{"paper_id":"774d6b75-6180-4533-a321-0cfb7e872442","body_text":"Journal of Obstetrics and Gynaecology\nISSN: 0144-3615 (Print) 1364-6893 (Online) Journal homepage: www.tandfonline.com/journals/ijog20\nMetformin enhances epithelial cell growth\ninhibition via the protein kinase–insulin-like\ngrowth factor binding protein-1 pathway\nXuping Shao, Changling Li, Junhui Liang & Li Changzhong\nTo cite this article: Xuping Shao, Changling Li, Junhui Liang & Li Changzhong (2024) Metformin\nenhances epithelial cell growth inhibition via the protein kinase–insulin-like growth factor\nbinding protein-1 pathway, Journal of Obstetrics and Gynaecology, 44:1, 2321651, DOI:\n10.1080/01443615.2024.2321651\nTo link to this article:  https://doi.org/10.1080/01443615.2024.2321651\n© 2024 The Author(s). Published by Informa\nUK Limited, trading as Taylor & Francis\nGroup\nView supplementary material \nPublished online: 11 Mar 2024.\n Submit your article to this journal \nArticle views: 2455\n View related articles \nView Crossmark data\n Citing articles: 4 View citing articles \nFull Terms & Conditions of access and use can be found at\nhttps://www.tandfonline.com/action/journalInformation?journalCode=ijog20\n\nGynaecoloGy\nJournal of obstetrics and GynaecoloGy\n2024, Vol. 44, no . 1, 2321651\nMetformin enhances epithelial cell growth inhibition via the protein kinase–\ninsulin-like growth factor binding protein-1 pathway\nXuping Shao a, changling li b, Junhui liang a,c and li changzhong a,d,e,f\nadepartment of Gynaecology, shandong Provincial Hospital, cheeloo c ollege of Medicine, shandong university, Jinan, shandong, china; \nbdepartment of obstetrics and Gynecology o utpatient clinic, the People’s Hospital of Pingyi c ounty, linyi, shandong, china; cdepartment of \nGynecology, shandong Provincial Hospital a ffiliated to shandong f irst Medical university, Jinan, shandong, china; ddepartment of obstetrics \nand Gynecology, shenzhen Hospital, Peking university, shenzhen, Guangdong, china; einstitute of obstetrics and Gynecology, shenzhen \nPKu-HKust Medical c enter, shenzhen, china; fshenzhen Key laboratory on technology for early d iagnosis of Major Gynecologic d iseases, \nshenzhen, china\nABSTRACT\nBackground:  a bnormal stromal-epithelial cell communication is a pathogenic mechanism in \nendometriosis, and metformin can modulate it. Insulin-like growth factor binding protein-1 (IGFBP1) \nplays a role in endometriosis, but the exact mechanism is unknown. IGFBP1 is reportedly a downstream \ntarget of metformin in some diseases. We aimed to investigate the role of IGFBP1 in endometriosis \ndevelopment, whether it is associated with abnormal communication, and whether metformin affects \nIGFBP1 expression.\nMethods: Patients who underwent surgical treatment for endometriosis or other diseases were enrolled. \nTen patients with ovarian-type endometriosis and eight patients each who underwent surgical treatment \nfor other lesions with or without endometriosis were selected, and their tissues taken for cell proliferation, \nwestern blotting, polymerase chain reaction, and knockdown experiments.\nResults: ectopic and eutopic stromal cells (e cScs and e uScs) lost their ability to inhibit epithelial cell \nproliferation, and IGFBP1 expression was downregulated in both groups of stromal cells compared to \nthat in normal stromal cells (nScs; 1.09 vs. 0.25, p = .0002 1.09 vs. 0.57, p = .0029). In an e cSc IGFBP1 \noverexpression model, the ability of ecScs to inhibit epithelial cell proliferation was enhanced (e dU \npositivity decreased from 38% to 25%, p = .0001). Furthermore, adenosine 5’-monophosphate-activated \nprotein kinase ( a MPK) phosphorylation was downregulated in ecScs and euScs compared to that in \nnScs (0.99 vs. 0.42, p = .0006/0.99 vs. 0.57, p = 0.0032). Treatment of e cScs with metformin increased \na MPK phosphorylation (0.47 vs. 1.04, p = .0107) while upregulating IGFBP1 expression (0.69 vs. 1.01, \np = .0164), whereas pre-treatment with an a MPK phosphorylation inhibitor abrogated metformin-induced \nIGFBP1 upregulation.