{"paper_id":"899b985d-7211-4ab6-92f9-aac8f0df0d32","body_text":"Hao et al. \nReproductive Biology and Endocrinology           (2022) 20:85  \nhttps://doi.org/10.1186/s12958-022-00955-w\nRESEARCH\nActivation of α7 nicotinic acetylcholine \nreceptor retards the development \nof endometriosis\nMeihua Hao1†, Xishi Liu1,2† and Sun‑Wei Guo1,2* \nAbstract \nBackground: Women with endometriosis have been shown to have a reduced vagal tone as compared with con‑\ntrols and vagotomy promoted while vagus nerve stimulation (VNS) decelerated the progression of endometriosis \nin mice. Extensive research also has shown that the activation of the cholinergic anti‑inflammatory pathway by VNS \nactivates α7 nicotinic acetylcholine receptor (α7nAChR), potently reducing inflammation. Yet whether α7nAChR plays \nany role in endometriosis is unknown. We evaluated its expression in normal endometrium, ovarian and deep endo‑\nmetriotic lesions, and evaluated its role in the development of endometriosis.\nMethods: Immunohistochemistry analyses of α7nAChR in endometriotic lesions as well as control endometrium, \nand quantification of tissue fibrosis by Masson trichrome staining were performed. Mouse experiments were con‑\nducted to evaluate the impact of α7nAChR activation or suppression on lesional progression and possible therapeutic \neffect. Finally, in vitro experiments were conducted to evaluate the effect of activation of α7nAChR on epithelial‑mes‑\nenchymal transition (EMT), fibroblast‑to‑myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM) \nand fibrogenesis in an endometriotic epithelial cell line and primary endometriotic stromal cells derived from ovarian \nendometrioma tissue samples.\nResults: Immunostaining of α7nAChR was significantly reduced in human endometriotic epithelial cells as com‑\npared with their counterpart in normal endometrium. Lesional α7nAChR staining levels correlated negatively with \nlesional fibrosis and the severity of dysmenorrhea. The α7nAChR agonist significantly impeded the development of \nendometriotic lesions in mouse models possibly through hindrance of EMT and FMT. It also demonstrated therapeu‑\ntic effects in mice with induced deep endometriosis. Treatment of endometriotic epithelial and stromal cells with an \nα7nAChR agonist significantly abrogated platelet‑induced EMT, FMT and SMM, and suppressed cellular contractility \nand collagen production.\nConclusions: α7nAChR is suppressed in endometriotic lesions, and its activation by pharmacological means can \nimpede EMT, FMT, SMM, and fibrogenesis of endometriotic lesions. As such, α7nAChR can be rightfully viewed as a \npotential target for therapeutic invention.\nTrial registration: Not applicable.\n© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco \nmmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.\nOpen Access\n†Meihua Hao and Xishi Liu contributed equally to this work.\n*Correspondence:  hoxa10@outlook.com\n1 Shanghai Obstetrics and Gynecology Hospital, Fudan University, \nShanghai 200011, China\nFull list of author information is available at the end of the article\n\nPage 2 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nIntroduction\nFeaturing the presence of endometrial-like tissues out -\nside the uterine cavity, endometriosis is an estrogen-\ndependent and debilitating disease, affecting 10% of \nwomen of reproductive age [1]. It negatively impacts \non the quality of life of affected patients and is a major \ncontributing cause of dysmenorrhea, chronic pelvic pain \nand infertility. Due to its poorly understood pathogen -\nesis and pathophysiology, its clinical management is still \nchallenging [1]. Although the new generation of GnRH \nantagonists has been proven to be effective for treating \nendometriosis-associated pain [2], cost appears to be a \nmajor barrier to effective management [3]. In addition, \nwell over 60% of endometriosis patients hold a negative \nattitude towards hormonal drugs in general [4], thus  it \nseems that the development of efficacious and economi -\ncal non-hormonal medical treatment is still an unmet \nneed waiting to be fulfilled [5–7].\nOver the years, it has been recognized that endome -\ntriosis also is a chronic inflammatory disease [8], featur -\ning NF-κB activation [9, 10] and increased production \nof pro-inflammatory cytokines and chemokines [11]. As \nsuch, anti-inflammatory therapy has attracted a great \ndeal of attention. Unfortunately, clinical trials on anti-\ninflammatory therapy so far have been or presumed to \nbe failed due to either lack of efficacy [12–14] or serious \nadverse events (such as AKR1C3 antagonist) [1] or both. \nHence, there is still a need for effective suppression of \ninflammation without causing serious collateral damage.\nIn the last two decades, there is accumulating evidence \nfor an intimate and intricate link between nervous and \nimmune systems. In particular, vagus nerves regulate \nnumerous central and peripheral processes through both \nafferent and efferent fibers [15], forming the cholinergic \nanti-inflammatory pathway (CAIP) [16–18]. The CAIP \ntheory stipulates that, first, the ascending afferent vagal \nnerve fibers can convey the presence of inflammatory \nmediators resulting from either pathogen invasion or tis -\nsue injury to inform the central nervous system (CNS), \nand, second, the CNS releases, through efferent nerves, \nneuromediators that act on immune cells and modulate \nlocal inflammation to restore local immune homeostasis \n[19, 20]. In particular, anti-inflammatory reflex signaling \ncan and has been proven to be enhanced by vagus nerve \nstimulation (VNS), resulting in significant reduction in \ncytokine production in a plethora of acute and progres -\nsive inflammatory conditions such as sepsis [21], asthma \n[22], rheumatoid arthritis [23], and stroke [24]. VNS also \nhas been reported to attenuate lipopolysaccharide (LPS) \ninduced acute lung injury by inhibiting neutrophil infil -\ntration and neutrophil extracellular traps (NETs) forma -\ntion [25].\nConsistent with the CAIP theory, we have previously \nshown that women with endometriosis have a reduced \nvagal tone as compared with controls [26]. In addition, \nvagotomy facilitates while VNS decelerates the lesional \nprogression in a mouse model of endometriosis [26]. \nRemarkably, non-invasive auricular VNS shows prom -\nising therapeutic effect, as evidenced by significantly \nreduced lesion weight and retarded lesional progression \nconcomitant with improved hyperalgesia [26].\nIt has been known that vagal efferent fibers release \nacetylcholine (ACh) that can interact with α7-subunit \nnicotinic acetylcholine  receptor (α7nAChR) expressed \non tissue macrophages and other immune cells to sup -\npress the production and secretion of pro-inflammatory \ncytokines such as TNFα, IL-1β, and IL-6 [17]. Indeed, \nthe activation of the CAIP by VNS typically activates \nα7nAChR, potently reduces inflammation in peripheral \ntissues [27, 28].\nIn endometriosis, it is reported that α7nAChR \nis expressed in peritoneal fluid mononuclear cells \n(PFMCs) from patients with and without endometriosis \n[29]. Treatment with an α7nAChR agonist inhibited the \ngene and protein expression of IL-1β in PFMCs stimu -\nlated with LPS [29]. The activation of α7nAChR by an \nagonist significantly suppressed the formation of endo -\nmetriotic lesions, which was reversed with an α7nAChR \nantagonist [29].\nWe have shown recently that, compared with normal \nendometrium, the immunoreactivity against α7nAChR \nis significantly reduced in both stromal and epithelial \ncells in adenomyotic lesions [30]. However, whether its \nexpression is reduced in endometriosis has so far not \nbeen investigated. Nor do we know whether its modula -\ntion would have any effect on lesional development, and, \nif so, whether it can be a potential therapeutic target.