The Role Of Exosomes Secreted By Urethral Supporting Tissue In The Pathogenesis Of Female SUI

BackgroundAs a common chronic disease,Stress Urinary Incontinence(SUI)usually occurs in maternal,middle-aged and elderly women,which severely plagued the patients.In recent years,with the opening of the second child policy and the aging of the population,the number of SUI patients has increased dramatically,which has attracted the attention of the medical profession.However,the pathogenesis of the disease is not clear,and there is no ideal clinical prevention and treatment measures.The SUI is mainly caused by the weakness of urethral supporting tissue have been recognized,and the recent studies have shown that extra-cellular matrix(ECM)remodeling disorder of urethral supporting tissue micro-environment dominated by the fibroblast is the key to the pathogenesis of SUI,but the detailed mechanism is still in the exploratory stage.Exosome is a highly concerned inter-cellular communication,which involved in the formation and functional regulation of ECM micro-environment in various tissues.It is expected to provide a feasible direction for the study of the pathogenesis of SUI by investigating the expression of exosomes in urethral supporting tissues and their role in the pathogenesis of SUI.However,there are few related reports at home and abroad.ObjectiveThe purpose of this study was to clarify whether there is the regulation of exosome in the local micro-environment of urethral supporting tissue,to clarify whether there is abnormal expression of exosome in urethral supporting tissue in SUI patients,and to explore the effect of exosome on the function of fibroblasts in urethral supporting tissue at the cell level in vitro.Method1.Female urethral supporting tissues(about 2 cm from the urethral orifice)were collected and divided into SUI group and non-SUI group with 8 cases in each group.Age,menopausal time,times of pregnancy and delivery,mode of delivery and body mass index(BMI)were matched between the two groups.2.The exosomes from human urethral supporting tissues were extracted by the method of precipitation.The morphological characteristics,the distribution of the particle size and expression of CD63(exosomes' marker protein)were identified by electron microscopy,nanoparticle tracking analysis and Western blot.3.The quantity of exosomes extracted from unit mass of urethral supporting tissues in SUI and non-SUI groups were compared by nanoparticle tracking analysis and BCA protein quantification.4.Fibroblasts were co-cultured with urethral supporting tissues-derived exosomes,dividing into F+Sexo group(co-culture of fibroblasts and urethral supporting tissue-derived exosomes from SUI group),F+Nexo group(co-culture of fibroblasts and urethral supporting tissue-derived exosomes from non-SUI group)and F group(equates control group,co-culture of fibroblasts and culture medium).The effects of exosomes on the proliferation,migration and collagen synthesis of fibroblasts were analyzed by CCK-8 increment test,scratch test,protein immunoblotting and reverse transcriptase-polymerase chain reaction.Results1.Precipitation was successfully extracted from primary urethral supporting tissues.The transmission electron microscopy showed that the morphology of the sediment was round cup-shaped.Western-blot showed that the expression of CD63,a specific marker protein of the exosome,was positive.Nanosight analysis showed that the diameter of the sediment was mainly distributed between 50 and 200 nm.2.The protein content of primary urethral supporting tissues-derived exosomes in SUI group and non-SUI group was 1606.02ug/g and 1216.75 ug/g respectively,and the number of exosomes was 2219 x 10 ^ 8 particles/g and 982 x 10 ^ 8 particles/g,respectively.There were significant differences between the two groups.The protein content and the number of exosomes in SUI group were significantly higher than those in non-SUI group(all P<0.05).3.The increase rate of OD value of cells in the group of F+Sexo was 30%in 24 hours,52%in 48 hours and 214%in 72 hours;in the group of F+Nexo was 16%in 24 hours,32%in 48 hours and 105%in 72 hours.Statistical analysis showed that the OD values of cells at 24,48 and 72 hours increased in both F+Sexo and F+Nexo groups(all P<0.05).The OD values of cells at 24,48 and 72 hours in F+Sexo group were significantly higher than those in F+Nexo group(all P<0.05).4.Scratch healing rate of cells at 24h,48h and 72h were 59%,100%and 100%in control group,respectively;were 19%,26%and 27%in F+Sexo group and were 38%,61%and 77%in F+Nexo group.Statistical analysis showed that the scratch healing rates of F+Sexo group and F+Nexo group at 24h,48h and 72h were significantly lower than those of control group(all P<0.05).Scratch healing rates of F+Sexo group at 24h,48h and 72h were significantly lower than that in F+Nexo group(all P<0.05)5.The expression of collagen I and collagen III of cells in F+Sexo group was significantly higher than that in control group both in m RNA and protein levels(all P<0.05).While there weren't significant difference between F+Nexo group and control group of the expression of collagen I and collagen III of cells both in m RNA and protein levelsConclusion1.Exosomes were existed in the micro-environment of urethral supporting tissues.2.Urethral supporting tissue-derived exosomes are correlated with SUI.3.The exosomes from urethral supporting tissues may participate in the pathogenesis of SUI by regulating the proliferation,migration and collagen synthesis ability of fibroblasts.