\nConclusions: IGFBP1 mediates aberrant stromal-epithelial communication in endometriosis. Metformin \ncan upregulate IGFBP1 expression in e cScs by activating a MPK, and upregulated IGFBP1 enhances the \ninhibition of epithelial cell proliferation. IGFBP1 is expected to be a therapeutic target for endometriosis.\nPLAIN LANGUAGE SUMMARY\nInsulin-like growth factor binding protein 1 (IGFBP1) is a protein that regulates cell growth and \nproliferation and is expressed at abnormal levels in patients with endometriosis. In some cases, metformin \nhas been shown to modulate the expression of this protein. Here, we investigated the role of IGFBP1 in \nendometriosis development, whether it is associated with abnormal communication, and whether \nmetformin affects IGFBP1 expression in endometrial cells. We found that downregulation of IGFBP1 in \nendometriosis diminished the ability of stromal cells to inhibit the proliferation of epithelial cells through \ninhibition of the protein kinase B and extracellular regulated protein kinase pathways. In addition, \nmetformin upregulated IGFBP1 expression by activating adenosine 5’-monophosphate-activated protein \nkinase, suggesting that IGFBP1 may be one of the potential targets for drug therapy for endometriosis.\nIntroduction\na pproximately, 190 million women globally are affected by \nendometriosis during their lifetime (at least until menopause) \naccording to the World Bank’s 2017 population estimates \n(Horne and Missmer 2022). endometriosis affects metabolism \nin various tissues, including the liver and adipose tissues, \nleading to systemic inflammation (Taylor et  al. 2021). a pro -\ngressive understanding of the disease’s nature has expanded \ntreatment options, and the potential of some drugs for treat -\ning endometriosis is being explored.\n© 2024 t he a uthor(s). Published by i nforma uK limited, trading as taylor & f rancis Group\nCONTACT changzhong li  15168888909@163.com , lichangzhong@pkuszh.com  d epartment of Gynaecology, shandong Provincial Hospital, cheeloo c ollege \nof Medicine, shandong university, Jinan, shandong, china\n supplemental data for this article can be accessed online at https://doi.org/10.1080/01443615.2024.2321651.\nhttps://doi.org/10.1080/01443615.2024.2321651\nt 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, \ndistribution, 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 \nManuscript in a repository by the author(s) or with their consent.\nKEYWORDS\nendometriosis; metformin; \na MPK-IGFBP1; stromal–\nepithelial communication; \nPI3K-aKT\nARTICLE HISTORY\nReceived 19 o ctober 2023\na ccepted 14 f ebruary 2024\n\n2 X. SHao e T al.\nThe growth and differentiation of healthy endometrial epi -\nthelial cells are regulated by stromal cells ( a rnold et  al. 2001), \nwhile a loss of this ability has been implicated in endometri -\nosis. Some factors secreted by ectopic stromal cells, including \nWnt family member 2 (Wnt2), are involved in this aberrant \ncommunication (Zhang et  al. 2015).\nInsulin-like growth factor-binding protein 1 (IGFBP1) is asso -\nciated with endometriosis development, although the exact \nmechanism remains unclear. This study aimed to elucidate this \nmechanism. IGFBP1 is primarily secreted by stromal cells of the \nsecretory endometrium and decreases during endometriosis \n(Meola et  al. 2010, Shih et  al. 2022). It competes with the \ninsulin-like growth factor (IGF) receptor for IGFs, affecting cell \nproliferation, growth, differentiation, apoptosis, migration, inva-\nsion, and adhesion in normal and tumour cells (lin et  al. 2021). \nDuring