Effect And Mechanism Of Nrf2 On Pelvic Tissue Repair Of Stress Urinary Incontinence Mice Induced By Mechanical Trauma

Part ?: Establishment and evaluation of stress urinary incontinence mouse model induced by mechanical traumaObjective: A mouse model of female stress urinary incontinence(SUI)was established via vaginal distention(VD),then,the effective duration of SUI mouse model was studied,and the optimum time-point for histological and molecular biological study of pelvic tissues of SUI mice was determined.Materials and Methods: Female C57BL/6 mice were randomly divided into 6 groups: NC group without treatment;the other 5 groups were VD1 d,VD3d,VD7 d,VD14d and VD28 d group,all mice in these 5 groups were subjected to VD to establish SUI mouse model and urodynamic analysis was carried out on day 1,3,7,14 and 28 after VD,respectively.After urodynamic analysis,all mice were sacrificed and anterior vaginal walls were harvested.Then,expression levels of collagen fibers in the anterior vaginal walls of mice in each group were detected by Masson staining,and mRNA expression levels of MMP-2 and MMP-9 were determined by real-time fluorescence quantitative PCR.Results: The MBC,ALPP and BLPP values of mice in the VD1 d,VD3d and VD7 d group were significantly lower than those in the NC group(all P<0.05).The ALPP and BLPP values of mice in the VD14 D group were significantly lower than those in the NC group(all P<0.05),but there were no statistical differences in MBC,ALPP and BLPP values of mice between VD28 d group and NC group(all P>0.05).While,the ALPP and BLPP values of mice in the VD14 d group were significantly higher than those in the VD7 d group(all P<0.05).Besides,the results of histopathological and molecular biological analyses of anterior vaginal wall showed that mRNA expression levels of MMP-2 and MMP-9 in the VD1 d group were increased when compared with NC group(all P<0.05),but there was no significant difference in collagen fiber contents between VD1 d and NC group(P>0.05).While,contents of collagen fibers in anterior vaginal walls of mice in the VD3 d and VD7 d group were significantly reduced,and mRNA levels of MMP-2 and MMP-9 were significantly increased when compared with NC group(all P<0.05).In addition,compared with NC group,contents of collagen fibers in anterior vaginal walls of mice in the VD14 d group were significantly decreased,but significantly increased than those in the VD7d group;similarly,mRNA expression levels of MMP-2and MMP-9 were significantly decreased,but significantly decreased than those in the Vd7d group(all P<0.05).There were no significant differences in collagen fibers contents,and mRNA levels of MMP-2 and MMP-9 in anterior vaginal walls of mice between NC and VD28d group(all P<0.05).Conclusion: The urodynamic parameters and abnormities of extracellular matrix(ECM)metabolism of SUI mouse model induced by VD were begun to repair from day 14 after VD,and day 7 after VD is the optimal time-point for histological and molecular biological studies of VD-induced SUI mouse model.Part ?: The effect and mechanism of Nrf2 on pelvic tissue repair of stress urinary incontinence mice induced by mechanical traumaObjective: The mouse model of SUI was established with wild-type and Nrf2-knockout mice,and the cell model of mechanical damage was established with normal,Nrf2-silencing and Nrf2-overexpressing L929 cells.Then,the effect and mechanism of Nrf2 and related antioxidative pathways on the repair of pelvic tissue injury of SUI mice caused by mechanical trauma were explored;Materials and Methods: In the animal experiment part,the wild type(WT)and Nrf2-Knockout(KO)female C57BL/6 mice were randomly divided into 4 groups,respectively: NC Group(WT-NC,KO-NC)without treatment;Sham group(WT-Sham,KO-Sham),the mice were subjected to sham surgery;VD7d Group(WT-VD7 