Estrogen inhibits the differentiation of fibroblasts induced by high stiffness matrix by enhancing DNMT1 expression

Estrogen inhibits the differentiation of fibroblasts induced by high stiffness matrix by enhancing DNMT1 expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Estrogen inhibits the differentiation of fibroblasts induced by high stiffness matrix by enhancing DNMT1 expression Zhihan Zhao, Guotao Huang, Yong He, Xiaohu Zuo, Wuyue Han, Li Hong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2549183/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction and Hypothesis: Pelvic organ prolapse(POP) is a multifactorial connective tissue disorder caused by damage to the supporting structures of the pelvic floor. Evidence from several studies suggests that anterior vaginal wall stiffness is higher in patients with POP, but the mechanisms involved remain unknown. Methods Tissue from the anterior vaginal wall of patients with POP or other benign diseases was obtained. The modulus of elasticity of the anterior vaginal wall was measured using a microindenter. Cells were cultured in vitro on acrylamide gels of different stiffness and treated with DNMT1 inhibitor, microtubule polymerisation inhibitor and estrogen. Western blot or immunohistochemical staining was performed to detect DNA Methyltransferase 1, α-smooth muscle actin(α-SMA) expression, and connective tissue growth factor(CTGF) expression. Conclusion Estrogen can inhibit high stiffness matrix-induced fibroblast differentiation, by enhancing DNMT1 expression. This study may help to elucidate the complex crosstalk between fibroblasts and their surrounding matrix under healthy and pathological conditions and provide new insights into the options for material-related therapeutic applications. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Pelvic organ prolapse(POP) is a multifactorial connective tissue disorder caused by damage to the supporting structures of the pelvic floor. Epidemiology shows that the prevalence of symptomatic POP in China is 9.1%[ 1 ]. The lives of POP patients are severely impacted and with significant global ageing, the prevention and treatment of POP is receiving increasing attention[ 2 ]. To date, the pathogenesis of pelvic organ prolapse is still unknown. One of the main risk factors for POP is stress injury during which extracellular matrix remodelling occurs[ 3 ]. Fibroblasts are widespread in connective tissue and are the main cells regulating the composition of the extracellular matrix, their differentiation into myofibroblasts is a key feature of wound healing in soft tissues such as the vagina[ 4 ]. Several studies have demonstrated that the microenvironment of vaginal tissue in patients with POP is more rigid than that of healthy vagina[ 5 – 7 ]. When fibroblasts grow in a stiffer environment, some of them differentiate into myofibroblasts, marked by α-SMA, CTGF and type I collagen production[ 8 ]. The prolonged presence of myofibroblasts results in excessive collagen deposition, tissue contracture and dysfunction[ 9 ]. DNA methylation is known to play an important role in the maintenance of normal development and biological function[ 10 ]. Covalent methylation of DNA cytosines promotes transcriptional repression in most cases. The importance of DNA methylation is reflected in the development of many diseases that are due to hypermethylation or hypomethylation of DNA and inappropriate temporal or spatial regulation[ 11 ]. The maintenance of methylation patterns during cell replication is mediated by DNA methyltransferase1, which catalyzes the transfer of methyl from S-adenosylmethionine to hemimethylated DNA[ 12 ]. Estrogen plays an important role in the development of POP. Estrogen deficiency may contribute to the development of POP as it has a profound effect on pelvic collagen synthesis, and the strength of connective tissue[ 13 ]. In vitro studies have found that estrogen improves the mechanical properties of connective tissue and inhibits excessive and abnormal remodelling of the fibrous extracellular matrix in response to mechanical stimulation and stretching[ 14 ]. It has been observed that low estrogen levels in women undergoing vaginal surgery can affect the outcome of the procedure, and women with persistently low estrogen levels have a slower recovery due to impaired wound healing[ 15 ]. The addition of estrogen to the surgical site may promote pelvic floor tissue repair. In summary, estrogen has positive implications for the treatment of pelvic floor organ prolapse, but the mechanisms that may be involved are unclear. Our study provides preliminary evidence that estrogen can inhibit high stiffness matrix-induced fibroblast differentiation by enhancing DNMT1 expression, and that the conversion of mechanical to biological signals involves the involvement of cellular microtubules. 2. Methods And Treatment 2.1 Cell Culture and Treatment All experiments were performed using L929 cells, a mouse fibroblast, purchased from the China Type Culture Centre (Wuhan, China) and cultured in RPMI Medium 1640 medium (Gibco, America) at 10% serum concentration (Yeasan, China). Cells were passaged or cryopreserved after they reached approximately 80% confluence. To inhibit DNMT1 expression, cells were cultured in medium for 24h and then replaced with medium containing Decitabine(5’-Aza) (5umol/L, MedChemExpress, China). To inhibit myosin ATPase and microtubule assembly, cells were incubated on gel for 6 h and then replaced with medium containing nocodazole (5umol/L, MedChemExpress, China) for 5 h. Afterwards, the medium was changed and the cells continued to be incubated for 24 h. To observe the effect of estrogen on fibroblast differentiation, cells were incubated in gel for 24 h and then replaced with medium containing estrogen (10 − 7 mol/L Sigma, America) medium for 24h. 2.2 Human Specimen To investigate the differences in myofibroblast expression in the anterior vaginal wall between patients with POP and normal women, patients with Grade III prolapse who underwent hysterectomy and pelvic floor reconstruction were the experimental group and patients who underwent total hysterectomy for other benign gynaecological conditions were the control group, with whole layers of anterior vaginal wall tissue taken during surgery. The experiment was approved by the Medical Ethics Committee of the Wuhan University People's Hospital under the approval number WDRY2021-K001 and the patients signed an informed consent form. The experiments with human samples conformed to the guidelines set by the Declaration of Helsinki Patient information is shown in Table 1 . Table 1 Patient Informations Variable POP non-POP Age 56 55 52 57 56 52 54 50 52 56 Parity 3 2 4 4 3 4 2 2 1 3 BMI 27.4 28.3 28.8 25.4 26.6 26.2 25.9 27.4 26.1 28.4 Diagnosis Prolapse of the anterior vaginal wall Uterine prolapse Uterine prolapse Uterine prolapse Prolapse of the anterior vaginal wal Adenomyosis of the uterus Multiple leiomyomas of the uterus Multiple leiomyomas of the uterus Adenomyosis of the uterus Adenomyosis of the uterus POP-Q III III III III III - - - - - 2.3 ECM Surface Micro-Stiffness The mechanical properties of ECM were measured by microindentation for measuring the microhardness of biological tissue surfaces. Samples were indented in PBS with a radius of 95 m, a stiffness of 0.5 N/m and a loading and unloading cycle of 3 seconds using PIUMA, an instrument that provides mechanical properties such as microscale nanoindentation for biological material tissues and cells. The effective Young's modulus was calculated automatically by the PIUMA software based on the load and displacement curves.Five samples were included in the POP and control groups, and the experiment was repeated three times for each sample. 2.4 Western blot 2.4.1Western blot of the human anterior vaginal wall The anterior vaginal wall was removed intraoperatively and immediately placed in an EP tube filled with PBS for further processing. The mucosal epithelial layer and epithelial tissue were removed with surgical scissors, preserving the mucosal lamina propria and the muscularis, cut into fine pieces with scissors, weighed on an electronic balance, and lysis solution (RIPA Buffer, Cocktail, phosphorylase inhibitor, PMSF) was added per 100 mg/1 mL, mixed into a homogenate using a homogenizer, and centrifuged at 12000g for 15 minutes in an ice bath, and the supernatant was taken as protein. The BCA method was used to determine the protein concentration for subsequent experiments. 2.4.2 Western blot of L929 cells Cells were harvested using a spatula and washed three times with pre-cooled phosphate buffered saline (PBS). The cells were then lysed in lysate, centrifuged at 12,000g for 15 minutes, and the supernatant was aspirated to collect the proteins. bca assay was used to determine protein concentrations for subsequent experiments. The antibody used was a rabbit polyclonal antibody (pAb) against DNMT1 (purchased from Cell Signaling Technology). Rabbit polyclonal antibody against α-SMA, mouse polyclonal antibody against GADPH, rabbit polyclonal antibody against Collagen1, rabbit polyclonal antibody against Collagen3, rabbit polyclonal antibody against CTGF, polyclonal antibody against β-actin (purchased from Proteintech). 