{"paper_id":"b78e0a9e-c9c6-4e6a-849d-335096b1e712","body_text":"Research paper\nThe protective effects of Azilsartan against hypoxia in\nendometrial stromal cells: an implication in\nendometriosis\n Keywords\nendometriotic lesions, HIF-1α, angiotensin II receptor, hypoxia, Endometriosis, Azilsartan (AZL)\n Abstract\nIntroduction\nEndometriosis is a reproductive disorder closely associated with hypoxia stress. Increasing evidences\nhave implied the important roles of angiotensin II (ANG II) receptors in the pathophysiology of\nendometriosis. Thus, we speculated that Azilsartan (AZL), an ANG II receptor blocker, might have an\neffective function in controlling endometriosis.\nMaterial and methods\nThe endometriotic animal model was established in female SD rats (6-8 weeks old, 200-220 g). Rats\nwere divided into sham group and endometriosis (EMS) group. Rats in EMS group were anaesthetized\nby halothane and a mid-ventral incision was performed to expose the bowels. Human endometrial\nstromal cell line T-HESC  was used for the in vitro assays. The T-HESC cells were cultured in DMEM-\nF12 mediumwith 10% fetal bovine serum (FBS, Hyclone), and 4 mM L-glutamine, 0.25% HEPES plus\nnecessary antibiotics (Sigma-Aldrich, USA), at 37°C in a humidified atmosphere of 5% CO2.\nResults\nThe results show that upregulation of ANG II type 1 (AT1) receptor was observed in the endometriotic\nrat models. Treatment with AZL prevented the development of endometriotic lesions and suppressed\nthe expressions of HIF-1α and cyclooxygenase 2 (COX-2) in endometriotic rats. In vitro assays proved\nthat hypoxia-induced proliferation, migration, and invasion of T-HESC cells were attenuated by AZL.\nAZL inhibited the expression levels of hypoxia-inducible factor-1α (HIF-1α), COX-2, and prostaglandin\nE2 (PGE2) production in hypoxia-induced T-HESC cells. Overexpression of HIF-1α blocked the effects\nof AZL on T-HESC cells in response to hypoxia.\nConclusions\nAZL showed therapeutic function on endometriosis through inhibiting hypoxia-induced cell\nproliferation, migration, and invasion of T-HESC cells via HIF-1α/COX-2/PGE2 signaling.\n Explanation letter\nDear Editorial Board,  \n    Thank you and the reviewers for the generous comments on the manuscript (AMS-14327-2022-01)\ntitled “The protective effects of Azilsartan against hypoxia in endometrial stromal cells: an implication\nin endometriosis”. We have gone over all the points that were raised and have edited the manuscript\nto address all the concerns of the editor and the reviewers. I attached an improved version of the\nmanuscript. All the changes made are highlighted in red. Thank you in advance for your continued\nconsideration and support of our manuscript. We truly appreciate the opportunity to submit this\nrevision.\nPoint by point reply to comments:\nReview 1:\nThis is a very interesting paper by Chen et al. about the effects of Azilsartan, a blocker of ANG II\nreceptor, on controlling endometriosi. Overall the article is well written, the experiments are well done\nand the conclusions are supported by the experimental data. However, there are a couple of points\nPreprint\n\nthat in my opinion need to be addressed before publication:\nMajor points:\n1.\tIn Figure 7A, the authors only examined the levels of COX-2 at the mRNA levels. Protein levels of\nCOX-2 should be measured by western blot analysis and the results should be included in this Figure. \nAnswer: Thank you. As per recommendation, protein levels of COX-2 were measured with Western\nblot analysis. The results have been included in Figure 7B. \n2.\tThe authors should clarify the rationale of the choice of the concentrations of Azilsartan used in\nthis study. \nAnswer: Thank you. The concentrations used in this study are based on previous studies. To address\nyour concerns, the following articles have been cited in the revised manuscript. \n[11]. Pan B, Zheng L, Fang J, Lin Y, Lai H, Gao J, Pan W, Zhang Y, Ni K, Lou C, He D. Azilsartan\nSuppresses Osteoclastogenesis and Ameliorates Ovariectomy-Induced Osteoporosis by Inhibiting\nReactive Oxygen Species Production and Activating Nrf2 Signaling. Front Pharmacol.\n2021;12:774709.\n[13]. Dong Q, Li Y, Chen J, Wang N. Azilsartan Suppressed LPS-Induced Inflammation in U937\nMacrophages through Suppressing Oxidative Stress and Inhibiting the TLR2/MyD88 Signal Pathway.\nACS Omega, 2021;6(1):113-118.\nMinor points: \n3.\tEnglish editing needs to be done before publication.\nAnswer: Thank you. To address your concerns, we have invited a native English speaker in our field to\nrevise the spelling and grammar of this manuscript. All grammatical errors have been eliminated.\nCorrections are marked in red in the revised manuscript. \n4.\tScale bars are missing in Figure 1. \nAnswer: Thank you. Scale bars have been added in Figure 1\n5.\tMolecular weights are missing in Figure 5 and Figure 8.\nAnswer: Thank you. Molecular weights have been added in Figure 5, Figure 7B, and Figure 8.