\nIn this study, we hypothesized that, as in adenomyosis, \nα7nAChR expression is reduced in endometriotic lesions, \nperhaps more so in deep endometriosis (DE) as compared \nwith ovarian endometrioma (OE). We also hypothesized \nthat modulation of α7nAChR by pharmacological means \nwould impact on lesional development. If this were the \ncase, we would further hypothesize that the activation \nof α7nAChR by pharmacological means may have thera -\npeutic potentials. Moreover, we hypothesized the arrest \nKeywords: α7 nicotinic acetylcholine receptor, Endometriosis, Epithelial‑mesenchymal transition, Fibroblast‑to‑\nmyofibroblast transdifferentiation, Fibrogenesis, Smooth muscle metaplasia\n\nPage 3 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nin lesional progression rendered by α7nAChR activation \nmay be through the inhibition of epithelial-mesenchymal \ntransition (EMT), fibroblast-to-myofibroblast transdiffer-\nentiation (FMT), smooth muscle metaplasia (SMM) and \nfibrogenesis—the molecular events known to occur in \nlesional progression [31–33]. This study was designed to \ntest these hypotheses.\nMaterials and methods\nStudy participants\nWe recruited 17 patients with laparoscopically and his -\ntologically diagnosed OE and 14 DE patients, who vis -\nited Shanghai OB & GYN Hospital, Fudan University, \nover a time span from March, 2015 to March, 2017. We \nrecruited these patients who were about to undergo lapa-\nroscopy due to OE or DE per gynecological and imaging \nexamination. None of them had taken any anti-platelet, \nhormonal, oral contraceptives, or other medications at \nleast 3 months prior to the surgery.\nFor controls, we recruited 18 age-matched patients \nwho came to our hospital to undergo colposcopy or loop \nelectrosurgical excision procedure (LEEP) treatment \ndue to cervical intraepithelial neoplasia. None of them \nhad any previous gynecological disorders and symp -\ntoms, or any evidence for endometriosis or adenomyo -\nsis per sonographic examination. The medical records of \nall recruited patients, including clinical symptoms and \npathological reports, were carefully reviewed. Written \ninformed consent was obtained from all patients. This \nstudy was approved by the ethics review board of Shang -\nhai Obstetrics and Gynecology Hospital, Fudan Univer -\nsity (approved on March 11, 2015, on file).\nAnimals and chemicals\nAll experiments were performed in the in-house animal \nfacility in accordance with the guidelines of the National \nResearch Council’s Guide for the Care and Use of Labo -\nratory Animals [34] and approved by the institutional \nexperimental animals review board of Shanghai OB/GYN \nHospital, Fudan University. A total of 60 female Balb/C \nmice, about 6–8  weeks old and about 18–21  g in body -\nweight, were purchased from Shanghai LingChang Labo -\nratory Animal Center (Shanghai, China) and used for this \nstudy. Among them, 20 were randomly selected as donors \nthat contributed uterine fragments, and the remaining 40 \nwere recipients.\nThe PNU-282987 and substance P (SP) were purchased \nfrom Sigma-Aldrich (St Louis, MO, USA), and methylly -\ncaconitine citrate (MLA) from Abcam (Cambridge, UK). \nPNU-282987, MLA and SP were all dissolved in 0.9% \nsterile saline. As such, the control group used saline as a \nmock treatment.\nInduction of endometriosis\nThe mouse model of endometriosis was established \nby intraperitoneal injection of uterine fragments as \nwe described previously [33, 35]. The inclusion of the \nfull-thickness of uterine tissues appeared to be more \nadvantageous in establishing endometriosis, as shown \nin baboons [36] and mouse [37]. Briefly, after one week \nof acclimatization, donor mice were injected i.m. with \n150 μg/kg estradiol benzoate (Animal Medicine Factory, \nHangzhou, China) twice within a week. The donor mice \nwere sacrificed and their uteri were harvested on the day \nof induction. The harvested uterine tissues were rinsed \ntwice with sterile saline and then cut into pieces by a pair \nof scissors, making sure that the maximal diameter of \nthe fragment was smaller than 1 mm. Uterine fragments \nfrom one donor mouse were injected intraperitoneally to \ntwo recipient mice.\nMouse experimental protocols\nIn mouse Experiment 1, we evaluated the effect of \nα7nAChR agonist (PNU-282987) and antagonist methyl -\nlycaconitine (MLA) on lesional development. PNU-\n282987 is a highly selective α7nAChR agonist and MLA \nis a selective and potent antagonist of α7nAChR. The \ndoses of these drugs were determined based on the con -\nversion of dosages used in other experiments [38, 39].\nA total of 24 female Balb/C mice were divided ran -\ndomly into 3 groups of equal sizes. PNU-282987 (2 mg/\nkg/day) and MLA (2  mg/kg/day) were administrated \nvia Alzet osmotic pumps (model 1004, DURECT Corp, \nCupertino, CA, USA) one week before the induction \nof endometriosis. The osmotic pump ensured consist -\nent and controlled release of its contents for 4  weeks. \nFor control mice, the same pumps containing an equal \namount sterile saline only were administrated one week \nbefore the induction of endometriosis. For all mice, their \nbodyweight was recorded weekly (every 7 days) and the \nhotplate test was administered right before the induction \nprocedure and then weekly until sacrifice. Three weeks \nafter induction, all mice were sacrificed. Their abdomi -\nnal cavity was immediately opened up, and all visible \nendometriotic lesions were carefully excised from the \nsurrounding tissue, washed thrice in order to remove \nthe mucosa and connective tissue. The lesion weight was \nmeasured 24 h after extraction and aspiration in order to \nminimize or eliminate any difference in water content, as \nreported previously [40]. Cystic appearance was observed \nin lesions [41]. The endometriotic tissue samples were \nthen processed for quantification, immunohistochemis -\ntry (IHC) and Masson trichrome staining. We note that \nfor the mouse model we used, the lesion weight seems \nto be a more reliable outcome measure than the number \n\nPage 4 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nof  lesions, since typically we observed that 2 or more \nlesions could be conjoined together, making the count a \nbit challenging.\nTo see whether PNU-282987 has any therapeutic \npotential to treat DE, we conducted mouse Experi -\nment 2. We used an established mouse model of DE as \ndescribed previously [40]. Briefly, one day before the \ninduction of endometriosis, Alzet osmotic pumps (model \n1004, DURECT Corp) were inserted in the nape of the \nneck in all mice to infuse substance P (100  μg/kg/day). \nFour weeks after the induction procedure, DE model was \nestablished. Sixteen Balb/C mice were randomly divided \ninto two equal-sized groups, the control and the PNU-\n282987 groups. Mice in PNU-282987 group received \nPNU-282987 (2  mg/kg/day) [38] via Alzet osmotic \npumps and mice in the control group only received \nequal amount sterile saline. The bodyweight and hotplate \nlatency were evaluated every week. Three weeks after the \nPNU-282987 treatment, all mice were sacrificed and all \nlesions were excised and processed for quantification, \nIHC and Masson trichrome staining.\nHotplate test\nThe hotplate test was performed with a Hot Plate Instru -\nment (RB-200, Chengdu Techman Software Co. Ltd., \nChengdu, China) to assess the extent of hyperalgesia \nas reported previously [42, 43]. Briefly, a metal plate of \n19  cm × 19  cm in size was heated to a constant tem -\nperature of 55.0 ℃ ± 0.1 ℃. A plastic cylinder was set on \nthe hotplate. Mice were brought into the cylinder and \nallowed to acclimatize for 10  min before the test. The \nlatency to respond to thermal stimulus was defined as \nthe time (in seconds) elapsed from the moment when the \nmouse was inserted into the cylinder until it began to lick \nits hind paws or jolted or jumped off the hotplate. Each \nmouse was tested only once in each session. The latency \nwas calculated as the mean of 2 readings recorded at an \ninterval of 24 h. The evaluator was unaware of the group \nidentify of the mouse she was evaluating, and was practi -\ncally blinded.