| Pelvic floor disorder (PFD) mainly including pelvic organ prolapse (POP) and stress urinary incontinence (SUI), is a worldwide common disease in elder women. PFD is down descent of pelvic organ, such as uterus and vagina, caused by the weakening of the pelvic floor support tissues. Though PFD won’t endanger lives, advanced POP severely affects the quality of life (QOL) and brings serious inconveniences in daily activities, and harms patients’ mental health.Although the exact underlying mechanism of PFD pathogenesis remains unknown, the dysfunctions in extracellular matrix (ECM) composition in which collagen plays an essential role form the foundation of molecule and biochemical metabolism for PFD. Numerous studies have shown that in pelvic floor support tissues like cardinal ligament, uterosacral ligament and vaginal wall, collagen content reduced significantly. Pelvic floor support tissues mainly composed of dense connective tissue and collagen is the main component. Connective tissues rich in collagen provide robustness and elasticity, providing support to the stability and plasticity of the vagina. Type I collagen is associated with hardness, and Type III collagen is associated with organizational flexibility. PFD, similar with other connective tissue diseases, originates in collagen changes at the molecular level. Fibroblasts are the main component and cell types in the pelvic floor connective tissues, possess the mechanoresponsive capacity, and directly response to the surrounding mechanical stimulation environment. Fibroblasts can secrete collagen and other ECM content, have an important role in maintaining the homeostasis, repair and reconstruction in pelvic floor. In the normal body, fibroblasts in pelvic floor connective tissues regulate collagen synthesis and catabolism and make both achieve a balance level. When the balance between collagen synthesis and catabolism is destroyed, the content and structure of collagen change, resulting in collagen dysfunction. The dysfunction in collagen causes a series of pathophysiological changes in pelvic floor support tissues that could eventually lead to PFD. Thus, the occurrence of collagen metabolism dysfunction in pelvic floor fibroblasts is closely related to POP, and is also the basis to study the pathogenesis of PFD at molecular biology level.Due to the experience of natural physiological process of pregnancy and child delivery, the pelvic floor support tissues are in a constantly changing environment of complex mechanics. Studies have shown that the number of times of pregnancy and vaginal delivery history is an important risk factor in the pathogenesis of PFD. In addition, long-term constipation and weight lifting are also closely related to the incidence of PFD. Studies have shown that the biomechanical properties of the tissues and cells in pelvic floor support had abnormal changes in patients with PFD. The occurrence of PFD is related to the increased intra-abdominal pressure (IAP) or direct effect of mechanical compression on the pelvic floor support tissues. Despite there are evidence to support the important role of mechanical force in the pathogenesis of PFD the specific molecular mechanism linking physical force with PFD remains unclear.In pelvic floor support tissues of patients with POP patients, tissues exhibit a high-level of oxidative stress (OS) status. Oxidative damage markers 8-OHdG and 4-HNE is significantly increased and antioxidase GPxl reduced in uterosacral ligament (USL). Above evidence suggests that OS is closely associated with the pathogenesis of PFD.Therefore, in this study, we used human uterosacral ligament fibroblast (hUSLF) representing pelvic floor fibroblast to establish mechanical load model and OS model. We tested the collagen metabolism changes of the two models, and we applied antioxidant N-acetyl-L-cysteine (NAC) treatment to fibroblasts and observed the antioxidative effect on collagen metabolism. This study is aimed to further investigate the roles and relationship between mechanical force and OS in the pathogenesis of PFD, discover the pathophysiological mechanism of PFD, and provide a theoretical basis of the prevention and treatment for PFD.Part I The role of mechanical strain in collagen metabolism in human pelvic floor fibroblastsObjectiveObservation of the content changes of collagen and collagen-related metabolic enzymes and regulatory factors of hUSLF loaded with mechanical strain. Investigation of the role of mechanical force in PFD-related collagen metabolism dysfunction.MethodsUSL tissue samples were taken from 