Metformin enhances epithelial cell growth inhibition via the protein kinase-insulin-like growth factor binding protein-1 pathway

Background:  a bnormal stromal-epithelial cell communication is a pathogenic mechanism in endometriosis, and metformin can modulate it. Insulin-like growth factor binding protein-1 (IGFBP1) plays a role in endometriosis, but the exact mechanism is unknown. IGFBP1 is reportedly a downstream target of metformin in some diseases. We aimed to investigate the role of IGFBP1 in endometriosis development, whether it is associated with abnormal communication, and whether metformin affects IGFBP1 expression.

Patients who underwent surgical treatment for endometriosis or other diseases were enrolled. Ten patients with ovarian-type endometriosis and eight patients each who underwent surgical treatment for other lesions with or without endometriosis were selected, and their tissues taken for cell proliferation, western blotting, polymerase chain reaction, and knockdown experiments.

ectopic and eutopic stromal cells (e cScs and e uScs) lost their ability to inhibit epithelial cell proliferation, and IGFBP1 expression was downregulated in both groups of stromal cells compared to that in normal stromal cells (nScs; 1.09 vs. 0.25, p = .0002 1.09 vs. 0.57, p = .0029). In an e cSc IGFBP1 overexpression model, the ability of ecScs to inhibit epithelial cell proliferation was enhanced (e dU positivity decreased from 38% to 25%, p = .0001). Furthermore, adenosine 5’-monophosphate-activated protein kinase ( a MPK) phosphorylation was downregulated in ecScs and euScs compared to that in nScs (0.99 vs. 0.42, p = .0006/0.99 vs. 0.57, p = 0.0032). Treatment of e cScs with metformin increased a MPK phosphorylation (0.47 vs. 1.04, p = .0107) while upregulating IGFBP1 expression (0.69 vs. 1.01, p = .0164), whereas pre-treatment with an a MPK phosphorylation inhibitor abrogated metformin-induced IGFBP1 upregulation.

IGFBP1 mediates aberrant stromal-epithelial communication in endometriosis. Metformin can upregulate IGFBP1 expression in e cScs by activating a MPK, and upregulated IGFBP1 enhances the inhibition of epithelial cell proliferation. IGFBP1 is expected to be a therapeutic target for endometriosis. PLAIN LANGUAGE SUMMARY Insulin-like growth factor binding protein 1 (IGFBP1) is a protein that regulates cell growth and proliferation and is expressed at abnormal levels in patients with endometriosis. In some cases, metformin has been shown to modulate the expression of this protein. Here, we investigated the role of IGFBP1 in endometriosis development, whether it is associated with abnormal communication, and whether metformin affects IGFBP1 expression in endometrial cells. We found that downregulation of IGFBP1 in endometriosis diminished the ability of stromal cells to inhibit the proliferation of epithelial cells through inhibition of the protein kinase B and extracellular regulated protein kinase pathways. In addition, metformin upregulated IGFBP1 expression by activating adenosine 5’-monophosphate-activated protein kinase, suggesting that IGFBP1 may be one of the potential targets for drug therapy for endometriosis.

a pproximately, 190 million women globally are affected by endometriosis during their lifetime (at least until menopause) according to the World Bank’s 2017 population estimates (Horne and Missmer 2022). endometriosis affects metabolism in various tissues, including the liver and adipose tissues, leading to systemic inflammation (Taylor et  al. 2021). a pro - gressive understanding of the disease’s nature has expanded treatment options, and the potential of some drugs for treat - ing endometriosis is being explored. © 2024 t he a uthor(s). Published by i nforma uK limited, trading as taylor & f rancis Group CONTACT changzhong li [email protected] , [email protected] d epartment of Gynaecology, shandong Provincial Hospital, cheeloo c ollege of Medicine, shandong university, Jinan, shandong, china supplemental data for this article can be accessed online at https://doi.org/10.1080/01443615.2024.2321651. https://doi.org/10.1080/01443615.2024.2321651 t his is an o pen a ccess article distributed under the terms of the c reative c ommons a ttribution license ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. t he terms on which this article has been published allow the posting of the a ccepted Manuscript in a repository by the author(s) or with their consent.