2.5 Immunohistochemistry Paraffin-embedded whole vaginal tissue sections were dewaxed at 65°C and subjected to antigen repair in citrate buffer solution (0.01 mol/L, pH 6.0) for 30 min at 100°C. After being blocked with 5% bovine serum albumin (BSA) for 1 h, the sections were incubated with anti-α-SMA or anti-DNMT1 antibodies at 4°C overnight. After incubation with biotin-labelled secondary antibodies for 1 hour at 37°C, stains were then added and all stained slides were observed under an Olympus BX50 microscope. 2.6 Immunofluorescence L929 cells were seeded on strain plates and treated as indicated in each experiment, followed by fixation with 4% formaldehyde for 30 min and blocking with 5% Bovine Serum Albumin (BSA) for 1 h. Cells were then incubated with anti-DNMT11 (1:100) and anti-α-SMA (1:100) overnight at 4°C. Next, cells were further incubated with FITC-conjugated goat anti-rabbit IgG (1:200, antgene, China). 4', 6'-Diamidino-2-phenylindole (DAPI, antgene, China) was used to stain cell nuclei. Complete images of the cells were taken using a fluorescent microscope (Olympus IX71) and analysed by ImageJ. 2.7 CCK8 assay Cell viability was estimated by CCK-8 assay (Seven, China). Approximately 10 4 cells were inoculated in 96-well plates with 100 µl of medium per well. After 24 h of incubation, different doses of Decitabine or nocodazole were added, respectively. Each well was incubated for 2 h with 10 µg CCK-8 solution protected from light before absorbance at 450 nm was measured using an enzyme marker. 2.8 Preparation of Polyacrylamide Gel (PA Gel) Substrates The polyacrylamide gel (PA gel) substrate was prepared according to Justin R et al. by varying the ratio of acrylamide to methacrylamide to adjust the stiffness of the PA gel to 4 kpa and 40 kpa. The prepared liquid was mixed and added to parallel glasses spaced 1.0 mm apart and left to set for about 1 hr. The PA gels were washed 2–3 times with PBS, covered with photocrosslinker (Sulfo-SANPA, abcam, 0.5 mg/ml in PBS) and irradiated with UV light for 1 h. The PA gels were washed 2–3 times with PBS to remove the excess photocrosslinker. Collagen I (15 ug/mL, Biosharp) was spread on the surface of the gel and placed in a 4°C chamber overnight. The following day, the collagen solution is poured off, washed 2–3 times with PBS to remove excess collagen, dried overnight and the collagen-coated PA gel is ready for cell inoculation the following day. 2.9 Microarray data archive Expression profiles for the GSE18224 array were retrieved from the GEO database. GSE18224 collected the left ventricular transcriptome of four mice from the Affymetrix RAE 430 2.0 GeneChip array testing each combination of three factors: sex, genotype, and surgical modality. The expression profile of the database was based on the GPL 1261 platform. Series matrix files and data table header descriptions for this database were downloaded from the GEO database to screen for genes with significant correlation to DNMT1. 2.10 Statistical analysis All data were presented as the mean ± SD and performed using SPSS version 21.0 software (IBM Corporation, Armonk, NY, USA). Differences between two groups were determined using Student’s t test. P < 0.05 were considered statistically significant. 3. Results 3.1 Mechanical and biological differences between patients with POP and control. The tissue of the anterior vaginal wall was taken intraoperatively and the mechanical properties of the vaginal wall were measured using a microindenter. The results showed that the Young's modulus of the anterior vaginal wall tissue was significantly higher in the POP patients (Table 2 ). Western blot results revealed that the expression of α-SMA, a marker of myofibroblast, in the anterior vaginal wall was significantly higher in POP patients (Fig. 1 a-c). We also examined the expression of Collagen I and III in the POP and control groups. Western blot and immunofluorescence results displayed no significant difference in the expression of CollagenI and increased expression of CollagenIII in the POP group (Figur1d-h). Table 2 Young's modulus of the anterior vaginal wall Feature ECM-Healthy ECM-POP p Value Effective Young modulus (kPa) 7.26 10.54 0.0044 3.2 Extracellular matrix stiffness affects fibroblast to myofibroblast transformation To investigate the effect of extracellular matrix stiffness on fibroblast phenotype, we prepared PA gels with different stiffnesses (Table 3 ). L929 was grown on the gels for 24 h and morphological observations were made under an inverted microscope (Fig. 2 a). The cells showed polymorphic morphology such as star-shaped and long spindle-shaped after 24 h on the high stiffness gels, whereas growth on the low stiffness gels was mainly round. Aspect ratio calculations showed that the cells cultured on the high stiffness gels was significantly higher (Fig. 2 b,c). Western Blot and immunofluorescence results showed that α-SMA expression was increased in cells cultured in high stiffness gels (Fig. 2 d-f). Table 3 PA gels Preparation Acrylamide % Bisacrylamide% Acrylamide from 40%stock solution (ml) Bis-acrylamide from 2% stock solution (ml) Water (ml) E ± St. Dev. (kPa) 4 0.3 1 1.5 7.5 2.24 ± 0.58 8 0.48 2 1 2.4 5.6 40.4 ± 2.39 3.3 Extracellular matrix stiffness regulates fibroblast to myofibroblast transformation via DNMT1 The key role of epigenetic mechanisms (DNA methylation and demethylation, histone modification and RNA-based mechanisms) in mediating the regulation of smooth muscle phenotype induced by environmental signals has been suggested, but whether DNMT1 is involved in the fibroblast to myofibroblast transformation and or regulated by extracellular matrix stiffness has not been investigated. Therefore, we first examined DNMT1 expression in the anterior vaginal wall of POP patients and controls. Western blot results showed reduced DNMT1 expression in the anterior vaginal wall of POP patients (Fig. 3 a,b), and immunohistochemistry verified these results (Fig. 3 c). Subsequently, we cultured L929 in different stiffness gels for 48h, the western blots showed that the cells had significantly lower DNMT1 levels after 48h incubation on high stiffness gels (Fig. 3 d,f) and immunofluorescence verified these results (Fig. 3 g). To investigate whether extracellular matrix stiffness affects the transformation of fibroblasts to myofibroblasts via DNMT1, we added DNMT1 inhibitors to the low stiffness gel medium. We tested the chemosensitivity of L929 to Decitabine by the CCK8 assay, with a gradient of Decitabine concentrations ranging from 0.5 to 10 umol/L and a final treatment concentration of 5umol/L. The results showed a significant decrease in DNMT1 expression and an increase in α-SMA, CTGF expression in the DNMT1 inhibitor group (Fig. 3 e). 3.4 Cells transform physical signals into biological signals through the microtubule cytoskeleton How extracellular matrix stiffness translates physical signals into intracellular biological signals is unclear. Cellular microtubules are known to have kinetic properties of aggregation and depolymerization and play an important role in the maintenance of cell morphology, cell division, signal transduction and material transport. Therefore, we retrieved the expression profiles of the GSE18224 array from the GEO database and used R software to screen for genes with significant relevance to DNMT1. The results showed that the microtubule-associated proteins Rmdn1, Mast2 and DNMT1 were significantly correlated (Fig. 4 a). We therefore treated the cells with Nocodazole, a rapid and reversible inhibitor of microtubule polymerisation, and we tested the chemosensitivity of L929 to Nocodazole by the CCK8 assay. We observed significant morphological differences in the cells in the different stiffness gels(Fig. 4 b,c). Western blot results showed that Nocodazole treatment eliminated the down-regulation of DNMT1 expression induced by high stiffness gels and reduced α-SMA, CTGF expression (Figur4d,e) suggesting a potential role for the microtubule cytoskeleton in the regulation of DNMT1 expression by gel stiffness. 3.5 Estrogen partially inhibits fibroblast differentiation induced by high stiffness gel Estrogen plays an important role in the development of POP, but the mechanisms involved are unclear. We therefore explored whether there is an inhibitory effect of estrogen on high stiffness-induced fibroblast transformation. We added estradiol to the culture medium and assayed the chemosensitivity of L929 to estradiol treatment by CCK8. The concentration gradient of estradiol was 10 − 8 ~ 10 − 5 mol/L, and the final treatment concentration was 10 − 7 mol/L. The results showed that the cells cultured in the high stiffness gels showed a reduction in transformation and a decrease in the aspect ratio of the cells after estrogen treatment (Fig. 5 a,b). The western blot and immunofluorescence showed that the expression of DNMT1 was increased and myofibroblast markers α-SMA and CTGF were reduced in cells cultured in high stiffness gels after treatment with estradiol, indicating an inhibitory effect on the transformation of fibroblasts into myofibroblasts (Fig. 5 c-f). 