Correlation Between The Role Of Fibulin-5 In TGF-signaling Pathway And The Pathogenesis Of POP In Female Patients In Xinjiang Region

Objective: Pelvic organ prolapse(POP)prevalence rate is high,is the serious influence women's physical and mental health and quality of life of the common diseases At present the cause of the lack of perfect treatment and prevention measures Our previous studies have found that the prevalence of Xinjiang Uighur women POP obviously higher than that of the Han,han prevalence has difference,TGF-? and Fibulin5 vaginal wall tissue of patients with POP Uighur quantity of protein expression was lower than those of normal people,and the POP group Fibulin5 structures are expressed in the vaginal wall in the racial differences,differences were statistically significant,but the POP exact pathogenesis is not fully clear This project intends to use Uygur female POP resources to study the protein expression levels of extracellular matrix TGF-?,TGFR I/II,Smad2/3,Fibulin-5 and other molecules in vaginal wall tissue by immunohistochemistry,RT-PCR,Western-blot and other methods for Uygur patients with relatively high incidence of POP.Cell experiments and animal experiments were conducted to enhance or inhibit the key molecules in the TGF-?/Smad signaling pathway in vaginal wall fibroblasts respectively,and to observe the changes in the expression levels of Fibulin-5 and other extracellular matrix.Thus,it provides a new possible way for the diagnosis,treatment and prevention of POP to find out the genes that play a key role in the occurrence and development of POP.Methods:(1)From March 2015 to June 2018,the anterior vaginal wall tissue of 36 Uygur patients with POP vaginal hysterectomy was selected as the experimental group without pelvic organ prolapse.The anterior vaginal wall tissue of 15 Uygur women undergoing hysterectomy for other benign gynecological diseases was used as the control group.The relative expression levels of extracellular matrix proteins such as Fibulin-5,Smad2/3,TGF-?1 ? TGF-?RI and TGF-?RII in the anterior vaginal wall of each group were determined by Immunohistochemical real-time fluorescence quantitative assay(RT-PCR)and Western blot.(2)in vitro cells experiment: choose ? POP patients with vaginal anterior wall tissue in parallel to the original generation culture,through the TGF-?1 enhancer(TGF-?1 expression plasmid transfection)and TGF-?1,TGF-?RI/II,Smad3 inhibitors after the intervention,using Western blot and fluorescence quantitative PCR from the level of protein analysis and comparison between groups of POP related extracellular matrix proteins: The expression of fibrin(Fibulin-5),lysyl oxidase(LOX),Elastin,matrix metalloproteinase-2(MMP-2)and MMP-9 protein were investigated.(3)Animal experiment: A total of 45 prolific,aged SD female rats(9 rats for pre-experiment)were selected,and the SD rats were purchased from the Animal Experimental Center of Xinjiang Medical University,Certificate No.: SCXK(new)2016-003 ? Model preexperiment: 9 prolific and old(female,500-550g)SD rats were randomly divided into three groups: 1)control group;2)POP model 1: vaginal dilation + negative pressure suction + LOX inhibitor group;3)the POP model 2: vaginal expansion + pressure suction+ elastase +lox inhibitor injection group Starting from the day 1 build mould until the end of the experiment,every measuring MOPQ index: the level of perineum uplift The perineal body height and perineal body length,Vaginal diameter,length of vagina Vaginal bulging level,and make records Through MOPQ quantitative indicators and test evaluation indexes of each building,choose a method of building the best effect for formal experiment?Formal experiment: select 36 SD rats(female,500-550 g),the adaptability of feeding after a month on average is divided into 4 groups of normal control group,the POP model group,POP + TGF-? recombinant protein intervention group,POP + Fibulin5 recombinant protein intervention group at the end of the experiment were put to death four groups quickly stripped of rat vaginal wall tissue of rats,then used scissors to cut into small pieces(about 1 cm3),take 1 piece of tissue 4 fixed in 10% neutral formaldehyde overnight for subsequent HE and masson staining observation,the rest of the organization in-80° ?The expression of POP-related extracellular matrix proteins in the vaginal wall tissues of rats in each group was detected by RT-PCR,and verified by WB test.Results:(1)Clinical experiment: immunohistochemical results: in the experimental group,Compared with the control group,the expression of extracellular matrix protein SMAD2/3?Fibulin5?TGF-?1?TGF-?R??TGF-?R? and other proteins decreased significantly,difference was statistically significant(P<0.05);RT-PCR experimental results show that in the experimental group,The expression of extracellular matrix protein TGF-?1?TGF-?RI ? TGF-?RII ? Fibulin-5 m RNA genes decreased significantly,And although the expression of SMAD2/3 protein decreased,However,there was no significant difference compared with the control group(P > 0.05);Western blot experiment showed that the expression of extracellular matrix protein TGF-?1?TGF-?RI?TGF-?RII?Fibulin-5?p-smad2/3 in experimental group decreased significantly,difference was statistically significant(P<0.05);And the two groups SMAD2/3 protein expression,(P>0.05).