| Introduction:Stress urinary incontinence(SUI) is one of the most common urogynecologic diseases.The clinical definition of SUI provided by the International Continence Society(ICS) is:the involuntary leakage of urine during increased abdominal pressure (e.g.coughing,sneezing,laughing,etc),in the absence of detrusor contraction.Recently, there is intense interest in the etiology of SUI,which is a result of the growing awareness of the enormous impact that these conditions have on quality of life and the increasingly large segment of the population.The etiology of SUI is still not completely understood.Pregnancy and childbirth are considered to be the most important risk factors.The persistent increased pelvic pressure,which is a result of hormone changes and increased uterine during pregnancy, leads to the damage of pelvic tissue.During vaginal delivery,the extremely stretched pelvic tissue resulted in the structure changes of muscles and supporting tissue around urethra,and ultimately induces SUI.Additionally,SUI is associated with the number and situation of parity,menopause,age,body mass index,family history,pelvic surgery history,education,chronic cough,constipation,smoking,and diabetes etc.Recently,genetic research on the pelvic supporting tissue attracts more attention. Connective tissue is the most important part of pelvic supporting tissue,which maintains the flexibility and toughness of pelvic floor.Collagen secreted by fibroblast is the major compartment of connective tissue.It is reported that collagenolytic activity might be related with the occurrence of SUI.The calpains are a family of calcium-dependent cysteine proteases that are widely expressed in almost all the organs. Calpains have been implicated in basic cellular processes including cytoskeletal rearrangements,different signal transduction pathways,and cell apoptosis.In pathological status,abnormal elevation of Ca2+ activates Calpains,which accelerate the degradation of various cytoskeleton and proteases,and ultimately induces the tissue damages.A long list of disease states has been claimed to involve disregulation of the calpain system,such as muscular dystrophy,cataract,Alzheimer s disease,type 2 diabetes,ischaemic brain damage,poor wound healing,and rheumatoid arthritis.Calpains play an important role in the metabolism of muscle fiber,collagen,and extracellular matrix;therefore it is supposed that calpain is an essential factor in the pathogenesis of SUI.And this supposition has been verified by some investigators.The calpains are defined as a unique class of intracellular calcium dependent cysteine proteases.The most well characterized calpains are the ubiquitously expressedμ-calpain and m-calpain.The terms,μ-calpain and m-calpain refer to a requirement for micromolar and millimolar concentrations of calcium respectively to confer catalytic activity in vivo.Theμ-calpain and m-calpain isoforms function as heterodimers consisting of a large catalytic subunit(80 kD) and a common regulatory subunit(28 kD).In pathological conditions,calcium imbalance is often accompanied with the proteolysis of calpain substrate.It is confirmed by in vitro tests that the elevation of cellular calcium activates the calpains and results in the proteolysis of proteins. Calpastatin is a protein that specifically inhibits the proteolytic activity ofμ-calpain and m-calpain.When activated by calcium,calpain is combined with calpastatin rapidly and the activity is inhibited;hence the proteolysis of substrate is specific.In this study,we investigated the expression and distribution of calpain/calpastatin in urethral surrounding tissue of SUI patients,explored the role of calpain/calpastatin in the pathogenesis of SUI,and provided a novel approach to the therapy of SUI.METHODS:The expression of calpain-1,calpain-2 and calpastatin was detected with an EnVision immunohistochemistry assay in 20 paraffin-embedded samples of normal urethral surrounding tissue(control group) and 20 samples of SUI patients(SUI group). SUI was defined according to the International Continence Society guidelines,and all subjects had not taken hormone drugs in the past 3 months.Inclusion criteria of the control group are:no symptoms of incontinence;gynecological exam without vaginal wall or uterine prolapse;no hormone drugs in the past 3 months;non-estrogen related diseases confirmed by pathological examination.RESULTS:Immunohistochemical evaluation incorporated both the intensity and distribution of staining.The expression level of calpain-1,Calpain-2 and calpastatin in epithelial cells,fibroblasts,smooth muscle cells were semi-quantified by the H-score.(1) The expression and distribution of calpain-1 in urethral surrounding tissue Calpain-1 was expressed in epithelial cells and smooth muscle cells of both SUI and control group,but not in fibroblasts.In epithelial cells,the expression was mainly in nuclei,membrane and cytoplasm,while in the smooth muscle cells calpain-1 was expressed in both cytoplasm and membrane.The expression level of calpain-1 in epithelial cells of SUI and control group was 2.71±0.45 and 2.41±0.38,respectively, in smooth muscle cells was 2.01±0.40 and 2.09±0.43.In both epithelial tissue and smooth muscle tissue,the comparison between the two groups had no significant difference(p＞0.05,p＞0.05).(2) The expression and distribution of calpain-2 in urethral surrounding tissue Calpain-2 was expressed in epithelial cells and smooth muscle cells of both SUI and control group, but not in fibroblast.In epithelial cells,the expression was mainly in nuclei,membrane and cytoplasm,while in the smooth muscle cells calpain-2 was expressed in both cytoplasm and membrane.The expression level of calpain-2 in epithelial tissue of SUI and control group was 3.07±0.36 and 2.67±0.47,respectively,in smooth muscle tissue was 2.50±0.42 and 2.08±0.51.In both epithelial and smooth muscle tissue,the comparison between the two groups all had significant difference(p＜0.05,p＜0.05).(3) The expression and distribution of calpastatin in urethral surrounding tissue Calpastatin was expressed in epithelial cells and smooth muscle cells of both SUI and control group,but not in fibroblast.In epithelial cells,the expression was mainly in nuclei,membrane and cytoplasm,while in the smooth muscle cells calpastatin was expressed in both cytoplasm and membrane.The expression level of calpastatin in epithelial tissue of SUI and control group was 2.91±0.42 and 3.23±0.39 respectively,in smooth muscle tissue was 2.69±0.43 and 3.09±0.45 respectively.In both epithelial and smooth muscle tissue,the comparison between the two groups had significant difference(p＜0.05,p＜0.05).Conclusion:1.Compared with control group,the expression of calpain-2 in urethral surrounding tissue of SUI patients was significant increased.It suggested the increased expression of calpain-2 promote the incidence of SUI.2.Compared with control group,the expression of calpastatin in urethral surrounding tissue of SUI patients was significant decreased.It suggested the decreased expression of calpastatin protect them from SUI.3.The imbalance expression of calpain/calpastatin may be involved in the occurrence of stress urinary incontinence.Calpastatin might play an important role in blocking the occurrence and development of stress urinary incontinence;consequently,calpain inhibitor may be a potential approach to the therapy of SUI.
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