4. Discussion According to a recent cross-sectional study, the prevalence of symptomatic pelvic organ prolapse in Chinese women is 9.6%[ 1 ]. One of the risk factors is transvaginal delivery[ 16 ], which can produce a huge stretch on the vaginal tissues by increasing intra-abdominal pressure, thus affecting the function of fibroblasts leading to changes in collagen metabolism, altering the strength of the tissues and eventually prolapse. Another important factor is menopause, which rapidly decreases estrogen production in post-menopausal women, resulting in atrophy, thinning and inflammation of the vagina, which deprives the pelvic floor of the ability to compensate for the trauma associated with childbirth, there by weakening the support network[ 13 ]. In addition, it has been shown that estrogen can influence the level of LOXL1[ 17 ] and thus the occurrence of POP. The connective tissue at the site of prolapse is stiffer and exhibits a higher modulus of elasticity compared to healthy tissue, similar to the characteristics of sarcoid tissue[ 18 ]. Several studies have suggested that the abnormal extracellular matrix in prolapsed tissue may maintain POP by regulating the differentiation of vaginal fibroblasts to myofibroblasts[ 19 – 21 ]. Fibroblasts can sense an increase in environmental stiffness and differentiate into myofibroblasts, a process known as fibroblast myofibroblast transformation[ 22 ]. The marker for myofibroblasts is α-SMA, which accelerates tissue repair during the repair phase of tissue damage, but sustained activation can cause pathological deposition of extracellular matrix and loss of tissue elasticity[ 23 ]. It has been shown that α-SMA levels in the anterior vaginal wall are significantly higher in patients with POP [ 18 ], suggesting that fibroblasts are relatively quiescent under physiological conditions and that myofibroblasts have a higher abundance in POP patients, but the mystery in between remains to be explored. The main treatment for POP is currently surgery. Natural (allograft, autograft and xenograft) and synthetic implants (permanent and biodegradable) have been introduced to surgically correct POP[ 24 , 25 ]. Synthetic permanent polypropylene (PP) mesh has been used for the surgical repair of POP, which provides mechanical support to the pelvic floor by inducing a foreign body response. However, PP mesh has been associated with clinical complications, such as the use of polypropylene mesh that is stiffer than native tissue for vaginal reconstruction, which can further compromise the mechanical properties of the vagina[ 7 , 26 ]. In animal models it has been shown that the mechanical properties of the vagina deteriorate after implantation of stiffer patches, suggesting that not only the vaginal wall but also the stiffness of the mesh can affect the biological properties of the vagina[ 7 ]. In addition, it has been suggested that the increased risk of pelvic floor disorders after menopause may be related to estrogen deficiency and changes in estrogen receptor expression[ 15 ]. Women with persistently low estrogen levels show delayed recovery due to impaired wound healing[ 27 ]. Therefore, the addition of estrogen to the surgical site may promote fibroblast proliferation and collagen synthesis, thereby promoting pelvic floor tissue repair[ 28 ]. In summary, abnormal stiffness in the anterior vaginal wall may maintain POP by regulating fibroblast to myofibroblast differentiation, and estrogen has an irreplaceable role in the treatment of POP. It is therefore clinically important to investigate the fate regulation of fibroblasts by extracellular matrix stiffness and the therapeutic mechanisms of estrogen in POP. Our results found that the anterior vaginal wall of POP patients had a significantly higher elastic modulus than controls, i.e. increased extracellular matrix stiffness, and a significant increase in α-SMA expression in vaginal wall tissue, suggesting a higher abundance of vaginal wall myofibroblasts, but the relationship between extracellular matrix stiffness and fibroblast differentiation remains to be explored. In recent years, there has been evidence that epigenetic mechanisms in vascular smooth muscle cells play a key role in mediating environmental stiffness signalling to induce cell phenotype regulation. However, understanding of the epigenetic regulation triggered by mechanical stimulation of fibroblasts is limited. Here, we report the effects of microenvironmental stiffness on fibroblast differentiation mediated by DNMT1. DNMT1 is a mechanosensitive protein and our study identifies a fundamental role for DNMT1 as a downstream component of mechanosensors in how fibroblasts perceive their physical microenvironment. When the matrix stiffens in elasticity, cells produce less DNMT1, leading to a decrease in DNA methylation levels. The detailed mechanotransduction mechanism by which ECM stiffness regulates DNMT1 awaits further characterisation, but our experimental results suggest that the process involves the polymerisation of microtubules. These findings have potential implications for the study of mechanotransduction and highlight the importance of DNMT1 in mediating the interaction between fibroblast and stromal stiffness. In addition, postmenopausal estrogen reduction is an important risk factor for the development of POP, and it has been shown that estrogen enhances the efficacy of vaginal pelvic floor electrical stimulation (PFS) for pelvic organ dysfunction and delays its recurrence[ 29 ]. We therefore investigated the possibility that estrogen inhibits fibroblast differentiation. We were encouraged to find that estrogen treatment increased DNMT1 expression and decreased α-SMA and CTGF expression in fibroblasts. However, there are some limitations to this study. Firstly, we have not yet determined how physical signals are translated into biological signals through cellular microtubule aggregation. Our preliminary study found that the integrin family and Hippo family may be involved in this process. Second, how estrogen regulates DNMT1 expression remains to be explored, and preliminary studies suggest that estrogen may affect DNMT1 expression through interaction with estrogen receptor2(ER2). Our study suggests a potential mechanism for fibroblast differentiation by showing that when mechanosensitive proteins (in our case DNMT1) receive signals from the mechanical microenvironment, the cells respond by changes in their stiffness, and thus offers new ideas and possibilities for the treatment of patients with POP and the selection of stiffness-related properties of implant materials. This article may contribute to a better understanding of the role of fibroblasts in pelvic organ prolapse and how to optimise surgical approaches, such as the choice of implant material stiffness. 5. Declaration Conflict of Interest Statement We declare that we have no conflict of interest. Each Author Participations Zhihan Zhao &Li Hong contributed to the conception of the study and performed the experiments. Guotao Huang & Yong He to analysis and manuscript preparation; Xiaohu Zuo & Wuyue Han helped perform the analysis with constructive discussions. The abstract has 163 words and the text has 3,303 words. • Ethics approval and consent to participate The experiment was approved by the Medical Ethics Committee of the Wuhan University People's Hospital under the approval number WDRY2021-K001 and the patients signed an informed consent form. All methods were carried out in accordance with relevant guidelines and regulations. The experiments with human samples conformed to the guidelines set by the Declaration of Helsinki. • Consent for publication Not applicable • Availability of data and materials The datasets generated and/or analysed during the current study are available in the GEO database repository, [ GSE18224] • Competing interests Not applicable • Funding The National Key Research and Development Program of China( 2018YFC200220) The National Natural Science Foundation of China (grant nos. 81971364） • Authors' contributions Zhihan Zhao &Li Hong contributed to the conception of the study and performed the experiments. Guotao Huang & Yong He to analysis and manuscript preparation; Xiaohu Zuo & Wuyue Han helped perform the analysis with constructive discussions. • Acknowledgements Not applicable References Pang, H., et al., A nationwide population-based survey on the prevalence and risk factors of symptomatic pelvic organ prolapse in adult women in China - a pelvic organ prolapse quantification system-based study. Bjog, 2021. 128(8): p. 1313-1323. Groenendijk, A.G., et al., Contribution of primary pelvic organ prolapse to micturition and defecation symptoms. Obstet Gynecol Int, 2012. 2012: p. 798035. Blomquist, J.L., et al., Pelvic floor muscle strength and the incidence of pelvic floor disorders after vaginal and cesarean delivery. Am J Obstet Gynecol, 2020. 222(1): p. 62.e1-62.e8. Tomasek, J.J., et al., Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol, 2002. 3(5): p. 349-63. 