16 cases of non-POP and non-SUI patients with benign gynecologic disease during hysterectomy. USL tissue fresh samples were immediately used to primary culture of fibroblasts. We use 3-8 generations of fibroblasts to establish mechanical force loading model. The frequency of 5333 με (plate deformation is 4 mm) mechanical strain is 0.1 Hz, and the loading lasted for 4 h. We used quantitative Real-time PCR (qRT-PCR) and Western Blot to detect the expression of type Ⅰ, type Ⅲ collagen content and related metabolic enzymes MMP-2, MMP-9 and transforming growth factor TGF-β1 which promotes ECM synthesis. Flow cytofluorometry were used to test the apoptosis of hUSLF. The expression and distribution of fluorescence-stained cytoskeletal protein F-actin was observed by fluorescence microscopy. We observed the influence of mechanical strain on collagen metabolism, cell apoptosis and cytoskeleton in pelvic floor fibroblasts, and testified the relationship between mechanical strain and the pathogenesis of PFD.Results1. Both mRNA and protein levels of type Ⅰ and type Ⅲ collagen significantly decreased in hUSLF loaded with mechanical strain (p<0.05).2. In fibroblasts loaded with mechanical strain, MMP-2 and MMP-9, degrading collagen and secreted by fibroblasts remarkably increased (p<0.05); and the expression of TGF-β1, which promoting collagen synthesis and stability and secreted by fibroblasts obviously decreased (p<0.05).3. The percentage of fibroblast apoptosis increased after mechanical loading (p< 0.05).4. Cytoskeleton arranged regularly but was slack, and the expression of skeletal protein declined with mechanical loading.Conclusions1. The mechanical strain disturbed the synthesis of typeⅠ and type Ⅲ collagen, destroyed the balance between synthesis and degradation of collagen, resulting in collagen content in human pelvic floor fibroblasts.2. The mechanical strain promoted cell apoptosis and the amount of fibroblasts declined. Large mechanical strain resulted in the relaxation of cytoskeleton which transmits and transforms the mechanical stimulations, and the reduction of cytoskeletal protein.3. The collagen metabolism abnormalities in human pelvic floor fibroblasts was associated with mechanical strain.Part Ⅱ The role of oxidative stress in collagen metabolism of human pelvic floor fibroblastsObjectiveObservation of the content changes of collagen and collagen-related metabolic enzymes and regulatory factors in hUSLF with high-level OS in the presence or absence of antioxidant NAC. Investigation of the role of high levels of OS within the body and cells in PFD-related collagen metabolism dysfunction.MethodsThe USL tissue samples were the same as the samples in the Part Ⅰ. USL tissue samples were taken from 16 cases of non-POP and non-SUI patients with benign gynecologic disease during hysterectomy. USL tissue fresh samples were immediately used to primary culture of fibroblasts. We use 3-8 generations of fibroblasts to establish oxidative stress model. We used 0.4 mM H2O2 to treat fibroblasts for 4 hours in the presence or absence of 10 mM antioxidant NAC pretreatment. Oxidative damage markers ROS and 8-OHdG levels were used to testify whether the model is successful. We used qRT-PCR and Western Blot to detect the expression of type Ⅰ, type Ⅲ collagen content and related metabolic enzymes MMP-2, MMP-9 and transforming growth factor TGF-β1 which promotes ECM synthesis. Flow cytofluorometry were used to test the apoptosis of hUSLF. The expression and distribution of fluorescence-stained cytoskeletal protein F-actin was observed by fluorescence microscopy. We observed the influence of intracellular high level of OS on collagen metabolism, cell apoptosis and cytoskeleton in pelvic floor fibroblasts, and testified the relationship between pelvic floor tissue and intracellular high OS levels in body and the pathogenesis of PFD.Results1. In high level of OS model of hUSLF, both of oxidative damage markers ROS and 8-OHdG content significantly increased compared with those in control fibroblasts.2. Both mRNA (p<0.001) and protein levels (p<0.01) of type I and type III collagen significantly decreased in hUSLF with high OS level.3. MMP-2 and MMP-9, degrading collagen and secreted by fibroblasts remarkably increased (p<0.01); and the expression of TGF-β1, which promoting collagen synthesis and stability and secreted by fibroblasts obviously decreased (p<0.001) in high level of OS in hUSLF.4. The percentage of fibroblast apoptosis increased in high level of OS in hUSLF (p< 0.001).5. Cytoskeleton arranged irregularly and was slack, and