The Effect Of Calpain 2/integrin β1/TGF-β1 Signaling Pathway In The Treatment Of Stress Urinary Incontinence By Electrical Stimulation

Background: Stress urinary incontinence（SUI）is a disease that seriously affects the quality of life of middle-aged and elderly people,causing economic burdens.In particular,with the comprehensive liberalization of the two-child policy and the aging of the population,SUI has become an issue that cannot be ignored to affect the quality of life of women.Electrical stimulation（ES）is effective in treatment for SUI,but the mechanism is unclear.Objective: The purpose of this study was to verify the effect of ES on the treatment of SUI through inducing collagen synthesis in fibroblasts,and to explore the relevant mechanism.Part ?:ES can enhance collagen expression and inhibit cell apoptosis in fibroblastsMethods: Select female wild-type C57BL/6 sterile mice aged 8-10 weeks.The mouse SUI model was established using vaginal distension（VD）.PES was administered to some mice at 50 Hz for 7 days,and 15 min each day.Then all mice divided into 3 groups according to different management:Untreated group（WT-NC）,SUI model（WT-VD）,SUI model + pelvic electrical stimulation group（WT-VD + PES）,all the mice ere treated with electrical stimulation 15 min for 7 days on the second day at 50 hz frequency,following by testing the Maximum cystometric capacity（MCC）and leak point pressure（LPP）,to evaluate the therapeutic effect of electrical stimulation on SUI mice.Subsequently,the mice were sacrificed and the anterior vaginal wall tissues were collected for immunofluorescence,TUNEL detection and Masson staining to evaluate the apoptosis and collagen expression.In addition,L929 cells were treated with ES on different parameters to screen the optimal parameters of cell ES（100mv/mm、2h）,and the expression of collagen under the optimal parameters was detected by western blotting.Results: 1.After PES treatment of the SUI model mice,both MCC and LPP were significantly increased in wild-type SUI model mice;2.After PES treatment of the SUI model mice,the Collagen ? and Ⅲ expression in anterior vagina wall were increased,and the apoptosis rate was declined;3.The proliferative activity of L929 cells under 200mv/mm ES showed a continuous decline at the time points of 2 h,4 h and 6h.The proliferation activity of mouse fibroblasts under 50mv/mm showed a continuous increasing trend at the time points of 2 h,4 h and 6h.However,the proliferation activity of mouse fibroblasts under the ES intensity of 100mv/mm was the highest at 2 h,and then decreased at 4 h and 6 h.There was no significant difference between the proliferation activity of mouse fibroblasts under the electric stimulation intensity of 50mv/mm at 4 h and that under the ES intensity of 100mv/mm at 2 h.4.After ES,the mice fibroblast proliferation activity increased,and the expression collagen ? and Ⅲ also increased;Coclusion: 1.We verified that PES can enhance the expression of collagens in the anterior wall of the vagina of mice,thus improving the MCC and LPP;2.Appropriate intensity of ES can promote collagen expression in mouse fibroblasts;3.ES at 100mv/mm for 2 h can significantly improve the proliferation activity of mouse fibroblasts,which can be used as the optimal parameter in this study.Part II: ES activates the calpain 2 protein through increasing intracellular Ca₂₊ concentrationMethods: Select female wild-type and Cav 3.2 knockout-type C57BL/6 sterile mice aged 8-10 weeks.The mouse SUI model was established using vaginal distension（VD）.PES was administered to some mice at 50 Hz for 7 days,and 15 min each day.Then all mice divided into 6 groups according to different management:Untreated group（WT-NC,KO-NC）,SUI model（WT-VD,KO-VD）,SUI model + pelvic floor electrical stimulation group（WT-VD + PES,KO-VD+PES）,all the mice ere treated with electrical stimulation 15 min for 7 days on the second day at 50 Hz frequency,following by testing the MCC and LPP,to evaluate the therapeutic effect of electrical stimulation on SUI mice.Subsequently,the mice were sacrificed and the anterior vaginal wall tissues were collected for immunofluorescence,TUNEL detection and Masson staining to evaluate the apoptosis and collagen expression.In addition,L929 cells were treated with Ca₂₊-free medium or mibefradil before electrical stimulation,then the concentration of Ca₂₊ was detected by Fluo-3AM,and the expression of calpain 2 was detected by western blotting and immunofluorescence.Results: 1.After PES treatment,MCC and LPP of the SUI mouse significantly improved.However,when Cav 3.2 was knocked out,the improvement of MCC and LPP induced by PES was greatly inhibited in SUI mice;2.After PES treatment,the expression of collagen fiber of in the anterior wall of the vagina had an unapparent change in Cav 3.2 knockout SUI mice;3.After ES expose,the intracellular Ca₂₊ concentration was significantly increased in L929 cells.When Ca₂₊ channels were inhibited or ES in Ca₂₊-free medium,intracellular Ca₂₊ concentration did not change significantly;4.After PES treatment,the calpain 2 was significantly activated in the anterior