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Tomaszewski, J., et al., [Collagen type III biosynthesis by cultured pubocervical fascia fibroblasts surrounding mono and multifilament polypropylene mesh after estrogens and tamoxifen treatment]. Ginekol Pol, 2010. 81(7): p. 493-500. Abdelbary, A.M., et al., Combined Vaginal Pelvic Floor Electrical Stimulation (PFS) and Local Vaginal Estrogen for Treatment of Overactive Bladder (OAB) in Perimenopausal Females. Randomized Controlled Trial (RCT). Urology, 2015. 86(3): p. 482-6. Additional Declarations No competing interests reported. Supplementary Files originalimages.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2549183","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":183643862,"identity":"3ff6624f-133c-49dd-b2f3-6332b03b2de0","order_by":0,"name":"Zhihan Zhao","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhihan","middleName":"","lastName":"Zhao","suffix":""},{"id":183643863,"identity":"5ca0f249-c4e6-4590-807b-fb7d3cccffe5","order_by":1,"name":"Guotao Huang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guotao","middleName":"","lastName":"Huang","suffix":""},{"id":183643864,"identity":"100966df-c1b6-4fb6-a22b-41664cb3d018","order_by":2,"name":"Yong He","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"He","suffix":""},{"id":183643865,"identity":"382a780e-54f1-470c-89f6-85b7a07a4715","order_by":3,"name":"Xiaohu Zuo","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaohu","middleName":"","lastName":"Zuo","suffix":""},{"id":183643866,"identity":"fc2e06c0-beaa-4fdc-acd7-dae967e0609d","order_by":4,"name":"Wuyue Han","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wuyue","middleName":"","lastName":"Han","suffix":""},{"id":183643867,"identity":"57484e5a-ae93-47ef-870f-892c9ecc6750","order_by":5,"name":"Li Hong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYDCCAwxsIEqGjb0BzGBsIFYLDxvPAVK1MEgkEKmF7/YBtgcfd9Ty8Em+MXvMw2Aju+EA87MH+LRInktgN5x55jgPm3SOuTEPQ5rxhgNs5gb4tBicYWCT5m07BtSSu02ah+Fw4oYDPGwSxGmRPAvS8p9oLTVAZbwgLQcIa5EEapGc2QZUxpP/TXKOQbLxzMNsZni18AG1SHxsq5OTbz+WJvGmwk6273jzM7xaGBj4PwCJwzB3AjEzfvUwUEecslEwCkbBKBiZAABwSD8nER3aWwAAAABJRU5ErkJggg==","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Hong","suffix":""}],"badges":[],"createdAt":"2023-02-04 06:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2549183/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2549183/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34597924,"identity":"dc6dccf8-9441-49ce-bb04-486e54c26454","added_by":"auto","created_at":"2023-03-21 15:54:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594473,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in the expression of myofibroblast-related proteins between pop patients and controls (a) α-SMA immunohistochemical staining for control and POP patients; (b-c) Western blotting of α-SMA protein in control and POP patients.(d-g) Western blotting of CollagenI and CollagenIII protein in control and POP patients. (h) Immunofluorescence of CollagenI and CollagenIII protein in control and POP patients.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2549183/v1/a63bb1087a0778ed41a672dd.png"},{"id":34597926,"identity":"487508f8-b453-4feb-ac45-5b24ee534d46","added_by":"auto","created_at":"2023-03-21 15:54:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":510356,"visible":true,"origin":"","legend":"\u003cp\u003eExtracellular matrix stiffness affects fibroblast-to-myofibroblast transformation (a)Flow chart of medium gel production. (b-c) Orthotopic microscope for cell culture; (d-e) Western blotting results of α-SMA protein. (f) Immunofluorescent staining was performed in indicated cells using α-SMA(red) and DAPI (blue)(3 independent experiments; at least 10 cells were observed) .\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2549183/v1/4b2ebe1db2adcf464523f96b.png"},{"id":34598483,"identity":"02c0daad-a6ed-4a59-9cdf-d3d4836a5cca","added_by":"auto","created_at":"2023-03-21 16:02:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":639922,"visible":true,"origin":"","legend":"\u003cp\u003eExtracellular matrix stiffness regulates fibroblast-to-myofibroblast transition through DNMT1 (a-c) Western blot and immunohistochemical staining of DNMT1 protein in the anterior vaginal wall of control and POP patients; (d,f) Expression of DNMT1 in cells cultured on gel with different stiffness.(e,f) The protein levels of these genes were detected by Western blot. Cells were treated with 5 '-AZA or PBS as control group and cultured on soft PA gel.(g)L929 cells were immunostained with anti-DNMT1 (red) and DAPI (blue) and then visualised using a co-polymer microscope.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2549183/v1/b1de2a0da91739e49c7c2ad0.png"},{"id":34597925,"identity":"1db17a75-5ccf-4087-b011-755aa9bf2efc","added_by":"auto","created_at":"2023-03-21 15:54:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":537312,"visible":true,"origin":"","legend":"\u003cp\u003eCells convert physical signals to biological signals through the microtubule cytoskeleton(a) Gene correlation analysis showed that DNMT1 expression was significantly correlated with genes related to microtubule polymerization; (b,c) The morphological differences of cells cultured with high stiffness gel and added with Nocodazole were observed under forward microscope.(d,e) The protein levels of these genes were detected by Western blot. Cells were treated with 5 '-AZA or PBS as control group and cultured on soft PA gel.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2549183/v1/c6a718a17ad857bf94f931f3.png"},{"id":34598484,"identity":"e8605e0f-3a9e-480b-a3da-63ac5a5cdbbe","added_by":"auto","created_at":"2023-03-21 16:02:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":487974,"visible":true,"origin":"","legend":"\u003cp\u003eEstrogen partially inhibits fibroblast differentiation induced by high stiffness gel （a，b）The cell morphology was observed under a forward microscope, and the fiber cells composed of 40kpa gel + estrogen were mostly round in shape, with a lower aspect ratio. (c-e) Western blotting results of 40kpa group and 40kpa+ estrogen group. (f)L929 cells were immunostained with anti-DNMT1 (red) and DAPI (blue) and then visualised using zhe microscope.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2549183/v1/8de39c362f56f256dbf45ee0.png"},{"id":42483309,"identity":"c3a3c269-5e57-41af-8791-6f909a97106a","added_by":"auto","created_at":"2023-09-01 09:07:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2046378,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2549183/v1/32d2fbf9-0a5b-4ae6-8ee3-f702d43eb813.pdf"},{"id":34598485,"identity":"ed3c2a0b-be31-491e-8546-4e9fb44dd542","added_by":"auto","created_at":"2023-03-21 16:02:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2717849,"visible":true,"origin":"","legend":"","description":"","filename":"originalimages.docx","url":"https://assets-eu.researchsquare.com/files/rs-2549183/v1/d11e1036133579916926d6d1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Estrogen inhibits the differentiation of fibroblasts induced by high stiffness matrix by enhancing DNMT1 expression","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePelvic organ prolapse(POP) is a multifactorial connective tissue disorder caused by damage to the supporting structures of the pelvic floor. Epidemiology shows that the prevalence of symptomatic POP in China is 9.1%[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The lives of POP patients are severely impacted and with significant global ageing, the prevention and treatment of POP is receiving increasing attention[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To date, the pathogenesis of pelvic organ prolapse is still unknown.\u003c/p\u003e \u003cp\u003eOne of the main risk factors for POP is stress injury during which extracellular matrix remodelling occurs[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Fibroblasts are widespread in connective tissue and are the main cells regulating the composition of the extracellular matrix, their differentiation into myofibroblasts is a key feature of wound healing in soft tissues such as the vagina[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Several studies have demonstrated that the microenvironment of vaginal tissue in patients with POP is more rigid than that of healthy vagina[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. When fibroblasts grow in a stiffer environment, some of them differentiate into myofibroblasts, marked by α-SMA, CTGF and type I collagen production[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The prolonged presence of myofibroblasts results in excessive collagen deposition, tissue contracture and dysfunction[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDNA methylation is known to play an important role in the maintenance of normal development and biological function[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Covalent methylation of DNA cytosines promotes transcriptional repression in most cases. The importance of DNA methylation is reflected in the development of many diseases that are due to hypermethylation or hypomethylation of DNA and inappropriate temporal or spatial regulation[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The maintenance of methylation patterns during cell replication is mediated by DNA methyltransferase1, which catalyzes the transfer of methyl from S-adenosylmethionine to hemimethylated DNA[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEstrogen plays an important role in the development of POP. Estrogen deficiency may contribute to the development of POP as it has a profound effect on pelvic collagen synthesis, and the strength of connective tissue[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In vitro studies have found that estrogen improves the mechanical properties of connective tissue and inhibits excessive and abnormal remodelling of the fibrous extracellular matrix in response to mechanical stimulation and stretching[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It has been observed that low estrogen levels in women undergoing vaginal surgery can affect the outcome of the procedure, and women with persistently low estrogen levels have a slower recovery due to impaired wound healing[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The addition of estrogen to the surgical site may promote pelvic floor tissue repair. In summary, estrogen has positive implications for the treatment of pelvic floor organ prolapse, but the mechanisms that may be involved are unclear.