The Role Of GPx1 In The Pathogenesis Of Female Pelvic Organ Prolapse

Objective:1.To explore the ECM alterations and oxidative stress status in Uterosacral ligament(USL)tissue samples of POP;2.To investigate the changes of cell apoptosis,oxidative stress status and ECM alterations in human uterosacral ligament fibroblasts(hUSLFs)after mechanical strain loading,in order to explore the role of mechanical strain in the pathogenesis of POP;3.To study the role of GPxl in protecting against oxidative damage and ECM remodeling in hUSLFs induced by mechanical force.Methods:Part ?:USL tissue samples were taken from 39 patients undergoing hysterectomy with POP(test group)which were divided into two groups:POP ?-?(17 cases)and POP???(22 cases)and also 20 cases without POP(control group).The collagen fibers in each group were detected by Masson trichrome staining;the expression of GPx1 and the related ECM metabolic regulatory factors were examined with Western blot and qRT-PCR;immunohistochemical staining(IHC)was used to detect the expression of 8-OHdG,4-HNE and GPx1.Part ?:USL tissue samples were taken from 10 cases of non-POP patients with CIN ? or carcinoma in situ disease during hysterectomy and were immediately sent to laboratory for primary culturing of fibroblasts.After identification by immunohistochemistry staining,the 4?8 generations of fibroblasts were used for subsequent experiments;Part ?:To construct a lentiviral overexpression vector with GPxl as the target gene(LV-GPxl).After amplification,conversion,extraction,lentivirus packaging and quality testing,the LV-GPxl vectors were used to transfect hUSLFs.Stably transfected cells were selected by puromycin and the expression level of GPxl protein after transfection in hUSLFs was tested by Western blot;Part IV:To establish cytomechanics model in vitro.The fibroblasts were divided into three groups:GPxl overexpression transfection group(GT),mock-vehicle transfectoion group(MT)and non-transfection group(NT).Cells in each group were treated with mechanical loading using four-point bending device.Cells were collected after treatment.JC-1 fluorescent dye and flow cytofluorometry were used to test the changes of mitochondrial membrane potential(MMP)and cell apoptosis rate in hUSLFs.The fluorescent probe H2DCF-DA and immunofluorescent staining were applied to detect the oxidative status.We also observed the expressions of ECM protein and the related regulator factors with Western blot and qRT-PCR.Results:Part ?:Compared to control group,the content of collagen fibers and the expression of Collagen ??Collagen ??Elastin and TIMP-2 in USL tissues significantly reduced with the increased degree of POP,while the expression of MMP-2 and MMP-9 was significantly up-regulated;POP groups exhibited a significantly higher expression of 8-OHdG and 4-HNE,and a lower expression of GPxl when comparing with control group.Part ?:The fibroblasts mainly presented with long spindle-shaped and a few of them also appeared irregular triangles or polygons in light microscope.Cells connected to each other and formed into a network structure.The immunohistochemical staining of cells showed that vimentin was positive and cytokeratin was negative;Part?:Results showed that the positive clones encoded by LV-GPxl vector were consistent with the sequence of target gene GPxl.The optimal transfection conditions by lentivirus for hUSLFs included normal medium with 5 ?g/ml Polybrene,MOI 30.0.8 ug/ml puromycin was tested as the appropriate concentration to select stably transfected cells.Western blot test showed that the protein level of GPxl was significantly higher in hUSLFs after tansfection of LV-GPxl vector when compared with normal cells.Part IV:Compared with the non-stretched cells,the cells pretreated by 5333 ?×4 h mechainical strain exhibited higher cell apoptosis and oxidative damage,less protein and mRNA levels of COL1A1,COL3A1,Elastin,TIMP-2 and TGF-?1,and higher protein and mRNA levels of MMP-2,MMP-9;while under the same condition of applying 5333 ?×4 h mechainical strain,compared with the non-transfection and mock-vehicle groups,we found that GPxl-overexpression can not only protect hUSLFs from cell apoptosis,oxidative and collagen damage induced by mechanical stimulation,but also improve the remodeling of ECM.Conclusions:The USL tissue from POP patients presented metabolic disorder and remodeling of the ECM and imbalance between pro-oxidants and antioxidants;Over-expression of GPxl protected hUSLFs against OS damage and ECM remodeling induced by the mechanical stimulation,together with our previous data,we concluded that mechanical strain caused abnormalities in ECM metabolism,apoptosis via inducing high OS in fibroblasts may be involved in the pathogenesis of POP,and GPxl played an important role in regulating this process.Antioxidation effect may provide a novel therapeutic strategy for POP treatment.