| Objective: Pelvic organ prolapse(POP)is a major health problem that affects the quality of life of many women.It is caused by a variety of reasons,such as damage,aging,relaxation of pelvic floor support structure,and then the bulge of uterus and vaginal wall.With the decrease of pelvic organs,the symptoms of bladder,intestine and prolapse appeared.In addition to physical symptoms,it also has a negative impact on women’s mental health.It is a common disease in elderly women.As far as the predisposing factors are concerned,it is related to pregnancy and childbirth,but the specific pathogenesis is still unclear.As we all know,pelvic floor supporting tissue includes fascia,ligament and connective tissue around the pelvic floor,which plays an important role in pelvic support system.The quantity,structure and tissue of extracellular matrix(ECM)are the key factors of tissue stability.The anatomical and functional integrity of the pelvic floor is closely related to the pelvic floor connecting stent,which ensures the mechanical support and organ stability.Ligaments and muscles hang the vagina and uterus from the pelvis.This structure can be effectively compared with the structure of suspension bridge: when the muscles contract,the ligaments will stretch,making the pelvic organs form and firm.If the tissue does not have enough collagen skeleton to support it,the ligament will anchor on an unformed mass and the whole structure will collapse downward.More and more studies show that POP is related to the change of extracellular matrix.Some people think that the production of collagen and elastin in POP has changed.Some studies suggest that if the number of collagen fibers in the tissue is reduced,it can reduce the supporting force of the basin ligament,fascia and other tissues.However,it is not clear whether these changes are the result of pelvic floor dysfunction,or more precisely the cause of pelvic floor dysfunction.Collagen usually exists in the form of insoluble fiber,which is involved in maintaining tissue integrity.In addition,it can resist high tensile strength,which is an important factor to determine the toughness of connective tissue.The connective tissue of pelvic floor mainly includes collagen I(Col I)and collagen III(Col III).Collagen and matrix metalloproteinases(MMP)play a key role in the pathophysiological process of POP,because the conversion of Col I and Col III and the simultaneous activation of MMPs can be observed in the vaginal wall.It has been reported that the content of Col I and Col III in POP is decreased.Studies show that the increase of collagen degradation is the main reason for the decrease of collagen content in pop.P44 / 42 signaling pathway,namely ERK(extracellular signal regulated kinase)signaling pathway,is a member of MAPK signaling pathway.It has been proved that the increase of ERK1/2 activation leads to the increase of smooth muscle cell proliferation.It has also been proved that the decreased expression of Col I and Col III in POP vaginal tissue may be due to MAPK and NF-κ B signaling pathways involved in collagen synthesis and degradation.Transforming growth factor-β1(TGF-β1)is a known stimulator for fibroblasts to produce ECM protein,and mediates the response of fibroblasts to mechanical stress.The induced signal pathway is mainly mediated by Smads protein,but the current research shows that TGF-β1 can also be independent of Smads signal.In addition to the classical TGF-β1/Smad signaling pathway,TGF-β1 can also be activated by non Smad signaling pathways,such as MAPK signaling pathway,PI3K/Akt signaling pathway,etc.We speculate that P44/42 MAPK pathway may be involved in the mechanism of pelvic floor support tissue changes in patients with pelvic organ prolapse.In order to further study whether P4442 MAPK pathway is involved in TGF-β1 on the metabolism of CTGF and collagen in pelvic floor support structure,we designed and completed the following experiments.Methods: Using Western blot,immunohistochemistry and RT-PCR methods,25 cases of POP group and 25 cases of control group were selected,TGF-β1,P44/42,P-P44/42,Collagen I,Collagen III,MMP9,TIMP1 and CTGF were detected and compared between the two groups.The primary culture of fibroblasts from the cardinal ligament of uterus was carried out by adherence method and enzyme digestion method,and the fibroblasts were identified by immunocytochemistry.The fibroblasts were treated with TGF-β1 and its inhibitor SB431542.The proliferation and apoptosis of fibroblasts were detected by MTT Assay and flow cytometry.The expressions of P442,P-P442,CTGF,Collagen I,Collagen III and related proteins were detected by WB assay,the m RNA expressions of CTGF and Collagen-related protein were detected by RT-PCR,the secretion of CTGF,Collagen I and Collagen III were detected by Elisa assay.The fibroblasts were treated with P44/42 inhibitor PD98059,then exogenous TGF-β1 was added,cell proliferation and apoptosis were detected by MTT assay and flow cytometry assay,CTGF and collagen-related proteins were detected by WB assay,and m RNA expression was detected by RT-PCR.Flexcell-4000 flexible substrate loading system was used to mechanically stretch the primary 3-4 generation uterine cardinal ligament fibroblasts,and the cytoskeleton was observed by phalloidin staining.The fibroblasts were stretched by 10% mechanical stretch for 12 hours and 24 hours,TGF-β1,P44/42,P-P44/42,CTGF,Collagen I,Collagen III protein were detected by WB,and m RNA expression was detected by RT-PCR.The fibroblasts were treated with P44/42 inhibitor PD98059 and the fibroblasts were stretched by 10% mechanical stretch for 12 hours.The expressions of P44/42,P-P44/42,CTGF,Collagen I,MMP9 and TIMP1 were detected by WB and RT-PCR.Results: The expression of TGF-β1 and P-P44/42 in cardinal ligament of uterus in POP group was lower than that in control group.In POP group,the expression of MMP9 was higher than that in control group,but the expression of CTGF,Collagen I,Collagen III and TIMP1 was lower than that in control group.Compared with the control group,fibroblast proliferation and apoptosis were increased,and the relative expression of P-P44/42 was increased in primary culture and TGF-β1with 5 ng/ml exogenous addition,the expression of CTGF,Collagen I,Collagen III and Timp1 increased,while the expression of MMP9 decreased.Sb431542 could decrease the proliferation and apoptosis of fibroblasts,and the expression of P-P44/42 decreased,the expression of CTGF,Collagen I,Collagen III and TIMP1 decreased,while the expression of MMP9 increased.PD98059,an inhibitor of P44/42,was used to treat fibroblasts before TGF-β1 treatment.PD98059 could decrease the effect of TGF-1 on fibroblast proliferation and increase apoptosis,the expression of CTGF,Collagen I,Collagen III and TIMP1 decreased,while the expression of MMP9 increased.The TGF-β1,P-P44/42,CTGF and Collagen I expression were increased in fibroblasts treated with 0.5Hz,10% mechanical stretch for 12 and 24 hours,but there was no significant difference between the two groups.The expression of Collagen III did not change after 12 hours of mechanical stretch,but increased after 24 hours of mechanical stretch.P44/42 inhibitor PD98059 could inhibit the proliferation of CTGF and Collagen I and increase the expression of MMP9 in fibroblasts treated with 10% mechanical stretch for 12 hours.Conclusion: 1.The expression of P-P44/42,TGF-β1,CTGF,Collagen I,Collagen III and Timp1 in the cardinal uterine ligament of patients with POP was lower,while the expression of MMP9 was higher than control group.2.TGF-β1 can regulate the proliferation,apoptosis and the metabolism of connective tissue growth factor,Collagen and their related proteins through P44/42 MAPK signal pathway.3.The the mechanical stretch of certain intensity plays a positive regulation on the metabolism of CTGF and Collagen in fibroblasts,and the expression of CTGF and Collagen can be regulated by P44/42 MAPK signal pathway,a certain intensity of mechanical stretch on the pelvic floor support tissue to promote Collagen metabolism of the protective role. |
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