d,KOVD7d)and VD14 d Group(WT-VD14 d,KO-VD14d),the mice were subjected to VD,and then urodynamic analysis was carried out on day 7 and 14 after VD,respectively.After that,all mice were sacrificed and anterior vaginal walls were harvested.The next,apoptosis rate and the protein expression levels of extracellular matrix(ECM)components,Nrf2 antioxidant signaling,TGF-?1/Smad3 signaling,and MMPs/TIMPs related proteins in the anterior vaginal walls of mice were determined by TUNEL assay and Western Blot analysis.Besides,levels of 8-OHd G,4-HNE and MDA,and activities of CAT,GSH-PX and T-SOD were determined via immunohistochemical staining and corresponding detection kits.In the cell experiment part,cell proliferative activity and ROS level of normal L929 cells were detected by CCK-8 assay and DCF fluorescent probe after administrated with different levels of mechanical strain(0,1333,5333?strain),then parameters of mechanical damage were determined.The next,normal,Nrf2-silencing and Nrf2-overexpressing L929 cells were subjected to 0 and 5333?strain of mechanical strain,respectively,then oxidative damage of cells in each group was determined by immunofluorescence.Besides,normal and Nrf2-overexpressing L929cells were treated with 0 and 5333?strain of mechanical strain,respectively,and then protein expression levels of GPx1,Mn SOD,TIMP-2 and COL1A1 were detected using Western Blot analysis.In addition,normal and exogenous TGF-?1-pretreated L929 cells were subjected to 0 and 5333?strain of mechanical strain,respectively,and then protein levels of p-Smad2/3,TIMP-2 and COL1A1 were determined using Western blot analysis.Lastly,normal and Nrf2-overexpressing L929 cells were treated with 5333?strain of mechanical strain,then nuclear translocation of Smad2/3 was detected by immunofluorescence,and normal L929 cells without treatment was used as negative control.Results: There were no significantly differences in all experimental indicators among WT-NC,KO-NC,WT-Sham and KO-Sham groups(all P>0.05).When compared with WT-NC and KO-NC Group respectively,the ALPP values of mice in the WT-VD7 d and KO-VD7 d group were significantly decreased,respectively(all P<0.05);levels of 8-OHd G,4-HNE and MDA in anterior vaginal walls of mice in the WT-VD7 d and KOVD7 d group were significantly increased,respectively(all P<0.05);protein levels of GPx1,HO-1,Bcl2,COL1A1,COL3A1,elastin,?-SMA,TGF-?1,p-Smad3,TIMP-2 and TIMP-3,and activities of GSH-PX,CAT and T-SOD in anterior vaginal walls of mice in the WT-VD7 d and KO-VD7 d group were significantly decreased,respectively(all P<0.05);and apoptosis rate and protein levels of Bax,cleaved-caspase-3,cleavedcaspase-9,MMP-2 and MMP-9 in anterior vaginal walls of mice in the WT-VD7 d and KO-VD7 d group were significantly decreased,respectively(all P<0.05).When compared with WT-VD7 d group,the ALPP values,protein levels of GPx1,HO-1,Bcl2,COL1A1,COL3A1,elastin,?-SMA,TGF-?1,p-Smad3,TIMP-2,TIMP-3,and activities of GSHPX,CAT,T-SOD in anterior vaginal walls of mice in the WT-VD14 d group were significantly increased(all P<0.05);but levels of 8-OHd G,MDA,apoptosis and protein levels of cleaved-caspase-3,cleaved-caspase-9,MMP-2,MMP-9 in anterior vaginal walls of mice were significantly decreased(all P<0.05).When compared with WT-VD7 d and WT-VD14 d Group respectively,the ALPP values of mice,protein levels of GPx1,Mn SOD,Bcl2,COL1A1,?