endometriosis; metformin; a MPK-IGFBP1; stromal– epithelial communication; PI3K-aKT ARTICLE HISTORY Received 19 o ctober 2023 a ccepted 14 f ebruary 2024 2 X. SHao e T al. The growth and differentiation of healthy endometrial epi - thelial cells are regulated by stromal cells ( a rnold et  al. 2001), while a loss of this ability has been implicated in endometri - osis. Some factors secreted by ectopic stromal cells, including Wnt family member 2 (Wnt2), are involved in this aberrant communication (Zhang et  al. 2015). Insulin-like growth factor-binding protein 1 (IGFBP1) is asso - ciated with endometriosis development, although the exact mechanism remains unclear. This study aimed to elucidate this mechanism. IGFBP1 is primarily secreted by stromal cells of the secretory endometrium and decreases during endometriosis (Meola et  al. 2010, Shih et  al. 2022). It competes with the insulin-like growth factor (IGF) receptor for IGFs, affecting cell proliferation, growth, differentiation, apoptosis, migration, inva- sion, and adhesion in normal and tumour cells (lin et  al. 2021). During gestation, decidualised endometrial stromal cells highly express IGFBP1, which regulates the growth of extravillous tro - phoblast cells, a highly migratory cell subset important in embryogenesis. Trophoblasts are more invasive in vitro, wherein IGFBP1 expression is inhibited (Irwin et  al. 1999). Progesterone induces IGFBP1 expression by endometrial stromal cells in ani - mals. High IGFBP1 levels impede endometrial epithelial cell proliferation by suppressing IGF action (Murphy and Ghahary 1990, Seppälä et  al. 1994). excess IGFBP1 impairs the rapid divi - sion of breast cancer cells by inhibiting the interaction between IGF1 and IGF receptor 1 (Figueroa et  al. 1993). We hypothesised that abnormal IGFBP1 expression in stromal cells in individuals with endometriosis causes abnormal stromal–epithelial cell communication. We examined whether abnormal IGFBP1 levels contribute to the diminished ability of stromal cells to regulate epithelial cells. Metformin can target Wnt2 and thereby alleviate abnormal stromal–epithelial communication (Zhang et  al. 2010). Metformin increases serum IGFBP1 levels in patients with polycystic ovary syndrome (Pawelczyk et  al. 2004). In lung cancer, metformin potentiates the effect of solamargine in upregulating IGFBP1 expression (Tang et  al. 2017b). Thus, we aimed to examine the potential effect of metformin on IGFBP1 expression in endometrial stromal cells and elucidate its underlying mechanism.