vaginal wall of wild-type SUI mouse.However,when Cav 3.2 was knocked out,the expression of calpain 2 shown no significant change;5.ES can activate calpain 2,but when the calcium channel was inhibited by mibefradil,there was no significant change in the expression of calpain 2 protein before and after electrical stimulation.Conclusion: 1.ES can mediate extracellular Ca₂₊ of L929 cells flowing into through the calcium channels on the cell membrane,and increase the intracellular Ca₂₊ concentration,thereby activating intracellular Ca₂₊ dependent protein calpain 2;2.ES may inhibit cell apoptosis in pelvic floor tissues of SUI model mice by Cav 3.2;3.ES may promote the synthesis of collagen in mouse pelvic floor tissues through Cav 3.2;4.The knockout of Cav 3.2 gene in mice inhibited the activation of calpain 2 protein by electrical stimulation,suggesting that ES may activate calpain 2 through Cav 3.2.Part Ⅲ: ES activates integrin β1 through calpain 2Methods:Silencing calpain 2 gene of L929 ccells.L929 cells were treated with a strength of 100mv/mm ES for 2h and divided into six groups:CON,ES,sh-capn2,shcapn2 + ES,sh-control,sh-control + ES.The expression of integrin β1 was detected by immunofluorescence assay,and the expression of integrin β1 and Talin 1 protein and m RNA were detected by western blotting and q RT-PCR respectively.Results: 1.The expression of integrin β1 of L929 cells was significantly increased after ES.However,after silencing calpain 2 gene,the expression of integrin 1 protein was significantly decreased,and its expression after ES was significantly lower than that after ES in normal L929 cells 2.ES activated calpain 2 and then increased talin 1 protein（47 k Da）in L929 cells.However,after silencing calpain 2 gene,the expression of the cleaved talin 1 protein was significantly reduced,and its expression after ES was significantly lower than that after ES in normal L929 cellsConclusion: 1.ES induced the expression of integrin β1,increasing the cleavage of Talin 1 to increase its active head end content;2.ES activated calpain 2,which cleaved the head end of Talin 1 protein,and then activated integrin β1.Part IV: ES up-regulated the expression of collagens through the integrin β1/TGF-β1 pathwayMethods: After Silence/over-expression of integrin β1gene in L929 cells.And L929 cells were treated with a strength of 100mv/mm ES for 2h.Then all cells were divided into six groups:CON、ES、sh-itgb1/LV-itgb1、sh-itgb1+ES/LV-itgb1+ES、shcontrol/LV-control、sh-control+ES/LV-control+ES.Then the expression of TGF-β1 was detected by immunofluorescence,and the protein and m RNA of TGF-β1,Collagen ? and Collagen Ⅲ were detected by western blotting and q RT-PCR respectively.Results: 1.The expression of TGF-β1 was significantly improved by ES.After silencing integrin β1 gene,the expression of TGF-β1 significantly decreased,and its expression after ES was significantly lower than that of normal L929 cells after ES;2.The expression of collagen ? and Ⅲ significantly improved by ES.After silencing integrin β1 gene,the expression of collagen ? and Ⅲsignificantly decreased,and its expression after ES was significantly lower than that of normal L929 cells after ES;3.The expression of TGF-β1 was significantly improved by ES.When the integrin β1 gene was overexpressed,the expression of TGF-β1 was significantly increased,and its expression after ES was significantly higher than that of normal L929 cells after ES;4.The expression of collagen ? and Ⅲ significantly improved by ES.When the integrin β1 was overexpressed,the expression of collagen ? and Ⅲ was significantly increased,and its expression after ES was significantly higher than that of normal L929 cells after ES.Coclusion: 1.Integrin β1 is closely related to TGF-β1,and the expression of integrin β1 affects the expression of TGF-β1 and collagens;2.Previous studies have confirmed that TGF-β1 can enhance collagen expression through Smad protein.Therefore,in the process of ES,integrin β1 was activated,thereby directly enhancing the expression of TGF-β1,following by promoting collagen expression through the TGF-β1/Smad signaling pathway.Summary: 1.The collagens in the anterior vaginal wall of SUI model mouse decreased and the apoptosis rate increased,which may be one of the reasons for the decrease of MCC and LPP in SUI mouse;2.Appropriate ES can promote the collagen synthesis of the anterior vaginal wall of SUI mice,thus achieving the purpose of treating SUI mice;3 Appropriate ES can promote the expression of collagens,its possible mechanism is: ES promote extracellular Ca2 + flowing into the cells,improve the concentration of intracellular Ca2 +,activating calcium-dependent protein calpain 2,and then the activated calpain 2 cleaved talin 1 protein to activate integrin β1 protein,following by activating the TGF-β1 / Smad signaling pathway,ultimately promoting the expression of collagens.4.This study clarified the relevant mechanism of ES promoting collagen expression,and provided a new theoretical basis for the clinical use of ES in the treatment of SUI and a new insight for improving the treatment effect of SUI by PES.