gestation, decidualised endometrial stromal cells highly \nexpress IGFBP1, which regulates the growth of extravillous tro -\nphoblast cells, a highly migratory cell subset important in \nembryogenesis. Trophoblasts are more invasive in vitro, wherein \nIGFBP1 expression is inhibited (Irwin et  al. 1999). Progesterone \ninduces IGFBP1 expression by endometrial stromal cells in ani -\nmals. High IGFBP1 levels impede endometrial epithelial cell \nproliferation by suppressing IGF action (Murphy and Ghahary \n1990, Seppälä et  al. 1994). excess IGFBP1 impairs the rapid divi -\nsion of breast cancer cells by inhibiting the interaction between \nIGF1 and IGF receptor 1 (Figueroa et  al. 1993). We hypothesised \nthat abnormal IGFBP1 expression in stromal cells in individuals \nwith endometriosis causes abnormal stromal–epithelial cell \ncommunication. We examined whether abnormal IGFBP1 levels \ncontribute to the diminished ability of stromal cells to regulate \nepithelial cells.\nMetformin can target Wnt2 and thereby alleviate abnormal \nstromal–epithelial communication (Zhang et  al.  2010). \nMetformin increases serum IGFBP1 levels in patients with \npolycystic ovary syndrome (Pawelczyk et  al. 2004). In lung \ncancer, metformin potentiates the effect of solamargine in \nupregulating IGFBP1 expression (Tang et  al. 2017b). Thus, we \naimed to examine the potential effect of metformin on \nIGFBP1 expression in endometrial stromal cells and elucidate \nits underlying mechanism.\nMethods\nMaterials\nc ollagenase Ia and metformin were purchased from Sigma–\na ldrich l td. (St l ouis, Mo , USa ). Trypsin, Dulbecco’s modified \neagle’s medium/nutrient mixture F-12 (DMeM/F12; 1:1) \nmedium, and charcoal-stripped foetal bovine serum (FBS) \nwere purchased from Gibco (Billings, MT, USa ). Rabbit \nanti-human IGFBP1, a MPK, phospho- a MPK, protein kinase B \n(aKT), phospho- aKT, extracellular regulated protein kinase \n(eRK), and phospho-eRK were purchased from a bcam \n(c ambridge, M a, USa ). Rabbit anti-human β-actin and  \nGaPDH primary antibodies and goat anti-rabbit horseradish \nperoxidase-conjugated secondary antibodies were purchased \nfrom Proteintech (Wuhan, china). c ompound c (an  \na MPK inhibitor) was purchased from Medchemexpress \n(Shanghai, china).\nPatients and tissue samples\nThis was an experimental study in which ectopic and eutopic \nendometrium of ovarian-type endometriosis and normal \nendometrium of non-endometriosis were collected from \npatients who underwent surgical treatment from June 2022 \nto June 2023 at the Department of Gynaecology of Shandong \nProvincial Hospital. endometriosis was defined as the pres -\nence of endometrial-like tissue outside the uterine cavity, and \novarian-type endometriosis was defined as ectopic endome -\ntrium colonising the ovaries.\nThe inclusion and exclusion criteria were as follows:\nInclusion criteria: (1) patients with ovarian-type endometri -\nosis clearly diagnosed by pathology, patients diagnosed with \novarian-type endometriosis without comorbidity of \noestrogen-dependent diseases, such as adenomyosis, uterine \nfibroids, endometrial polyps, and endometrial cancer, and \nnon-endometriosis patients without the combination of the \nabove oestrogen-dependent diseases (control group).\n(2) a ll participants were premenopausal and had regular \nmenstrual cycles. a ll samples were collected during the secre -\ntory phase of the menstrual period based on menstrual his -\ntory and histological assessment.\nexclusion criteria: (1) c ombination of oestrogen-progestin \ndysregulation caused by other reproductive endocrine-related \ndiseases, such as polycystic ovary syndrome.\n(2) c omorbid oestrogen-related malignant tumours, such \nas endometrial cancer and breast cancer or previous relevant \nmedical history.\n(3) Received hormone therapy within 6 months prior to \nsurgery.