\nReview 2:\nThe authors showed therapeutic function of Azilsartan on endometriosis through inhibiting hypoxia-\ninduced cell proliferation, migration, and invasion of T-HESC cells via HIF-1α/COX-2/PGE2 signaling\nThe manuscript need improvement in many ways.\n- Add clearly the hypothesis, aims and goals of this work to the last paragraph to your introduction.\nAnswer: Thank you. The following content has been added to the last paragraph of the “Introduction”\nsection to clarify the aims of this work:\n“However, whether AZL possesses a protective effect against endometriosis is still unknown. We\nspeculated that AZL might have an effective function in controlling endometriosis. Here, we studied its\neffects on endometriosis development in vivo and hypoxia-induced metastasis of endometrial stromal\ncells in vitro”.\n- Methods should have appropriate citation of references. Check and revise them properly.\nAnswer: Thank you. As per recommendation, the following articles have been cited in the “Materials\nand Methods” section of the revised manuscript. \n[11]. Pan B, Zheng L, Fang J, Lin Y, Lai H, Gao J, Pan W, Zhang Y, Ni K, Lou C, He D. Azilsartan\nSuppresses Osteoclastogenesis and Ameliorates Ovariectomy-Induced Osteoporosis by Inhibiting\nReactive Oxygen Species Production and Activating Nrf2 Signaling. Front Pharmacol.\n2021;12:774709.\n[12] Y. Wang, M. Zhang, R.Bi, et al. ACSL4 deficiency confers protection against ferroptosis-mediated\nacute kidney injury. Redox Biol, 51(2022):102262. \n[13]. Dong Q, Li Y, Chen J, Wang N. Azilsartan Suppressed LPS-Induced Inflammation in U937\nMacrophages through Suppressing Oxidative Stress and Inhibiting the TLR2/MyD88 Signal Pathway.\nACS Omega, 2021;6(1):113-118.\n[14] Y. Han, X. Qian, T. Xu, Y. Shi, Carcinoma-associated fibroblasts release microRNA-331-3p\ncontaining extracellular vesicles to exacerbate the development of pancreatic cancer via the\nSCARA5-FAK axis. Cancer Biol Ther, 23(2022):378-392.\n--Materials and methods are poorly written, many details are missed\nAnswer: Thank you. As per recommendation, more information and references have been added to\nthe “Materials and Methods” section. \nPreprint\n\n--primer design should be added.\nAnswer: Thank you. The following content has been added to the \"Materials and methods\" section:\n“The following primers were used: \nMMP-2 (forward: 5′ -GATACCCCTTTGACGGTAAGGA-3′, reverse: 5′ -\nCCTTCTCCCAAGGTCCATAGC-3′); MMP-9 (forward: 5′-ACGCACGACGTCTTCCAGTA-3′, reverse:\n5′ -CCACCTGGTTCAACTCACTCC-3′); PCNA (forward: 5′ -CCTGCTGGGATATTAGCTCCA-3′,\nreverse: 5′ -CAGCGGTAGGTGTCGAAGC-3′); HIF-1α (forward: 5′-\nTGACTGTGCACCTACTATGTCACTT-3′,\nreverse: 5′-GGTCAGCTGTGGGTAATCCACTC-3′);\nCOX-2 (forward: 5′-TGACTGTGCACCTACTATGTCACTT-3′,\nreverse: 5′-GGTCAGCTGTGGGTAATCCACTC-3′);\nGADPH (forward: 5′-GCACCGTCAAGGCTGAGAAC-3′,\nreverse: 5′-ATGGTGGTGAGACGCCAGT-3′)”.\n--catalog number for all antibodies\nAnswer: Thank you. Catalog numbers for antibodies have been added to the manuscript.\n“Membranes were incubated with rabbit anti-HIF-1α diluted in blocking buffer (1: 500; #ab179483,\nAbcam Cambridge, MA) overnight at 4°C and then incubated with secondary goat-anti-rabbit antibody\n(1: 3000; #ab150077, Abcam Cambridge, MA) for 1 h at room temperature”.\n--kits and reagents should be added\nAnswer: Thank you. The resources of kits and reagents have been added to the revised manuscript.\n--Details of ELISA method.\nAnswer: Thank you. To address your concern, the following content has been added to the revised\n“Materials and Methods” section. \n“Briefly, 50 μl of each standard or sample was added into the appropriate wells, followed by adding 50\nμl Biotin-labeled antibody working solution into each well.\nAfter incubation for 45 min at 37°C, each well was washed 3 times, followed by 0.1 mL of SABC\nworking solution being added into each well for 30 min at 37 ℃. 90 ml of TMB substrate was then\nadded and incubated at 37°C in the dark for 20 minutes. The reaction was then stopped by adding 50\nμL of stop solution to each well. Results were then read at 450 nm within 20 minutes”.\nReview 3:\nIn the study titled “The protective effects of Azilsartan against hypoxia in endometrial stromal \ncells: an implication in endometriosis”, the authors report the pharmacological function of Azilsartan, a\nblocker of ANG II receptor, in controlling endometriosis. Firstly, they found that ANG II type 1 receptor\n(AT1R) was upregulated in endometriotic rat models. Treatment with Azilsartan prevented the\ndevelopment of endometriotic lesions and suppressed the expression of HIF-1α and COX-2 in\nendometriotic rats. In vitro assays proved that hypoxia-induced proliferation, migration, and invasion of\nT-HESC cells were attenuated by Azilsartan. Azilsartan inhibited the expression levels of HIF-1α,\nCOX-2, and PGE2 production in hypoxia-induced T-HESC cells. Finally, they report that the\ntherapeutic function of Azilsartan on endometriosis is mediated by the HIF-1α/COX-2/PGE2 signaling\npathway. \nThe topic of the paper is interesting although needs some adjustments.