(2)In vitro cell experiment: 1.Successfully isolated,purified,identified and primary cultured human anterior vaginal wall fibroblasts,and passed to the fourth generation for subsequent experiments.2.The expression of POP-related extracellular matrix proteins in human anterior vaginal fibroblasts was observed after different interventions,such as enhancement agent and inhibitor.The expressions of Fibulin-5 and LOX were significantly up-regulated after TGF-?1 enhancement agent intervention(P<0.05),and were significantly down-regulated after three inhibitors treatment(P < 0.05).The expression of Elastin is basically the same as that of fibulin-5 and LOX.However,there was no statistical significance in the expression of MMP-2 and MMP-9 after various interventions(P > 0.05).(3)Animal experiments: 1.The ideal and stable pelvic floor organ prolapse(POP)model of SD rats was designed and established successfully.2.After modeling,the height and diameter of the perineum increased significantly,while the length of the perineum decreased significantly.However,after TGF-? or Fibuin-5interference,the perineal height and vaginal diameter decreased,while the perineal length increased.3.RT-PCR results showed that the expressions of TGF-?,Fibulin-5,LOX and Elastin in POP model group were significantly decreased compared with the blank control group.Compared with the POP model group,the expression of Fibulin-5 and LOX genes were significantly increased after the intervention of recombinant TGF-? and recombinant Fibulin-5,with statistical significance(P < 0.05).WB results showed that the MMP-2gene expression was increased,and the expression of fibuin-5,p-Smad2/3,TGF-?,LOX and Elastin genes were decreased.After the intervention of recombinant TGF-? and recombinant Fibulin-5,the expression of MMP-2 gene decreased,and the expression of Fibuin-5,p-Smad2/3,TGF-?,Lox,Elastin gene increased.4.Recombinant TGF-? and Recombinant Fibulin-5 interference could not affect the expression of Smad2/3 and MMP-9 proteins.5.HE staining showed that the collagen fibers and smooth muscle bundles in the subsquamous mucosa of the vaginal wall in the control group were arranged in regular order.In the lamina propria of mucosa,slender elastic fibers can be seen faintly among the collagen fibers,extending from the muscular layer to the basement membrane.There were a few chronic inflammatory cells in the interstitium.In the transverse section of vaginal wall of the model group,the collagen fiber space under the squamous epithelium was widened and arranged slightly disorderly,while the smooth muscle fiber bundles were partly thinned,partly broken and arranged irregularly,and some were intermixed with the collagen fibers.In the lamina propria of mucosa,the elastic fibers were faintly broken,reduced and disorderly arranged.Some mucosal epithelium and stroma were scattered with more acute and chronic inflammatory cells.Compared with the model group,the irregular arrangement of collagen fibers and smooth muscle bundles in TGF-?recombinant protein intervention group and Fibulin-5 recombinant protein intervention group were improved,the shape and arrangement of elastic fibers in the submucosal lamina propria were fine,and the infiltration degree of inflammatory cells was reduced.Masson staining results showed that the collagen fibers and smooth muscle tissues in the squamous epithelium of the control group were arranged neatly,and the focal smooth muscle was slightly broken.In the model group,the submucosal collagen fibers were hyperplasia and disorderly arranged,and the smooth muscle bundles were reduced,partially broken and irregularly arranged.Compared with model group,TGF-?recombinant protein intervention group and Fibulin-5 recombinant protein intervention group had reduced collagen hyperplasia and disorder,increased the number of smooth muscle,and reduced the breakage.Conclusion:(1)The number of functional elastic fibers in pelvic floor connective tissue decreased in POP patients,among which TGF-?1and Fibulin-5 were fibrogenic cytokines;TGF-? signaling pathway showed that TGF-?1promoted TGF-?RI and TGF-?RII expression,and by activating Smad2/3 activity,TGF-?1 and Fibulin-5 expression was significantly decreased in the tissues of POP patients.The expression of TGF-?RI,TGF-?RII and p-Smad2/3 decreased.These results indicate that TGF-? signaling pathway is involved in the pathological process of POP.However,it is necessary to further study which cytokines and genes are involved in regulating the TGF-?/ Smad signaling pathway through in vitro cell experiments and animal experiments.(2)The expression of fibulin-5,LOX and Elastin varied with the change of TGF-?1.TGF-?1 may play a key regulatory role in the metabolism of ECM such as fibroblast collagen or Elastin in vaginal wall,and may be involved in the occurrence and development of POP.The expression of MMP-2 and MMP-9 is relatively stable,which may be related to the disease state,race and heredity.They are not active in vitro and may require related activation.(3)Mechanical damage can disarrange the collagen fibers and smooth muscle tissue in the vaginal squamous epithelium submucosa,and cause focal smooth muscle rupture;In the model group,the submucosal collagen fibers were hyperplasia and disorderly arranged,and the smooth muscle bundles were reduced,partially broken and irregularly arranged.Inflammatory cells infiltrate,The establishment of the model resulted in increased MMP-2 gene expression,and the expressions of TGF-?,Fibulin-5,LOX,and Elastin are significantly reduced,leading to the occurrence of POP.After the intervention of recombinant TGF-? or recombinant Fibulin-5,the MMP-2 gene expression decreased and expression levels of Fibulin-5,TGF-? and LOX genes were significantly increased,and the POP microenvironment was reversed.It is expected to be a new targeted therapeutic approach for etiology.