\n(4) Taking hormonal contraceptives within 6 months prior \nto surgery.\n(5) Hormonal birth control device placed in the uterine cavity.\na total of 26 patients were included in this experiment; the \nexperimental group contained 18 patients with ovarian-type \nendometriosis. The ectopic endometrium located in the ovaries \nof 10 patients with ovarian-type endometriosis and the intra -\nuterine eutopic endometrium of 8 patients with ovarian-type \nendometriosis were collected; the normal endometrium of \neight patients with non-endometriosis (control group) was also \ncollected. The mean age of the experimental group was \n43.23 ± 5.07 years (mean ± standard deviation), and that of the \ncontrol group was 44.83 ± 5.62 years (mean ± standard deviation).\nPrimary cell culture\nendometrial cells were isolated as previously described \n(Zhang et  al. 2010). endometriotic cysts in the ovaries (ecto -\npic stromal cells [e cScs]), eutopic stromal cells (e uScs; endo -\nmetriomas), and normal stromal cells (nScs; patients without \nendometriosis) were cultured in complete culture media \n(DMeM/F12 [1:1]) supplemented with 10% charcoal-stripped \nFBS and 1% penicillin/streptomycin (Hyclone, l ogan, UT,  \nUSa ) at 37 °c with 5% co 2. The medium was replaced  \nevery 2–3 d. The cultured cells were identified using  \nimmunohistochemistry with mouse anti-human vimentin  \n\nJoURnal oF  oBSTe TRIcS  anD  GynaecoloGy 3\nantibodies (Zhang et  al. 2010). The purity of stromal cells \nwas >98%.\na well-differentiated human endometrioid adenocarcinoma \nepithelial cell line (Ishikawa; c entral laboratory of Shandong \nProvincial Hospital) was grown in a complete culture medium.\nCell proliferation assay\nThe 5-ethynyl-2’-deoxyuridine (e dU) assay was performed to \nassess the effect of IGFBP1 overexpression and metformin \ntreatment on Ishikawa cell proliferation, using the c ell light \nedU a pollo 567 in Vitro Imaging Kit (RiboBio c o., l td., \nGuangzhou, china) following the manufacturer’s instructions. \nBy removing the upper layer of the non-contact co-culture \nsystem, proliferation in the lower layer was assayed. Images \nwere captured using a ZeISS inverted fluorescence micro -\nscope (ZeISS, Germany), and the results were analysed using \nthe ImageJ software (version 2.3.0, https://imagej.nih.gov/).\nHTS Transwell-96 plates with 0.4-µm pore polyester mem -\nbrane inserts ( c orning, ny , USa ) were used in c ell c ounting. \nISK cells (3 × 103/well) were seeded in 96-well plates. The stro -\nmal cells were added to the upper chamber to co-culture \nwith ISK cells for specific times (0, 24, 48, and 72 h), and then, \nc ell c ounting Kit-8 (Medchemexpress, Shanghai, china) was \nused for incubation at 2 h in the dark. The absorbance value \nwas measured at 450 nm.\nEnzyme-linked immunosorbent assay (ELISA)\na n elISa Kit (elabscience Biotechnology, Wuhan, china) was \nused to determine IGFBP1 levels secreted by nScs, e uScs, \nand e cScs following the manufacturer’s instructions with \nsupernatants from cells cultured for 72 h. The mean of dupli -\ncate readings for the standard and sample was calculated, \nand the average zero standard optical density was deducted \nfrom the readings. a four-parameter logistic curve was plot -\nted on a log–log axis, with standard concentrations and opti -\ncal density values on the x- and y-axes, respectively, to \ndetermine sample concentrations.\nTransfection\nl entiviral vectors for IGFBP1 overexpression and a negative \ncontrol lentivirus, both expressing eGFP , were purchased from \nGenechem (Shanghai, china). one group of cells was trans -\nfected with the lentivirus overexpressing IGFBP1 (IGFBP1-l V \ngroup), whereas the other was transfected with an empty \nlentivirus (nc–l V group). The multiplicity of infection was \n50% in both groups. l entivirus was introduced to the medium \nthe following day when the cells had reached 40% conflu -\nence. The lentivirus-containing medium was replaced with \nfresh media after 24 h. a fter 72 h, the transfection efficiency \nwas estimated using an inverted phase/fluorescence micro -\nscope (ZeISS, Germany).