\n----Full names of abbreviations should be provided.\nAnswer: Thank you. All the full names of abbreviations have been shown where they appeared the\nfirst time in the revised manuscript: \n“angiotensin II (ANG II), Azilsartan (AZL), ANG II type 1 (AT1), cyclooxygenase 2 (COX-2), hypoxia-\ninducible factor-1α (HIF-1α), prostaglandin E2 (PGE2), Azilsartan (AZL), endometriosis (EMS),\nproliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP), Cell counting kit-8 (CCK-8),\nEnzyme-linked immunosorbent assay (ELISA), analysis of variance (ANOVA)”...\n----English needs to be reviewed to eliminate typos.\nAnswer: Thank you. To address your concerns, we have invited a native English speaker in our field to\nrevise the spelling and grammar of this manuscript. All grammatical errors have been eliminated.\nCorrections are marked in red in the revised manuscript. \n----References should be updated.\nAnswer: Thank you. To address your concerns, references have been updated in the revised\nmanuscript. Corrections are marked in red in the References list. \n----Introduction on “Azilsartan” is not sufficient. \nAnswer: Thank you. To address your concerns, the following content has been added to the\nPreprint\n\n“Introduction” section in the revised manuscript to introduce Azilsartan:\n“Azilsartan (AZL) is an AT1 receptor blocker that is used for the treatment of hypertension. Recently,\nseveral studies have demonstrated the protective benefits of AZL in a wide range of diseases. For\nexample, Liu et al. showed that AZL suppressed inflammatory response by increasing e-NOS\nphosphorylation [9]. Furthermore, it inhibited hydroperoxide-induced oxidative stress in endothelial\ncells [10]. However, whether AZL possesses a protective effect against endometriosis is still unknown.\nWe speculated that AZL might have an effective function in controlling endometriosis. Here, we\nstudied the effects of AZL on endometriosis development in vivo and hypoxia-induced metastasis of\nendometrial stromal cells in vitro”.\n----Ethical approval should be mentioned in the “Materials and Methods” section.\nAnswer: Thank you. Ethical approval has been added to the “Materials and Methods” section in the\nrevised manuscript. \n“The protocol of this study was approved by the Ethical Committee of the General Hospital of Ningxia\nMedical University”.\n----It’s unknown how many mice were used in each group of the experiment.\nAnswer: Thank you. Rats were divided equally into the sham group and the endometriosis (EMS)\ngroup (ten rats for each group).\nResponse letter20220530.docx\nPreprint\n\nTitle: The protective effects of Azilsartan against hypoxia in endometrial stromal \ncells: an implication in endometriosis \nAbstract  \nEndometriosis is a reproductive disorder closely associated with hypoxia stress. \nIncreasing evidences have implied  the important roles  of angiotensin II (ANG II)  \nreceptors in the pathophysiology of endometriosis.  Thus, we speculated that \nAzilsartan (AZL), an ANG II receptor  blocker, might have an effective function in \ncontrolling endometriosis.  We investigated the effects of AZL on endometriosis \ndevelopment in vivo . The results show that upregulation of ANG II  type 1 (AT1)  \nreceptor was observed in the endometriotic rat models. Treatment with AZL \nprevented the development of endometriotic lesions and suppressed the expressions of \nHIF-1α and cyclooxygenase 2 (COX -2) in endometriotic rat s. In vitro assays proved \nthat hypoxia -induced proliferation, migration , and invasion of T-HESC cells were \nattenuated by AZL. AZL inhibited the  expression levels of hypoxia-inducible \nfactor-1α (HIF-1α), COX-2, and prostaglandin E2 (PGE2) production in \nhypoxia-induced T-HESC cells . Overexpression of HIF -1α blocked the effects of \nAZL on T-HESC cells  in response to hypoxia . In conclusion, AZL showed \ntherapeutic function on endometriosis through inhibiting hypoxia -induced cell \nproliferation, migration , and invasion of T-HESC cells  via HIF -1α/COX-2/PGE2 \nsignaling.  \nKeywords: Endometriosis, Azilsartan (AZL), angiotensin II receptor, endometriotic  \nlesions, hypoxia, HIF-1α \nPreprint\n\n1. Introduction \nEndometriosis is a reproductive disorder that occurs  in women of reproductive \nage [1]. It is manifested by the  formation of endometrial tissues outside the uterus, \nwith the symptoms of infertility, dysmenorrhea, and long-term pelvic pain  [2]. It is \ncrucial to fully understand the pathogenesis of endometriosis for  developing proper \ntherapeutic approaches. Over the past decades, hypoxia has been shown to play a key \nrole in regulating numerous important processes in the development of endometriosis  \n[3]. In p articular, endometrial stromal cells  undergo epigenetic modulation under \nhypoxic conditions, since they normally reside in an oxygen-enriched environment [4]. \nIt is evident that hypox ia is one of the major factors responsible for the metastasis of \nendometrial stromal cells.  