Materials c ollagenase Ia and metformin were purchased from Sigma– a ldrich l td. (St l ouis, Mo , USa ). Trypsin, Dulbecco’s modified eagle’s medium/nutrient mixture F-12 (DMeM/F12; 1:1) medium, and charcoal-stripped foetal bovine serum (FBS) were purchased from Gibco (Billings, MT, USa ). Rabbit anti-human IGFBP1, a MPK, phospho- a MPK, protein kinase B (aKT), phospho- aKT, extracellular regulated protein kinase (eRK), and phospho-eRK were purchased from a bcam (c ambridge, M a, USa ). Rabbit anti-human β-actin and GaPDH primary antibodies and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Proteintech (Wuhan, china). c ompound c (an a MPK inhibitor) was purchased from Medchemexpress (Shanghai, china). Patients and tissue samples This was an experimental study in which ectopic and eutopic endometrium of ovarian-type endometriosis and normal endometrium of non-endometriosis were collected from patients who underwent surgical treatment from June 2022 to June 2023 at the Department of Gynaecology of Shandong Provincial Hospital. endometriosis was defined as the pres - ence of endometrial-like tissue outside the uterine cavity, and ovarian-type endometriosis was defined as ectopic endome - trium colonising the ovaries. The inclusion and exclusion criteria were as follows: Inclusion criteria: (1) patients with ovarian-type endometri - osis clearly diagnosed by pathology, patients diagnosed with ovarian-type endometriosis without comorbidity of oestrogen-dependent diseases, such as adenomyosis, uterine fibroids, endometrial polyps, and endometrial cancer, and non-endometriosis patients without the combination of the above oestrogen-dependent diseases (control group). (2) a ll participants were premenopausal and had regular menstrual cycles. a ll samples were collected during the secre - tory phase of the menstrual period based on menstrual his - tory and histological assessment. exclusion criteria: (1) c ombination of oestrogen-progestin dysregulation caused by other reproductive endocrine-related diseases, such as polycystic ovary syndrome. (2) c omorbid oestrogen-related malignant tumours, such as endometrial cancer and breast cancer or previous relevant medical history. (3) Received hormone therapy within 6 months prior to surgery. (4) Taking hormonal contraceptives within 6 months prior to surgery. (5) Hormonal birth control device placed in the uterine cavity. a total of 26 patients were included in this experiment; the experimental group contained 18 patients with ovarian-type endometriosis. The ectopic endometrium located in the ovaries of 10 patients with ovarian-type endometriosis and the intra - uterine eutopic endometrium of 8 patients with ovarian-type endometriosis were collected; the normal endometrium of eight patients with non-endometriosis (control group) was also collected. The mean age of the experimental group was 43.23 ± 5.07 years (mean ± standard deviation), and that of the control group was 44.83 ± 5.62 years (mean ± standard deviation). Primary cell culture endometrial cells were isolated as previously described (Zhang et  al. 2010). endometriotic cysts in the ovaries (ecto - pic stromal cells [e cScs]), eutopic stromal cells (e uScs; endo - metriomas), and normal stromal cells (nScs; patients without endometriosis) were cultured in complete culture media (DMeM/F12 [1:1]) supplemented with 10% charcoal-stripped FBS and 1% penicillin/streptomycin (Hyclone, l ogan, UT, USa ) at 37 °c with 5% co 2. The medium was replaced every 2–3 d. The cultured cells were identified using immunohistochemistry with mouse anti-human vimentin JoURnal oF oBSTe TRIcS anD GynaecoloGy 3 antibodies (Zhang et  al. 2010). The purity of stromal cells was >98%. a well-differentiated human endometrioid adenocarcinoma epithelial cell line (Ishikawa; c entral laboratory of Shandong Provincial Hospital) was grown in a complete culture medium. Cell proliferation assay The 5-ethynyl-2’-deoxyuridine (e dU) assay was performed to assess the effect of IGFBP1 overexpression and metformin treatment on Ishikawa cell proliferation, using the c ell light edU a pollo 567 in Vitro Imaging Kit (RiboBio c o., l td., Guangzhou, china) following the manufacturer’s instructions. By removing the upper layer of the non-contact co-culture system, proliferation in the lower layer was assayed. Images were captured using a ZeISS inverted fluorescence micro - scope (ZeISS, Germany), and the results were analysed using the ImageJ software (version 2.3.0, https://imagej.nih.gov/). HTS Transwell-96 plates with 0.4-µm pore polyester mem - brane inserts ( c orning, ny , USa ) were used