\nImmunohistochemistry\nTissue samples were fixed in 4% paraformaldehyde (w/v) \nand then paraffin embedded. Serial 4-μm sections were \nobtained from each block. Hematoxylin and eosin stain -\ning were performed to further confirm pathological diag -\nnosis. Immunohistochemistry analysis for α7nAChR \n(rabbit polyclonal antibody, 1:200, Abcam, Cambridge, \nUK), E-cadherin (rabbit monoclonal antibody, 1:100, \nCell Signaling Technology, Boston, MA, USA), α-smooth \nmuscle actin (α-SMA) (rabbit polyclonal antibody, 1:100, \nAbcam), and smooth muscle myosin heavy chain (SM-\nMHC) (rabbit polyclonal antibody, 1:100, Abcam) and \ndesmin (mouse monoclonal antibody, 1:100, Abcam) was \nperformed in subsequent slides via a polymeric/enzy -\nmatic HRP method.\nDeparaffinization and dehydration procedures were \nperformed as previously described [44]. Slides were \nimmersed in citrate buffer and heated at 98 °C pressure \ncooker for 30 min, then cooled down to room tempera -\nture. The tissue was blocked in goat serum for 30 min at \nroom temperature and then incubated at 4  °C with pri -\nmary antibodies overnight. HRP conjugated secondary \nantibodies were applied at room temperature for 30 min \nthe following day. Positive staining was visualized using \n3, 3’-diaminobenzidine (JieHao Biological Technology, \nShanghai, China) and counterstained with hematoxy -\nlin (JieHao). Images were obtained with the microscope \n(Olympus BX53, Olympus, Tokyo, Japan) fitted with a \ndigital camera (Olympus DP73, Olympus). Five randomly \nselected images at 400 × magnification of each sample \nwere taken to obtain a mean optical density (MOD) value \nand the mean density of staining intensity was acquired \nby Image-Pro Plus 6.0 (Media Cybernetics Inc, Bethesda, \nMD, USA). Staining was defined via color intensity. \nBriefly, a color mask was made, and then the mask was \napplied equally to all images. Subsequent measurement \nreadings were obtained. Immunohistochemical param -\neters were assessed in the staining area, including inte -\ngrated optical density (IOD), total stained area (S), and \nthe MOD, which was defined as MOD = IOD/S. MOD \nwas equivalent to the mean intensity of staining across \nall evaluated areas. To minimize any bias, the scorer was \nunaware of the group identify of the mouse she was eval -\nuating, and was practically blinded.\nFor positive controls, human breast cancer tissues \nwere used for E-cadherin, mouse liver tissues for α-SMA, \nhuman adenomyotic tissue samples for desmin and SM-\nMHC, and mouse lung tissues for α7nAChR. For nega -\ntive controls, mouse endometriotic lesions were used \nwith IgG from rabbit or mouse serum instead of primary \nantibodies. The representative photomicrographs for \nnegative and positive staining controls are shown in Sup -\nplementary Figure S1.\nMasson trichrome staining\nMasson trichrome staining was performed for detect -\ning collagen and fibers in endometriotic tissue samples \nas previously described [45]. Briefly, tissue sections were \ndeparaffinized in xylene and rehydrated in a gradient \nalcohol series, then were immersed in Bouin’s solution \n(saturated picric acid 75 ml, 10% (w/v) formalin solution \n25 ml and acetic acid 5 ml) at 37 °C for 2 h. Sections were \nstained using the Masson’s Trichrome Staining kit (Baso, \nWuhan, China) following the manufacturer’s instructions \nand used as previously reported. In Masson staining, \n\nPage 5 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nthose stained in blue were ollagen fibers, while those in \nred were muscle fibers. The areas of the collagen fiber \nlayer stained in blue in proportion to the entire field of \nthe ectopic endometrium were calculated by the Image \nPro-Plus 6.0 as the percentage of fibrosis.\nEndometriotic epithelial cells and primary endometriotic \nstromal cells\nThe immortalized human endometriotic epithelial cell \nline (11Z), established by Professor Anna Strazinski-\nPowitz using SV40-antigen [46], was kindly provided \nby Dr. Jung-Hye Choi of Kyung Hee University, Seoul, \nKorea. Cells were cultured in RPMI 1640 medium (Gibco \nLaboratories, Life Technologies, Grand Island, NY, USA) \nsupplemented with 5% fetal bovine serum (FBS) (Gibco \nLaboratories), 100  IU/mL penicillin G, 100  mg/mL \nstreptomycin and 2.5 mg/mL Amphotericin B (Hyclone, \nLogan, UT, USA). Human endometriotic stromal cells \n(HESCs) were primarily cultured as reported in our pre -\nvious work [31]. Briefly, the OE tissues were washed with \nDMEM/F-12 medium and minced into small pieces of \nabout 1  mm3 in size. Tissues were digested with 0.2% col-\nlagenase II (Sigma-Aldrich) in a shaking bed for 1.5 h at \n37 °C. The digested tissues were filtrated through a sterile \n76-mm then a 37-mm filter. The filtrated cells were cen -\ntrifuged and suspended in DMEM/F-12 medium sup -\nplemented with 10% FBS, 100 IU/mL penicillin, 100 mg/\nmL streptomycin and 2.5  mg/mL Amphotericin B, and \nseeded into a 25-cm 2 cell culture flask and incubated at \n37  °C in 5%  CO2 in air. Cells with different treatments \nwere used for quantitative real-time RT-PCR, Western \nblot, invasion assay, scratch test, cell immunofluores -\ncence, cell contraction assay and collagen assay.\nRNA isolation and quantitative real‑time PCR\nTotal RNA was isolated from 11Z cells and HESCs using \nTRIzol (Invitrogen, Carlsbad, CA, USA). cDNA synthe -\nsis was performed using the reverse transcription kit \n(Takara, Takara Bio, Inc., Otsu, Shiga, Japan). The abun -\ndance of mRNA was evaluated by real-time PCR using \nSYBR Premix Ex Taq (Takara). Expression values were \nnormalized to the geometric mean of GAPDH measure -\nments. The names of genes and their primers are listed in \nTable 1.\nWestern blot analysis\nCells were washed twice with PBS and lysed on ice \nwith Radio-Immunoprecipitation Assay (RIPA) buffer \n(Thermo Fisher, Waltham, MA, USA) containing 1% pro-\ntease inhibitor cocktail (Sigma). Protein concentration \nwas determined using bicinchoninic acid (BCA) protein \nquantitative analysis kit (Beyotime, Shanghai, China). \nProteins were loaded on a 6%-12% SDS-PAGE, and \ntransferred to polyvinyl difluoride (PVDF) membranes \n(Sigma-Aldrich). The membranes were incubated at 4℃ \novernight with the primary antibodies for E-cadherin \n(1:1000, Cell Signaling Technology), α-SMA (1:1000, \nAbcam), LOX (1:1000, Abcam), SM-MHC (1:1000, \nAbcam), desmin (1:1000, Abcam), GAPDH (1:1000, Bey -\notime) and β-tubulin (1:1000, Cell Signaling Technology). \nThe membranes were incubated with HRP labeled sec -\nondary antibodies for 1 h at room temperature, and the \nsignals were developed with enhanced chemilumines -\ncence (ECL) reagents (Millipore, Burlington, MA, USA) \nand digitized on Image Quant LAS 4000 mini. Image \nquantification was carried out with Quantity One soft -\nware (Bio-Rad, Hercules, California, USA).