Yulinzhu Decoction Modulates the Mechanical Properties of POI Rats Ovarian Granulosa Cells via Microfluidic Chip Technology

Yulinzhu Decoction Modulates the Mechanical Properties of POI Rats Ovarian Granulosa Cells via Microfluidic Chip Technology | 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 Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Yulinzhu Decoction Modulates the Mechanical Properties of POI Rats Ovarian Granulosa Cells via Microfluidic Chip Technology Qingqing Yang, Xin Ruan, Pengxu Wang, Jifeng Ren, Xiaoying Dong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6794919/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 The ovarian follicle, composed of an oocyte surrounded by multiple layers of granulosa cells, relies on the structural stability of granulosa cells for efficient substance exchange. In this study, we investigated the mechanical properties of granulosa cells in a rat model of primary ovarian insufficiency (POI) using microfluidic chip technology. We found that POI rats exhibited increased ovarian fibrosis and a higher number of atretic follicles, which disrupted the homeostasis of the ovarian microenvironment and altered the mechanical properties of granulosa cells. YAP (Yes-associated protein), a mechanosensitive transcription factor, was identified as a key regulator of granulosa cell morphology through mechanical signaling. Yulinzhu decoction, a traditional Chinese medicine formula, improved the ovarian microenvironment and modulated YAP signaling, thereby enhancing the mechanical properties and function of granulosa cells. Our findings propose a novel mechanism by which Yulinzhu decoction regulates the mechanical properties of granulosa cells in POI rats through YAP modulation. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Primary ovarian insufficiency (POI) is characterized by diminished ovarian reserve function and compromised oocyte quality [ 1 ] . The ovary’s basic functional unit, the follicle, progresses through five developmental stages: primordial, primary, secondary, antral, and mature follicles. Primordial follicles consist of an oocyte surrounded by a layer of flat granulosa cells. As follicles develop into primary follicles, these flat granulosa cells transition into cuboidal cells. Further development into secondary follicles is marked by the proliferation of granulosa cells into a bilayered structure [ 2 ] . In the antral follicle stage, granulosa cells differentiate into mural granulosa cells (MGCs) and cumulus cells (CCs), forming a stable cubic structure around the oocyte by establishing gap junctions [ 3 ] . Oocytes and granulosa cells interact through gap junctions. Granulosa cells secrete AMH, E2, and FSH receptors to regulate oocyte development, while oocytes promote the proliferation and differentiation of granulosa cells through gap junctions [ 4 ] . The stable mechanical properties of granulosa cells, such as their cubic structure and adhesion, play a crucial role in this interaction. The cytoskeleton within granulosa cells and the extracellular matrix outside form a stable three-dimensional structure, regulating cell morphology and function, including proliferation, migration, and adhesion [ 5 ] . As ovarian function declines, granulosa cell cytoskeletal reconstruction occurs, characterized by F-actin rearrangement and downregulation of focal adhesion proteins, which are detrimental to follicle development and may lead to follicle atresia [ 6 ] . These changes are regulated by mechanical signals in the microenvironment, such as tissue stiffness and cell contraction, which can influence cellular responses at the molecular level. YAP serves as a mechanosensitive transcription factor, acting as a nuclear hub for mechanical signals and requiring Rho GTPase activity and cytoskeletal contractility for its regulation [ 7 ] . Recent studies have highlighted the relationship between cellular mechanical properties and disease progression. Microfluidic chips have emerged as a valuable tool for studying mechanical properties, with most research focusing on cancer mechanisms [ 8 ] . However, studies on ovarian granulosa cells are limited. Our research team previously demonstrated that Yulinzhu decoction, a traditional Chinese medicine formula, improved ovarian microcirculation by reducing blood flow resistance in the ovarian artery, thereby enhancing ovarian reserve function [ 9 ] . Further studies revealed that Yulinzhu decoction regulated levels of HIF1α, CX43, and glycolytic genes and proteins, promoting granulosa cell proliferation and energy metabolism, and improving follicle development impaired by deoxyvinyl cyclohexene (VCD) [ 10 ] . Building on these findings, this study investigates the mechanical properties of ovarian granulosa cells in POI rats using microfluidic chip technology and explores the mechanism by which Yulinzhu decoction regulates granulosa cell growth and development through YAP1. Results Yulinzhu Decoction Enhances Ovarian Function and Morphology in POI Rats To assess the effects of Yulinzhu decoction on ovarian tissues in POI rats, we established a POI model by injecting rats with VCD and subsequently administered Yulinzhu decoction via oral gavage. Ovarian tissue function and morphology were evaluated using ELISA and Masson staining. ELISA results indicated that E2 and AMH levels decreased, while FSH levels increased in the VCD-treated group compared to the control, confirming successful POI modeling. In contrast, Yulinzhu treatment restored E2 and AMH levels and reduced FSH levels, demonstrating its ability to improve ovarian function. Masson staining revealed collagen fiber accumulation and fibrotic changes in ovarian tissues of VCD-treated rats, with increased atretic follicles. Yulinzhu treatment significantly reduced collagen deposition and the number of atretic follicles, indicating its efficacy in mitigating ovarian fibrosis and restoring ovarian microenvironment homeostasis (Figure 1). Altered Elastic Modulus of Granulosa Cells Based on Microfluid Chip Technology To investigate the impact of ovarian tissue fibrosis on force stimulation signal transduction, we isolated primary granulosa cells from 3-week-old rats and characterized them using FSHR immunostaining, achieving high-purity ovarian granulosa cells. ELISA results revealed decreased estrogen secretion in granulosa cells from the VCD group, which was restored by Yulinzhu treatment, indicating enhanced hormone secretion function. Microfluidic chips were employed to measure the elasticity of granulosa cells. Based on the average cell diameter (19-20μm), microfluidic channels were designed with a height greater than 20 μm to accommodate cell entry. The chips measured deformation width, cell diameter, and crossing distance to calculate the elastic modulus. Results showed that VCD-treated cells exhibited increased channel crossing distance, reduced elastic modulus, and more pronounced deformation, indicative of compromised cell elasticity. Conversely, Yulinzhu treatment shortened the crossing distance, increased the elastic modulus, and reduced deformation, highlighting its restorative effects on granulosa cell elasticity(Figure2). Changes in Mechanical Structure and Characteristics of Granulosa Cells Phalloidin staining was used to explore the relationship between cell elasticity and cytoskeleton integrity. Results showed depolymerized cytoskeletons with reduced microfilament numbers and compromised structure in the VCD group. Yulinzhu treatment promoted cytoskeleton reorganization, increased microfilament density, and partially restored cytoskeleton integrity. Scratch experiments further examined the impact of these cytoskeletal changes on cell mobility. Initial scratch areas were consistent across groups. At 12 hours, VCD-treated cells exhibited larger scratch areas and slower migration. Yulinzhu treatment significantly accelerated cell migration at 36 and 60 hours, demonstrating its ability to enhance granulosa cell mobility (Figure 3). Cytoskeleton Contraction in Response to Force Stimulation to Affects YAP1 Nuclear Translation Subsequently, we will study how mechanical stimulation signals are transmitted through the cytoskeleton. Previous studies have found that YAP1 as a mechanical stimulation sensitive factor plays a role in traditional Chinese medicine in mechanical stimulation conduction. Therefore, we use immunofluorescence and Western Blotting to measure the localization and protein level of YAP1. Immunofluorescence staining results show that YAP1 is expressed in both the cytoplasm and the nucleus, and most of them are concentrated in the nucleus in control group. YAP1 in the VCD group showed a significant reduction in nuclear localization. In the VCD+YLZ group, YAP1 showed a significant increase in nuclear localization. Western Blotting results analysis showed that YAP1 expression level in the VCD group decreased; but the YAP1 expression level in the VCD+YLZ group increased. Subsequently verify whether the cytoskeleton affects the nuclear localization and protein level expression of YAP1. So IC50 was calculated through the CCK-8. 