-SMA,TGF-?1,p-smad3,and activities of GSH-PX,CAT,TSOD in anterior vaginal walls of mice in the KO-VD7 d and KO-VD14 d Group were all significantly reduced,respectively(all P<0.05);but the levels of apoptosis,8-OHd G,4-HNE,MDA and protein levels of Bax,cleaved-caspase-3,cleaved-caspase-9,MMP-2and MMP-9 in anterior vaginal walls of mice in the KO-VD7d and KO-VD14d group were all significantly increased,respectively(all P<0.05).In addition,1333?strain of mechanical strain promoted cell proliferation and ROS production,and 5333?strain of mechanical strain inhibited cell proliferation and increased ROS levels in L929 cells(all P<0.05).Nrf2-silencing and-overexpressing aggravated and alleviated 5333?strain of mechanical strain-induced oxidative damage in L929 cells,respectively(all P<0.05).Besides,Nrf2-overexpressing increased protein levels of GPx1 and Mn SOD,and alleviated 5333?strain of mechanical strain-induced decreases of TIMP-2 and COL1A1protein expressions(all P<0.05).Similarly,exogenous TGF-?1-pretreatment increased pSmad2/3 protein levels,and alleviated 5333?strain of mechanical strain-induced decreases of TIMP-2 and COL1A1 protein expressions(all P<0.05).In addition,Nrf2-overexpressing reduced 5333?strain of mechanical strain-induced inhibition of Smad2/3nuclear translocation.Conclusion: Nrf2 activates antioxidative cascades,and then up-regulates TGF-?1 signaling,reduces oxidation damage,apoptosis and ECM metabolic abnormalities caused by mechanical trauma,and promotes the ECM deposition in pelvic connective tissues and the rise of ALPP values in SUI mice.These suggest that Nrf2 is contribute to the repair process of pelvic connective tissue injury caused by mechanical trauma in SUI mice.Part ?: The effect of Nrf2 antioxidative pathway activator on pelvic tissue repair of stress urinary incontinence mice induced by mechanical traumaObjective: SUI mice were treated with Punicalagin(PUN),and the effect of Nrf2 antioxidative pathway activator on the repair of pelvic tissue injury in SUI mice caused by mechanical injury was preliminarily explored.Materials and Methods: Female C57BL/6 mice were randomly divided into 5 groups: NC group without treatment;the remaining 4 groups were VD,VD+PUN2.5,VD+PUN5 and VD+PUN10 group,the mice in these 4 groups were subjected to VD to establish SUI model,and then intragastric administrated with saline and 2.5,5,10 mg/kg of PUN everyday with 1 times/day,respectively.The urodynamic analysis was performed on day 7 after VD.Then,all mice were sacrificed and anterior vaginal walls were harvested.The next,collagen fiber content and protein levels of ECM components,TGF-?1/Smad3 and Nrf2/ARE signaling in anterior vaginal walls of mice were detected by Masson staining and western blot analysis.Besides,8-OHd G levels in anterior vaginal walls of mice were determined by immunohistochemical staining.Results: When compared with VD group,the ALPP values of mice and protein levels of COL1A1 in anterior vaginal walls of mice in the VD+PUN10 group were significantly increased(all P<0.05).Besides,contents of collagen fibers and protein levels of COL3A1,?-SMA,TGF-?1,p-Smad3,Nrf2,GPx1 and Mn SOD in anterior vaginal walls of mice in the VD+PUN5 and VD+PUN10 group were significantly increased than those in the VD group(all P<0.05);while,8-OHd G levels in anterior vaginal walls of mice in the VD+PUN5 and VD+PUN10 group were lower than that of VD group(all P<0.05).In addition,when compared with VD group,collagen fiber contents and TGF-?1 protein levels in anterior vaginal walls of mice in the VD+PUN2.5 group were significantly increased(all P<0.05);although,the ALPP values of mice,protein levels of COL1A1,COL3A1,?-SMA,TGF-?1,P-Smad3,Nrf2,GPx1,Mn SOD,and 8-OHd G levels in anterior vaginal wall of mice in the VD+PUN2.5 group were increased,but all of these increases were of no statistical significance(all P>0.05).Conclusion: Nrf2 antioxidative signaling activator up-regulates Nrf2/ARE and TGF-?1/Smad3 signaling,then reduces oxidative damage,promotes collagen and smooth muscle actin deposition in the anterior vaginal wall and periurethral tissues,and increases the ALPP value of mice.These suggest that antioxidative drugs targeting Nrf2 have therapeutic effect on SUI mice and their pelvic tissue injury caused by mechanical trauma.