the expression of skeletal protein declined in high level of OS in hUSLF.6. Antioxidant NAC inhibited the elevation of intracellular ROS and 8-OHdG levels in high OS model of hUSLF.7. Antioxidant NAC prevented high OS caused decrease of mRNA and protein levels of type I and type III collage (p<0.01), MMP-2 and MMP-9 content increase (p< 0.05), TGF-β1 expression decline (p< 0.01), cell apoptosis increase and cytoskeletal proteins reduce in OS model of hUSLF.Conclusions1. Intracellular high OS level disturbed the synthesis of type I and type III collagen, destroyed the balance between synthesis and degradation of collagen, resulting in collagen content in human pelvic floor fibroblasts.2. High OS promoted cell apoptosis and the amount of fibroblasts declined. Intracellular high OS resulted in the relaxation of cytoskeleton which transmits and transforms the mechanical stimulations, and the reduction of cytoskeletal protein.3. The collagen metabolism abnormalities in human pelvic floor fibroblasts was associated with intracellular high OS level.4. Antioxidant NAC could effectively inhibit high OS level in human pelvic floor fibroblasts, and antioxidant NAC could also reduce large mechanical load-induced high OS level in fibroblasts.Part III The interrelationship between mechanical strain and oxidative stress in collagen metabolism abnormalities of pelvic floor fibroblastsObjectiveGive mechanical strain loading model antioxidant NAC pretreatment. We observed in the presentation of NAC, the content changes of collagen and collagen- related metabolic enzymes and regulatory factors. Investigation of the interrelation-ship between mechanical strain and high levels of OS within the body and cells in PFD-related collagen metabolism abnormalities. To explore the possible physiopa-thological mechanism of PFD.MethodsThe USL tissue samples were the same as the samples in the Part I. USL tissue samples were taken from 16 cases of non-POP and non-SUI patients with benign gynecologic disease during hysterectomy. USL tissue fresh samples were immediately used to primary culture of fibroblasts. We use 3-8 generations of fibroblasts to establish mechanical strain loading model. We treat fibroblasts with NAC as pretreatment before establishing the fibroblast model, Oxidative damage markers ROS and 8-OHdG levels were used to detect the level of OS in cells. We used qRT-PCR and Western Blot to detect the expression of type I, type III collagen content and related metabolic enzymes MMP-2, MMP-9 and transforming growth factor TGF-β1 which promotes ECM synthesis. Flow cytofluorometry were used to test the apoptosis of hUSLF. The expression and distribution of fluorescence-stained cytoskeletal protein F-actin was observed by fluorescence microscopy. We observed the changes of collagen metabolism, cell apoptosis and cytoskeleton in pelvic floor fibroblasts, and testified the interrelationship between mechanical strain and high OS level and figure out their roles in pelvic floor fibroblasts collagen abnormalities in pathogenesis of PFD.Results1. In mechanical loading model of hUSLF, oxidative damage markers ROS and 8-OHdG content significantly increased compared with those in non-strain fibroblasts.2. Antioxidant NAC inhibited the elevation of intracellular ROS and 8-OHdG levels in mechanical strain loading model of hUSLF.3. Antioxidant NAC prevented mechanical strain induced decrease of mRNA and protein levels of type I and type III collage (p<0.05), MMP-2 and MMP-9 content increase (p<0.05) and TGF-β1i expression decline (p<0.05) in mechanical strain loading model of hUSLF.4. Antioxidant NAC inhibited the increase of fibroblast apoptosis rate in mechanical strain loading model in hUSLF (p<0.05).5.Antioxidant NAC made the expression of cytoskeletal protein increase in mechanical strain loading model in hUSLF.Conclusions1. Mechanical strain induced intracellular high OS level in hUSLF.2. Antioxidant NAC could effectively inhibit high OS level in human pelvic floor fibroblasts, and antioxidant NAC could also reduce large mechanical load-induced high OS level in fibroblasts.3. Antioxidant NAC prevented mechanical load and high OS caused decreased type I and type III collagen content, increased cellular apoptosis rate and decreased cytoskeletal protein expression.4. The external mechanical strain load on human pelvic floor support tissue fibroblasts induced intracellular high OS level, and caused collagen metabolism abnormalities and reduced collagen content, which could probably lead to PFD. |
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