1μM cytochalasin B was selected for subsequent experiments. Immunofluorescence results showed that YAP1 nuclear localization was reduced in the inhibitor group. WB results showed that YAP1 protein expression was also significantly reduced in the inhibitor group. This suggests that mechanical stimulation signals may cause the contraction of the cytoskeleton, thereby affecting the nuclear transfer of YAP1 and thus affecting the downstream related signaling pathways (Figure 4). DISCUSSION Application Value of Microfluidic Chip Technology in Cell Mechanics Research This study innovatively uses microfluidic chip technology to quantify the mechanical characteristics of ovarian granulosa cells. Compared with traditional atomic force microscopes or optical tweezers, microfluidic chips have the advantages of high throughput and strong repeatability, providing a new technical platform for cell mechanics research. The microfluidic device comprises two arrays of microchannels, each with 40 microchannels, which can be used to quantify the physical properties of suspended and soft globular cells. The prepared sample can be pushed into the microfluidic device at a constant speed by a syringe pump. Under normal circumstances, approximately 40 cells can be captured in one experiment. In this experiment, we quantified the cellular elasticity of granulosa cells without loss by capturing the entire cells in the microchannel. Each restriction microchannel is configured in a linear restriction. Its width is from Win at its entrance to Wout at its exit. Win should be large enough to cover the entire cell size range to help the cell enter, while Wout should be small enough to capture the whole cell. Driven by constant water pressure from the inlet of the channel, cells captured along the channel deformed due to direct contact with the side wall of the channel, as shown in Figure 5. Here, we define a limiting angle θ to reflect the rate of change of microchannel width along channel length. The value of θ should be small enough to approximate the shape of the captured cells to the top and bottom spheres that were cut off. We set the channel length L channel to be large enough to ensure a smaller θ, and the relationship is θ°= tan − 1 ((Win−Wout)/(2L channel)). Furthermore, we represent the cell position as the distance from the entrance of the microchannel and refer to it as "through length" as L. Considering that the size of the microchannel depends to a large extent on the size of the target cells, we measured the entire cell diameter of normal granules and senescent granules. Cells were digested by trypsin and their diameters were quantified in bright field. We obtained normal granule cells, aged granule cells and granule cells after Yulinzhu decoction intervention as 19.04 (N=10), 18.59 (N=10) and 18 (N=10), respectively. Based on these measured cell sizes, we configured the size of the restricted microchannels to Win=30μm, Wout=4μm, and L channel=300μm, such that θ≈2.5°. Based on this, the whole-cell elasticity of cells captured in the microchannel was finally calculated [11] . This study used a microfluidic chip to measure the elastic modulus of granule cells, and then explored the mechanism by which Yulinzhu decoction regulates the structure of granule cell skeleton from the perspective of cellular mechanics. It is worth noting that this study applies this technology to the study of the mechanical properties. Yulinzhu Decoction Improves the Multi-Target Effect of Mechanics of Granulosa Cells. As a classic famous prescription for the treatment of infertility, Yulinzhu is derived from the "Jingyue Quanshu". The whole prescription is composed of Siwu Decoction and Sijunzi Decoction, as well as four Chinese medicine that are good for kidney. It can promote metabolism and blood circulation. Among them, Siwu Decoction replenishes blood, Sijunzi Decoction replenishes qi, which establish a solid material foundation for ovarian development. In addition, the traditional Chinese medicine to nourish the kidney and ultimately exerts the effect of nurturing and giving birth to children. Through animal experiments and cell experiments, the research team found that Yulinzhu can improve the oxidative stress state of the ovary, promote the proliferation of granulosa cells and follicle development. Animal experiments showed that Yulinzhu decoction significantly reduced the degree of ovarian fibrosis in POI rats (Massone staining), while regulating serum hormone levels (FSH↓, E2↑, AMH↑), providing a more suitable mechanical microenvironment for granulosa cells [12] . Cell growth is affected by the surrounding microenvironment, which induces changes in the internal structure of the tissue to respond. As the supporting structure of cells, the cytoskeleton plays a key role in maintaining the morphological and mechanical properties of cells. The actin skeleton of cells is highly dynamic, the polymerization and depolymerization of actin are important determinants of cell structure formation [13] . Deadline polymerization of peripheral actin in cells to drive the formation of flaky pseudopods in the frontier of motile cells, synergistically with cytoskeleton rearrangement to help cell migration [14]. Cell experiments show that after modeling, the mechanical properties of granulosa cells were found to have poor elasticity, slowed down migration, and depolymerized the cytoskeleton. The medicine-containing serum of Yulinzhu can reverse the VCD-induced F-actin depolymerization and restore the cubic structure of the cytoskeleton. Further microfluidic chips and further scratch experiments showed that the elastic modulus of granulosa cells increased after Yulinzhu intervention and the migration ability improved, indicating that it can fully restore the mechanical properties of the cells. This change in mechanical properties may be closely related to changes in the internal microenvironment of ovarian tissue and damage to the skeleton structure of the granulosa cells [15] . The cytochalasin B inhibition experiment further confirmed that skeleton integrity directly affects YAP1 nuclear localization, suggesting that the cytoskeleton-YAP1 axis may be a key regulatory pathway. Yulinzhu Decoction Mediates Mechanical Signaling Mechanism through YAP1 YAP (Yes-associated protein) is a key transcriptional co-activator, which plays an important role in regulating organ size, tissue regeneration, stem cell self-renewal, and tumor occurrence and development. In physiological states, the activity of YAP is strictly regulated by the Hippo pathway, and its phosphorylation state determines its retention or translocation in the nuclear region. When the Hippo pathway is inactivated, the dephosphorylated YAP is translocated into the nucleus and binds to transcription factors such as TEAD to activate downstream target gene expression, promote cell proliferation and inhibit apoptosis [16] , and participate in the construction of the cytoskeleton. So it was further verified through relevant cell experiments. In terms of proliferation, YAP/TAZ can synergize with other transcription factors or chromatin remodeling complexes to promote the expression of proliferation-related genes [17-18] . In anti-apoptosis, YAP/TAZ also regulates the expression of various anti-apoptosis-related genes, including inhibitor of apoptosis (IAP) and B cell lymphoma (BCL-2) family genes, thereby enhancing cell survival under physiological or pathological