The Effect And Molecular Mechanism Of Mechanical Stretch In Pelvic Organ Prolapse

Objective1.To investigate the expression of PI3K-Akt-FOXO1 pathway in uterocardinal and uterosacral ligament of POP,and to explore the role of oxidative injury induced by delivery in POP.2.Basing on a cell model established by cyclic mechanical stretch,to explore whether the mechanical stretch play a dual effect on pelvic support fibroblasts,and further study the mechanism of POP that mechanical strain induces apoptosis and senescence,and reduces collagen type Ⅰ production via regulated PI3K-Akt-FOXO1 signaling pathway-mediated OS injury.MethodsPart Ⅰ:The present study was approved by the ethics committee of Renmin Hospital of Wuhan University.All of the samples were collected from Renmin Hospital of Wuhan University from 2013.1 to 2015.1.Of the 56 women who underwent hysterectomy,the 20 who were diagnosed with stage Ⅲ or Ⅳ POP were assigned to the POP group and the 36 without POP were assigned to the control group.Of the control group,16 patients without POP were used to develop primary cultures of hUSLFs.Specimens were taken from uterosacral ligaments and fibroblasts were cultured and purified as described previously.TUNEL was used to detect apoptosis.Masson’s Trichrome Staining and Immunochemistry staining were used to detect the expression and morphous of collagen and elastin.Immunochemistry staining was used to detect the level of 8-OHdG,4-HNE in uterocardinal and uterosacral ligament.The expression of Akt,p-Akt,FOXO1 p-FOXO1,GPx1,Mn-SOD were detected by western-blot.Part Ⅱ:The mechanical injury cell model was established by cyclic mechanical stretch(CMS)and identified.The PI3K/Akt specific inhibitor LY294002(20 μM)dissolved in dimethyl sulfoxide was added to cell culture 30 min prior to the application of mechanical strain.Depending on whether the mechanism stretch or LY294002 was used,four experimental groups were seted up.:① Group 1:4mm 4h 0.5Hz(-),LY294002(-).② Group2:4mm 4h 0.5Hz(+)、LY294002(-);③Group3:4mm 4h 0.5Hz(-)、LY294002(+).④ Group4:4mm 4h 0.5Hz(+),LY294002(+).H2DCF-DA was used to detect ROS level.The level of intracellular 8-OHdG was assayed by Cell immunofluorescence assay.Cell senescence was assayed by β-galactosidase(SA-[β-gal)staining.Apoptosis was detected by Flow cytometry.The expression of Akt,p-Akt,FOXO1,p-FOXO1,GPx1,Mn-SOD were detected by western-blot.The mRNA expression of collagen I,GPx1,Mn-SOD were detected by Real-time PCR.Part Ⅲ:Basing on a cell model established by cyclic mechanical stretch,depending on the mechanism stretch parameter,three experimental groups were seted up.① CON group served as the non-mechanical stretch control.② 1mm 4h 0.5Hz group.③4mm 4h 0.5Hz group.Cells growth and viability were assayed by CCK8 after different mechanical stretch stimulation.ResultsPart Ⅰ:The percentage of apoptosis and the level of 8-OHdG and 4-HNE in POP group were higher in pelvic tissues of POP than in the control group(P<0.001).The collagen fibers are much more loosen,and significantly less in the POP group.The elastin fibers in the POP group become shortening,concentrate in appearance as well as disorganized rearrangement.And-the immunuoactivity of elastin in the POP group was significantly lower than in the control group.p-Akt/Akt,p-FOXOl/FOXO1 GPx1 and Mn-SOD levels in pelvic tissues were significantly higher in POP group than control group(P<0.001).Part Ⅱ:Investigation indicated the apoptosis and senescence percentage in cells exposed to 4mm 4h 0.5Hz was markedly higher,the level of intracellular ROS and 8-OHdG were significantly increased compared with control group(P<0.05).The protein expression levels of COL1A1 were significantly reduced in the 4mm 4h 0.5Hz group(P<0.05).4mm 4h 0.5Hz mechanical strainwas applied to fibroblasts,Akt was activated within 10 min and remained so for 1 h,and p-Akt levels returned to basal levels in 3 h(P<0.01).FOXO1 was phosphorylated for 30 min following exposure to mechanical strain,and returned to basal levels within 4 h(P<0.001).In order to investigate the association between the phosphorylation of FOXO1 and mechanical strain-activated Akt,hUSLFs were incubated with the PI3K/Akt signaling inhibitor LY294002(20 μM)for 30 min prior to exposure to mechanical strain.It was observed that Akt phosphorylation was blocked and FOXO1 phosphorylation was significantly inhibited(P<0.05).The present study demonstrated that the mRNA(P<0.01)and protein(P<0.01)expression levels of the antioxidases GPx1 and Mn-SOD in fibroblasts were significantly decreased following loading with 4mm 4h 0.5Hz strain,this also resulted in an increased intracellular ROS level(P<0.001).To further investigate the role of the PI3K/Akt/FOXO1 signaling pathway in regulating OS,hUSLFs were incubated with PI3K/Akt inhibitor LY294002 prior to exposure to mechanical strain.It was observed that the expression levels of GPxl and Mn-SOD were significantly increased as Akt activation was blocked following loading with mechanical strain(P<0.01).The levels of intracellular ROS dropped evidently(P<0.001).Part III:After cells were loaded by different mechanical stretch,we found that cells viability were better in 1mm 4h 0.5Hz group than control group.However,it was worse in 1mm 4h 0.5Hz group(P<0.05).Conclusions1.PI3K/Akt/FOXO1 signaling pathway was phosphorylated by some mechanical stretch stimulation in pelvic,such as pregnancy and childbirth,which would reduce the expression of GPx1 and Mn-SOD,induce oxidative stress injury and depress the expression of collagen and elastin fibers.2.FOXO1 is a key downstream target of the PI3K-Akt pathway,excessive mechanical stretch would hyperactivate Akt,which would phosphorylate FOXO1 transcription factors.Phosphorylation of FOXO1 factors by Akt triggers the rapid relocalization of FOXO proteins from the nucleus to the cytoplasm and it will lose the transcription activity.So Akt hyper-activation leads to a sustained inhibition of FOXO transcription factors,which normally up-regulate the expression of antioxidant proteins,including Mn-SOD,catalase and glutathione peroxides,which results in a decreased ability to scavenge ROS and disturb the balance between oxidation and antioxidation.The present study indicates that mechanical strain results in PI3K/Akt-mediated OS,which is key in the pathogenesis of POP and may have potential as a therapeutic strategy for POP.3.The mechanical stretch play a dual effect on pelvic support fibroblasts,moderate mechanical stretch could stimulate fibroblast proliferation,but excessive mechanical stretch would injure fibroblast.