| IntroductionStress urinary incontinence (SUI) is defined as involuntary leakage of urine on increased abdominal pressure.SUI is a serious common chronic disease affecting the life quality of the middle-aged and old women. Childbirth damage and the low level of estrogen are two main risk factors of SUI, however, its pathogenesis is still unclear. According to the well-recognized SUI pathogenesis theories, that is, the integral theory and hammock hypothesis, the imperfection of the structure and function of the urethral support tissues including the anterior vaginal wall leads to the occurrence of SUI.The main composition of urethral support tissues is connective tissue, which is composed of a large number of extracellular matrix (ECM) with collagen as the main component and a small amount of cells. Among them, fibroblasts play a core role in mediating ECM metabolism, however, the specific control approach is unknown. Mitogen-activated protein kinases (MAPKs) cell signaling pathways is the main signaling pathway of cell damage and stress reaction. MAPK is also the main kinase of estrogen receptor cell signaling pathways. Among them, P44/42MAPK signaling pathways play a key role in differentiation, proliferation and synthesis of ECM of fibroblasts in varies human tissues. In this study, to explore the correlation of phosphorylated P44/42MAPK (p-P44/42MAPK) expression and SUI, immunohistochemical method was applied to localize the p-P44/42MAPK in female urethral support tissues, and western blot method was employed to observe the expression differences of p-P44/42MAPK in female urethral support tissues between SUI patients and controls. To explore the impact of P44/42MAPK signaling pathway on the occurrence of SUI, through in vitro studies on fibroblasts from female urethral support tissues, RT-PCR and western blot method were adopted to investigate the intervention of P44/42MAPK specificity inhibitor on fibroblasts collagen synthesis.Materials and Methods:1. Clinical research:Ten women with SUI (study group) and ten age matched women at the same period (control group) hospitalized in women’s hospital of Zhejiang university medical college were recruited from January2010to January2011. The anterior vaginal wall (urethral support tissues) was collected during operations due to urinary incontinence (SUI group) or vaginal anterior wall cyst and cervical cancer (control group). Vaginal wall with the size of0.5*1cm2that is1-2cm below the cervix were derived as study specimens. The immunohistochemical En Vision staining was carried out to localize the p-P44/42MAPK protein in the fibroblasts of the periurethral tissues. The Western blot assay was conducted to investigate the expression of p-P44/42MAPK protein in the periurethral tissues.2. Cell experiment:The fibroblasts model of the female urethral support tissues were constructed in vitro by fibroblasts culture of fresh specimens of female urethral support tissues. The mRNA and protein expression level of collagen Ⅰ/Ⅲ were detected by Real-time RT-PCR and Western blot. After the treatment of P44/42 MAPK inhibitor PD98059, the collagen Ⅰ/Ⅲ were detected again to confirm the intervention of PD98059on synthesis of collagen Ⅰ/Ⅲ.Results1. The immunohistochemical En Vision staining showed that p-P44/42MAPK protein expressed strongly in the fibroblasts, squamous epithelial cells, endothelial cells of urethral support tissue, mainly in the nucleus and cytoplasm.2. T test and χ2test demonstrated that the age, body weight, menstruation and BMI were similar between SUI group and control group. In the SUI group the expression of p-P44/42MAPK protein was21.65±7.04and40.44±18.95. In the control group the expression of p-P44/42MAPK protein was71.93±15.01and76.54±15.32. The expression of p-P44/42protein decreased significantly in the periurethral tissues in the SUI group.3. After primary culture of periurethral tissues of women, the mRNA and protein expression of collagen I was9.82±0.13and12.O8±0.95, and after the treatment of PD98059, the mRNA and protein expression of collagen I decreased to7.08±0.11and8.68±0.84(P<0.05). After primary culture of periurethral tissues of women, the mRNA and protein expression of collagen Ⅲ was8.16±0.11and13.72±1.47, and after the treatment of PD98059, the mRNA and protein expression of collagen III decreased to6.00±0.07and10.94±1.76(P<0.05).Conclusion1. p-P44/42MAPK protein expressed in the periurethral tissues of women.2. The expression of p-P44/42decreased significantly in the periurethral tissues of women with SUI compared to the controls, suggesting the correlation between expression of p-P44/42MAPK and the occurrence of SUI 3. The expression of collagen Ⅱ/Ⅲ decreased significantly with the administration of P44/42MAPK inhibitor, indicating that down-regulation of P44/42MAPK signaling pathways in the periurethral tissues may decrease collagen synthesis, disturb the metabolism of ECM and lead to the occurrence of SUI. |
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