\u003c/p\u003e \u003cp\u003eOur study provides preliminary evidence that estrogen can inhibit high stiffness matrix-induced fibroblast differentiation by enhancing DNMT1 expression, and that the conversion of mechanical to biological signals involves the involvement of cellular microtubules.\u003c/p\u003e"},{"header":"2. Methods And Treatment","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell Culture and Treatment\u003c/h2\u003e \u003cp\u003eAll experiments were performed using L929 cells, a mouse fibroblast, purchased from the China Type Culture Centre (Wuhan, China) and cultured in RPMI Medium 1640 medium (Gibco, America) at 10% serum concentration (Yeasan, China). Cells were passaged or cryopreserved after they reached approximately 80% confluence. To inhibit DNMT1 expression, cells were cultured in medium for 24h and then replaced with medium containing Decitabine(5\u0026rsquo;-Aza) (5umol/L, MedChemExpress, China). To inhibit myosin ATPase and microtubule assembly, cells were incubated on gel for 6 h and then replaced with medium containing nocodazole (5umol/L, MedChemExpress, China) for 5 h. Afterwards, the medium was changed and the cells continued to be incubated for 24 h. To observe the effect of estrogen on fibroblast differentiation, cells were incubated in gel for 24 h and then replaced with medium containing estrogen (10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e mol/L Sigma, America) medium for 24h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Human Specimen\u003c/h2\u003e \u003cp\u003eTo investigate the differences in myofibroblast expression in the anterior vaginal wall between patients with POP and normal women, patients with Grade III prolapse who underwent hysterectomy and pelvic floor reconstruction were the experimental group and patients who underwent total hysterectomy for other benign gynaecological conditions were the control group, with whole layers of anterior vaginal wall tissue taken during surgery. The experiment was approved by the Medical Ethics Committee of the Wuhan University People's Hospital under the approval number WDRY2021-K001 and the patients signed an informed consent form. The experiments with human samples conformed to the guidelines set by the Declaration of Helsinki Patient information is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient Informations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003ePOP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c11\" namest=\"c7\"\u003e \u003cp\u003enon-POP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e27.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e28.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiagnosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProlapse of the anterior vaginal wall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUterine prolapse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUterine prolapse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUterine prolapse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProlapse of the anterior vaginal wal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAdenomyosis of the uterus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMultiple leiomyomas of the uterus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMultiple leiomyomas of the uterus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAdenomyosis of the uterus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAdenomyosis of the uterus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePOP-Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 ECM Surface Micro-Stiffness\u003c/h2\u003e \u003cp\u003eThe mechanical properties of ECM were measured by microindentation for measuring the microhardness of biological tissue surfaces. Samples were indented in PBS with a radius of 95 m, a stiffness of 0.5 N/m and a loading and unloading cycle of 3 seconds using PIUMA, an instrument that provides mechanical properties such as microscale nanoindentation for biological material tissues and cells. The effective Young's modulus was calculated automatically by the PIUMA software based on the load and displacement curves.Five samples were included in the POP and control groups, and the experiment was repeated three times for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blot\u003c/h2\u003e \u003cp\u003e2.4.1Western blot of the human anterior vaginal wall\u003c/p\u003e \u003cp\u003eThe anterior vaginal wall was removed intraoperatively and immediately placed in an EP tube filled with PBS for further processing. The mucosal epithelial layer and epithelial tissue were removed with surgical scissors, preserving the mucosal lamina propria and the muscularis, cut into fine pieces with scissors, weighed on an electronic balance, and lysis solution (RIPA Buffer, Cocktail, phosphorylase inhibitor, PMSF) was added per 100 mg/1 mL, mixed into a homogenate using a homogenizer, and centrifuged at 12000g for 15 minutes in an ice bath, and the supernatant was taken as protein. The BCA method was used to determine the protein concentration for subsequent experiments.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Western blot of L929 cells\u003c/h2\u003e \u003cp\u003eCells were harvested using a spatula and washed three times with pre-cooled phosphate buffered saline (PBS). The cells were then lysed in lysate, centrifuged at 12,000g for 15 minutes, and the supernatant was aspirated to collect the proteins. bca assay was used to determine protein concentrations for subsequent experiments.\u003c/p\u003e \u003cp\u003eThe antibody used was a rabbit polyclonal antibody (pAb) against DNMT1 (purchased from Cell Signaling Technology). Rabbit polyclonal antibody against α-SMA, mouse polyclonal antibody against GADPH, rabbit polyclonal antibody against Collagen1, rabbit polyclonal antibody against Collagen3, rabbit polyclonal antibody against CTGF, polyclonal antibody against β-actin (purchased from Proteintech).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin-embedded whole vaginal tissue sections were dewaxed at 65\u0026deg;C and subjected to antigen repair in citrate buffer solution (0.01 mol/L, pH 6.0) for 30 min at 100\u0026deg;C. After being blocked with 5% bovine serum albumin (BSA) for 1 h, the sections were incubated with anti-α-SMA or anti-DNMT1 antibodies at 4\u0026deg;C overnight. After incubation with biotin-labelled secondary antibodies for 1 hour at 37\u0026deg;C, stains were then added and all stained slides were observed under an Olympus BX50 microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescence\u003c/h2\u003e \u003cp\u003eL929 cells were seeded on strain plates and treated as indicated in each experiment, followed by fixation with 4% formaldehyde for 30 min and blocking with 5% Bovine Serum Albumin (BSA) for 1 h. Cells were then incubated with anti-DNMT11 (1:100) and anti-α-SMA (1:100) overnight at 4\u0026deg;C. Next, cells were further incubated with FITC-conjugated goat anti-rabbit IgG (1:200, antgene, China). 4', 6'-Diamidino-2-phenylindole (DAPI, antgene, China) was used to stain cell nuclei. Complete images of the cells were taken using a fluorescent microscope (Olympus IX71) and analysed by ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 CCK8 assay\u003c/h2\u003e \u003cp\u003eCell viability was estimated by CCK-8 assay (Seven, China). Approximately 10\u003csup\u003e4\u003c/sup\u003e cells were inoculated in 96-well plates with 100 \u0026micro;l of medium per well. After 24 h of incubation, different doses of Decitabine or nocodazole were added, respectively. Each well was incubated for 2 h with 10 \u0026micro;g CCK-8 solution protected from light before absorbance at 450 nm was measured using an enzyme marker.