\nImmunofluorescence\nThe endometriotic epithelial cells (11Z) were seeded \ninto 24-well plates and treated with buffer (PBS), acti -\nvated platelets, PNU-282987 or both activated platelets \nand PNU-282987 for 3  days. HESCs were seeded into \n24-well plates and co-cultured with PBS, activated plate -\nlets, PNU-282987 or both activated platelets and PNU-\n282987 for 3 days or 12 days, depending on the purpose \nof the experiment. Then cells were washed with PBS \ntwice, fixed with 10% formalin (w/v), suspended in 0.5% \nTriton X-100 for 15  min, and blocked in normal goat \nserum, followed by incubation with the primary anti -\nbodies. For 11Z cells, E-cadherin (1:100, CST), α-SMA \n(1:100, Abcam), F-actin (1:100, Abcam) were used to \nevaluate the EMT and FMT, respectively. HESCs were \nincubated with α-SMA (1:100, Abcam), oxytocin recep -\ntor (OTR) (1:100, Abcam), SM-MHC (1:100, Abcam) and \ndesmin (1:100, Abcam) overnight at 4  °C in darkness. \nAfter washing, cells were incubated at 37 °C for 1 h with \nAlexa Flour 488-conjugated goat anti-mouse IgG (1:200, \nTable 1 List of primers used in the real‑time RT‑PCR analysis\nGene name Sequence\nE‑cadherin forward 5’‑ GCA GTT CTG CCA GAG AAA CC‑3’\nreverse 5’‑TGG ATC CAA GAT GGT GAT GA‑ 3’\nα‑SMA forward 5’‑CTG ACA GAG GCA CCA CTG AA‑3’\nreverse 5’‑CAT CTC CAG AGT CCA GCA CA‑3’\nVimentin forward 5’ ‑TTG ACA ATG CGT CTC TGG CAC‑3’\nreverse 5’ ‑CCT GGA TTT CCT CTT CGT GGAG‑3’\nCCN2 forward 5’‑ GCC CTG ACC CAA CTA TGA TG‑3’\nreverse 5’‑ CAG AGA CGA CTC TGC TTC TC‑3’\nLOX forward 5’ ‑TGG TGG ATC CAG ATG TTT GA‑3’\nreverse 5’‑ GTT GGT TGG GAG ACT TTG GA‑3’\nGAPDH forward 5’ ‑GTC TTC CTG GGC AAG CAG TA‑3’\nreverse 5’‑ CTG GAC AGA AAC CCC ACT TC‑3’\n\nPage 6 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nCST) or Alexa Fluor 647 goat anti-rabbit (CST, 1:200) \nand then washed with PBS twice and the nuclei was \nstained with DAPI (Sigma-Aldrich). Images of cells were \nobtained by a laser scanning confocal microscope (Leica \nTCS SP5 Confocal Microscope, Leica, Solms, Germany). \nThe experiments were performed in duplicate.\nPreparation of platelets\nThe platelets preparation procedure consists of two cen -\ntrifuge steps, and performed as previously described [47]. \nBriefly, 20 ml of peripheral blood samples each from 20 \nhealthy male volunteer donors, who had no known dis -\nease and had not taken any medication 3 months proior \nto the blood drawing, were collected using collection \ntubes containing 3.2% citrate solution. The platelet-rich \nplasma (PRP) was obtained by centrifugation at 1000 rpm \nfor 10  min at room temperature. Supernatant was col -\nlected, and centrifuged at 3,500  rpm for 10  min. About \n2 ×  107 platelets were collected from 1 ml of blood. The \nplatelet pellets were suspended in RPMI 1640 or DMEM/\nF12 media and a total of 2 ×  107 platelets/ml was added \ninto the cell-culture dishes. Platelets were activated \nusing thrombin 0.5 U/mL (T8885, Sigma-Aldrich). Plate -\nlets were removed from cells by sterile PBS washing as \nreported previously [47].\nScratch assay\nThe migratory ability of 11Z cells was assessed by the \nscratch assay as described previously [31]. Briefly, 11Z \ncells were cultured in 6-well plate (Corning, Tewksbury, \nMA, USA) and were allowed to grow to confluence. Then \na 100 μL-sterile pipette tip was used to make a scratch \nhorizontally. Serum-free cell culture medium and dif -\nferent solutions were added into the 6-well plate after \nwashing with PBS thrice. Images were taken by a micro -\nscope (Olympus, Tokyo, Japan) at 0, 12 and 24 h after the \nscratch. The distance of each edge of 11Z cells was meas -\nured with Image Pro-Plus software 6.0 (Media Cybernet -\nics, Inc, Bethesda, MD, USA). The assay was replicated \n4 times, and the mean and standard deviation were \ncalculated.\nInvasion assay\nBiocoat 24-well Matrigel transwells were used in this \nassay as we reported previously [31]. Briefly, the Matrigel \nmatrix and serum-free RPMI 1640 (Thermo Fisher, \nWaltham, MA, USA) culture medium were mixed in a \nratio of 1:8, and then 50 μL of the mixture was added to \na 24-well transwell insert and solidified in a 37 °C incu -\nbator for 30 min to form a thin gel layer. 11Z cells were \ndetached by using 0.25% Trypsin–EDTA solution, and \nthe cell density was adjusted to 5 ×  105 cells/mL. Two \nhundred μL of cell solution was poured into the top of \nthe filter membrane in a transwell insert, and 600 μL \nof media with or without treatment was added into the \nbasolateral side, then the cells were incubated at 37  °C \nin 5%  CO2 air for 48  h. Transwell inserts were fixed by \n95% alcohol, crystal violet stained, and counted under a \nmicroscope. Cells were imaged underneath an inverted \nmicroscope and counted in different fields of view to \nobtain an average number of cells. The invasion index \nwas defined to be the mean counts of the infiltrated cells \nunder × 200 magnification.\nCell contraction assay\nCell contractility in  vitro with different treatments was \nevaluated by cell contraction assay (Cell Biolabs, San \nDiego, CA, USA) according to the manufacturer’s intro -\nductions as we reported previously [48]. All solutions \nwere kept on ice throughout the entire experiment. Three \nhundred forty μL of neutralization solution, 2.46  mL \n5 × medium and 9.54  mL collagen solution were mixed \nwell in a centrifuge tube to prepare for the cold collagen \ngel working solution. Cells were harvested and resus -\npended in DMEM/F12 medium at 2–5 ×  106 cells/mL. \nThe collagen lattice was prepared by mixing 2 parts of \ncell suspension and 8 parts of cold collagen gel working \nsolution. 24-well plates (Corning, Tewksbury, MA, USA) \nwere coated with 0.5 mL of the cell-collagen mixture per \nwell and incubated at 37℃ for one hour. After collagen \npolymerization, 1.0 mL of culture medium with different \ntreatments was added atop each collagen gel lattice. The \ncultures were incubated for 72 h and collagen gels were \ngently released from the sides of the culture dishes. The \ncollagen gel size change was measured with a ruler at 3, \n24 and 48 h after released.\nSoluble collagen assay\nSircol soluble collagen assay (Biocolor, Carrickfergus, \nUK) was used to evaluate the amount of soluble collagens \nproduced by HESCs (n = 7) after co-cultured with dif -\nferent treatments for 72  h following the manufacturer’s \nintroductions as we previously reported [48]. The cell \nculture medium was collected and then centrifuged to \ndiscard the particulate materials. Low protein binding \n1.5 mL conical microcentrifuge tubes (Eppendorf, Ham -\nburg, Germany) were used to mix 1.0 mL of cell culture \nsupernatant and 200 μL of cold Collagen Isolation and \nConcentration Reagent (Biocolor). DEME/F12 medium \nwas used as blank controls and the mixture was incu -\nbated overnight at 4℃. Tubes were centrifuged at 12,000 \nr.p.m for 10 min without delay the next day and a micro -\npipette was used to slowly remove 1,000 μL of superna -\ntant from each tube. Then 1.0 mL of Sircol Dye Reagent \n(Biocolor) and 100 μL sample were added to each tube. \nThe tubes were capped and placed in a gentle mechanical \n\nPage 7 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nshaker for 30 min, and then were transferred to a micro -\ncentrifuge and spun at 12,000 r.p.m. for 10 min. The tubes \nwere carefully inverted and drained. Seven hundred and \nfifty μL of ice-cold Acid-Salt Wash Reagent (Biocolor) \nwas gently added to the collagen-dye pellet and tubes \nwere centrifuged at 12,000 r.p.m for 10  min. The wash \nwas drained into a waste container and the residual fluid \nwas carefully removed from the tip of the tubes using \ncotton wool buds. Two hundred and fifty μL of Alkali \nReagent were added to the reagent blanks, standards \nand samples, and after that a vortex mixer was used for \nthorough mixing. Then, 200 μL of each sample was trans-\nferred to individual wells of a 96 micro-well plate. The \nabsorbance value at 555 nm was measured within 2 h by \na microplate reader (Biotek, Winooski, VT, USA) and col-\nlagen concentrations were read from the standard curve.