conditions [19] . In terms of cell differentiation, YAP/TAZ performs two distinct functions as a worker cell and a helper cell [20] . The results of this study show that YAP1 is mainly localized to the nucleus in the control group. After using VCD, YAP1 nuclear localization in the cells decreased and YAP1 expression decreased. This shows a synchronous change with the reduction of elastic modulus. After treating Yulinzhu with medicine-containing serum, western blotting showed an increase in total YAP1 protein expression. It shows that Yulinzhu can improve the distribution and expression of YAP1 in granulosa cells to a certain extent, suggesting that YAP1 may be a key effector molecule for mechanical properties. Further explore whether the changes in the nuclear localization and protein expression of YAP1 are affected by the changes in the cytoskeleton. Therefore, after using cytochalasin B to inhibit the cytoskeleton, it was found that the nuclear localization of YAP1 was reduced and the expression of YAP1 was also reduced, which indicated that changes in the cytoskeleton may affect the nuclear localization and protein expression of YAP1 And in turn affects the growth and development of granule cells. The existence of the "Cytoskeleton-YAP1 mechanical conduction axis" was confirmed, which provides a modern biological basis for traditional Chinese medicine to nourish kidneys and promote blood circulation to improve ovarian function. To sum up, this study innovatively applied microfluidic chip technology to quantify granulosa cell mechanics, demonstrating its advantages over traditional methods. Yulinzhu decoction improved ovarian fibrosis, hormone levels, and cytoskeletal integrity, enhancing granulosa cell elasticity and migration. The cytoskeleton-YAP1 axis was identified as a key regulatory pathway, providing a biomechanical basis and lays the foundation for subsequent in-depth research for Yulinzhu’s therapeutic effects in POI. However, there are still some limitations in this study, such as small sample size, short experimental period, and the regulatory mechanism of cell mechanical properties has not been fully understood. Future research can further explore the regulatory role of Yulinzhu on the mechanical properties of POI granules through related genes and proteomics, which will further improve the research system in this field, and also provide more scientific basis for the diagnosis and treatment of POI. STAR★Methods Key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclona anti-YAP1 Proteintech Cat# 13584-1-AP Cytochalasin B MCE Cat# HY-16928 Rabbit Anti-FSH receptor Polyclonal Antibody Bioss Cat# bs-20658R PMSG Solarbio Cat# P9970 Rabbit monoclonal anti-GAPDH MCE Cat# HY-P80137 FITC Proteintech Cat# SA00003-2 Rhodamine -Phalloidin Lablead Cat# G0063 DMSO Solarbio Cat# D8370 VCD Sigma Cat# 94956 Rat E2 ELISA Kit RayBio Cat# EIAR-E2 Rat E2 Elisa Kit ImmunoWay Cat# KE1818 Rat AMH Elisa Kit ImmunoWay Cat# KE1760 Rat FSH Elisa Kit ImmunoWay Cat# KE1449 Masson dye liquid set Beijing Kaiyue Biotechnology Co, Ltd. Cat# K1011 Experimental models SD rats 3w/8-10w Beijing Vital River Laboratory Animal Technology Co, Ltd. Software and algorithms Graphpad Prism Graphpad software https://www.graphpad.com/ Image J NIH https://imagej.net/ Experimental Methods Animal Models and Treatments Experimental Animals: Three-week-old and 8-10-week-old female Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed in a specific pathogen-free (SPF) facility with a 12-hour light/dark cycle (lights on at 7:00 AM). All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University (CCMU). POI Model Establishment and Treatment: Control Group: Rats were gavaged with normal saline daily for 6 weeks (1 day off per week). VCD Group: Rats were intraperitoneally injected with deoxyvinylcyclohexene (VCD) at a dose of 160 mg/kg per day (VCD dissolved in sesame oil at a ratio of 3:97 VCD to sesame oil) for 15 days. After confirming the POI model through serum E2 and AMH levels, rats were gavaged with normal saline for 6 weeks (1 day off per week). VCD+YLZ Group: Based on the "Equivalent Dosage Rating Table of Human and Animal Body Surface Area," POI rats were administered a traditional Chinese medicine dose of 30.24 g/kg daily according to their body weight for 6 weeks (1 day off per week). Cell Model Drug Treatment: VCD Group: VCD stock solution (2 mmol/L) was diluted to 0.5 mmol/L and applied for 12 hours. VCD+YLZ Group: Yulinzhu decoction-containing serum was diluted to a 5% concentration and applied for 48 hours. The concentration and duration were determined based on previous research by the team. Experimental Procedures Hormone Level Detection: Enzyme-linked immunosorbent assay (ELISA) was used to measure hormone levels in rat serum and cell supernatants. Samples and reagents were added according to kit instructions, and the optical density (OD) was measured using a microplate reader. Hormone levels were calculated based on standard curves. Detection of Ovarian Tissue Fibrosis Ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were dewaxed and dehydrated in graded xylene and anhydrous ethanol, followed by staining with potassium dichromate, iron hematoxylin, Lichun red acid fuchsin, phosphomolybdic acid, and aniline blue. Sections were then dehydrated and sealed. Collagen fibers appeared blue, while muscle fibers, cellulose, and red blood cells appeared red under optical microscopy. Microfluidic Chip Measurement of Cell Elastic Modulus Cell suspensions (1 ml) of the control, VCD, and 5% YLZ groups were prepared at a concentration of 5×10⁴/ml. Cell diameters were measured under an optical microscope to determine the specifications of the microfluidic chip. The chip was placed on a microscope stage, and cell suspensions were injected into the chip channels using a syringe pump at constant pressure. The deformation of cells as they passed through the channels was recorded, and the elastic modulus was calculated based on the measured diameters and distances. Cytoskeleton Staining to Assess Structural Integrity When cells reached 80% confluence, they were digested, resuspended, and seeded into 24-well plates. After fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100, cells were stained with a 1:100 dilution of rhodamine-phalloidin for 20 minutes. Stained cells were observed under a fluorescence microscope after washing with PBS. Cell Migration Assay Cells were digested with 0.25% trypsin, resuspended, and seeded in six-well plates. When cells reached a density of approximately 4×10⁵ cells per well, uniform scratches were made using a 10 μl pipette tip. The width of the scratches was controlled to approximately 0.5-1 mm. After washing with sterile PBS to remove debris, cells were treated with drugs. Images were taken at 0, 12, 36, and 60 hours under an inverted microscope. Cell migration was calculated as follows: Cell migration = (initial intercellular distance - 0-hour intercellular distance) / 0-hour intercellular distance. CCK-8 Assay for Inhibitor Concentration Screening: Cells were digested, resuspended, and seeded in 96-well plates. After 24 hours of incubation at 37°C with 5% CO₂, cells were treated with different concentrations of inhibitors (0.1 μM, 1 μM, 10 μM). After 3 hours, 10 μL of CCK-8 solution was added to each well. After incubation for 1-2 hours, the absorbance (OD) was measured at 450 nm using a microplate reader. Cell viability was calculated as: Viability (%) = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%. The IC50 value was determined using GraphPad Prism software. Western Blot Analysis of YAP1 Protein Expression: Ovarian granulosa cells were lysed with Ripa buffer, and protein samples were collected and quantified. SDS-PAGE gels were prepared, and proteins were electrophoresed and transferred to a PVDF membrane. YAP1 protein expression was detected using specific primary and secondary antibodies, and bands were quantified using an imaging analysis system. Quantification and statistical analysis Data were analyzed using GraphPad Prism version 8(GraphPad, La Jolla, CA, USA). Results are presented as mean ± SD. Statistical significance was set at p< 0.05. Statistical details of the experiment can be found in the legend. Declarations Acknowledgments The authors would like to thank the School of Bioengineering, Capital University of Medical Sciences, for the support in microfluidic chip analysis technology. This study was supported by the Natural Science Foundation of Beijing (7212162). Author contributions Y.Q., R.J. and D.X. were involved in the research design. All authors were involved in the research implementation and data analyses. Y.Q. wrote the paper. Declaration of interests The authors declare no competing interests. Funding declaration This work was supported by the Beijing Natural Science Foundation (Grant No. 7212162). References Pellicer N, Cozzolino M, Diaz-García C, et al. Ovarian rescue in women with premature ovarian insufficiency: facts and fiction. Reprod Biomed Online. 