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Preparation of Polyacrylamide Gel (PA Gel) Substrates\u003c/h2\u003e \u003cp\u003eThe polyacrylamide gel (PA gel) substrate was prepared according to Justin R et al. by varying the ratio of acrylamide to methacrylamide to adjust the stiffness of the PA gel to 4 kpa and 40 kpa. The prepared liquid was mixed and added to parallel glasses spaced 1.0 mm apart and left to set for about 1 hr. The PA gels were washed 2\u0026ndash;3 times with PBS, covered with photocrosslinker (Sulfo-SANPA, abcam, 0.5 mg/ml in PBS) and irradiated with UV light for 1 h. The PA gels were washed 2\u0026ndash;3 times with PBS to remove the excess photocrosslinker. Collagen I (15 ug/mL, Biosharp) was spread on the surface of the gel and placed in a 4\u0026deg;C chamber overnight. The following day, the collagen solution is poured off, washed 2\u0026ndash;3 times with PBS to remove excess collagen, dried overnight and the collagen-coated PA gel is ready for cell inoculation the following day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Microarray data archive\u003c/h2\u003e \u003cp\u003eExpression profiles for the GSE18224 array were retrieved from the GEO database. GSE18224 collected the left ventricular transcriptome of four mice from the Affymetrix RAE 430 2.0 GeneChip array testing each combination of three factors: sex, genotype, and surgical modality. The expression profile of the database was based on the GPL 1261 platform. Series matrix files and data table header descriptions for this database were downloaded from the GEO database to screen for genes with significant correlation to DNMT1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and performed using SPSS version 21.0 software (IBM Corporation, Armonk, NY, USA). Differences between two groups were determined using Student\u0026rsquo;s t test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Mechanical and biological differences between patients with POP and control.\u003c/h2\u003e \u003cp\u003eThe tissue of the anterior vaginal wall was taken intraoperatively and the mechanical properties of the vaginal wall were measured using a microindenter. The results showed that the Young's modulus of the anterior vaginal wall tissue was significantly higher in the POP patients (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Western blot results revealed that the expression of α-SMA, a marker of myofibroblast, in the anterior vaginal wall was significantly higher in POP patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c). We also examined the expression of Collagen I and III in the POP and control groups. Western blot and immunofluorescence results displayed no significant difference in the expression of CollagenI and increased expression of CollagenIII in the POP group (Figur1d-h).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eYoung's modulus of the anterior vaginal wall\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeature\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eECM-Healthy\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eECM-POP\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep Value\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEffective Young modulus (kPa)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.26\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.54\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0044\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Extracellular matrix stiffness affects fibroblast to myofibroblast transformation\u003c/h2\u003e \u003cp\u003eTo investigate the effect of extracellular matrix stiffness on fibroblast phenotype, we prepared PA gels with different stiffnesses (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). L929 was grown on the gels for 24 h and morphological observations were made under an inverted microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The cells showed polymorphic morphology such as star-shaped and long spindle-shaped after 24 h on the high stiffness gels, whereas growth on the low stiffness gels was mainly round. Aspect ratio calculations showed that the cells cultured on the high stiffness gels was significantly higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). Western Blot and immunofluorescence results showed that α-SMA expression was increased in cells cultured in high stiffness gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePA gels Preparation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcrylamide %\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBisacrylamide%\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcrylamide from 40%stock solution (ml)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBis-acrylamide\u003c/p\u003e \u003cp\u003efrom 2% stock\u003c/p\u003e \u003cp\u003esolution (ml)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003cp\u003e(ml)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE ± St. Dev.\u003c/p\u003e \u003cp\u003e(kPa)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e2.24 ± 0.58\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e40.4 ± 2.39\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Extracellular matrix stiffness regulates fibroblast to myofibroblast transformation via DNMT1\u003c/h2\u003e \u003cp\u003eThe key role of epigenetic mechanisms (DNA methylation and demethylation, histone modification and RNA-based mechanisms) in mediating the regulation of smooth muscle phenotype induced by environmental signals has been suggested, but whether DNMT1 is involved in the fibroblast to myofibroblast transformation and or regulated by extracellular matrix stiffness has not been investigated. Therefore, we first examined DNMT1 expression in the anterior vaginal wall of POP patients and controls. Western blot results showed reduced DNMT1 expression in the anterior vaginal wall of POP patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b), and immunohistochemistry verified these results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Subsequently, we cultured L929 in different stiffness gels for 48h, the western blots showed that the cells had significantly lower DNMT1 levels after 48h incubation on high stiffness gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,f) and immunofluorescence verified these results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). To investigate whether extracellular matrix stiffness affects the transformation of fibroblasts to myofibroblasts via DNMT1, we added DNMT1 inhibitors to the low stiffness gel medium. We tested the chemosensitivity of L929 to Decitabine by the CCK8 assay, with a gradient of Decitabine concentrations ranging from 0.5 to 10 umol/L and a final treatment concentration of 5umol/L. The results showed a significant decrease in DNMT1 expression and an increase in α-SMA, CTGF expression in the DNMT1 inhibitor group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cells transform physical signals into biological signals through the microtubule cytoskeleton\u003c/h2\u003e \u003cp\u003eHow extracellular matrix stiffness translates physical signals into intracellular biological signals is unclear. Cellular microtubules are known to have kinetic properties of aggregation and depolymerization and play an important role in the maintenance of cell morphology, cell division, signal transduction and material transport. Therefore, we retrieved the expression profiles of the GSE18224 array from the GEO database and used R software to screen for genes with significant relevance to DNMT1. The results showed that the microtubule-associated proteins Rmdn1, Mast2 and DNMT1 were significantly correlated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). We therefore treated the cells with Nocodazole, a rapid and reversible inhibitor of microtubule polymerisation, and we tested the chemosensitivity of L929 to Nocodazole by the CCK8 assay. We observed significant morphological differences in the cells in the different stiffness gels(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c). Western blot results showed that Nocodazole treatment eliminated the down-regulation of DNMT1 expression induced by high stiffness gels and reduced α-SMA, CTGF expression (Figur4d,e) suggesting a potential role for the microtubule cytoskeleton in the regulation of DNMT1 expression by gel stiffness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Estrogen partially inhibits fibroblast differentiation induced by high stiffness gel\u003c/h2\u003e \u003cp\u003eEstrogen plays an important role in the development of POP, but the mechanisms involved are unclear. We therefore explored whether there is an inhibitory effect of estrogen on high stiffness-induced fibroblast transformation. We added estradiol to the culture medium and assayed the chemosensitivity of L929 to estradiol treatment by CCK8. The concentration gradient of estradiol was 10\u003csup\u003e− 8\u003c/sup\u003e~ 10\u003csup\u003e− 5\u003c/sup\u003e mol/L, and the final treatment concentration was 10\u003csup\u003e− 7\u003c/sup\u003e mol/L. The results showed that the cells cultured in the high stiffness gels showed a reduction in transformation and a decrease in the aspect ratio of the cells after estrogen treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). The western blot and immunofluorescence showed that the expression of DNMT1 was increased and myofibroblast markers α-SMA and CTGF were reduced in cells cultured in high stiffness gels after treatment with estradiol, indicating an inhibitory effect on the transformation of fibroblasts into myofibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAccording to a recent cross-sectional study, the prevalence of symptomatic pelvic organ prolapse in Chinese women is 9.6%[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One of the risk factors is transvaginal delivery[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which can produce a huge stretch on the vaginal tissues by increasing intra-abdominal pressure, thus affecting the function of fibroblasts leading to changes in collagen metabolism, altering the strength of the tissues and eventually prolapse. Another important factor is menopause, which rapidly decreases estrogen production in post-menopausal women, resulting in atrophy, thinning and inflammation of the vagina, which deprives the pelvic floor of the ability to compensate for the trauma associated with childbirth, there by weakening the support network[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, it has been shown that estrogen can influence the level of LOXL1[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and thus the occurrence of POP. The connective tissue at the site of prolapse is stiffer and exhibits a higher modulus of elasticity compared to healthy tissue, similar to the characteristics of sarcoid tissue[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Several studies have suggested that the abnormal extracellular matrix in prolapsed tissue may maintain POP by regulating the differentiation of vaginal fibroblasts to myofibroblasts[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e–\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Fibroblasts can sense an increase in environmental stiffness and differentiate into myofibroblasts, a process known as fibroblast myofibroblast transformation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The marker for myofibroblasts is α-SMA, which accelerates tissue repair during the repair phase of tissue damage, but sustained activation can cause pathological deposition of extracellular matrix and loss of tissue elasticity[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been shown that α-SMA levels in the anterior vaginal wall are significantly higher in patients with POP [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], suggesting that fibroblasts are relatively quiescent under physiological conditions and that myofibroblasts have a higher abundance in POP patients, but the mystery in between remains to be explored.\u003c/p\u003e\u003cp\u003eThe main treatment for POP is currently surgery. Natural (allograft, autograft and xenograft) and synthetic implants (permanent and biodegradable) have been introduced to surgically correct POP[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Synthetic permanent polypropylene (PP) mesh has been used for the surgical repair of POP, which provides mechanical support to the pelvic floor by inducing a foreign body response. However, PP mesh has been associated with clinical complications, such as the use of polypropylene mesh that is stiffer than native tissue for vaginal reconstruction, which can further compromise the mechanical properties of the vagina[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In animal models it has been shown that the mechanical properties of the vagina deteriorate after implantation of stiffer patches, suggesting that not only the vaginal wall but also the stiffness of the mesh can affect the biological properties of the vagina[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, it has been suggested that the increased risk of pelvic floor disorders after menopause may be related to estrogen deficiency and changes in estrogen receptor expression[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Women with persistently low estrogen levels show delayed recovery due to impaired wound healing[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, the addition of estrogen to the surgical site may promote fibroblast proliferation and collagen synthesis, thereby promoting pelvic floor tissue repair[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn summary, abnormal stiffness in the anterior vaginal wall may maintain POP by regulating fibroblast to myofibroblast differentiation, and estrogen has an irreplaceable role in the treatment of POP. It is therefore clinically important to investigate the fate regulation of fibroblasts by extracellular matrix stiffness and the therapeutic mechanisms of estrogen in POP.\u003c/p\u003e\u003cp\u003eOur results found that the anterior vaginal wall of POP patients had a significantly higher elastic modulus than controls, i.e. increased extracellular matrix stiffness, and a significant increase in α-SMA expression in vaginal wall tissue, suggesting a higher abundance of vaginal wall myofibroblasts, but the relationship between extracellular matrix stiffness and fibroblast differentiation remains to be explored. In recent years, there has been evidence that epigenetic mechanisms in vascular smooth muscle cells play a key role in mediating environmental stiffness signalling to induce cell phenotype regulation. However, understanding of the epigenetic regulation triggered by mechanical stimulation of fibroblasts is limited. Here, we report the effects of microenvironmental stiffness on fibroblast differentiation mediated by DNMT1. DNMT1 is a mechanosensitive protein and our study identifies a fundamental role for DNMT1 as a downstream component of mechanosensors in how fibroblasts perceive their physical microenvironment. When the matrix stiffens in elasticity, cells produce less DNMT1, leading to a decrease in DNA methylation levels. The detailed mechanotransduction mechanism by which ECM stiffness regulates DNMT1 awaits further characterisation, but our experimental results suggest that the process involves the polymerisation of microtubules. These findings have potential implications for the study of mechanotransduction and highlight the importance of DNMT1 in mediating the interaction between fibroblast and stromal stiffness. In addition, postmenopausal estrogen reduction is an important risk factor for the development of POP, and it has been shown that estrogen enhances the efficacy of vaginal pelvic floor electrical stimulation (PFS) for pelvic organ dysfunction and delays its recurrence[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We therefore investigated the possibility that estrogen inhibits fibroblast differentiation. We were encouraged to find that estrogen treatment increased DNMT1 expression and decreased α-SMA and CTGF expression in fibroblasts. However, there are some limitations to this study. Firstly, we have not yet determined how physical signals are translated into biological signals through cellular microtubule aggregation. Our preliminary study found that the integrin family and Hippo family may be involved in this process. Second, how estrogen regulates DNMT1 expression remains to be explored, and preliminary studies suggest that estrogen may affect DNMT1 expression through interaction with estrogen receptor2(ER2). Our study suggests a potential mechanism for fibroblast differentiation by showing that when mechanosensitive proteins (in our case DNMT1) receive signals from the mechanical microenvironment, the cells respond by changes in their stiffness, and thus offers new ideas and possibilities for the treatment of patients with POP and the selection of stiffness-related properties of implant materials. This article may contribute to a better understanding of the role of fibroblasts in pelvic organ prolapse and how to optimise surgical approaches, such as the choice of implant material stiffness.\u003c/p\u003e"},{"header":"5. Declaration","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that we have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEach Author Participations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhihan Zhao \u0026amp;Li Hong\u0026nbsp;contributed to the conception of the study and performed the experiments.\u003c/p\u003e\n\u003cp\u003eGuotao Huang \u0026amp; Yong He to analysis and manuscript preparation;\u003c/p\u003e\n\u003cp\u003eXiaohu Zuo \u0026amp; Wuyue Han helped perform the analysis with constructive discussions.\u003c/p\u003e\n\u003cp\u003eThe abstract has 163 words and the text has 3,303 words.\u003c/p\u003e\n\u003cp\u003e\u0026bull;\u0026nbsp;Ethics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe experiment was approved by the Medical Ethics Committee of the Wuhan University People\u0026apos;s Hospital under the approval number WDRY2021-K001 and the patients signed an informed consent form. All methods were carried out in accordance with relevant guidelines and regulations. The experiments with human samples conformed to the guidelines set by the Declaration of Helsinki.