\nNon-contact co-culture in transwell plates\nWe used stromal and Ishikawa cells from passages 1–3. \nRegular 24-well and Transwell plates with 0.4-µm pore  \npolyester membrane inserts were purchased from c orning \n(ny, USa ). Stromal cells were seeded into regular twenty-four-\nwell plates and cultured in serum-free DMeM/F-12 (1:1). a fter \nadhesion, the cells were pre-treated for 3 d with DMeM/F12 \n(1:1) containing 2% FBS and 20 μM metformin; based on clin -\nical pharmacokinetics, 20 μM is the accepted clinically equiva -\nlent in-vitro dose (Isoda et  al. 2006, Graham et  al. 2011). \nSubsequently, metformin-treated stromal cells were tryp -\nsinised and seeded in the upper chamber of a Transwell \n24-well culture plate, and Ishikawa cells were seeded in the \nlower chamber, with 0.1 and 0.6 ml of 2% charcoal-stripped \nFBS in the upper and lower chambers, respectively. a fter 3 d, \nthe upper chamber was detached; cell proliferation in the \nlower chamber was examined using the e dU assay, or pro -\nteins were extracted for western blotting.\nTotal protein extraction and western blotting\nTotal protein was extracted using radio-immunoprecipitation \nassay lysis buffer (Beyotime, Shanghai, china) containing 1% \nPMSF reagent (Beyotime, Shanghai, china) and a 1% phos -\nphatase inhibitor cocktail (Medchemexpress, Shanghai, china). \nDenatured total proteins were electrophoresed on a 12% \npolyacrylamide gel and transferred onto polyvinylidene fluo -\nride membranes. Blocking was performed for 1 h in 5% bovine \nserum albumin (Solarbio, Beijing, china), and membranes \nwere incubated overnight at 4 °c with the indicated rabbit \nanti-human antibodies (1:3000). Subsequently, the mem -\nbranes were incubated with HRP-conjugated goat anti-rabbit \nsecondary antibodies (1:5000) for 1 h. The blots were visual -\nised using the Beyoecl Moon chemiluminescence kit \n(Beyotime, Shanghai, china) on the a mersham Imager 680 \nimager. Grey values were analysed using the ImageJ software. \nBand densities were normalised to that of β-actin or GaPDH.\nAMPK phosphorylation assay\nc ells were inoculated in culture plates and left to adhere \nbefore serum starvation in serum-free DMeM/F-12 (1:1) for \n16 h. The cells were treated for 1 h with or without 40 μM \nc ompound c (Xue et  al. 2013); 20 μM metformin was added, \nand the cells were maintained for 3 d. We determined a MPK \nphosphorylation and IGFBP1 expression by western blotting \nand polymerase chain reaction (PcR), respectively.\nRNA extraction and reverse transcription quantitative \nPCR (RT-qPCR)\nTotal Rna was extracted using the TRIzol reagent. one micro -\ngram of Rna was reverse-transcribed into complementary \nDna (cDna ) using transcription reagents from Vazyme. \nPrimers for RT-qPcR were designed by Takara Bio (Shiga, \nJapan), and the homo gene primer sequences are shown in \nTable 1 . RT-qPcR was conducted using a 20-μl reaction mix -\nture containing 2 μl of cDna. IGFBP1 expression was assessed \nusing 2 × chamQ Sy BR qPcR Master Mix (Vazyme, Shanghai, \nchina) on the Roche a pplied Science lightc ycler 480 II (Roche \nl td, Germany). The internal reference was β-actin, and the \n\n4 X. SHao e T al.\nrelative IGFBP1 expression was evaluated using the \n2-ΔΔ c T method.