\nStatistical analysis\nThe comparison of distributions of continuous variables \nbetween or among 2 or more groups was made using \nthe Wilcoxon and Kruskal test, respectively. Pearson or \nSpearman rank correlation coefficient was used when \nevaluating correlations between 2 variables when both \nvariables were continuous or when at least 1 variable was \nordinal. To see whether there is a trend for the α7nAChR \nstaining levels or the extent of lesional fibrosis as a func -\ntion of the dysmenorrhea severity, Jonckheere’s trend test \nwas used. Multiple linear regression analysis was used \nto identify factors associated with the hotplate latency. \nTo evaluate the difference in scratch assay results and in \ncellular contractility, linear regression analyses were per -\nformed. P values of less than 0.05 were considered sta -\ntistically significant. All computations were made with R \n4.1.2 [49] (www.r- proje ct. org).\nResults\nReduced α7nAChR staining in both OE and DE lesions\nWe first evaluated the α7nAChR staining in normal endo-\nmetrium from controls and in OE and DE lesions. The \ncharacteristics of these recruited subjects are listed in \nTable 2 Characteristics of recruited patients with ovarian endometriomas, deep endometriosis and without (controls). Kruskal–\nWallis test was used for age while for other data Fisher exact test was used\nAbbreviations: NA not applicable, NS not significant, SD Standard deviation, Rasrm revised American Society of Reproductive Medicine classification\nNA not applicable\nVariable Control (n = 18) Ovarian Endometriomas \n(n = 17)\nDeep Endometriosis (n = 14) P‑value\nAge (in years)\n Mean ± S.D 36.3 ± 7.0 35.6 ± 7.4 38.3 ± 5.4 0.53\n Median (Range) 36(26–48) 35(25–48) 38(27–47\nMenstrual phase\n Proliferative Secretory 12 (66.7%) 6 (33.3%) 6 (35.3%) 11 (64.7%) 5 (35.7%) 9 (64.3%) 0.11\nParity\n 0 4 (22.2%) 6 (35.3%) 4 (28.6%)\n 1 13 (72.2%) 8 (47.1%) 10 (71.4%) 0.39\n ≥ 2 1 (5.6%) 3 (17.6%) 0 (0.0%)\nrASRM score\n Mean ± S.D NA 41.6 ± 21.7 74.1 ± 22.2 NA\n Median (Range) 40 (20–112) 75 (46–114)\nrASRM stage\n I 0 (0.0%) 0 (0.0%)\n II NA 0 (0.0%) 0 (0.0%) NA\n III 7 (41.2%) 0 (0.0%)\n IV 10 (58.8%) 14 (100%)\nSeverity of dysmenorrhea\n None 18 (100%) 10 (58.8%) 0 (0.0%)\n Mild 0 (0.0%) 6 (35.3%) 4 (28.6%) 8.3 ×  10–11\n Moderate Severe 0 (0.0%) 0 (0.0%) 1 (5.9%) 0(0.0%) 1 (7.1%) 9 (64.3%)\nCo‑occurrence with uterinefibroids\n No Yes 18 (100%) 0 (0.0%) 13 (76.5%) 4 (23.5%) 8 (57.1%) 6 (42.9%) 0.006\nCo‑occurrence with adenomyosis\n No Yes 18 (100%) 0 (0.0%) 16 (94.1%) 1 (5.9%) 11 (78.6%) 3 (21.4%) 0.06\n\nPage 8 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nTable 2. We found a robust immunostaining of α7nAChR \nin normal endometrium, especially in glandular epithe -\nlium, while its staining was weaker in the stromal compo-\nnent (Fig. 1A). Similarly, in endometriotic lesion samples \nfrom patients with OE and DE, the α7nAChR staining \nwas also seen mostly in the epithelial component, mainly \nin cell membrane (Fig. 1A). In stark contrast with the nor-\nmal endometrium, however, the staining was significantly \nFig. 1 The immunohistochemistry analysis of α7nAChR in ectopic endometrium from patients with ovarian endometrioma (OE) and deep \nendometriosis (DE) as compared to normal endometrium from control subjects. A Representative immunostaining results for α7nAChR staining in \nnormal endometrium from control subjects and ectopic endometrium from patients with OE and DE (left panel), along with boxplots summarizing \nthe staining data (right panel). B Representative images of Masson trichrome staining in normal endometrium from control subjects and ectopic \nendometrium from patients with OE and DE (left panel), along with boxplots summarizing the staining data (right panel). Scatter plots show the \nrelationship between α7nAChR staining levels and the extent of lesional fibrosis C, and between α7nAChR staining levels and rASRM scores D. \nThe green, blue and red dots represent control participants, OE patients, and DE patients, respectively. The boxplots showing the relationship \nbetween α7nAChR staining levels and severity of dysmenorrhea E, and between severity of dysmenorrhea and the extent of lesional fibrosis F. \nMagnification = 400 × , scale bar = 50 μm. Symbols for statistical significance levels: NS: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001 \n\nPage 9 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nreduced in both OE and DE lesions in glandular epithe -\nlium (both p-values ≤ 0.003; Fig.  1A). As reported pre -\nviously [50], Masson trichrome staining indicates that \nthe extent of lesional fibrosis was significantly higher in \nboth OE and DE lesions as compared to control endo -\nmetrium (both p-values ≤ 4.3 ×  10–9 ; Fig.  1B), especially \nin DE lesions. Multiple linear regression on α7nAChR \nstaining levels incorporating age, parity, the menstrual \nphase at which the tissue sample was collected, and \nco-occurrence of uterine fibroids indicated that both \nOE and DE lesions were significantly associated with \nreduced α7nAChR staining (p = 0.0002 and p = 0.0009, \nFig. 2 Modulation of α7nAChR affects the development of endometriosis. A Dynamic changes in mean bodyweight in mice from the \nControl, PNU‑282987 and MLA groups. n = 8 in each group. B Boxplot summarizing the lesion weight among the 3 different treatment groups. \nRepresentative immunostaining results for E‑cadherin C and α‑SMA D in endometriotic lesions from Control, PNU‑282987 and MLA mice (left \npanel), along the boxplots summarizing the staining data (right panel). E Representative images of Masson trichrome staining in endometriotic \nlesions in the three groups of mice (left panel), along with the boxplot summarizing the staining data (right panel). Magnification = 400 × , scale \nbar = 50 μm. Symbols for statistical significance levels: NS: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001 \n\nPage 10 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nrespectively; R2 = 0.39). Multiple linear regression on \nthe extent of tissue fibrosis incorporating age, parity, \nmenstrual phase, co-occurrence of uterine fibroids, and \nα7nAChR staining indicated that α7nAChR staining lev -\nels was negatively associated with (p = 0.0007), while OE \nand DE lesions were positively associated with the extent \nof fibrosis (p = 3.0 ×  10–10  and p = 2.4 ×  10–10 , respec -\ntively; R2 = 0.85).\nWe found that the α7nAChR staining levels in eutopic \nand ectopic endometrium correlated negatively the \nextent of fibrosis (r = -0.72, p = 4.0 ×  10–9 ; Fig.  1C). \nLesional α7nAChR staining levels also correlated nega -\ntively the rASRM scores (r = -0.46, p = 0.009; Fig. 1D). In \naddition, the lesional α7nAChR staining levels correlated \nnegatively with the severity of dysmenorrhea (Spear -\nman’s r = -0.45, p = 0.012; p = 0.012 by Jonckheere’s trend \ntest; Fig.  1E). The extent of lesional fibrosis correlated \npositively with the severity of dysmenorrhea (Spearman’s \nr = 0.68, p = 2.8 ×  10–5 ; p = 8.8 ×  10–5  by Jonckheere’s \ntrend test; Fig. 1F).\nModulation of α7nAChR affects the development \nof endometriosis\nGiven the apparent expression of α7nAChR in nor -\nmal endometrium and its aberration in endometriotic \nlesions, we wondered whether α7nAChR would partici -\npate in the development of endometriosis. We carried \nout a mouse experimentation to see whether activation \nor inhibition of α7nAChR would affect the development \nof endometriosis.\nWe randomly divided 24 female Balb/C mice into three \nequal-sized groups: Control group, PNU-282987 (an \nα7nAChR agonist) group, and MLA group (an α7nAChR \ninhibitor). One week before the induction of endome -\ntriosis, osmotic pumps were inserted into these 3 groups \nof mice to infuse, in a controlled manner, either sterile \nsaline, PNU-282987 or MLA for 4  weeks. Three weeks \nafter the induction, endometriotic lesions were carefully \nexcised and analyzed.