Interestingly, the expression of HIF -1α, a crucial \nresponder to hypoxia stress, is upregulated in the ectopic endometrial stromal cells [5]. \nThese findings provide evidence for the participation of hypoxia in the modulation of \nendometrial stromal cells, which represents a crucial mechanism of endometriosis. \nAngiotensin II (ANG II) , an octapeptide, is one of the biologically active \ncomponents of  the renin-angiotensin system  [6]. Previous studies have shown that \nANG II may cause reduced blood flow and lead  to hypoxic conditions in several \norgans, thereby participat ing in various diseases . Marciante et al . [7] reported that \nANG II is involved in the development of hypertension and cognitive decline \nmediated by chronic intermittent hypoxia . It induces hypoxia in the kidney via both \nnon-hemodynamic and hemodynamic mechanisms. ANG II has been found to elicit \nits multiple actions through its specific receptors , type 1 (AT1) and type 2 (AT2). \nTherefore, targeting either one  is an effective  approach for blocking the effects of \nANG II.  \nIncreasing evidences have implied that ANG II receptors may participate in the \npathophysiology of endometriosis. Several studies have proven that ANG II receptor  \nblockers have the capacity to control the develo pment of endometriosis. For instance, \nan ANG II receptor blocker, losartan, suppresses the formation of endometriotic \nlesions in experimental endometriotic rats  [8]. Azilsartan (AZL) is an AT1 receptor \nblocker that is used for the treatment of hypertension. Recently, several studies have \nPreprint\n\ndemonstrated the protective benefits of AZL in a wide range of diseases. For example, \nLiu et al. showed that AZL suppressed inflammatory response by increasing e-NOS \nphosphorylation [9]. Furthermore, it inhibited hydroperoxide-induced oxidative stress \nin endothelial cells [10]. However, whether AZL possesses a protective effect against \nendometriosis is still unknown. We speculated that AZL might have an effective \nfunction in controlling endometriosis.  Therefore, we studied its effects on \nendometriosis development in vivo  and hypoxia-induced metastasis of endometrial \nstromal cells in vitro.  \n2. Methods and materials  \n2.1 Animal model \nThe endometriotic animal model was established in female SD rats (6 -8 weeks \nold, 200 -220 g). Rats were divided equally into the sham group and endometriosis \n(EMS) group, and the AZL+EMS group (ten rats for each  group). Rats in the EMS \ngroup were anaesthetized with halothane and a mid-ventral incision was performed to \nexpose the bowels. The protocol of this study was approved by the Ethical Committee \nof the General Hospital of Ningxia Medical University . Then, the right uterine horn \nwas removed and longitudinally opened using scissors. After  cutting into 4 mm 2 \npieces, the fragments were sutured to the intestine mesentery. The incision was closed, \nand the rats were kept for 4 weeks  to induce endometriosis . Rats in th e sham group \nwere subjected to a similar surgery without transplantation of the uterine tissue. Rats \nin the EMS group were randomly divided into 2 groups: in the EMS model group, rats \nwere orally treated with an equal volume of normal saline solution; in t he AZL \ntreatment group, rats were orally treated with AZL (3.0 mg/kg body weight) [11] for \n21 days after surgery. \n2.2 Immunofluorescence \nThe endometriotic lesions were separated, fixed in 4% paraformaldehyde, \nembedded in paraffin , and cut into sections for the immunofluorescence assay . The \nsections were incubated with blocking buffer 5% BSA at room temperature for 2 h , \nand then incubated with anti -AT1 antibody (dilut ed in  1:200; Abcam) at 4°C \novernight, and then incubated with AlexaFluor 488 -conjugated secondary antibody \nPreprint\n\n(1:200; Invitrogen, Carlsbad, CA)  for1 h at 37°C. Finally, the  immunofluorescence \nresults were analyzed using a confocal fluorescence microscope(Nikon, Japan). \n2.3RT-PCR analysis \nThe mRNA levels of relevant genes proliferating cell nuclear antigen (PCNA), matrix \nmetalloproteinase (MMP)-2, MMP-9, HIF-1α, and COX-2 in total RNAs from \nendometriotic lesions or cultured cells were assessed using qRT-PCR with a Prime \nScriptRT reagent kit (Takara, Japan) and SYBR Green I kit (Takara) on a Bio-Rad \nIQ5 Real-Time System (Bio-Rad, Hercules, CA). The β-actin was applied as an \ninternal control. The relative expression of target genes was analyzed using the 2–ΔΔCt \napproach [12]. The following primers were used:  \nMMP-2 (forward: 5′ -GATACCCCTTTGACGGTAAGGA-3′, reverse: 5′ - \nCCTTCTCCCAAGGTCCATAGC-3′); MMP-9 (forward: \n5′-ACGCACGACGTCTTCCAGTA-3′, reverse: 5′ \n-CCACCTGGTTCAACTCACTCC-3′); PCNA (forward: 5′ \n-CCTGCTGGGATATTAGCTCCA-3′, reverse: 5′ \n-CAGCGGTAGGTGTCGAAGC-3′); HIF-1α (forward: 5′- \nTGACTGTGCACCTACTATGTCACTT-3′, \nreverse: 5′-GGTCAGCTGTGGGTAATCCACTC-3′); \nCOX-2 (forward: 5′-TGACTGTGCACCTACTATGTCACTT-3′, \nreverse: 5′-GGTCAGCTGTGGGTAATCCACTC-3′); \nGADPH (forward: 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse: \n5′-ATGGTGGTGAGACGCCAGT-3′).  \n2.4 Culture of endometrial stromal cells \nHuman endometrial stromal cell line T -HESC (ATCC, Manassas, VA) was used \nfor the in vitro  assays. The T -HESC cells were cultured in DMEM -F12 medium \n(Hyclone, Logan, UT) with 10% fetal bovine serum (FBS, Hyclone), and 4 m M \nL-glutamine (Sigma -Aldrich, St. Louis, MO), 0.25% HEPES plus necessary \nantibiotics ( Sigma-Aldrich, USA ), at 37°C in a humidified atmosphere of 5% CO 2. \nHypoxia exposure was conducted at 1% O 2, and 5% CO 2 using a ProOx C21 \nnitrogen-induced hypoxia system ( BioSpherix, Red Field, NY). For the AZL \nPreprint\n\ntreatment group [13], T -HESC cells were pre -treated with 5 μM AZL, followed by \nhypoxia exposure for 48 h.  \n2.5 Cell transfection  \nThe full-length cDNA sequence of HIF-1α was linked to the pcDNA3.0 vector to \nconstruct the pcDNA3.0 -HIF-1α. Afterward, cell transfection of T -HESC cells with \npcDNA3.0-HIF-1α or pcDNA3.0 was performed utilizing the lipofectamine3000 \nreagent (Thermo Fisher Scientific, Massachusetts, USA) . Finally, the cells were \nharvested at 48h after transfection to detect the transfection efficiency. \n2.6 Western blot  \nWestern blot was carried out to assess protein expression  of HIF-1α in T -HESC \ncells after transfection. The cellular lysates were loaded in SDS-PAGE to separate the \ntarget protein,  followed by transfer to  the polyvinylidene difluoride (PVDF) \nmembrane. Membranes were incubated with rabbit anti -HIF-1α diluted in blocking \nbuffer (1: 500; #ab179483, Abcam Cambridge, MA ) overnight at 4°C and then \nincubated with secondary goat -anti-rabbit antibody (1: 3000; #ab150077, Abcam \nCambridge, MA) for 1 h at room temperature.  Finally, protein  bands were detected \nwith ECL Plus  reagent (Thermo Fisher Scientific) and analyzed using Image J \nsoftware.  \n2.7 Cell counting kit-8 (CCK-8) assay  \nCell viability of T-HESC cells was evaluated by employing a CCK-8 kit (#C0037, \nBeyotime Biotechnology, Shanghai, China ). Briefly, T -HESC cells (5000 cells per \nwell) were maintained in a 96-well plate and subjected to hypoxia exposure for 0, 24, \n48, and 72 h with or without 5 μM AZL. Then CCK-8 reagent (10μl) was added to \neach well and maintained for another 4 h at 37°C. Finally, the detection of absorbance \nat 450nm was performed using a Microplate Reader (Bio-Rad). \n2.8 Transwell assay \nThe migrative and invasive capacity of T -HESCs were measured with the \ntranswell assay. Briefly, T -HESCs (2 × 10 4 cells/well) were seeded in the upper \nchambers in a serum-free medium and subjected to hypoxia exposure for 0, 24, 48, \nand 72 h with or without 5 μM AZL. Meanwhile, a serum-containing medium  was \nPreprint\n\nadded to the lower chambers . The inserts were coated with or without the Matrigel. \nThe non-migrating or non-invading cells on inserts were cleansed with a cotton swab \nafter a 24-hour incubation period. The cells that had migrated or invaded through the \ninserts were stained with DAPI. The images from 5 random fields were obtained with \nan Inversion Microscope (Zeiss, Germany) [14]. \n2.9 Enzyme-linked immunosorbent assay (ELISA)  \nELISA was used to examine the PGE2 level in the supernatant of T-HESC cells using \na commercial kit (#ab176480, Abcam Cambridge, MA). Briefly, 50 μl of each \nstandard or sample was added into the appropriate wells, followed by adding 50 μl of \nbiotin-labeled antibody working solution into each well. After incubation for 45 min \nat 37°C, each well was washed 3 times, followed by 0.1 mL of streptavidin conjugate \n(SABC) working solution being added into each well for 30 min at 37 ℃. 90 ml \noftetramethylbenzidine (TMB) substrate was then added and incubated at 37°C in \ndark for 20 minutes. The reaction was then stopped by adding 50 μL of stop solution \nto each well. Results were then read at 450 nm within 20 minutes. \nThe absorbance at 450 nm was detected using the spectrophotometer (Bio-Rad). \n2.10 Statistical analysis \nAll statistical testing was performed using GraphPad Prism 5 software. The data \nare expressed as the mean ±standard errors of mean ( S.E.M.)with three repeats. \nStatistically significant differences were determined using a one-way analysis of \nvariance (ANOVA). \n3. Results \n3.1 Up-regulation of the AT1 receptor in endometriotic rat models \nThrough the IFC assay, there was a significant increase in AT1 expression in the \nendometriotic lesions from the EMS group compared to the endometrial tissues from \nthe control group (Figure 1A). Consistent with the IFC results, the mRNA levels of \nAT1 were upregulated in  the endometriotic lesions from  the endometriosis rats \n(Figure 1B).  \n3.2 The impact of AZL on endometriotic lesions formation in experimental \nendometriosis rats \nPreprint\n\nAs shown in figure 2,  AZL treatment caused a significant reduction in the size of \nendometriotic lesions  compared to  that in  the EMS group models, implying that it \nprevented the development of endometriotic lesions.  \n3.3 AZL regulated the proliferation- and metastasis-related genes expression in \nendometriosis models \nAs illustrated by RT -PCR, rats from  the EMS group exhibited significant  PCNA \nincreases in endometriotic lesions, which was attenuated by AZL treatment. The \nincreased mRNA levels of MMP-2 and MMP-9 in the EMS group were also reduced \nafter AZL administration (Figures 3). \n3.4 AZL suppressed the  expressions of HIF-1α and COX-2 in endometriosis \nmodels \nWe detected significant HIF-1α and COX-2 mRNA levels elevations in endometriotic \nlesions from rats in  the EMS group (Figures 4A and 4B). Administration of AZL \nattenuated the upregulation of both HIF-1α and COX-2 in the endometriotic lesions.  \n3.5 AZL inhibited cell proliferation of T-HESC cells in response to hypoxia \nThe protein and mRNA expression levels of AT1 were elevated in T-HESC cells after \nhypoxia exposure in a time-dependent manner (Figures 5A and 5B). Cell proliferation \nof T-HESC cells  was dramatically increased after hypoxia exposure for 48 h. AZL \ntreatment attenuated the hypoxia-induced cell proliferation of T-HESC cells (Figure \n5C).  \n3.6 AZL modulated the cell migration and invasion of T-HESC cells in response \nto hypoxia \nIn F igure 6A, we confirm that hypoxia induced the migration capacity of T-HESC \ncells, which could be attenuated by AZL. Meanwhile, the enhanced invasive capacity \nof T-HESC cells was also found to be alleviated after AZL treatment (Figure 6B).  \n3.7 AZL suppressed COX-2 expression and PGE2 production in T-HESC cells in \nresponse to hypoxia \nWe next evaluated changes in the COX -2 expression, and the results show that \nthe COX -2 mRNA level was upregulated after hypoxia exposure. Treatment with \nAZL effectively blocked the elevated mRNA (Figure 7A) and protein levels  (Figure \nPreprint\n\n7B) of COX -2 in hypoxia -induced T-HESC cells . In addition, ELISA showed that \nhypoxia exposure also caused increased PGE2 production. However, the increased \nPGE2 level was attenuated by AZL treatment (Figure 7C). \n3.8 HIF-1α mediated the effects of AZL in T-HESC cells in response to hypoxia \nHIF-1α expression was  also upregulated in T-HESC cells exposed to  hypoxia. \nAZL reduced the expression of HIF -1α against hypoxia induction, as shown by \nWestern blot (Figure 8A). To further confirm the role of HIF -1α, \nHIF-1α-overexpressing T-HESC cells  were constructed through transfection with \npcDNA3.0-HIF-1α. As confirmed by Western blot, transfection efficiency with \npcDNA3.0-HIF-1α in T-HESC cells  was successful with a 4.5 -fold increase in \nHIF-1α expression (Figure 8B).  \nTransfection with pcDNA3.0 -HIF-1α elevated the  AZL-caused decrease  in \nproliferation of T-HESC cells  (Figure 8C). The inhibitory effects of AZL on \nmigration and invasive capacit ies were reversed by HIF -1α overexpression (Figures \n8D and  8E). In addition, the decreased COX -2 mRNA and PGE 2 levels in \nAZL-treated T-HESC cells were increased after transfection with pcDNA3.0 -HIF-1α \n(Figures 8F and 8G).  \n4. Discussion \nIt has been reported that  the renin-angiotensin system (RAS) participates in \nendometriosis progression. For instance, ANG II regulates COX-2 expression , thus \npromoting the proliferation of endometrial tissue in endometriosis rats. AT1 and AT2 \nreceptors are located in endometrial stromal cells and their protein levels are \nincreased in endometriotic lesions . The AT1 receptor  regulates the development of \nendometriosis by promoting the cell proliferati ve and migration capacities of stromal \ncells and preventing stromal cells from undergoing apoptosis [15]. Tanshinone IIA \nwas reported to contribute to regulating endometriosis progression by decreasing the \nexpressions of estradiol (E2), ANG II, and the AT2 receptor [16]. Particularly, several \nstudies have found that the ANG II receptor blockers have the capacity to repress the \ndevelopment of endometriosis. Losartan, a n ANG II receptor blocker, was found to \nsuppress the implant growth of experimental endometriosis rats [8]. Here, we found \nPreprint\n\nthat the AT1 receptor expression was upregulated in the endometriotic lesions from \nendometriosis rats. Treatment with AZL, an AT1 receptor blocker, caused significant \nreductions in the sizes of the endometriotic lesions.  \nCurrently, endometriosis is widely accepted to be associated with an implantation \ntheory [17]. Ectopic implantation of endometrial tissues may be initiated during the \nmenstrual cycle  through the exoteric fallopian  tube. Furthermore, the uncontrolled \nmetastasis and cell proliferation of endometrial cells  facilitate the development of \nendometriotic lesions [18]. It is generally accepted that preventin g the metastasis of \nendometrial stromal cells may ameliorate endometriosis [19-21]. In this study, we \nfound that the rats from the EMS group exhibited significant increases in PCNA (for \nproliferation), MMP-2, and MMP-9 (for metastasis) levels, which could be attenuated \nby AZL. It is well established that hypoxia acts as a potent risk factor for epigenetic \nregulation of certain genes involved in differentiation, proliferation, survival, \nmigration, and angiogenesis in endometri al cells . It t hereby facilitates the \nimplantation and progression of ectopic endometriotic lesions  [22, 23]. Here we used \na hypoxia-induced in vitro model of endometriosis in endometrial stromal T-HESC \ncells. We found that AT1 receptor expression  was upregulated in T-HESC cells after \nhypoxia exposure in a time -dependent manner. Treatment with AZL alleviated the \nhypoxia-induced increase in proliferative, migration, and invasi ve capacities  of \nT-HESC cells.  \n Researchers have increasingly discovered that hypoxic stress is one of the most \ncritical driving forces for the development of ectopic endometriotic tissues. Multiple \nstudies over the past years have found that there is a crucial association between the \naberrant expression of HIF-1α and endometriosis. Higher expression levels of HIF-1α \nin endometriosis patients relative to those in the ectopic endometria of women  \nwithout endometriosis were observed  [5]. Compared to stage I/II endometriosis, \nelevated serum HIF-1α levels were observed in stage III/IV endometriosis, indicating \nthat HIF-1α may be a biomarker for patients with severe endometriosis [24]. It is well \nvalidated that HIF -1α led to the increased COX-2 expression , and thus PGE 2 \nover-production [25]. It has been demonstrated that PGE2 stimulates the dysregulation \nPreprint\n\nof steroidogenic acute regulatory protein s, vascular endothelial growth factors, and \nfibroblast growth factors in endometriotic stromal cells . In turn,  endometrial and \nendothelial cell proliferation is induced and this results in estrogen production in the \nendometriotic tissue [26]. Considering their multipotent effects,  the \nHIF-1α/COX-2/PGE2 pathway is considered a master  regulator of endometriosis . \nTherefore, we evaluated the overall hypothesis that the augmentation of \nHIF-1α/COX-2/PGE2 could be involved in endometriosis. We found that the \nupregulated expression levels of HIF -1α and COX -2, and PGE 2 production in \nendometriotic lesions and/or hypoxia -induced endometriotic stromal cells , were \nrepressed by AZL. Overexpression of HIF -1α blocked the effects of AZL on \nhypoxia-induced endometriotic stromal cells.  \nIn conclusion, we provide evidence to show the therapeutic function of AZL on \nendometriosis with a novel mechanism of inhibiting hypoxia-induced cell \nproliferative, migration, and invasi ve capacities of endometriotic stromal cells . \nFurthermore, all these phenomena were attributed to the  inhibition of \nHIF-1α/COX-2/PGE2 signaling.  \nAcknowledgement \nThis study was supported by the “Ningxia Natural Science Foundation  \n(2022AAC03494)”.  \nReferences \n[1] T. Tanbo, P. Fedorcsak, Endometriosis -associated infertility: aspects of \npathophysiological mechanisms and treatment options, Acta Obstet Gynecol Scand, \n96 (2017) 659-667. \n[2] P.T.K. Saunders, A.W. Horne, Endometriosis: Etiology, pathobiology, and \ntherapeutic prospects, Cell, 184 (2021) 2807-2824. \n[3] M.H. Wu, K.Y. Hsiao, S.J. Tsai, Hypoxia: The force of endome triosis, J Obstet \nGynaecol Res, 45 (2019) 532-541. \n[4] W.N. Li, M.H. Wu, S.J. Tsai, HYPOXIA AND REPRODUCTIVE HEALTH: The \nrole of hypoxia in the development and progression of endometriosis, Reproduction, \n161 (2021) F19-F31. \nPreprint\n\n[5] M.H. Wu, K.F. Chen, S.C. Lin, C.W. Lgu, S.J. Tsai, Aberrant expression of leptin \nin human endometriotic stromal cells is induced by elevated levels of hypoxia \ninducible factor-1alpha, Am J Pathol, 170 (2007) 590-598. \n[6] S.J. Forrester, G.W. Booz, C.D. Sigmund, T.M. Coffman, T. Kawai , V. Rizzo, R. \nScalia, S. Eguchi, Angiotensin II Signal Transduction: An Update on Mechanisms of \nPhysiology and Pathophysiology, Physiol Rev, 98 (2018) 1627-1738. \n[7] A.B. Marciante, B. Shell, G.E. Farmer, J.T. Cunningham, Role of angiotensin II in \nchronic intermittent hypoxia -induced hypertension and cognitive decline, Am J \nPhysiol Regul Integr Comp Physiol, 320 (2021) R519-R525. \n[8] B. Cakmak, T. Cavusoglu, U. Ates, A. Meral, M.C. Nacar, O. Erbas, Regression \nof experimental endometriotic implants in a rat model with the angiotensin II receptor \nblocker losartan, J Obstet Gynaecol Res, 41 (2015) 601-607. \n[9] J.Lei, M.He, L.Xu, C.He, J.Li, W.Wang , Azilsartan Prevented AGE -Induced \nInflammatory Response and Degradation of Aggrecan in Human Chondrocytes \nthrough Inhibition of Sox4. J. Biochem. Mol. Toxicol, 35(2021) e22827. \n[10] H.Liu, P .Mao, J.Wang, T.Wang, C .H.Xie, Azilsartan, an angiotensin II type 1 \nreceptor blocker, attenuates tert -butyl hydroperoxide -induced endothelial cell injury \nthrough inhibition of mitochondrial dysfunction and anti -inflammatory activity. \nNeurochem Int, 94(2016):48–56. \n[11]. Pan B, Zheng L, Fang J, Lin Y, Lai H, Gao J, Pan W, Zhang Y, Ni K, Lou C, He \nD. Azilsartan Suppresses Osteoclastogenesis and Ameliorates Ovariectomy -Induced \nOsteoporosis by Inhibiting Reactive Oxygen Species Production and Activating Nrf2 \nSignaling. 