\nImmunohistochemistry\nParaffin sections of ectopic and eutopic endometrial tissue \nfrom patients with ovarian-type endometriosis and normal \nendometrial tissue from patients without endometriosis were \ncollected. The sections were incubated at 65 °c for 2 h, depa -\nraffinised with xylene, and hydrated with ethanol. a ntigen \nrepair was performed using high-pressure boiling, followed \nby blocking of endogenous peroxidase blocker (ZSGB, Beijing, \nchina) and incubation with anti-IGFBP1 primary antibody \n(Proteintech, Wuhan, china) (1:200) at 4 °c overnight. next, \nsections were incubated with HRP-labelled secondary anti -\nbody (ZSGB, Beijing, china) for 30 min. c olour was developed \nusing the diaminobenzidine (D aB) substrate kit (ZSGB, Beijing, \nchina) for 1 to 2 min, and nuclei were re-stained with \nhaematoxylin.\nStatistical analysis\nStatistical analyses were conducted using the GraphPad Prism \nsoftware (version 9.0, http://www.graphpad.com). a ll experi -\nments were independently repeated thrice, and the data are \nreported as mean ± standard deviation. The normality of the \ndata was assessed using the Shapiro–Wilk test. The test and \ncontrol groups were compared using Student’s t-test or a \none-way analysis of variance followed by Dunnett’s multiple \ncomparison test. Statistical significance was set at p < .05.\nResults\nNSCs inhibited Ishikawa cell proliferation, whereas \nEuSCs and EcSCs lost this ability\nThe edU assay and ccK-8 (expressed as percentages) revealed \nthat e uScs and e cScs lost their ability to suppress Ishikawa \ncell proliferation to varying degrees compared to nScs ( Fig. \n1a, B, and c ).\nIGFBP1 expression in EuSCs and EcSCs of patients with \nendometriosis was downregulated\nIGFBP1 mRna and protein levels in e uScs and e cScs were \nlower than those in nScs ( Fig. 2(a and B) ). Further, low IGFBP1 \nexpression levels were detected in both e uScs and e cScs \n(Fig. 2( c ))\nQuantification of IGFBP1 secretion in stromal cells\nelISa showed that IGFBP1 secretion by nScs (1.8 ng/ml) was \napproximately 3.8 and 10 times higher than that in e uScs \n(0.47 ng/ml) and e cScs (0.18 ng/ml), respectively ( Fig. S1 ).\nConstruction of IGFBP1 overexpression vector\nWe hypothesised that the effects of differences in stromal \ncells on Ishikawa cell proliferation are related to IGFBP1 levels. \necScs with low IGFBP1 expression were selected for the sub -\nsequent construction of an IGFBP1 overexpression model. \nWestern blotting and RT-qPcR demonstrated IGFBP1 overex -\npression in e cScs ( Fig. S2 ).\nHigh IGFBP1 expression in EcSCs increased their ability \nto inhibit Ishikawa cell proliferation\nWe co-cultured IGFBP1-overexpressing e cScs with Ishikawa \ncells and performed the e dU assay to assess the effect on cell \nproliferation. e cScs with high IGFBP1 expression showed \nenhanced inhibition of Ishikawa cell proliferation. Metformin-\n|stimulated and IGFBP1-overexpressing e cScs inhibited \nIshikawa cell proliferation to a similar extent ( Fig. S3 ).\nDecreased AMPK phosphorylation in EuSCs and EcSCs\na MPK phosphorylation was reduced in euScs and ecScs, \nwhereas total a MPK protein expression in the two cell types \nwas similar to that in nScs ( Fig. S4 ).\nMetformin treatment increases AMPK phosphorylation \nin EcSCs and upregulates IGFBP1 expression\nMetformin treatment increased a MPK phosphorylation in \necScs and upregulated IGFBP1 expression ( Fig. S5a and B) . \nWhen a MPK phosphorylation was inhibited using c ompound \nc, metformin lost its ability to increase IGFBP1 expression in \nthese cells.\nMetformin inhibits ERK and AKT phosphorylation in \nIshikawa cells through IGFBP1 upregulation in EcSCs\nTo explore whether metformin inhibited epithelial cell prolif -\neration through IGFBP1 expressional upregulation, we per -\nformed further experiments using Ishikawa cells. Western \nblotting showed consistent inhibition of aKT and eRK phos -\nphorylation ( Fig. S6 ).