasma shuttle of Nrf2 in CCSM cells were evaluated.After transfected with Nrf2 siRNA or control siRNA,Nrf2-ARE pathway,cell viability,the level of oxidation stress and cell apoptosis were evaluated.Then Akt and MAPK pathway in CCSM cells were determinated.Results1.Culture of ADSCs and isolation of ADSCs-derived EXOsThe primary cultured ADSCs are irregular fusiform or angular.The results of fat and osteogenic induction differentiation showed that there were many red fat particles appearing in the cells under the condition of fat induction and the red-nodular bone nodules were found in the cells under the condition of osteogenesis induction.The results of flow cytometry showed that ADSCS expression CD90 and CD29 and did not express CD45.The result of NTA showed that most of the particles are in the range of EXOs diameter(<200 nm),The diameter of the most content of EXOs is 76nm,the concentration is 9.76*108;The transmission electron microscopy(TEM)revealed particles were round-shaped vesicles with double layer membrane structure and diameters were about 100nm,and found that the positive expression of CD63 and CD81 protein were detected by western blot,not expressed the calnexin protein detected in the cell endoplasmic reticulum.2.ADSCs-EXOs ameliorate erectile dysfunction of type 2 diabetic ratAfter high-fat diet and low-dose STZ intraperitoneal injection,the probability of success diabetic rat model was 100%.The result of tissue frozen sections showed that after intracavernous injection ADSCs-EXOs,the number of them gradually decreased,the decline became apparent in the 21st day,and only a small number of ADSCs-EXOs remaining in corpus cavernosum tissue at 28d.Diabetes mellitus consistently resulted in erectile dysfunction in rat model,which reflected by significantly decreased ICP/MAP ratio in the PBS treated diabetic rats compared to the normal control rats.After diabetic rats treatment with EXOs or ADSCs,a significant increase of ICP/MAP ratio,the expression of CD31 and a-SMA,the smooth muscle/collagen ratio,the expression of Bcl-2 were seen compared to the diabetic rats treated with PB.The numbers of endothelial cells and smooth muscle cells apoptosis and the expression of caspase-3 were markedly lower in EXOs or ADSCs-treated diabetic rats than in diabetic rats treated with PBS.The result of DHE staining in frozen section showed that the red fluorescence intensity increased significantly in diabetic rat corpus cavernosum tissue and CCSM cells;compared with the PBS group,4 weeks after ADSCs or ADSCs-EXOs treatment,the red fluorescence intensity in diabetic rat corpus cavernosum tissue and CCSM cells is significantly reduced;ADSCs-EXOs treatment showed no statistical difference compared with ADSCs therapy.The expression of Nrf2-ARE pathway proteins in the corpus cavernosum tissue were detected by western blot and immunohistochemistry,the results showed that the expression of Nrf2,HO-1 and NQO-1 in penile tissue of diabetic rats were increased;Compared with the PBS group,4 weeks after ADSCs or ADSCs-EXOs treatment,the expression of Nrf2,HO-1 and NQO-1 in penile tissue of diabetic rats were increased significantly.3.Nrf2 has a key role in reducing oxidative stress mediated CCSM cells apoptosis in vitroThe passage cells were spindle and arranged swirling or "peak-valley" like when the cell density is high.The cell immunofluorescence results showed that about 97%cells showed green fluorescence in cytoplasm as positive for smoothelin expression,and about 96.5%cells showed red fluorescence in cytoplasm as positive for smoothelin expression.PKH26-labeled ADSCs-EXOs cocultured with CCSM cells for 12h,and the result showed that the red granule samples in CCSM cells were clustered in the cytoplasm,indicating that in vitro,ADSCs-EXOs could be taken into CCSM cells.Different concentrations of ADSCs-EXOs pretreatment CCSM cells for 24-48h,then treated with 200uM H2O2,the results of flow cytometry and western blot showed that H2O2 could significantly induce CCSM cells apoptosis,and 50ug/ml ADSCs-EXOs can significantly inhibit CCSM cells apoptosis induced by H2O2.the result of DHE staining showed that ADSCs-EXOs obviously decreased the red fluorescence density of DHE in CCSM cells.4.Nrf2 plays a key role in reducing oxidative stress mediated CCSM cells apoptosis in ADSCs-EXOs.With different concentrations of ADSCs-EXOs pretreatment CCSM cells for 24-48h,with 200uM H2O2 treatment for another 12h.The results of western blot and QPCR showed that 50ug/ml ADSCs-EXOs could significantly promote expression of Nrf2,HO-1 and NQO-1 at both mRNA and protein levels in H2O2 induced CCSM cells;We further showed that ADSCs-EXOs increased the expression of nuclear Nrf2 but decreased that of cytosolic Nrf2.Consistently,cell immunofluorescence staining showed the expression of Nrf2 in CCSM cells without treatment was very low,and H2O2 could promote the expression of Nrf2,mainly concentrated in cytoplasm.However,remarkable nuclear translocation of Nrf2 was observed after 50ug/ml ADSCs-EXOs treated.The result showed that Nrf2 silencing obviously increased the red fluorescence optical density in CCSM cells.After transfected with si-RNA-Nrf2,the result of western blot showed that Nrf2 deficiency markedly abrogated the up-regulation of Nrf2,HO-1 and NQO-1 in ADSCs-EXOs-treated CCSM cells under oxidation stress.In addition,CCK-8 result showed that Nrf2 deficiency significantly abrogated the up-regulation of cell activity caused by ADSCs-EXOs under oxidation stress.The results of western blot for apoptosis-related proteins showed that interfering Nrt2 significantly increased the ratio of Bax/Bcl-2 and the expression of Caspase3,the decreasing ratio of Bax/Bcl-2 and the expression of Caspase3 with ADSCs-EXOs treatment were disturbed with Nrf2 silencing,After treated with different concentrations of ADSCs-EXOs,200 uM H2O2 treatment for another 4h,DHE staining detected oxidative stress in CCSM cells,the result showed that Nrf2 silencing obviously increased the red fluorescence density of DHE in CCSM cells,ADSCs-EXOs induced the decreasing of red fluorescence density was significantly dampened with Nrf2 silencing.Furthermore,ADSCs-EXOs induced the activation of SOD and CAT was significantly dampened with Nrf2 silencing.Akt and MAPK pathway proteins were detected by western blot,and found that ADSCs-EXOs significantly promoted the expression of p-Akt,p-ERK1/2,p-p38 and p-JNk proteins.Conclusion1.ADSCs from primary culture was obtained and ADSCs-EXOs can be obtained by ultracentrifugation from supernatants of ADSCs culture.2.ADSCs-EXOs can be partially lodged in the penile tissue after injection,and can be taken into CCSM cells.ADSCs-EXOs reduce the apoptosis of CCSM cells in corpus cavernosum tissue,thereby improving ED of diabetic rats3.ADSCs-EXOs activated the Nrf2-ARE pathway,and significantly reduced oxidative stress in diabetic rat corpus cavernosum tissue and CCSM cells.4.In vitro,CCSM cells were obtained by primary culture using the tissue block and overspeed adherent centrifugation method,and the CCSM cells in vitro culture could take in ADSCs-EXOs.ADSCs-EXOs can activate nrf2-ARE pathway and reduce CCSM cells apoptosis mediated by oxidative stress.Nrf2 activation is required for the protective effect of ADSCs-EXOs on CCSM cells apoptosis mediated by oxidative stress.5.Phosphorylations of Akt and MAPK pathways were the molecular mechanism of Nrf2 activation in CCSM cells induced by ADSCs-EXOs under oxidation stress. |
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