in c ell c ounting. ISK cells (3 × 103/well) were seeded in 96-well plates. The stro - mal cells were added to the upper chamber to co-culture with ISK cells for specific times (0, 24, 48, and 72 h), and then, c ell c ounting Kit-8 (Medchemexpress, Shanghai, china) was used for incubation at 2 h in the dark. The absorbance value was measured at 450 nm. Enzyme-linked immunosorbent assay (ELISA) a n elISa Kit (elabscience Biotechnology, Wuhan, china) was used to determine IGFBP1 levels secreted by nScs, e uScs, and e cScs following the manufacturer’s instructions with supernatants from cells cultured for 72 h. The mean of dupli - cate readings for the standard and sample was calculated, and the average zero standard optical density was deducted from the readings. a four-parameter logistic curve was plot - ted on a log–log axis, with standard concentrations and opti - cal density values on the x- and y-axes, respectively, to determine sample concentrations. Transfection l entiviral vectors for IGFBP1 overexpression and a negative control lentivirus, both expressing eGFP , were purchased from Genechem (Shanghai, china). one group of cells was trans - fected with the lentivirus overexpressing IGFBP1 (IGFBP1-l V group), whereas the other was transfected with an empty lentivirus (nc–l V group). The multiplicity of infection was 50% in both groups. l entivirus was introduced to the medium the following day when the cells had reached 40% conflu - ence. The lentivirus-containing medium was replaced with fresh media after 24 h. a fter 72 h, the transfection efficiency was estimated using an inverted phase/fluorescence micro - scope (ZeISS, Germany). Non-contact co-culture in transwell plates We used stromal and Ishikawa cells from passages 1–3. Regular 24-well and Transwell plates with 0.4-µm pore polyester membrane inserts were purchased from c orning (ny, USa ). Stromal cells were seeded into regular twenty-four- well plates and cultured in serum-free DMeM/F-12 (1:1). a fter adhesion, the cells were pre-treated for 3 d with DMeM/F12 (1:1) containing 2% FBS and 20 μM metformin; based on clin - ical pharmacokinetics, 20 μM is the accepted clinically equiva - lent in-vitro dose (Isoda et  al. 2006, Graham et  al. 2011). Subsequently, metformin-treated stromal cells were tryp - sinised and seeded in the upper chamber of a Transwell 24-well culture plate, and Ishikawa cells were seeded in the lower chamber, with 0.1 and 0.6 ml of 2% charcoal-stripped FBS in the upper and lower chambers, respectively. a fter 3 d, the upper chamber was detached; cell proliferation in the lower chamber was examined using the e dU assay, or pro - teins were extracted for western blotting. Total protein extraction and western blotting Total protein was extracted using radio-immunoprecipitation assay lysis buffer (Beyotime, Shanghai, china) containing 1% PMSF reagent (Beyotime, Shanghai, china) and a 1% phos - phatase inhibitor cocktail (Medchemexpress, Shanghai, china). Denatured total proteins were electrophoresed on a 12% polyacrylamide gel and transferred onto polyvinylidene fluo - ride membranes. Blocking was performed for 1 h in 5% bovine serum albumin (Solarbio, Beijing, china), and membranes were incubated overnight at 4 °c with the indicated rabbit anti-human antibodies (1:3000). Subsequently, the mem - branes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:5000) for 1 h. The blots were visual - ised using the Beyoecl Moon chemiluminescence kit (Beyotime, Shanghai, china) on the a mersham Imager 680 imager. Grey values were analysed using the ImageJ software. Band densities were normalised to that of β-actin or GaPDH. AMPK phosphorylation assay c ells were inoculated in culture plates and left to adhere before serum starvation in serum-free DMeM/F-12 (1:1) for 16 h. The cells were treated for 1 h with or without 40 μM c ompound c (Xue et  al. 2013); 20 μM metformin was added, and the cells were maintained for 3 d. We determined a MPK phosphorylation and IGFBP1 expression by western blotting and polymerase chain reaction (PcR), respectively. RNA extraction and reverse transcription quantitative PCR (RT-qPCR) Total Rna was extracted using the TRIzol reagent. one micro - gram of Rna was reverse-transcribed into complementary Dna (cDna ) using transcription reagents from Vazyme. Primers for RT-qPcR were designed by Takara Bio (Shiga, Japan), and the homo gene primer sequences are shown in Table 1 . RT-qPcR was conducted using a 20-μl reaction mix - ture containing 2 μl of cDna. IGFBP1 expression was assessed using 2 × chamQ Sy BR qPcR Master Mix (Vazyme, Shanghai, china) on