2023;46(3):543-565. Cox, E.; Takov, V. Embryology, Ovarian Follicle Development. In StatPearls; StatPearls Publishing: Treasure Island, FL, USA, 2020. Dompe C, Kulus M, Stefańska K, et al. Human Granulosa Cells-Stemness Properties, Molecular Cross-Talk and Follicular Angiogenesis.Cells. 2021;10(6):1396. Guo Yanyan, Zhang Yuxin, Lu Rui, et al. Research progress on proliferation and differentiation of granule cells in mammalian follicle development stage [J/OL]. Journal of Animal Husbandry and Veterinary Medicine,1-10[2025-03-04] Hao Y, Cheng S, Tanaka Y, Hosokawa Y, Yalikun Y, Li M. Mechanical properties of single cells: Measurement methods and applications. Biotechnol Adv. 2020;45:107648. Li Chunming, Zhou Jianhong. The process and related concepts of ovarian aging [J]. Journal of Practical Obstetrics and Gynecology, 2022, 38(02): 84-86. Dupont S,Morsut L,Aragon M, et al. Role of YAP/TAZ in mechanotransduction.Nature. 2011;474(7350):179-183. Lin Shuide. Cell mechanics microfluidic chip used for cancer diagnosis [J]. Medical Biomechanics, 2024, 39(S1): 277. Ai Beibei, Dou Na. 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Nat Rev Mol Cell Biol, 2006, 7(10):713-726 Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton.Science, 1993, 260(5111):1124-1127 Pan D. Hippo signaling in organ size control.Genes Dev. 2007;21(8):886-897. Jin Y,Xu J,Yin M X,et al.Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling.eLife,2013,2:e00999 Wang C,Yin M X,Wu W,et al.Taiman acts as a coactivator of Yorkie in the Hippo pathway to promote tissue growth and intestinal regeneration.Cell Discov,2016,2:16006 Dong J,Feldmann G,Huang J,et al.Elucidation of a universal size-control mechanism in Drosophila and mammals.Cell,2007,130:1120-1133 Zhong Z,Jiao Z,Yu F X.The Hippo signaling pathway in development and regeneration.Cell Rep,2024,43:113926 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-6794919","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472741232,"identity":"f249cc58-891b-44d6-9e26-541865a0a301","order_by":0,"name":"Qingqing Yang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qingqing","middleName":"","lastName":"Yang","suffix":""},{"id":472741233,"identity":"173db489-8f2e-4d67-814b-b7257e72bdbb","order_by":1,"name":"Xin Ruan","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Ruan","suffix":""},{"id":472741236,"identity":"afd469a9-0f3c-42ee-9287-09ad84b60a37","order_by":2,"name":"Pengxu Wang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Pengxu","middleName":"","lastName":"Wang","suffix":""},{"id":472741237,"identity":"c31c0131-d164-4c45-9792-b7586b6b1b78","order_by":3,"name":"Jifeng Ren","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jifeng","middleName":"","lastName":"Ren","suffix":""},{"id":472741238,"identity":"c3237eea-dd07-41b6-b3e8-9b8088d79f34","order_by":4,"name":"Xiaoying Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBAC+QbGBxAWe2Pjww/EaDE4wGzYAGbxHG42liBKCwNMi0R6mwAPUVrYm9kf/qi4Yzd/5sM2BgkGOzndBgJa5HsOMzbznHmWvOF2YtuDAoZkY7MDhKy5kX+wmbHtcLKBdGK7gQTDgcRthLUkMzb+BGqRn3mwTYKHWC0NvG2H7RhuMBKpxeDMYcbZPGcOJxicSQQGsgERfpFvb2b4+KPisL18+/GHDz9U2MkR9j4UJDZALCVSOQjYk6B2FIyCUTAKRhoAACPqR8BrNRVDAAAAAElFTkSuQmCC","orcid":"","institution":"Capital Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2025-06-01 09:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6794919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6794919/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84983253,"identity":"01b42e9b-4437-40cb-a452-eb5f6da1a70e","added_by":"auto","created_at":"2025-06-19 13:53:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1381711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in Ovarian Tissue Structure and Function Following Yulinzhu Decoction Administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Schematic diagram of animal modeling and oral gavage\u003c/p\u003e\n\u003cp\u003e(B)Levels of ovarian E2, AMH, and FSH examined by ELISA.（n=3）\u003c/p\u003e\n\u003cp\u003e(C)Masson staining of ovarian tissues. Representative images of Masson staining for collagen in ovaries of control, VCD, and VCD+YLZ rats. Blue areas (indicated by arrows) represent collagen fiber deposition. Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6794919/v1/ef698056e40ef6b55c5833e4.png"},{"id":84983256,"identity":"6a549c08-a45c-4544-8022-c4ad9bdb627f","added_by":"auto","created_at":"2025-06-19 13:53:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1614449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrofluidic chips measure elastic modulus of granulosa cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence staining for FSHR in granulosa cells from 3-week-old rats. Scale bar, 50μm.（n=3）\u003c/p\u003e\n\u003cp\u003e(B) Levels of E2 of granulosa cells measured by ELISA.（n=3）\u003c/p\u003e\n\u003cp\u003e(C) Design of microfluidic chip channel height based on cell diameter. Scale bar, 50 μm (n=10).\u003c/p\u003e\n\u003cp\u003e(D) Elastic modulus levels of granulosa cells captured by microfluidic chips. Scale bar, 50 μm (n-control=27, n-VCD=27, n-YLZ=14).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6794919/v1/f482717099ca07146a7f375e.png"},{"id":84983298,"identity":"98bdda64-6063-4f46-9670-64127e9b883d","added_by":"auto","created_at":"2025-06-19 13:53:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1198170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhalloidin Staining and Scratch Experiments to Assess Mechanical structure and Properties of Granulosa Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phalloidin staining to observe microfilament changes in the cytoskeleton, with red fluorescence indicating microfilaments and statistical analysis of fluorescence intensity. Scale bar, 50 μm (n=11).\u003c/p\u003e\n\u003cp\u003e(B) Scratch experiment to measure cell mobility, with the dotted line indicating the scratch areaScale bar, 50 μm \u0026nbsp;(n=6).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6794919/v1/e7280133d42aa3fd597d718e.png"},{"id":84983275,"identity":"b31af9e2-f8fb-40c8-9a05-e1857ea5fb48","added_by":"auto","created_at":"2025-06-19 13:53:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":828786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Relationship between YAP1 and Cytoskeleton\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Fluorescence staining of YAP1, green fluorescence is YAP1. Scale bar, 50μm (n=3)\u003c/p\u003e\n\u003cp\u003e(B) YAP1 protein expression level (n=6)\u003c/p\u003e\n\u003cp\u003e(C) IC50 examined by CCK-8 (n=3)\u003c/p\u003e\n\u003cp\u003e(D) Fluorescence staining of YAP1, green fluorescence is YAP1 in the inhibitor group. Scale bar, 50μm.(n=3)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6794919/v1/d8f742edae4ef202e5905c6f.png"},{"id":84983310,"identity":"b2b8359b-abc9-460b-8594-f058f7551753","added_by":"auto","created_at":"2025-06-19 13:53:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104641,"visible":true,"origin":"","legend":"\u003cp\u003eMicrofluidic chip diagram\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6794919/v1/8fd53ec33616353a46f117ac.png"},{"id":87334686,"identity":"b13734e9-a5f2-4a2a-a5b2-280b65cc7100","added_by":"auto","created_at":"2025-07-22 20:16:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5419270,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6794919/v1/7507def9-f2c0-4e38-a097-8d85ff35da80.pdf"},{"id":84983346,"identity":"98342d3e-e721-4353-b932-de8418beb2d7","added_by":"auto","created_at":"2025-06-19 13:53:30","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":64540046,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-6794919/v1/56b5a1a1f3041799487d101c.