\nThere was no significant difference in bodyweight \nbefore and after induction of endometriosis among the \n3 groups of mice (all p-values ≥ 0.22; Fig.  2A). Endome-\ntriosis lesions were harvested and all lesions appeared \nto be cystic as we showed in Supplementary Figure S2. \nCompared with the Control group, the lesion weight in \nmice receiving PNU-282987 was reduced by nearly 60% \n(39.3  mg vs. 88.8  mg, p = 0.038), whereas that of mice \nreceiving MLA was similar (p = 0.72; Fig.  2B). Similarly, \nthe number of lesions in PNU-282987 mice was reduced \nby 46.4% (1.88 ± 0.99, vs. 3.50 ± 1.20, p = 0.019), whereas \nthat of MLA mice was not significantly different from \nthe Control mice (5.0 ± 2.0, p = 0.12; Supplemental Fig -\nure S3). Endometriosis was confirmed and endometriotic \nepithelium was visualized by H&E staining as shown in \nSupplementary Figure S4.\nTo see whether α7nAChR modulation affects EMT in \nendometriosis or not, we also performed IHC analysis of \nE-cadherin and α-SMA. Compared with control mice, the \nlesional E-cadherin staining levels in glandular epithelial \ncells from the PNU group, but not the MLA group, were \nsignificantly elevated (p = 0.040 and p = 0.28, respec -\ntively; Fig.  2C). In contrast, the staining levels of α-SMA \nin the glandular epithelial component in the PNU mice, \nbut not the MLA mice, were significantly reduced as \ncompared with the control mice (p = 0.049 and p = 0.19, \nrespectively; Fig.  2D). Consistently, the extent of lesional \nfibrosis was significantly reduced in the PNU group, \nbut not the MLA group, as compared to control mice \n(p = 0.0006 and p = 0.72, respectively; Fig.  2E). Taken \ntogether, these data strongly suggest that the activation of \nα7nAChR would partially reverse the EMT in endome -\ntriosis, hindering lesional progression.\nTreatment with an α7nAChR agonist stalls lesional \nprogression in mouse with induced deep endometriosis\nTaking advantage of a mouse model of DE [33], we next \nevaluated the therapeutic potential, if any, of pharmaco -\nlogical activation of α7nAChR for DE. Sixteen mice were \ninduced with DE, and 4  weeks after the induction, they \nwere randomized to receive either PNU-282987 or saline \ntreatment for 3 weeks. All mice survived the experiment. \nThere was no significant difference in bodyweight before \nand after induction of endometriosis between the two \ngroups (all p-values > 0.20; Fig. 3A).\nRemarkably, compared with the control group, \nmice receiving PNU-282987 had their lesion weight \nreduced by 44.0% (261.8 ± 73.9 mg vs. 560.4 ± 115.0 mg, \np = 0.0019; Fig.  3B), even though no difference in num -\nber of lesions between the two groups (3.63 ± 1.19 vs. \n4.5 ± 1.51, p = 0.21; Supplemental Figure S5). There was \n(See figure on next page.)\nFig. 3 Therapeutic potentials of α7nAChR activation in treating deep endometriosis in mouse. A Dynamic changes in mean bodyweight in the \nControl and the PNU‑282987 groups. B Boxplots of the lesion weight from the two groups of mice. C Dynamic changes in mean hotplate latency, \ntested at the indicated time points, from the two groups of mice. Representative immunostaining results for E‑cadherin D, α‑SMA E, desmin F and \nSM‑MHC G in endometriotic lesions from Control and PNU‑282987 treated mice (left panel), along the boxplots summarizing the staining data \n(right panel). H Representative images of Masson trichrome staining in endometriotic lesions in Control and PNU‑282987 mice (left panel), along \nwith the boxplot summarizing the staining data (right panel). Magnification = 400 × , scale bar = 50 μm. n = 8 in both groups. Symbols for statistical \nsignificance levels: NS: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001\n\nPage 11 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nFig. 3 (See legend on previous page.)\n\nPage 12 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nno significant difference in hotplate latency prior to the \ninduction of endometriosis between the two groups (all \np-values > 0.27). When DE was induced 4  weeks after \ninduction, there was a significant reduction in latency \n(p = 0.0013; Fig.  3C). However, the mice receiving the \nPNU treatment had significantly longer latency at the \nend of the experiment than the control mice (16.9 ± 3.6 s \nvs. 13.6 ± 2.4 s; p = 0.042; Fig. 3C).\nTo see whether α7nAChR agonist treatment affects \nEMT and fibrogenesis in mice with induced DE, we per -\nformed IHC analysis of E-cadherin, α-SMA, desmin, \nSM-MHC as well as Masson trichrome staining. In mice \ntreated with PNU-282987, the lesional staining levels \nof E-cadherin in glandular epithelial cells were signifi -\ncantly elevated as compared with control mice (p = 0.021; \nFig.  3D). In contrast, the staining levels of α-SMA, \ndesmin and SM-MHC were all significantly decreased \nas compared with control mice (all 3 p-values ≤ 0.038; \nFig. 3E-G).\nConsistent with stalled EMT, FMT and SMM, we found \nthat the extent of fibrosis was significantly reduced in mice \ntreated with PNU-282987 as compared with controls \n(Fig. 3H). The extent of lesional fibrosis was negatively cor-\nrelated with the epithelial staining of E-cadherin (r = -0.64, \np = 0.007), positively with that stromal staining levels of \nα-SMA (r = 0.59, p = 0.016) and desmin (r = 0.67, p = 0.0045). \nIt also correlated marginally with the lesion weight (r = 0.47, \np = 0.066) and the latency (r = -0.46, p = 0.073).\nThus, after three weeks of treatment, mice with induced \nDE that were treated with PNU had significantly smaller \nlesions and longer latency as compared with untreated \ncontrol mice, most likely through arresting EMT, FMT, \nSMM and fibrogenesis.\nActivation of α7nAChR inhibits epithelial‑mesenchymal \ntransition in endometriotic epithelial cells induced \nby activated platelets\nWe previously found that activated platelets stimula -\ntion could facilitate EMT in endometriotic epithelial cells \n[31]. This prompted us to further examine whether the \nactivation of α7nAChR can stall EMT induced by activated \nplatelets. We first evaluated the gene expression levels of \nepithelial and mesenchymal makers known to be involved \nin EMT, and found that vimentin, Slug and PAI-1 were all \nsignificantly upregulated in endometriotic epithelial cells \nco-cultured with activated platelets (all p -values ≤ 0.046; \nFig.  4A). However, the platelet-induced upregulation of \nvimentin and Slug, but not PAI-1, was abrogated by PNU-\n282987 treatment (p  = 0.024, p = 0.011 and p = 0.092, \nrespectively; Fig.  4A). Consistently, Western blot analysis \nand immunofluorescent staining demonstrated the protein \nexpression of E-cadherin was significantly decreased while \nthat of α-SMA was significantly elevated in 11Z cells co-\ncultured with activated platelets (both p -values ≤ 0.0067; \nFig.  4B). However, α7nAChR activation by PNU-282987 \nattenuated platelet-induced E-cadherin inhibition while \nabrogated α-SMA expression induced by platelets (both \np-values ≤ 0.027; Fig. 4B).\nWe found that activation of α7nAChR attenuated the \nmigratory propensity of 11Z cells induced by platelets \n(p = 0.043; Fig.  4C). In addition, activation of α7nAChR \nsignificantly attenuated increased invasiveness of 11Z \ncells induced by platelets (both p-values ≤ 0.016; Fig. 4D).\nWe also examined the expression of E-cadherin, \nα-SMA and F-actin through immunofluorescence after \nco-culturing 11Z cells with buffer, activated platelets \nwith or without PNU-282987, or just PNU-282987 alone \nfor 3 days. We found that the E-cadherin expression was \nsignificantly reduced while α-SMA and F-action expres -\nsion were elevated when co-cultured with platelets, but \nPNU-282987 treatment attenuated platelet-induced sup -\npression of E-cadherin while abrogated the expression \nα-SMA and F-actin induced by platelets (Fig. 4E).\nTaken together, these data indicate that pharmacologi -\ncal activation of α7nAChR attenuated EMT induced by \nactivated platelets in endometriotic epithelial cells.