the Roche a pplied Science lightc ycler 480 II (Roche l td, Germany). The internal reference was β-actin, and the 4 X. SHao e T al. relative IGFBP1 expression was evaluated using the 2-ΔΔ c T method. Immunohistochemistry Paraffin sections of ectopic and eutopic endometrial tissue from patients with ovarian-type endometriosis and normal endometrial tissue from patients without endometriosis were collected. The sections were incubated at 65 °c for 2 h, depa - raffinised with xylene, and hydrated with ethanol. a ntigen repair was performed using high-pressure boiling, followed by blocking of endogenous peroxidase blocker (ZSGB, Beijing, china) and incubation with anti-IGFBP1 primary antibody (Proteintech, Wuhan, china) (1:200) at 4 °c overnight. next, sections were incubated with HRP-labelled secondary anti - body (ZSGB, Beijing, china) for 30 min. c olour was developed using the diaminobenzidine (D aB) substrate kit (ZSGB, Beijing, china) for 1 to 2 min, and nuclei were re-stained with haematoxylin. Statistical analysis Statistical analyses were conducted using the GraphPad Prism software (version 9.0, http://www.graphpad.com). a ll experi - ments were independently repeated thrice, and the data are reported as mean ± standard deviation. The normality of the data was assessed using the Shapiro–Wilk test. The test and control groups were compared using Student’s t-test or a one-way analysis of variance followed by Dunnett’s multiple comparison test. Statistical significance was set at p < .05.

NSCs inhibited Ishikawa cell proliferation, whereas EuSCs and EcSCs lost this ability The edU assay and ccK-8 (expressed as percentages) revealed that e uScs and e cScs lost their ability to suppress Ishikawa cell proliferation to varying degrees compared to nScs ( Fig. 1a, B, and c ). IGFBP1 expression in EuSCs and EcSCs of patients with endometriosis was downregulated IGFBP1 mRna and protein levels in e uScs and e cScs were lower than those in nScs ( Fig. 2(a and B) ). Further, low IGFBP1 expression levels were detected in both e uScs and e cScs (Fig. 2( c )) Quantification of IGFBP1 secretion in stromal cells elISa showed that IGFBP1 secretion by nScs (1.8 ng/ml) was approximately 3.8 and 10 times higher than that in e uScs (0.47 ng/ml) and e cScs (0.18 ng/ml), respectively ( Fig. S1 ). Construction of IGFBP1 overexpression vector We hypothesised that the effects of differences in stromal cells on Ishikawa cell proliferation are related to IGFBP1 levels. ecScs with low IGFBP1 expression were selected for the sub - sequent construction of an IGFBP1 overexpression model. Western blotting and RT-qPcR demonstrated IGFBP1 overex - pression in e cScs ( Fig. S2 ). High IGFBP1 expression in EcSCs increased their ability to inhibit Ishikawa cell proliferation We co-cultured IGFBP1-overexpressing e cScs with Ishikawa cells and performed the e dU assay to assess the effect on cell proliferation. e cScs with high IGFBP1 expression showed enhanced inhibition of Ishikawa cell proliferation. Metformin- |stimulated and IGFBP1-overexpressing e cScs inhibited Ishikawa cell proliferation to a similar extent ( Fig. S3 ). Decreased AMPK phosphorylation in EuSCs and EcSCs a MPK phosphorylation was reduced in euScs and ecScs, whereas total a MPK protein expression in the two cell types was similar to that in nScs ( Fig. S4 ). Metformin treatment increases AMPK phosphorylation in EcSCs and upregulates IGFBP1 expression Metformin treatment increased a MPK phosphorylation in ecScs and upregulated IGFBP1 expression ( Fig. S5a and B) . When a MPK phosphorylation was inhibited using c ompound c, metformin lost its ability to increase IGFBP1 expression in these cells. Metformin inhibits ERK and AKT phosphorylation in Ishikawa cells through IGFBP1 upregulation in EcSCs To explore whether metformin inhibited epithelial cell prolif - eration through IGFBP1 expressional upregulation, we per - formed further experiments using Ishikawa cells. Western blotting showed consistent inhibition of aKT and eRK phos - phorylation ( Fig. S6 ). o verall, stromal cells in patients with endometriosis had a reduced ability to inhibit the growth of epithelial cells than those from patients with no endometriosis. These cells showed differences in the levels of IGFBP1 and a MPK phos - phorylation; specifically, expression was low in e uScs and ecScs. Metformin stimulated a MPK phosphorylation and upregulated IGFBP1 expression in e uScs and e cScs. e cScs stimulated with metformin or IGFBP1 overexpression inhib - ited the aKT and eRK pathways in epithelial cells and enhanced the inhibitory effect on their growth.