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Yulinzhu Decoction Modulates the Mechanical Properties of POI Rats Ovarian Granulosa Cells via Microfluidic Chip Technology","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrimary ovarian insufficiency (POI) is characterized by diminished ovarian reserve function and compromised oocyte quality \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The ovary\u0026rsquo;s basic functional unit, the follicle, progresses through five developmental stages: primordial, primary, secondary, antral, and mature follicles. Primordial follicles consist of an oocyte surrounded by a layer of flat granulosa cells. As follicles develop into primary follicles, these flat granulosa cells transition into cuboidal cells. Further development into secondary follicles is marked by the proliferation of granulosa cells into a bilayered structure \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. In the antral follicle stage, granulosa cells differentiate into mural granulosa cells (MGCs) and cumulus cells (CCs), forming a stable cubic structure around the oocyte by establishing gap junctions \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOocytes and granulosa cells interact through gap junctions. Granulosa cells secrete AMH, E2, and FSH receptors to regulate oocyte development, while oocytes promote the proliferation and differentiation of granulosa cells through gap junctions \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The stable mechanical properties of granulosa cells, such as their cubic structure and adhesion, play a crucial role in this interaction. The cytoskeleton within granulosa cells and the extracellular matrix outside form a stable three-dimensional structure, regulating cell morphology and function, including proliferation, migration, and adhesion \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. As ovarian function declines, granulosa cell cytoskeletal reconstruction occurs, characterized by F-actin rearrangement and downregulation of focal adhesion proteins, which are detrimental to follicle development and may lead to follicle atresia \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. These changes are regulated by mechanical signals in the microenvironment, such as tissue stiffness and cell contraction, which can influence cellular responses at the molecular level. YAP serves as a mechanosensitive transcription factor, acting as a nuclear hub for mechanical signals and requiring Rho GTPase activity and cytoskeletal contractility for its regulation \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent studies have highlighted the relationship between cellular mechanical properties and disease progression. Microfluidic chips have emerged as a valuable tool for studying mechanical properties, with most research focusing on cancer mechanisms \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. However, studies on ovarian granulosa cells are limited. Our research team previously demonstrated that Yulinzhu decoction, a traditional Chinese medicine formula, improved ovarian microcirculation by reducing blood flow resistance in the ovarian artery, thereby enhancing ovarian reserve function \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Further studies revealed that Yulinzhu decoction regulated levels of HIF1α, CX43, and glycolytic genes and proteins, promoting granulosa cell proliferation and energy metabolism, and improving follicle development impaired by deoxyvinyl cyclohexene (VCD) \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Building on these findings, this study investigates the mechanical properties of ovarian granulosa cells in POI rats using microfluidic chip technology and explores the mechanism by which Yulinzhu decoction regulates granulosa cell growth and development through YAP1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eYulinzhu Decoction Enhances Ovarian Function and Morphology in POI Rats\u003c/p\u003e\n\u003cp\u003eTo assess the effects of Yulinzhu decoction on ovarian tissues in POI rats, we established a POI model by injecting rats with VCD and subsequently administered Yulinzhu decoction via oral gavage. Ovarian tissue function and morphology were evaluated using ELISA and Masson staining. ELISA results indicated that E2 and AMH levels decreased, while FSH levels increased in the VCD-treated group compared to the control, confirming successful POI modeling. In contrast, Yulinzhu treatment restored E2 and AMH levels and reduced FSH levels, demonstrating its ability to improve ovarian function. Masson staining revealed collagen fiber accumulation and fibrotic changes in ovarian tissues of VCD-treated rats, with increased atretic follicles. Yulinzhu treatment significantly reduced collagen deposition and the number of atretic follicles, indicating its efficacy in mitigating ovarian fibrosis and restoring ovarian microenvironment homeostasis (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAltered Elastic Modulus of Granulosa Cells Based on Microfluid Chip Technology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the impact of ovarian tissue fibrosis on force stimulation signal transduction, we isolated primary granulosa cells from 3-week-old rats and characterized them using FSHR immunostaining, achieving high-purity ovarian granulosa cells. ELISA results revealed decreased estrogen secretion in granulosa cells from the VCD group, which was restored by Yulinzhu treatment, indicating enhanced hormone secretion function.\u003c/p\u003e\n\u003cp\u003eMicrofluidic chips were employed to measure the elasticity of granulosa cells. Based on the average cell diameter (19-20\u0026mu;m), microfluidic channels were designed with a height greater than 20 \u0026mu;m to accommodate cell entry. The chips measured deformation width, cell diameter, and crossing distance to calculate the elastic modulus. Results showed that VCD-treated cells exhibited increased channel crossing distance, reduced elastic modulus, and more pronounced deformation, indicative of compromised cell elasticity. Conversely, Yulinzhu treatment shortened the crossing distance, increased the elastic modulus, and reduced deformation, highlighting its restorative effects on granulosa cell elasticity(Figure2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in Mechanical Structure and Characteristics of Granulosa Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhalloidin staining was used to explore the relationship between cell elasticity and cytoskeleton integrity. Results showed depolymerized cytoskeletons with reduced microfilament numbers and compromised structure in the VCD group. Yulinzhu treatment promoted cytoskeleton reorganization, increased microfilament density, and partially restored cytoskeleton integrity. Scratch experiments further examined the impact of these cytoskeletal changes on cell mobility. Initial scratch areas were consistent across groups. At 12 hours, VCD-treated cells exhibited larger scratch areas and slower migration. Yulinzhu treatment significantly accelerated cell migration at 36 and 60 hours, demonstrating its ability to enhance granulosa cell mobility (Figure 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytoskeleton Contraction in Response to Force Stimulation to Affects YAP1 Nuclear Translation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, we will study how mechanical stimulation signals are transmitted through the cytoskeleton. Previous studies have found that YAP1 as a mechanical stimulation sensitive factor plays a role in traditional Chinese medicine in mechanical stimulation conduction. Therefore, we use immunofluorescence and Western Blotting to measure the localization and protein level of YAP1. Immunofluorescence staining results show that YAP1 is expressed in both the cytoplasm and the nucleus, and most of them are concentrated in the nucleus in control group. YAP1 in the VCD group showed a significant reduction in nuclear localization. In the VCD+YLZ group, YAP1 showed a significant increase in nuclear localization. Western Blotting results analysis showed that YAP1 expression level in the VCD group decreased; but the YAP1 expression level in the VCD+YLZ group increased. Subsequently verify whether the cytoskeleton affects the nuclear localization and protein level expression of YAP1. So IC50 was calculated through the CCK-8. 