\nActivation of α7nAChR inhibits platelet‑induced FMT \nand collagen production in endometriotic stromal cells\nTo investigate whether activation of α7nAChR impacts \non platelet-induced FMT and collagen production in \nFig. 4 Activation of α7nAChR partially reverses platelet‑induced EMT in endometriotic epithelial cells. A Relative fold change in gene expression \nlevels of vimentin, SLUG and PAI‑1 in 11Z cells treated with buffer, platelets activated with or without PNU‑282987, or PNU‑282987 alone for 48 h \n(n = 4). Values are normalized to GAPDH expression. B Left panel: Detection of protein levels of E‑cadherin and α‑SMA by immunoblotting of \nlysate of 11Z cells treated as in (A). Right panel: Relative fold changes of the protein levels of E‑cadherin and α‑SMA (n = 3). C Migratory capacity, as \nevaluated by the scratch assay, of 11Z cells treated with buffer, platelets with or without PNU‑282987, or PNU‑282987 alone for 12 and 24 h. The cells \nwere photographed at 0 h, 12 and 24 h after scratch (n = 4). Fold change in migration distance was shown for different treatments as compared \nwith controls (buffer). D Representative images of the invaded 11Z cells in the transwell assay under treatment indicated (Magnification: X200). Cells \nwere added to the top of transwells coated with Matrigel and treated with buffer (Con), platelets with or without PNU‑282987 (PLT, PLT + PNU), \nor PNU‑282987 alone (PNU) for 48 h (n = 3). The total number of cells invaded to the bottom of transwell was then counted. Scale bar = 100 μm. \nE Representative immunofluorescence staining results of E‑cadherin, α‑SMA and F‑actin expression in 11Z cells under different treatments as \nindicated. Symbols for statistical significance levels: NS: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001\n(See figure on next page.)\n\nPage 13 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nFig. 4 (See legend on previous page.)\n\nPage 14 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nendometriotic stromal cells, we co-cultured HESCs \nwith buffer, activated platelets with or without PNU-\n282987, or PNU-282987 alone for 72  h. We evaluated \nthe expression levels of genes known to be involved in \nFMT. As expected, the gene expression levels of LOX, \nCCN2, α-SMA and COL1A1 were significantly ele -\nvated in HESCs co-cultured with activated platelets (all \np-values ≤ 0.012; Fig.  5A) [31]. However, treatment with \nPNU-282987 significantly abrogated the expression lev -\nels of LOX, CCN2 and α-SMA (all 3 p-values ≤ 0.02; \nFig. 5A), but not COL1A1 (p = 0.15; Fig. 5A). This result \nwas corroborated by Western blot analysis for LOX and \nα-SMA. The protein expression of LOX and α-SMA were \nincreased significantly (both p-values ≤ 0.0018; Fig.  5B), \nbut the α7nAChR activation by PNU-282987 abrogated \nthe LOX and α-SMA expression induced by platelets \n(both p-values ≤ 0.048; Fig. 5B).\nIn addition, we found that HESCs co-cultured with \nactivated platelets for 12 days displayed increased stain -\ning of α-SMA, OTR, SM-MHC and desmin, but this \nincrease was abrogated by α7nAChR activation (Fig.  5C). \nConsistently, the protein expression level of SM-MHC \nand desmin were significantly elevated in HESCs co-cul -\ntured with activated platelets (both p-values ≤ 3.0 ×  10–4 ; \nFig.  6A), but this overexpression was significantly abro -\ngated by activation of α7nAChR by PNU-282987 (both \np-values ≤ 0.023; Fig. 6A).\nConsistent with the above findings, we found that the \ncontractility and the collagen production were signifi -\ncantly increased in HESCs after co-cultured with acti -\nvated platelets for 72 h [31] but both the contractility and \nthe collagen production were significantly attenuated by \nα7nAChR activation (both p-values ≤ 0.049; Fig. 6B-C).\nTaken together, these data strongly indicate that activa-\ntion of α7nAChR inhibits platelet-induced FMT, SMM \nand collagen production in endometriotic stromal cells.\nDiscussion\nIn this study, we have shown that, similar to adenomyo -\nsis [30], the α7nAChR immunostaining is significantly \nreduced in endometriotic lesions, especially in DE lesions. \nThe staining levels of α7nAChR in lesional glandular epi -\nthelium are negatively correlated with the extent of lesional \nfibrosis and the severity of dysmenorrhea. The α7nAChR \nagonist significantly impeded the development of endome-\ntriotic lesions in mouse, likely through hindrance of EMT \nand FMT. In mice with induced DE, treatment with an \nα7nAChR agonist significantly reduced the lesion weight \nand improved the pain behavior, which were accompanied \nby the arrest of EMT, FMT, SMM and fibrogenesis. Treat-\nment of endometriotic epithelial cells with an α7nAChR \nagonist significantly abrogated platelet-induced EMT and \ninvasiveness. Treatment of endometriotic stromal cells \nwith an α7nAChR agonist also significantly attenuated \nplatelet-induced FMT and SMM, and suppressed cellu -\nlar contractility and collagen production. Taken together, \nthese results indicate that α7nAChR is suppressed in \nendometriotic lesions, and its induction by pharmacologi-\ncal means can impede EMT, FMT, SMM, and fibrogenesis \nof endometriotic lesions. As such, α7nAChR can be right-\nfully viewed as a potential target for therapeutic invention.\nOur finding of reduced lesional α7nAChR staining is \nconsistent with our previous report of depressed vagal \ntone in women with endometriosis [26]. Our finding \nthat α7nAChR activation by agonists stalls lesional pro -\ngression is also consistent with the reported therapeutic \npotential of α7nAChR agonist [29], and is in agreement \nwith our previous report that VNS impedes lesional pro -\ngression [26]. Our finding that α7nAChR activation sig -\nnificantly retarded the development of endometriosis \nmay explain as why nicotine or smoking has been iden -\ntified as a protective factor for endometriosis [51] and \nadenomyosis [52] since nicotine is a ligand for AChRs.\nWhat is puzzling is that treatment with MLA, an \nα7nAChR antagonist, did not seem to have much effect \non lesions. It is possible that dosage that we used in this \nstudy was not optimized to affect lesions. It is also pos -\nsible that there could be some redundant signaling path -\nways when α7nAChR is inhibited by an antagonist. For \nexample, muscarinic AChR1 (m1AChR) in the forebrain \nhas been reported to affect the neurons of the Medullary \nVisceral Zone (MVZ), which is the core of CAIP , to regu-\nlate systemic inflammation and immunity [53]. If endo -\nmetriosis results in reduced vagal tone, it is possible that \nboth α7nAChR and M1AChR are suppressed. If this is \nthe case, then further suppression of α7nAChR may not \nbe able to change much but its activation may be enough \nto activate the CAIP to exert desired therapeutic effects.\n(See figure on next page.)\nFig. 5 Activation of α7nAChR inhibits platelet‑induced FMT in primary human endometrial stromal cells (HESCs) derived from ovarian \nendometrioma tissue samples. A Relative fold change in gene expression levels of α‑SMA, LOX1, COL1A1 and CCN2 in HESCs treated with buffer \n(Con), activated platelets (PLT) with or without PNU‑282987 (PNU), or PNU‑282987 alone for 72 h (n = 4). B Left panel: Detection of protein \nlevels of α‑SMA and LOX1 by immunoblotting of lysate of HESCs treated with buffer (Con), activated platelets (PLT) with or without PNU‑282987 \n(PNU), or PNU‑282987 alone for 72 h. Right panel: Relative fold change of the protein levels of α‑SMA and LOX1 (n = 5). C Representative \nimmunofluorescence of SM‑MHC, desmin, α‑SMA and OTR expression in HESCs cells after indicated treatment for Day 12. Symbols for statistical \nsignificance levels: NS: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001\n\nPage 15 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nFig. 5 (See legend on previous page.)