\no verall, stromal cells in patients with endometriosis had a \nreduced ability to inhibit the growth of epithelial cells than \nthose from patients with no endometriosis. These cells \nshowed differences in the levels of IGFBP1 and a MPK phos -\nphorylation; specifically, expression was low in e uScs and \necScs. Metformin stimulated a MPK phosphorylation and \nupregulated IGFBP1 expression in e uScs and e cScs. e cScs \nstimulated with metformin or IGFBP1 overexpression inhib -\nited the aKT and eRK pathways in epithelial cells and \nenhanced the inhibitory effect on their growth.\nDiscussion\nWe found lower IGFBP1 expression in eutopic and ectopic \nendometriosis endometrium compared to normal endome -\ntrial tissues, especially in ectopic endometrium. \nTable 1. Homo gene primers for reverse transcription Pcr.\nGene f orward primer (5’–3’) r everse primer (3’–5’)\niGfbP1 aGccaa GGcaca GGaGacatc ttccaa GGGta Gac Gcacca G\nβ- a ctin t GGcaccca Gcacaat Gaa ctaa Gtcata Gtcc Gccta Gaa Gca\n\nJoURnal oF  oBSTe TRIcS  anD  GynaecoloGy 5\nDownregulating IGFBP1 in ectopic endometrial stromal cells \nreduced their ability to inhibit epithelial cell proliferation. \nBecause of the limited growth potential of primary epithelial \ncells, we used the Ishikawa cell line (Guzel et  al. 2011). When \nlentivirus was used to overexpress IGFBP1 in ectopic stromal \ncells, their ability to inhibit epithelial cell growth was \nenhanced. Further mechanistic studies revealed that IGFBP1 \nacts by inhibiting aKT and eRK-related proliferative signal -\nling pathways.\nMetformin is an insulin sensitiser that plays a role in mod -\nulating IGFBP1 levels (Tang et al. 2017b). Therefore, we treated \necScs with equivalent in-vitro concentrations of metformin \nand found that it upregulated IGFBP1. a dditionally, metformin \nis an agonist of a MPK and acts mainly by activating a MPK \n(Wang and Wei 2024). a MPK activation plays an important \nrole in various benign and malignant diseases in humans. It \nregulates cellular energy metabolism and is also a central \nnode in the regulation of malignant tumour progression (Xu \net  al. 2024). For example, in colon cancer, it is involved in \nautophagy induction, leading to inhibition of tumour cell \ngrowth (Zhou et  al. 2023). c onversely, its activation may be \nassociated with oncogenesis and drug resistance \ndevelopment (lin et  al. 2023). We observed reduced a MPK \nphosphorylation in both e uScs and e cScs, while metformin \ntreatment upregulated IGFBP1 expression and enhanced \na MPK phosphorylation. a fter pre-treatment with an a MPK \ninhibitor, IGFBP1 upregulation disappeared, while metformin \nlost its role in activating a MPK phosphorylation. Therefore, \nwe conjectured that metformin might exert its up-regulatory \neffect on IGFBP1 by activating a MPK.\nUrsolic acid and rhodopsin inhibit the growth of cancer \ncells, such as hepatocellular carcinoma and lung cancer cells, \nby increasing IGFBP1 expression ( yang et  al. 2016, Tang et  al. \n2017a). High IGFBP1 expression correlates with good \nrecurrence-free survival in patients with breast cancer (Wang \net  al. 2019). IGFBP1 levels decrease in the follicular fluid of \npatients with endometriosis, and its extent is linked to the \nseverity of the condition ( c unha-Filho et  al. 2003). o ur find -\nings are consistent with previous findings that aberrant \nIGFBP1 expression in patients with endometriosis is involved \nin aberrant stromal–epithelial cell communication (stromal \ncells have a diminished ability to inhibit epithelial cells). \nIncreasing IGFBP1 levels inhibited epithelial cell \nproliferation.\nFigure 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 \nand 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 -\ncantly 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).\n\n6 X. SHao e T al.