We found lower IGFBP1 expression in eutopic and ectopic endometriosis endometrium compared to normal endome - trial tissues, especially in ectopic endometrium. Table 1. Homo gene primers for reverse transcription Pcr. Gene f orward primer (5’–3’) r everse primer (3’–5’) iGfbP1 aGccaa GGcaca GGaGacatc ttccaa GGGta Gac Gcacca G β- a ctin t GGcaccca Gcacaat Gaa ctaa Gtcata Gtcc Gccta Gaa Gca JoURnal oF oBSTe TRIcS anD GynaecoloGy 5 Downregulating IGFBP1 in ectopic endometrial stromal cells reduced their ability to inhibit epithelial cell proliferation. Because of the limited growth potential of primary epithelial cells, we used the Ishikawa cell line (Guzel et  al. 2011). When lentivirus was used to overexpress IGFBP1 in ectopic stromal cells, their ability to inhibit epithelial cell growth was enhanced. Further mechanistic studies revealed that IGFBP1 acts by inhibiting aKT and eRK-related proliferative signal - ling pathways. Metformin is an insulin sensitiser that plays a role in mod - ulating IGFBP1 levels (Tang et al. 2017b). Therefore, we treated ecScs with equivalent in-vitro concentrations of metformin and found that it upregulated IGFBP1. a dditionally, metformin is an agonist of a MPK and acts mainly by activating a MPK (Wang and Wei 2024). a MPK activation plays an important role in various benign and malignant diseases in humans. It regulates cellular energy metabolism and is also a central node in the regulation of malignant tumour progression (Xu et  al. 2024). For example, in colon cancer, it is involved in autophagy induction, leading to inhibition of tumour cell growth (Zhou et  al. 2023). c onversely, its activation may be associated with oncogenesis and drug resistance development (lin et  al. 2023). We observed reduced a MPK phosphorylation in both e uScs and e cScs, while metformin treatment upregulated IGFBP1 expression and enhanced a MPK phosphorylation. a fter pre-treatment with an a MPK inhibitor, IGFBP1 upregulation disappeared, while metformin lost its role in activating a MPK phosphorylation. Therefore, we conjectured that metformin might exert its up-regulatory effect on IGFBP1 by activating a MPK. Ursolic acid and rhodopsin inhibit the growth of cancer cells, such as hepatocellular carcinoma and lung cancer cells, by increasing IGFBP1 expression ( yang et  al. 2016, Tang et  al. 2017a). High IGFBP1 expression correlates with good recurrence-free survival in patients with breast cancer (Wang et  al. 2019). IGFBP1 levels decrease in the follicular fluid of patients with endometriosis, and its extent is linked to the severity of the condition ( c unha-Filho et  al. 2003). o ur find - ings are consistent with previous findings that aberrant IGFBP1 expression in patients with endometriosis is involved in aberrant stromal–epithelial cell communication (stromal cells have a diminished ability to inhibit epithelial cells). Increasing IGFBP1 levels inhibited epithelial cell proliferation. Figure 1. normal stromal cells (nscs) inhibit the growth of ishikawa cells but not that of eutopic stromal cells (e uscs) and ectopic stromal cells (e cscs) (e du a and b, ccK-8 c ). nscs exert a suppressive effect on ishikawa cell proliferation compared to the controls; however, the effect of ecscs and euscs was not signifi - cantly different from that of the control ( p > .05). s cale bar: 50 μm. Values are mean ± sd ( n = 3; ns, not significantly different; *, p < .05; ****, p < .0001). 