\n\nPage 16 of 19Hao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \nMore remarkably, it is well-documented that women \nwith endometriosis often exhibit anxiety, depression and \ninsomnia [54– 57]. These psychological co-morbidities \nthemselves could activate the hypothalamic–pituitary–\nadrenal/sympatho-adreno-medullary  (HPA/SMA) axes, \nand accelerate the development of endometriosis through \nadrenaline receptor β2 [58, 59]. This, in turn, is likely to \ncause more pain, anxiety and depression, forming a vicious \ncycle [60]. Yet anxiety, depression, and insomnia are \nreported to be associated with reduced vagal tone as well, \nwhich further justifies for VNS therapy [61– 65]. Since \nactivation of the CAIP is largely through the activation \nof α7nAChR [18, 19], targeting α7nAChR for therapeutic \npurpose may achieve the goal of not only regressing endo-\nmetriotic lesions but also improving the overall wellbeing \nof the patients with endometriosis.\nOur study has several strengths. First, by evaluating \nα7nAChR immunoreactivity in both OE and DE lesions, \nalong with the extent of lesional fibrosis, we demon -\nstrated that differential α7nAChR staining in different \nsubtypes of endometriosis, which are known to have sub-\nstantial histological differences [50, 66]. Second, through \nthe use of in vitro and in vivo experimentations, we pro -\nvided several, interlocking pieces of evidence for the \ntherapeutic potentials of α7nAChR activation.\nOur study also has some notable limitations. First, in \nmouse Experiment 1, we did not specifically quantitate \nthe staining of either α7nAChR or m1AChR. However, \nPNU is known to be a specific agonist for α7nAChR [38, \n67], while MLA is known to be a specific antagonist [39]. \nYet quantification of m1AChR staining, perhaps in the \nMVZ may provide us with more information regarding \nFig. 6 Activation of α7nAChR partially reverses platelet‑induced FMT in HESCs. A Left panel: Detection of protein levels of SM‑MHC and desmin \nby immunoblotting of lysate of HESCs cells treated with buffer (Con), activated platelets (PLT) with or without PNU‑282987 (PNU), or PNU‑282987 \nalone for 12 days (n = 4). Right panel: Relative fold change in the protein levels of SM‑MHC and desmin. B Summary contractility results, in terms \nof diameter of the gel surface, for HESCs measured at 0, 3, 24 and 48 h after indicated treatment (n = 5). C The absorbance value at 570 nm (optical \ndensity, or OD) reflects the amounts of soluble collagens in the culture medium from cells treated as indicated (n = 7). Symbols for statistical \nsignificance levels: NS: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001\n\nPage 17 of 19\nHao et al. Reproductive Biology and Endocrinology           (2022) 20:85 \n \nthe suppressed CAIP in mouse with induced endome -\ntriosis. Second, while we have demonstrated the effect \nof α7nAChR activation on a few major molecular events, \nsuch as EMT, FMT, SMM and fibrogenesis, involved in \nlesional progression, it is possible that there could be \nother pathways in retarding the progression. For exam -\nple, thymic stromal lymphopoietin (TSLP) has been \nreported to be involved in the development of endome -\ntriosis [68–70], but α7nAChR has been shown to inhibit \nTSLP [71]. Future investigations are warranted to illumi -\nnate this issue.\nIn summary, we have shown in this study reduced \nα7nAChR in endometriotic lesions, especially in DE \nlesions. Through the impedance of EMT, FMT, SMM and \nfibrogenesis, pharmacological activation of α7nAChR \ndecelerates the lesional progression in mouse and dem -\nonstrates its therapeutic potentials in mice with induced \nDE. Thus, α7nAChR can be viewed as a potential target \nfor therapeutic invention.\nSupplementary Information\nThe online version contains supplementary material available at https:// doi. \norg/ 10. 1186/ s12958‑ 022‑ 00955‑w.\nAdditional file 1: SupplementaryFigure S1. Positive andnegative con‑\ntrols for immunohistochemistry. For positive controls, human breastcancer \ntissues were used for E‑cadherin, mouse liver tissues for α‑SMA, humanad‑\nenomyotic tissue samples for desmin and SM‑MHC, and mouse lung \ntissues for α7nAChR.For negative controls, mouse endometriotic lesions \nwere used. Magnification: 400×;Scale bar: 50 μm. Supplementary Figure \nS2. Cysticappearance of endometriotic lesions in mouse experiment 1. \nLesions seen fromExperiment 2 are similar. Supplementary Figure S3. \nThe number ofendometriotic lesions per mouse was evaluated from the \nControl, PNU‑282987 andMLA groups in mouse experiment 1. Symbols \nforstatistical significance levels: NS: p>0.05; *: p<0.05;**: p<0.01. Supple‑\nmentaryFigure S4. H&E staining from endometriotic lesions. Magnifica‑\ntion = 200×, scale bar=100 μm. Supplementary Figure S5. The number \nof endometrioticlesions per mouse was evaluated from the Control and \nPNU‑282987 groups in mouse experiment 2. NS: p>0.05. \nAcknowledgements\nWe thank Dr. Jung‑Hye Choi for providing the 11Z cell line. This research \nwas funded in part by grants 82071623 (SWG) and 81871144 (XL) from the \nNational Natural Science Foundation of China, an Excellence in Centers of \nClinical Medicine grant 2017ZZ01016 (SWG) from the Science and Technol‑\nogy Commission of Shanghai Municipality, and Clinical Research Plan grant \nSHDC2020CR2062B from Shanghai Shenkang Center for Hospital Develop‑\nment (SWG).\nAuthors’ contributions\nS.W.G. conceived and designed the study, performed data analysis and \ndata interpretation, and drafted the manuscript. M.H. performed all the \nexperiments and carried out initial data analysis. X.L. was involved in patient \nrecruitment and the data interpretation and discussion. S.W.G. and M.H. jointly \nprepared Figs. 1–6. All participated in the writing and approved the final ver‑\nsion of the manuscript. All authors read and approved the final manuscript. \nFunding\nNational Natural Science Foundation of China (82071623 to S.W.G.; 81871144 to \nX.S.L.), an Excellence in Centers of Clinical Medicine grant (2017ZZ01016) from \nthe Science and Technology Commission of Shanghai Municipality, and grant \nSHDC2020CR2062B from Shanghai Shen Kang Hospital Development Center.\nAvailability of data and materials\nThe de‑identified supporting data are available from the senior author upon \nwritten and reasonable request.\nDeclarations\nEthical approval and consent to participate\nThis study was approved by the Institutional Ethics Review Board of the \nShanghai OB/GYN Hospital, Fudan University. All tissue samples were obtained \nafter written, full and informed consent from recruited subjects.\nConsent to publication\nNot applicable.\nCompeting interests\nS.‑W.G. provides consultancy advice for MSD R&D, Chugai Pharmaceutical Co., \nand BioHaven Pharmaceuticals. All other authors have no conflicts to declare.\nAuthor details\n1 Shanghai Obstetrics and Gynecology Hospital, Fudan University, Shang‑\nhai 200011, China. 2 Shanghai Key Laboratory of Female Reproductive \nEndocrine‑Related Diseases, Fudan University, Shanghai, China. \nReceived: 13 January 2022   Accepted: 9 May 2022\nReferences\n 1. Saunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, and \ntherapeutic prospects. Cell. 2021;184:2807–24.\n 2. Barra F, Scala C, Ferrero S. Elagolix sodium for the treatment of women \nwith moderate to severe endometriosis‑associated pain. Drugs Today \n(Barc). 2019;55:237–46.\n 3. Vercellini P , Vigano P , Barbara G, Buggio L, Somigliana E, Luigi Mangiagalli’ \nEndometriosis Study G. Elagolix for endometriosis: all that glitters is not \ngold. Hum Reprod. 2019;34:193–9.\n 4. Burla L, Kalaitzopoulos DR, Metzler JM, Scheiner D, Imesch P . 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Cell Immunol. 2016;302:19–25.\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in pub‑\nlished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}