\nThe PI3K/aKT and MeK/eRK pathways mediate cell metab -\nolism, proliferation, survival, and angiogenesis. a ctivation of \nthe PI3K/aKT, mT oR, and Ras/Raf/MeK/eRK pathways pro -\nmotes the metabolic activity of cancer cells ( a sati et  al. 2016). \nInhibition of aKT-related pathways reduces endometrial epi -\nthelial cell proliferation ( yoo et  al. 2018), and the MeK/eRK1/2 \nsignalling pathway is involved in regulating endometrial epi -\nthelial cell growth ( chen et  al. 2018). We assessed whether \ninhibition of ectopic epithelial cells by IGFBP1 was related to \nthese pathways. Metformin treatment or IGFBP1 overexpres -\nsion in stromal cells inhibited aKT and eRK phosphorylation \nin Ishikawa cells, suggesting that IGFBP1-mediated inhibition \nof epithelial cell growth occurs via both a TK- and \neRK-associated pathways.\nThis study has some limitations. o ur experiments were \nperformed only in primary cells and cell lines derived from \npatients with endometriosis or endometrial carcinoma and \nnon-endometrial diseases and were not validated in animal \nmodels. We plan to address this in the future. Moreover, we \ndid not explore the specific targets of IGFBP1 in epithelial \ncells. Furthermore, our study is limited to theory and has not \nbeen confirmed in clinical practice; hence, clinical correlation \nanalysis between metformin treatment and pathological \nparameters of endometriosis is lacking.\nIn summary, IGFBP1 secretion from endometrial cells could \ninfluence epithelial cell proliferation. Metformin upregulated \nIGFBP1 expression in stromal cells by activating a MPK, which \nin turn exerted antiproliferative effects on the endometriotic \nepithelium ( Fig. S7 ).\nThis study revealed another mechanism through which \nmetformin affects stromal–epithelial crosstalk in endometrio -\nsis, laying a foundation for clinical research on the \ntherapeutic potential of metformin in managing endometrio -\nsis. In addition, IGFBP1 is expected to be another important \ntarget for the treatment of endometriosis.\nAcknowledgements\nWe thank all individuals who supported this study and participated in \nmultiple revisions of the manuscript. We would like to thank e ditage for \nenglish language editing.\nEthics statement\na ll women recruited provided written informed consent, and the utilisa -\ntion of human tissues was approved by the ethics Review Board of \nShandong Provincial Hospital, following the Declaration of Helsinki \n(a pproval number: SWy X: no . 2023-356).\nAuthorship contribution statement\nchangzhong li conceived the study, Xuping Shao designed and con -\nducted the experiments, analysed the data, and completed the first draft \nof the article, and Junhui liang helped in collecting human tissues and \nparticipated in the revision of the manuscript. a ll authors have reviewed \nand approved the final manuscript.\nDisclosure statement\nno potential conflict of interest was reported by the author(s).\nFunding\nThis work was supported by the Funding for the c onstruction of Key \nMedical Disciplines in Shenzhen under grant number SZXK027; Funding \nFigure 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 \nin 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 \nmean ± sd ( n = 3; **, p < .01; ***, p < .001; ****, p < .0001). ( c ) i mmunohistochemistry of iGfbP1 expression in both e uscs and e cscs.\n\nJoURnal oF  oBSTe TRIcS  anD  GynaecoloGy 7\nfor the Shenzhen ‘Healthcare Three Project’ under grant number \nSZSM202011016; and General program of Shenzhen Science and \nTechnology Innovation c ommission under grant number \nJcy J20220531094012027.\nData availability statement\nData will be made available on request.\nReferences\na rnold, J.T., et  al., 2001. endometrial stromal cells regulate epithelial cell \ngrowth in vitro: a new co-culture model. Human Reproduction (Oxford, \nEngland), 16 (5), 1–7.\na sati, V., et  al., 2016. 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