Effect Of HNPG On HUCMSCs Cells Transplantation Treating SUI Models Of SD Rats Via Regulating ER-β And Its Mechanism

Objects:1.To establish and identify hUCMSCs cells of hypoxia-reoxygenation models,and detect the influence of proliferation and apoptosis of hUCMSCs cells caused by oxygen-reoxygenation,and determine the protective effect of HNPG to hUCMSCs cells caused by hypoxia-reoxygenation injury,and investigate the possible mechanism of molecular biology.2.To establish and identify SUI models of SD rats by simulating pregnancy, dysplasia and ovariectomy,and to explore the possible molecular biological mechanism of the models.3.hUCMSCs cells were transplanted in urethral sphincters of SUI models of SD rats.The treating effect of the transplant of hUCMSCs cells were detected,and the promoting effect of HNPG on the transplant of hUCMSCs cells were examined and the possible mechanisms of molecular biology were investigated.Methods:1.Hypoxia-reoxygenation models of hUCMSCs cells and hUCMESCs^ER-βsiRNA cells were induced by Na₂S₂O₄ in vitro,which were radomly divided into control group,Na₂S₂O₄ group,E₂ group,HNPG group,PHTPP group and ER-βsiRNA group.The inhibited rates of proliferation were determined by MTT assay, distribution of cell cycle,apoptosis rate,reactive oxygen species and mitochondrial membrane potential were detected using FCM.SOD and CAT activity or GSH and MDA content were measured by ELISA method.The mRNA and protein expressions of ER-β,β-catenin,cyclin D1,bcl-2,bax and caspase-3 were assessed using RT-qPCR method and western blotting assay.2.hUCMSCs cells were transplanted in urethral sphincters of SUI models of SD rats, the researching rats were separated into control group,SUI group,hUCMSCs group,E₂ group,HNPG group,PHTPP group and ER-βsiRNA group.The clinical features of SUI models were examined by sneeze experiment,the urine dynamics indexes of SUI models were assessed using MBC,LPP and ALPP,the urethral sphincter histopathology characteristics were observed using hematoxylin-eosin（HE）staining and Masson staining.SOD and CAT activity or GSH and MDA content were measured using ELISA method.The mRNA and protein expressions of ER-β,β-catenin,cyclin D1,bcl-2,bax and caspase-3 were assessed using RT-qPCR method and western blotting assay.3.Comprehensive pathogenic SUI models of SD rats were established by simulating pregnancy,dysplasia and ovariectomies.The clinical features of SUI models were examined by sneeze experiment,the urine dynamics indexes of SUI models were assessed using MBC,LPP and ALPP,the urethral sphincter histopathology properties were observed using HE staining and Masson staining.SOD and CAT activity or GSH and MDA content were measured using ELISA method.The mRNA and protein expressions of ER-β,β-catenin,cyclin D1,bcl-2,bax, caspase-3,α-SMA,calponin and SM-MHC mRNA and protein were assessed using RT-qPCR and western blotting assay.Results:1.hUCMSCs cell models of hypoxia-reoxygenation were established by Na₂S₂O₄ in vitro.pSilencer 3.1-H1neo-ER-βwere restructured and stably transfected into hUCMSCs cells in vitro.hUCMSCs cells could be damaged by Na₂S₂O₄ via accumulating cell cycle in G1 phase,increasing apoptotic rate,enhancing intracellular ROS content,inactivating SOD and CAT,decreasing GSH, increasing MDA content,decreasing the mitochondrial membrane potential, down-regulating the expressions of mRNA and protein of ER-β,β-catenin,cyclin D1 and bcl-2,and up-regulating the expressions of mRNA and protein bax and caspase-3.HNPG could significantly reverse the damage caused by Na₂S₂O₄ through promoting proliferation,reducing the proportion of G1 phase cells, decreasing apoptosis rate and ROS levels,improving the SOD and CAT activity, enhancing the content of GSH,reducing MDA content,up-regulating the expressions of mRNA and protein of ER-β,cyclin D1 and bcl-2,down-regulating the expressions of mRNA and protein of bax and caspase-3.While PHPTPP and ER-βsiRNA could block the reversal effect of HNPG on the hUCMSCs cell models of hypoxia-reoxygenation.2.Comprehensive pathogenic SUI models of SD rats were established by simulating pregnancy,dysplasia and ovariectomies.The clinical indicators of sneeze experiment were positive,the urine dynamics indexes of MBC,LPP and ALPP significantly reduced,the urethral sphincter histopathologies of HE staining and Masson staining indicated the amount of musles and cell nucleus’were dramatically reduced,and muscular layers and the collagen fiber layers turned thinner,tissue staining became shallow,fiber gaps increased,tissue arrangement disordered and fractured,normal structure lost.ELISA assay showed SOD and CAT activity decreased,GSH content decreased,while MDA content increased. RT-qPCR method and western blotting assay indicated the expressions of mRNA and protein of ER-β,β-catenin,cyclin D1 and bcl-2 down-regulated,while the expressions of mRNA and protein bax and caspase-3 up-regulated.3.hUCMSCs cells were transplanted in urethral sphincters of SUI models of SD rats, which could significantly decreased the positive rates of sneeze test in SUI models of SD rats,increased the urine dynamics indexes of MBC,LPP and ALPP, significantly improved the urethral sphincter histopathological features.HNPG could promote the treating effect of hUCMSCs cell transplantation,reduce the content of ROS,increase SOD and CAT activity,enhance GSH content,reduce MDA content,up-regulate the expression of mRNA and protein of ER-β, β-catenin,cyclin D1,bcl-2,α-SMA,calponin and SM-MHC,down-regulate the expressions of mRNA and protein of bax and caspase-3.However,PHPTPP and ER-βsiRNA could block the promoting effects of HNPG on hUCMSCs cell transplantation treatment.Conclusion:1.Hypoxia-reoxygenation models of hUCMSCs cells could be induced by Na₂S₂O₄ in vitro,while HNPG could inverse the damage caused by Na₂S₂O₄,and the mechanism might be related to ER-βand wnt signaling pathways activated by HNPG,proliferation improved,oxidative stress caused by hypoxia-reoxygen- ation inhibited,apoptosis inhibited in hUCMSCs cells.2.SUI models of SD rats were established by simulating pregnancy,dysplasia and ovariectomies.The mechanism of SUI models of SD rats might be related to the expressions of ER-βand wnt signal pathway of urethral sphincters were inhibited and the expressions of ER-βandβ-catenin decreased,the proliferation of sphincter cells impressed,and apoptosis induced by oxidative stress.3.hUCMSCs cell transplantion demonstrated the treating effect to SUI models of SD rats.HNPG could promote the treating effects of hUCMSCs cell transplantion in urethral sphincters of SUI models of SD rats,could significantly improve the dynamics indexes of MBC,LPP and ALLP,reduce the sneeze test positive rate, enhance the urethral sphincter histopathological characteristics,and the mechanism might be related to activation ER-beta signaling pathways,inhibition of oxidative stress caused by hypoxia-reoxygenation,suppression of apoptosis of mitochondrial pathway in hUCMSCs cells.