1\u0026mu;M cytochalasin B was selected for subsequent experiments. Immunofluorescence results showed that YAP1 nuclear localization was reduced in the inhibitor group. WB results showed that YAP1 protein expression was also significantly reduced in the inhibitor group. This suggests that mechanical stimulation signals may cause the contraction of the cytoskeleton, thereby affecting the nuclear transfer of YAP1 and thus affecting the downstream related signaling pathways (Figure 4).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eApplication Value of Microfluidic Chip Technology in Cell Mechanics Research\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study innovatively uses microfluidic chip technology to quantify the mechanical characteristics of ovarian granulosa cells. Compared with traditional atomic force microscopes or optical tweezers, microfluidic chips have the advantages of high throughput and strong repeatability, providing a new technical platform for cell mechanics research. The microfluidic device comprises two arrays of microchannels, each with 40 microchannels, which can be used to quantify the physical properties of suspended and soft globular cells. The prepared sample can be pushed into the microfluidic device at a constant speed by a syringe pump. Under normal circumstances, approximately 40 cells can be captured in one experiment. In this experiment, we quantified the cellular elasticity of granulosa cells without loss by capturing the entire cells in the microchannel. Each restriction microchannel is configured in a linear restriction. Its width is from Win at its entrance to Wout at its exit. Win should be large enough to cover the entire cell size range to help the cell enter, while Wout should be small enough to capture the whole cell. Driven by constant water pressure from the inlet of the channel, cells captured along the channel deformed due to direct contact with the side wall of the channel, as shown in Figure 5. Here, we define a limiting angle \u0026theta; to reflect the rate of change of microchannel width along channel length. The value of\u0026nbsp;\u0026theta;\u0026nbsp;should be small enough to approximate the shape of the captured cells to the top and bottom spheres that were cut off. We set the channel length L channel to be large enough to ensure a smaller\u0026nbsp;\u0026theta;, and the relationship is\u0026nbsp;\u0026theta;\u0026deg;= tan\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e((Win\u0026minus;Wout)/(2L channel)). Furthermore, we represent the cell position as the distance from the entrance of the microchannel and refer to it as \u0026quot;through length\u0026quot; as L.\u003c/p\u003e\n\u003cp\u003eConsidering that the size of the microchannel depends to a large extent on the size of the target cells, we measured the entire cell diameter of normal granules and senescent granules. Cells were digested by trypsin and their diameters were quantified in bright field. We obtained normal granule cells, aged granule cells and granule cells after Yulinzhu decoction intervention as 19.04 (N=10), 18.59 (N=10) and 18 (N=10), respectively. Based on these measured cell sizes, we configured the size of the restricted microchannels to Win=30\u0026mu;m, Wout=4\u0026mu;m, and L channel=300\u0026mu;m, such that \u0026theta;\u0026asymp;2.5\u0026deg;. Based on this, the whole-cell elasticity of cells captured in the microchannel was finally calculated\u003csup\u003e\u0026nbsp;[11]\u003c/sup\u003e. This study used a microfluidic chip to measure the elastic modulus of granule cells, and then explored the mechanism by which Yulinzhu decoction regulates the structure of granule cell skeleton from the perspective of cellular mechanics. It is worth noting that this study applies this technology to the study of the mechanical properties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYulinzhu Decoction Improves the Multi-Target Effect of Mechanics of Granulosa Cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a classic famous prescription for the treatment of infertility, Yulinzhu is derived from the \u0026quot;Jingyue Quanshu\u0026quot;. The whole prescription is composed of Siwu Decoction and Sijunzi Decoction, as well as four Chinese medicine that are good for kidney. It can promote metabolism and blood circulation. Among them, Siwu Decoction replenishes blood, Sijunzi Decoction replenishes qi, which establish a solid material foundation for ovarian development. In addition, the traditional Chinese medicine to nourish the kidney and ultimately exerts the effect of nurturing and giving birth to children. Through animal experiments and cell experiments, the research team found that Yulinzhu can improve the oxidative stress state of the ovary, promote the proliferation of granulosa cells and follicle development.\u003c/p\u003e\n\u003cp\u003eAnimal experiments showed that Yulinzhu decoction significantly reduced the degree of ovarian fibrosis in POI rats (Massone staining), while regulating serum hormone levels (FSH\u0026darr;, E2\u0026uarr;, AMH\u0026uarr;), providing a more suitable mechanical microenvironment for granulosa cells\u003csup\u003e[12]\u003c/sup\u003e. Cell growth is affected by the surrounding microenvironment, which induces changes in the internal structure of the tissue to respond. As the supporting structure of cells, the cytoskeleton plays a key role in maintaining the morphological and mechanical properties of cells. The actin skeleton of cells is highly dynamic, the polymerization and depolymerization of actin are important determinants of cell structure formation \u003csup\u003e[13]\u003c/sup\u003e. Deadline polymerization of peripheral actin in cells to drive the formation of flaky pseudopods in the frontier of motile cells, synergistically with cytoskeleton rearrangement to help cell migration [14]. Cell experiments show that after modeling, the mechanical properties of granulosa cells were found to have poor elasticity, slowed down migration, and depolymerized the cytoskeleton. The medicine-containing serum of Yulinzhu can reverse the VCD-induced F-actin depolymerization and restore the cubic structure of the cytoskeleton. Further microfluidic chips and further scratch experiments showed that the elastic modulus of granulosa cells increased after Yulinzhu intervention and the migration ability improved, indicating that it can fully restore the mechanical properties of the cells. This change in mechanical properties may be closely related to changes in the internal microenvironment of ovarian tissue and damage to the skeleton structure of the granulosa cells \u003csup\u003e[15]\u003c/sup\u003e. The cytochalasin B inhibition experiment further confirmed that skeleton integrity directly affects YAP1 nuclear localization, suggesting that the cytoskeleton-YAP1 axis may be a key regulatory pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYulinzhu Decoction Mediates Mechanical Signaling Mechanism through YAP1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYAP (Yes-associated protein) is a key transcriptional co-activator, which plays an important role in regulating organ size, tissue regeneration, stem cell self-renewal, and tumor occurrence and development. In physiological states, the activity of YAP is strictly regulated by the Hippo pathway, and its phosphorylation state determines its retention or translocation in the nuclear region. When the Hippo pathway is inactivated, the dephosphorylated YAP is translocated into the nucleus and binds to transcription factors such as TEAD to activate downstream target gene expression, promote cell proliferation and inhibit apoptosis \u003csup\u003e[16]\u003c/sup\u003e, and participate in the construction of the cytoskeleton. So it was further verified through relevant cell experiments. In terms of proliferation, YAP/TAZ can synergize with other transcription factors or chromatin remodeling complexes to promote the expression of proliferation-related genes \u003csup\u003e[17-18]\u003c/sup\u003e. In anti-apoptosis, YAP/TAZ also regulates the expression of various anti-apoptosis-related