6 X. SHao e T al. The PI3K/aKT and MeK/eRK pathways mediate cell metab - olism, proliferation, survival, and angiogenesis. a ctivation of the PI3K/aKT, mT oR, and Ras/Raf/MeK/eRK pathways pro - motes the metabolic activity of cancer cells ( a sati et  al. 2016). Inhibition of aKT-related pathways reduces endometrial epi - thelial cell proliferation ( yoo et  al. 2018), and the MeK/eRK1/2 signalling pathway is involved in regulating endometrial epi - thelial cell growth ( chen et  al. 2018). We assessed whether inhibition of ectopic epithelial cells by IGFBP1 was related to these pathways. Metformin treatment or IGFBP1 overexpres - sion in stromal cells inhibited aKT and eRK phosphorylation in Ishikawa cells, suggesting that IGFBP1-mediated inhibition of epithelial cell growth occurs via both a TK- and eRK-associated pathways. This study has some limitations. o ur experiments were performed only in primary cells and cell lines derived from patients with endometriosis or endometrial carcinoma and non-endometrial diseases and were not validated in animal models. We plan to address this in the future. Moreover, we did not explore the specific targets of IGFBP1 in epithelial cells. Furthermore, our study is limited to theory and has not been confirmed in clinical practice; hence, clinical correlation analysis between metformin treatment and pathological parameters of endometriosis is lacking. In summary, IGFBP1 secretion from endometrial cells could influence epithelial cell proliferation. Metformin upregulated IGFBP1 expression in stromal cells by activating a MPK, which in turn exerted antiproliferative effects on the endometriotic epithelium ( Fig. S7 ). This study revealed another mechanism through which metformin affects stromal–epithelial crosstalk in endometrio - sis, laying a foundation for clinical research on the therapeutic potential of metformin in managing endometrio - sis. In addition, IGFBP1 is expected to be another important target for the treatment of endometriosis.

We thank all individuals who supported this study and participated in multiple revisions of the manuscript. We would like to thank e ditage for english language editing. Ethics statement a ll women recruited provided written informed consent, and the utilisa - tion of human tissues was approved by the ethics Review Board of Shandong Provincial Hospital, following the Declaration of Helsinki (a pproval number: SWy X: no . 2023-356). Authorship contribution statement changzhong li conceived the study, Xuping Shao designed and con - ducted the experiments, analysed the data, and completed the first draft of the article, and Junhui liang helped in collecting human tissues and participated in the revision of the manuscript. a ll authors have reviewed and approved the final manuscript. Disclosure statement no potential conflict of interest was reported by the author(s). Funding This work was supported by the Funding for the c onstruction of Key Medical Disciplines in Shenzhen under grant number SZXK027; Funding Figure 2. iGfbP1 expression in e uscs and e cscs of patients with endometriosis was downregulated. ( a ) IGFBP1 levels in e uscs and e cscs were lower than those in nscs, as determined using rt -qPcr. (b) iGfbP1 levels in e uscs and e cscs were lower than those in nscs, as determined using western blotting. Values are mean ± sd ( n = 3; **, p < .01; ***, p < .001; ****, p < .0001). ( c ) i mmunohistochemistry of iGfbP1 expression in both e uscs and e cscs. JoURnal oF oBSTe TRIcS anD GynaecoloGy 7 for the Shenzhen ‘Healthcare Three Project’ under grant number SZSM202011016; and General program of Shenzhen Science and Technology Innovation c ommission under grant number Jcy J20220531094012027. Data availability statement Data will be made available on request.

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