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Gong, Tanshinone IIA contributes to the pathogenesis of \nendometriosis via renin angiotensin system by regulating the dorsal root ganglion \naxon sprouting, Life Sci, 240 (2020) 117085. \n[17] P.R. Koninckx, A. Ussia, L. Adamyan, A. Wattiez, V. Gomel, D.C. Martin, \nPathogenesis of endometriosis: the genetic/epigenetic theory, Fertil Steril, 111 (2019) \n327-340. \n[18] P.C. Logan, P. Yango, N.D. Tran, Endometrial Stromal and Epithelial Cells \nExhibit Unique Aberrant Molecular Defects in Patients With Endometriosis, Reprod \nSci, 25 (2018) 140-159. \n[19] J. Wang, X. Yao, D.L. Wu, Y. Jin, Kallikrein -related peptidase 4 promotes \nmigration and invasion of endometrial stromal cells in endometriosis by inducing \nepithelial-mesenchymal transition, J Biol Regul Homeost Agents, 34 (2020) 93-100. \n[20] L. Cui, S. Chen, D. Wang, Q. 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Cetin, \nElevated Serum CD95/FAS and HIF -1alpha Levels, but Not Tie -2 Levels, May Be \nBiomarkers in Patients With Severe Endometriosis: A Preliminary Report, J Minim \nInvasive Gynecol, 23 (2016) 573-577. \n[25] M.H. Wu, S.C. Lin, K.Y. Hsiao, S.J. Tsai, Hypoxia -inhibited dual -specificity \nphosphatase-2 expression in endometriotic cells regulates cyclooxygenase -2 \nexpression, J Pathol, 225 (2011) 390-400. \n[26] Z.Z. Lai, H.L. Yang, S.Y. Ha, K.K. Chang, J. Mei, W.J. Zhou, X.M. Qiu, X.Q. \nWang, R. Zhu, D.J. Li, M.Q. Li, Cyclooxygenase -2 in Endometriosis, Int J Biol Sci, \n15 (2019) 2783-2797. \nFigure legends \nFigure 1 Upregulation of angiotensin II type 1 (AT1) in endometriotic rat model. \n(A) IFC for AT1 expression; Scale bar, 100 μm; (B) RT-PCR for mRNA level of AT1 \n(***, P<0.001 vs. vehicle group).  \nFigure 2 The impact of AZL on endometriotic lesions in experimental rats. \nSize of endometrioticlesions ( ***, P<0.001 vs. vehicle group; ##, P<0.01 vs. EMS \ngroup). \nFigure 3 The regulation of AZL on related-genes expression in rat endometriosis \nmodel. RT-PCR analysis for the mRNA levels of PCNA, MMP -2, and MMP-9 in \nendometriotic lesions (***, P<0.001 vs. vehicle group; ##, P<0.01 vs. EMS group). \nFigure 4 The inhibitory effects of AZL on HIF-1α and COX-2 expression in rat \nendometriosis model. RT-PCR analysis for the mRNA levels of HIF-1α and COX-2  \nin endometriotic lesions (***, P<0.001 vs. vehicle group; ##, P<0.01 vs. EMS group). \nFigure 5 The effects of AZL on cell proliferation of endometrial stromal cells in \nresponse to hypoxia for 12, 24, and 48 hours. (A) Western blot for AT1 expression; \n(B) RT -PCR for mRNA level of AT1 ; (C) CCK -8 assay for cell proliferation of \nT-HESC cells  (*, **, ***, P<0.05, 0.01, 0.001 vs. vehicle group ; ##, P<0.01 vs. \nHypoxia group).  \nFigure 6 The regulatory effects of AZL on cell migration and invasion of  \nendometrial stromal cells in response to hypoxia. (A) Transwell assay for cell \nPreprint\n\nmigration of T-HESC cells (migrated cells number per field) .(B) Transwell assay for \ncell invasion of T-HESC cells  (invaded cells number per field) ( ***, P<0.001 vs. \nvehicle group; ##, P<0.01 vs. Hypoxia group). \nFigure 7 The inhibitory effects  of AZL on COX-2 and PGE 2in endometrial \nstromal cells in response to hypoxia. (A) RT-PCR analysis for the mRNA level of \nCOX-2 in T-HESC cells. (B) Western blot analysis for the protein level of COX-2 in \nT-HESC cells. (C) ELISA kits to determine levels of PGE 2 in the supernatant samples \n(***, P<0.001 vs. vehicle group; ##, P<0.01 vs. Hypoxia group). \nFigure 8 HIF-1α mediated the effects of AZL in endometrial stromal cells in \nresponse to hypoxia. (A) Western blot for the expression of HIF-1α in T-HESC cells. \n(B) Western blot for the determination of transfection efficiency with \npcDNA3.0-HIF-1α. (C) CCK -8 assay for cell proliferation of T-HESC cells . (D) \nTranswell assay for cell migration of T-HESC cells (migrated cells number per field). \n(E) Transwell assay for cell invasion of T-HESC cells (invaded cells number per field) \n(F) RT-PCR analysis for the mRNA level of COX-2 in T-HESC cells. (G) ELISA kits \nto determine levels of PGE2in the supernatant samples(***, P<0.001 vs. vehicle group; \n##, P<0.01 vs. hypoxia group; &&, P<0.001 vs. hypoxia+AZL group). \n \n \nPreprint\n\nFigure 1\nPowered by TCPDF (www.tcpdf.org)\nPreprint\n\nFigure 2\nPowered by TCPDF (www.tcpdf.org)\nPreprint\n\nFigure 3\nPowered by TCPDF (www.tcpdf.org)\nPreprint\n\nFigure 4\nPowered by TCPDF (www.tcpdf.org)\nPreprint\n\nFigure 5\nPowered by TCPDF (www.tcpdf.org)\nPreprint\n\nFigure 6\nPowered by TCPDF (www.tcpdf.org)\nPreprint\n\nFigure 7\nPowered by TCPDF (www.tcpdf.org)\nPreprint\n\nFigure 8\nPowered by TCPDF (www.tcpdf.org)\nPowered by TCPDF (www.tcpdf.org)\nPreprint","source_license":"CC0","license_restricted":false}