genes, including inhibitor of apoptosis (IAP) and B cell lymphoma (BCL-2) family genes, thereby enhancing cell survival under physiological or pathological conditions \u003csup\u003e[19]\u003c/sup\u003e. In terms of cell differentiation, YAP/TAZ performs two distinct functions as a worker cell and a helper cell \u003csup\u003e[20]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe results of this study show that YAP1 is mainly localized to the nucleus in the control group. After using VCD, YAP1 nuclear localization in the cells decreased and YAP1 expression decreased. This shows a synchronous change with the reduction of elastic modulus. After treating Yulinzhu with medicine-containing serum, western blotting showed an increase in total YAP1 protein expression. It shows that Yulinzhu can improve the distribution and expression of YAP1 in granulosa cells to a certain extent, suggesting that YAP1 may be a key effector molecule for mechanical properties. Further explore whether the changes in the nuclear localization and protein expression of YAP1 are affected by the changes in the cytoskeleton. Therefore, after using cytochalasin B to inhibit the cytoskeleton, it was found that the nuclear localization of YAP1 was reduced and the expression of YAP1 was also reduced, which indicated that changes in the cytoskeleton may affect the nuclear localization and protein expression of YAP1 And in turn affects the growth and development of granule cells. The existence of the \u0026quot;Cytoskeleton-YAP1 mechanical conduction axis\u0026quot; was confirmed, which provides a modern biological basis for traditional Chinese medicine to nourish kidneys and promote blood circulation to improve ovarian function.\u003c/p\u003e\n\u003cp\u003eTo sum up, this study innovatively applied microfluidic chip technology to quantify granulosa cell mechanics, demonstrating its advantages over traditional methods. Yulinzhu decoction improved ovarian fibrosis, hormone levels, and cytoskeletal integrity, enhancing granulosa cell elasticity and migration. The cytoskeleton-YAP1 axis was identified as a key regulatory pathway, providing a biomechanical basis and lays the foundation for subsequent in-depth research for Yulinzhu\u0026rsquo;s therapeutic effects in POI.\u003c/p\u003e\n\u003cp\u003eHowever, there are still some limitations in this study, such as small sample size, short experimental period, and the regulatory mechanism of cell mechanical properties has not been fully understood. Future research can further explore the regulatory role of Yulinzhu on the mechanical properties of POI granules through related genes and proteomics, which will further improve the research system in this field, and also provide more scientific basis for the diagnosis and treatment of POI.\u003c/p\u003e\n\u003cp\u003eSTAR★Methods Key resources table\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eREAGENT or RESOURCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eSOURCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eIDENTIFIER\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eAntibodies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRabbit polyclona anti-YAP1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eProteintech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;13584-1-AP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eCytochalasin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eMCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;HY-16928\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRabbit Anti-FSH receptor Polyclonal Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eBioss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;bs-20658R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003ePMSG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eSolarbio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;P9970\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRabbit monoclonal anti-GAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eMCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;HY-P80137\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eFITC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eProteintech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;SA00003-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRhodamine -Phalloidin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eLablead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;G0063\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eDMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eSolarbio\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;D8370\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eVCD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eSigma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;94956\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRat E2 ELISA Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eRayBio\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;EIAR-E2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRat E2 Elisa Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eImmunoWay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;KE1818\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRat AMH Elisa Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eImmunoWay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;KE1760\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eRat FSH Elisa Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eImmunoWay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;KE1449\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eMasson dye liquid set\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eBeijing Kaiyue Biotechnology Co, Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eCat#\u0026nbsp;K1011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eExperimental models\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eSD rats\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003e3w/8-10w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003eBeijing Vital River Laboratory Animal Technology Co, Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eSoftware and algorithms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eGraphpad Prism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eGraphpad software\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003ehttps://www.graphpad.com/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2064%;\"\u003e\n \u003cp\u003eImage J\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6975%;\"\u003e\n \u003cp\u003eNIH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.0961%;\"\u003e\n \u003cp\u003ehttps://imagej.net/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Experimental Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal Models and Treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental Animals: Three-week-old and 8-10-week-old female Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed in a specific pathogen-free (SPF) facility with a 12-hour light/dark cycle (lights on at 7:00 AM). All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University (CCMU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePOI Model Establishment and Treatment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eControl Group: Rats were gavaged with normal saline daily for 6 weeks (1 day off per week).\u003c/p\u003e\n\u003cp\u003eVCD Group: Rats were intraperitoneally injected with deoxyvinylcyclohexene (VCD) at a dose of 160 mg/kg per day (VCD dissolved in sesame oil at a ratio of 3:97 VCD to sesame oil) for 15 days. After confirming the POI model through serum E2 and AMH levels, rats were gavaged with normal saline for 6 weeks (1 day off per week).\u003c/p\u003e\n\u003cp\u003eVCD+YLZ Group: Based on the \u0026quot;Equivalent Dosage Rating Table of Human and Animal Body Surface Area,\u0026quot; POI rats were administered a traditional Chinese medicine dose of 30.24 g/kg daily according to their body weight for 6 weeks (1 day off per week).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Model Drug Treatment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVCD Group: VCD stock solution (2 mmol/L) was diluted to 0.5 mmol/L and applied for 12 hours.