\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u0026bull;\u0026nbsp;Consent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u0026bull;\u0026nbsp;Availability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the\u0026nbsp;GEO database\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;repository, [\u0026nbsp;GSE18224]\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u0026bull;\u0026nbsp;Competing interests\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u0026bull;\u0026nbsp;Funding\u003c/p\u003e\n\u003cp\u003eThe National Key Research and Development Program of China( 2018YFC200220)\u003c/p\u003e\n\u003cp\u003eThe National Natural Science Foundation of China (grant nos. 81971364）\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u0026bull;\u0026nbsp;Authors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eZhihan Zhao \u0026amp;Li Hong\u0026nbsp;contributed to the conception of the study and performed the experiments.\u003c/p\u003e\n\u003cp\u003eGuotao Huang \u0026amp; Yong He to analysis and manuscript preparation;\u003c/p\u003e\n\u003cp\u003eXiaohu Zuo \u0026amp; Wuyue Han helped perform the analysis with constructive discussions.\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u0026bull;\u0026nbsp;Acknowledgements\u003cbr\u003e\u0026nbsp;Not applicable\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePang, H., et al., A nationwide population-based survey on the prevalence and risk factors of symptomatic pelvic organ prolapse in adult women in China - a pelvic organ prolapse quantification system-based study. Bjog, 2021. 128(8): p. 1313-1323.\u003c/li\u003e\n\u003cli\u003eGroenendijk, A.G., et al., Contribution of primary pelvic organ prolapse to micturition and defecation symptoms. Obstet Gynecol Int, 2012. 2012: p. 798035.\u003c/li\u003e\n\u003cli\u003eBlomquist, J.L., et al., Pelvic floor muscle strength and the incidence of pelvic floor disorders after vaginal and cesarean delivery. Am J Obstet Gynecol, 2020. 222(1): p. 62.e1-62.e8.\u003c/li\u003e\n\u003cli\u003eTomasek, J.J., et al., Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol, 2002. 3(5): p. 349-63.\u003c/li\u003e\n\u003cli\u003eMartins, P., et al., Biomechanical properties of vaginal tissue in women with pelvic organ prolapse. Gynecol Obstet Invest, 2013. 75(2): p. 85-92.\u003c/li\u003e\n\u003cli\u003eZhou, L., et al., Biomechanical properties and associated collagen composition in vaginal tissue of women with pelvic organ prolapse. J Urol, 2012. 188(3): p. 875-80.\u003c/li\u003e\n\u003cli\u003eFeola, A., et al., Deterioration in biomechanical properties of the vagina following implantation of a high-stiffness prolapse mesh. Bjog, 2013. 120(2): p. 224-232.\u003c/li\u003e\n\u003cli\u003eDarby, I.A., et al., Fibroblasts and myofibroblasts in wound healing. Clin Cosmet Investig Dermatol, 2014. 7: p. 301-11.\u003c/li\u003e\n\u003cli\u003eGrant, C.A., P.C. Twigg, and D.J. Tobin, Static and dynamic nanomechanical properties of human skin tissue using atomic force microscopy: effect of scarring in the upper dermis. Acta Biomater, 2012. 8(11): p. 4123-9.\u003c/li\u003e\n\u003cli\u003ePortela, A. and M. Esteller, Epigenetic modifications and human disease. Nat Biotechnol, 2010. 28(10): p. 1057-68.\u003c/li\u003e\n\u003cli\u003eQin, W., H. Leonhardt, and G. Pichler, Regulation of DNA methyltransferase 1 by interactions and modifications. Nucleus, 2011. 2(5): p. 392-402.\u003c/li\u003e\n\u003cli\u003eZhang, W., et al., Epigenetic Modifications in Cardiovascular Aging and Diseases. Circ Res, 2018. 123(7): p. 773-786.\u003c/li\u003e\n\u003cli\u003eLang, J.H., et al., Estrogen levels and estrogen receptors in patients with stress urinary incontinence and pelvic organ prolapse. Int J Gynaecol Obstet, 2003. 80(1): p. 35-9.\u003c/li\u003e\n\u003cli\u003eReddy, R.A., et al., Role of sex steroid hormones in pelvic organ prolapse. Menopause, 2020. 27(8): p. 941-951.\u003c/li\u003e\n\u003cli\u003eDeng, Z.M., et al., Advances in molecular mechanisms of pelvic organ prolapse (Review). Exp Ther Med, 2021. 22(3): p. 1009.\u003c/li\u003e\n\u003cli\u003ePoncet, S., et al., The expression and function of the endothelin system in contractile properties of vaginal myofibroblasts of women with uterovaginal prolapse. Am J Obstet Gynecol, 2005. 192(2): p. 426-32.\u003c/li\u003e\n\u003cli\u003eSflomos, G., et al., Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1. EMBO Mol Med, 2021. 13(3): p. e13180.\u003c/li\u003e\n\u003cli\u003eRuiz-Zapata, A.M., et al., Extracellular Matrix Stiffness and Composition Regulate the Myofibroblast Differentiation of Vaginal Fibroblasts. Int J Mol Sci, 2020. 21(13).\u003c/li\u003e\n\u003cli\u003eRuiz-Zapata, A.M., et al., Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates. Int Urogynecol J, 2013. 24(9): p. 1567-75.\u003c/li\u003e\n\u003cli\u003eWang, S., et al., Effects of mechanical stretching on the morphology and cytoskeleton of vaginal fibroblasts from women with pelvic organ prolapse. Int J Mol Sci, 2015. 16(5): p. 9406-19.\u003c/li\u003e\n\u003cli\u003eMeyer, S., et al., The contractile properties of vaginal myofibroblasts: is the myofibroblasts contraction force test a valuable indication of future prolapse development? Int Urogynecol J Pelvic Floor Dysfunct, 2008. 19(10): p. 1399-403.\u003c/li\u003e\n\u003cli\u003eNajafi, M., B. Farhood, and K. Mortezaee, Extracellular matrix (ECM) stiffness and degradation as cancer drivers. J Cell Biochem, 2019. 120(3): p. 2782-2790.\u003c/li\u003e\n\u003cli\u003eLiu, M., B. L\u0026oacute;pez de Juan Abad, and K. Cheng, Cardiac fibrosis: Myofibroblast-mediated pathological regulation and drug delivery strategies. Adv Drug Deliv Rev, 2021. 173: p. 504-519.\u003c/li\u003e\n\u003cli\u003eKalkan, U., et al., Native tissue repair versus mesh repair in pelvic organ prolapse surgery. Climacteric, 2017. 20(6): p. 510-517.\u003c/li\u003e\n\u003cli\u003eRichter, L.A. and A.I. Sokol, Pelvic Organ Prolapse---Vaginal and Laparoscopic Mesh: The Evidence. Obstet Gynecol Clin North Am, 2016. 43(1): p. 83-92.\u003c/li\u003e\n\u003cli\u003eLiang, R., et al., Vaginal degeneration following implantation of synthetic mesh with increased stiffness. Bjog, 2013. 120(2): p. 233-243.\u003c/li\u003e\n\u003cli\u003eCaruso, S., et al., Effects of ultralow topical estriol dose on vaginal health and quality of life in postmenopausal women who underwent surgical treatment for pelvic organ prolapse. Menopause, 2017. 24(8): p. 900-907.\u003c/li\u003e\n\u003cli\u003eTomaszewski, J., et al., [Collagen type III biosynthesis by cultured pubocervical fascia fibroblasts surrounding mono and multifilament polypropylene mesh after estrogens and tamoxifen treatment]. Ginekol Pol, 2010. 81(7): p. 493-500.\u003c/li\u003e\n\u003cli\u003eAbdelbary, A.M., et al., Combined Vaginal Pelvic Floor Electrical Stimulation (PFS) and Local Vaginal Estrogen for Treatment of Overactive Bladder (OAB) in Perimenopausal Females. Randomized Controlled Trial (RCT). Urology, 2015. 86(3): p. 482-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2549183/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2549183/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction and Hypothesis:\u003c/h2\u003e \u003cp\u003ePelvic organ prolapse(POP) is a multifactorial connective tissue disorder caused by damage to the supporting structures of the pelvic floor. Evidence from several studies suggests that anterior vaginal wall stiffness is higher in patients with POP, but the mechanisms involved remain unknown.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTissue from the anterior vaginal wall of patients with POP or other benign diseases was obtained. The modulus of elasticity of the anterior vaginal wall was measured using a microindenter. Cells were cultured in vitro on acrylamide gels of different stiffness and treated with DNMT1 inhibitor, microtubule polymerisation inhibitor and estrogen. Western blot or immunohistochemical staining was performed to detect DNA Methyltransferase 1, α-smooth muscle actin(α-SMA) expression, and connective tissue growth factor(CTGF) expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eEstrogen can inhibit high stiffness matrix-induced fibroblast differentiation, by enhancing DNMT1 expression. This study may help to elucidate the complex crosstalk between fibroblasts and their surrounding matrix under healthy and pathological conditions and provide new insights into the options for material-related therapeutic applications.\u003c/p\u003e","manuscriptTitle":"Estrogen inhibits the differentiation of fibroblasts induced by high stiffness matrix by enhancing DNMT1 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-21 15:54:53","doi":"10.21203/rs.3.rs-2549183/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7dbe4da1-9ddd-4eed-a5f9-751fc235aae3","owner":[],"postedDate":"March 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-01T08:59:17+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-21 15:54:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2549183","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2549183","identity":"rs-2549183","version":["v1"]},"buildId":"qQ7_6M8ijIrYJ9CiyUnPg","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}