\u003c/p\u003e\n\u003cp\u003eVCD+YLZ Group: Yulinzhu decoction-containing serum was diluted to a 5% concentration and applied for 48 hours. The concentration and duration were determined based on previous research by the team.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHormone Level Detection:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzyme-linked immunosorbent assay (ELISA) was used to measure hormone levels in rat serum and cell supernatants. Samples and reagents were added according to kit instructions, and the optical density (OD) was measured using a microplate reader. Hormone levels were calculated based on standard curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Ovarian Tissue Fibrosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were dewaxed and dehydrated in graded xylene and anhydrous ethanol, followed by staining with potassium dichromate, iron hematoxylin, Lichun red acid fuchsin, phosphomolybdic acid, and aniline blue. Sections were then dehydrated and sealed. Collagen fibers appeared blue, while muscle fibers, cellulose, and red blood cells appeared red under optical microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrofluidic Chip Measurement of Cell Elastic Modulus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell suspensions (1 ml) of the control, VCD, and 5% YLZ groups were prepared at a concentration of 5\u0026times;10⁴/ml. Cell diameters were measured under an optical microscope to determine the specifications of the microfluidic chip. The chip was placed on a microscope stage, and cell suspensions were injected into the chip channels using a syringe pump at constant pressure. The deformation of cells as they passed through the channels was recorded, and the elastic modulus was calculated based on the measured diameters and distances.\u003c/p\u003e\n\u003cp\u003eCytoskeleton Staining to Assess Structural Integrity\u003c/p\u003e\n\u003cp\u003eWhen cells reached 80% confluence, they were digested, resuspended, and seeded into 24-well plates. After fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100, cells were stained with a 1:100 dilution of rhodamine-phalloidin for 20 minutes. Stained cells were observed under a fluorescence microscope after washing with PBS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Migration Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were digested with 0.25% trypsin, resuspended, and seeded in six-well plates. When cells reached a density of approximately 4\u0026times;10⁵\u0026nbsp;cells per well, uniform scratches were made using a 10\u0026nbsp;\u0026mu;l pipette tip. The width of the scratches was controlled to approximately 0.5-1 mm. After washing with sterile PBS to remove debris, cells were treated with drugs. Images were taken at 0, 12, 36, and 60 hours under an inverted microscope. Cell migration was calculated as follows: Cell migration = (initial intercellular distance - 0-hour intercellular distance) / 0-hour intercellular distance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK-8 Assay for Inhibitor Concentration Screening:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were digested, resuspended, and seeded in 96-well plates. After 24 hours of incubation at 37\u0026deg;C with 5% CO₂, cells were treated with different concentrations of inhibitors (0.1\u0026nbsp;\u0026mu;M, 1\u0026nbsp;\u0026mu;M, 10\u0026nbsp;\u0026mu;M). After 3 hours, 10\u0026nbsp;\u0026mu;L of CCK-8 solution was added to each well. After incubation for 1-2 hours, the absorbance (OD) was measured at 450 nm using a microplate reader. Cell viability was calculated as: Viability (%) = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group)\u0026nbsp;\u0026times;\u0026nbsp;100%. The IC50 value was determined using GraphPad Prism software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis of YAP1 Protein Expression:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvarian granulosa cells were lysed with Ripa buffer, and protein samples were collected and quantified. SDS-PAGE gels were prepared, and proteins were electrophoresed and transferred to a PVDF membrane. YAP1 protein expression was detected using specific primary and secondary antibodies, and bands were quantified using an imaging analysis system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using GraphPad Prism version 8(GraphPad, La Jolla, CA, USA). Results are presented as mean \u0026plusmn; SD. Statistical significance was set at p\u0026lt; 0.05. Statistical details of the experiment can be found in the legend.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank the School of Bioengineering, Capital University of Medical Sciences, for the support in microfluidic chip analysis technology. This study was supported by the Natural Science Foundation of Beijing (7212162). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eY.Q., R.J. and D.X. were involved in the research design. All authors were involved in the research implementation and data analyses. Y.Q. wrote the paper.\u003c/p\u003e\n\u003ch2\u003eDeclaration of interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding declaration\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Beijing Natural Science Foundation (Grant No. 7212162).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePellicer N, Cozzolino M, Diaz-Garc\u0026iacute;a C, et al. Ovarian rescue in women with premature ovarian insufficiency: facts and fiction. Reprod Biomed Online. 2023;46(3):543-565.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCox, E.; Takov, V. Embryology, Ovarian Follicle Development. In StatPearls; StatPearls Publishing: Treasure Island, FL, USA, 2020.\u003c/li\u003e\n \u003cli\u003eDompe C, Kulus M, Stefańska K, et al. Human Granulosa Cells-Stemness Properties, Molecular Cross-Talk and Follicular Angiogenesis.Cells. 2021;10(6):1396.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGuo Yanyan, Zhang Yuxin, Lu Rui, et al. Research progress on proliferation and differentiation of granule cells in mammalian follicle development stage [J/OL]. Journal of Animal Husbandry and Veterinary Medicine,1-10[2025-03-04]\u003c/li\u003e\n \u003cli\u003eHao Y, Cheng S, Tanaka Y, Hosokawa Y, Yalikun Y, Li M. Mechanical properties of single cells: Measurement methods and applications. Biotechnol Adv. 2020;45:107648.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLi Chunming, Zhou Jianhong. The process and related concepts of ovarian aging [J]. Journal of Practical Obstetrics and Gynecology, 2022, 38(02): 84-86.\u003c/li\u003e\n \u003cli\u003eDupont S,Morsut L,Aragon M, et al. Role of YAP/TAZ in mechanotransduction.Nature. 2011;474(7350):179-183.\u003c/li\u003e\n \u003cli\u003eLin Shuide. 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In this study, we investigated the mechanical properties of granulosa cells in a rat model of primary ovarian insufficiency (POI) using microfluidic chip technology. We found that POI rats exhibited increased ovarian fibrosis and a higher number of atretic follicles, which disrupted the homeostasis of the ovarian microenvironment and altered the mechanical properties of granulosa cells. YAP (Yes-associated protein), a mechanosensitive transcription factor, was identified as a key regulator of granulosa cell morphology through mechanical signaling. Yulinzhu decoction, a traditional Chinese medicine formula, improved the ovarian microenvironment and modulated YAP signaling, thereby enhancing the mechanical properties and function of granulosa cells. Our findings propose a novel mechanism by which Yulinzhu decoction regulates the mechanical properties of granulosa cells in POI rats through YAP modulation.\u003c/p\u003e","manuscriptTitle":"Yulinzhu Decoction Modulates the Mechanical Properties of POI Rats Ovarian Granulosa Cells via Microfluidic Chip Technology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 13:53:11","doi":"10.21203/rs.3.rs-6794919/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":"e4bf5508-fe68-4019-9b01-1c1bc31d193a","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-22T20:08:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-19 13:53:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6794919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6794919","identity":"rs-6794919","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}