{"paper_id":"20546700-787d-4c47-aa6d-6175ef57c01b","body_text":"* Corresponding author: dedysyahrizal@unsyiah.ac.id   \n \nHyperbaric Oxygen (HBO) Reduce Expression of Hypoxia -\nInducible Factor -1 Alpha (HIF -1 alpha) and  Endometriotic \nTissue Size in  Mice Model of Endometriosis \nDedy Syahrizal1*, Cut Mustika2, Teuku Renaldi2, Mohammad G. Suryokusumo3, and Hendy Hendarto4  \n1 Department of Biochemistry,  Medical Faculty, Universitas Syiah Kuala, Banda Aceh, Indonesia \n2  Department of Public Health, Medical Faculty, Universitas Syiah Kuala, Banda Aceh, Indonesia \n3 Medical Hyperbaric Study Program, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia \n4 Department of Obstetrics and Gynaecology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia \nAbstract. Hypoxia in endometriosis will increase the expression of Hypoxia Inducible Factor-1alpha (HIF-\n1alpha) and its expression could be decreased by Hyperbaric Oxygen (HBO). This study aimed to analyze the \neffect of  HBO 2.4 ATA for 3x30 minutes per day for 10 days on the expression of HIF -1 alpha an d \nendometriotic tissue size on mice model of endometriosis. This study was an experimental laboratory study \nwith a separate pretest-posttest control group design. The mice were divided into three groups, the first was a \npretest control group (which describes the condition after endometrium transplantation ), the second was the \nendometriotic group that received hyperbaric oxygen, and the third was the endometriotic group that did not \nreceive hyperbaric oxygen therapy. The endometriosis implant size in the peritoneal tissue was assessed and \nthe immunohistochemistry examination was conducted to determine the expression of HIF -1 alpha . The \nendometriosis tissue size was reduced in the HBO group compared to the control and nonHBO group . The \nlowest expression of HIF-1 alpha was significantly found in HBO over the other group. The decrease of HIF-\n1 alpha expression mediates the reduction of size endometriotic tissue due to the therapy of HBO.  \nKeywords: hypoxia, endometriosis, hyperbaric oxygen (HBO), hypoxia-inducible Factor-1 alpha (HIF-1α). \n1 Introduction  \nHypoxic conditions occur in endometriosis. The hypoxic \nconditions are due to the dysregulation of immune cells \nand cytokines released by endometriotic tissue [1,2]. \nHypoxic conditions are known to cause an increase in the \nexpression of Hypoxia -Inducible Factor -1 alpha (HIF -1 \nalpha), this situation is also found in endometriosis tissue \n[3,4]. According to the previous study, HIF-1 alpha plays \na role in increasing the e xpression of estrogen receptor β \nwhich triggers the endometriosis [5]. This increase then \nleads to the expression of vascular endothelial growth \nfactor (VEGF), which is required by endometriosis tissue \nto carry out angiogenesis so that the network can \ncontinuously evolve  [6,7]. Based on this process, an \nattempt to decrease the expression of HIF -1 alpha is \npredicted to be a potential approach to treat \nendometriosis. \nHyperbaric oxygen (HBO) is a therapy that uses a \npressurized chamber to increase the amount of oxygen in \nthe blood. According to several studies, HBO may \ndecrease the expression of HIF -1 alpha because the \noxygen-rich atmosphere would increase the breakdown of \nHIF-1 alpha protein by the ubiquitin system. [8] \nConcerning endometriosis, HBO is predicted to decrease \nthe expression of HIF-1 alpha in the endometriosis tissue.  \n2 Materials and methods  \nThis study has been ethically approved by the Animal \nCare and Use Committee (ACUC) Faculty of Veterinary \nMedicine, Universitas Airlangga (Approvement No. 497-\nKE).  \n \n2.1 Animals \n \nTwenty-eight female mice of three -month-old were \nrandomly divided into three groups:  \nGroup 1: pretest control group which was examined \nimmediately after endometriosis implantation,  \nGroup 2: an endometriotic group that received hyperbaric \noxygen for ten days, \nGroup 3: an endometriotic group that did not receive \nhyperbaric oxygen therapy. \n \n2.2 Endometriosis modeling \n \nMice have given intramuscular cyclosporin A injection at \nthe dose of 10 milligrams per kilogram of body weight on \nthe first day to make the mice immune -deficiency. \nFurthermore, endometrial tissue formula was injected into \nthe mice intraperitoneally. The endometrial tissue was \ntaken from myometrium and adenometrium of \nadenomyosis patients. The tissue is then stored in \n© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons At tribution License 4.0\n(htt p://creativecommons.or g/licenses/b y/4.0/). \nE3S Web of Conferences 151, 01003 (2020) https://doi.org/10.1051/e3sconf/202015101003\n1st ICVAES 2019\n\n \n \nphosphate buffer saline (PBS). The tissue was washed two \ntimes with a centrifuge at a speed of 2500 rpm. The \nsupernatant was discarded then PBS, penicillin 200 IU/ml \nand streptomycin 20 0 mg/ml were added. Each mouse \nreceived 0.1 ml injection of the formula. Following the \nendometriosis tissue formula injection, the mouse \nreceived intramuscular estrogen injection in the thigh at a \ndose of 30 milligrams per kilogram of body weight on the \nfirst and fifth day after the endometriosis tissue formula \ninjection. On the fourteenth day, all the mice have become \nmodels of endometriosis.[9,10] \n \n2.3 Hyperbaric oxygen (HBO) \n \nMice of Group 2 were placed in a hyperbaric chamber a \nday after they became mod els of endometriosis. The \nprocedure was conducted by using 2.4 ATA hyperbaric \noxygen for three 30-minute periods with a five-minute air \nbreak between periods. The procedure used 100% of \noxygen at a flow rate of 8 -10 l/min. The hyperbaric \noxygen procedure w as conducted once a day for ten \ndays.[8] \n \n2.4 Histopathology examination \n \nLaparotomy was performed to collect the mice's \nendometriosis tissue. The tissue was then placed on a plate \ncontaining phosphate -buffered saline (PBS), fixed and \ndehydrated in acetone at a temperature of -200C for 20 \nhours. The tissue was purified with methyl benzoate and \nxylene and was impregnated with paraffin. The paraffin \nblock was then stored at a temperature of 40C. Each of the \nblocks was sliced into 100 μm-thick slices.[11] \n \n2.5 Determination of HIF-1 alpha expression \n \nThe determination of HIF -1 Alpha was conducted with \nimmunohistochemistry staining by using an anti -HIF-1 \nalpha antibody from Abcam (ab16066). The examination \nprocedure of HIF -1 Alpha expression was conducted  by \nthe standard listed on the kit. HIF-1 alpha expression was \nmeasured semiquantitatively with the modified Remmele \nmethod, where Immuno  Reactive Score (IRS) was the \nresult of multiplication between immunoreactive cell \npercentage score and immunoreactive cell color intensity. \nDatum of each sample was the IRS score observed on five \ndifferent viewing fields with 1000 -times \nmagnification.[12] \n \n2.6 Measurement of endometriosis tissue size \n \nMeasurement of endometriosis tissue size was conducted \nby using Image Ruster 2 software with 1-mm precision. \n \n2.7 Data analysis \n \nData are presented as mean ± standard deviation and the \ndifferences between gr oups were analyzed by using the \nANOVA test followed by LSD as a post hock test.  \n \n3 Results and Discussion \n3.1 Results \n \n3.1.1 HIF-1 alpha expression \n \nThe lowest HIF-1 alpha expression was found on the mice \ngroup that received hyperbaric oxygen. Visual description \nof the HIF-1 alpha expression is described in Figure 1. \n \n \nFigure 1. Comparison of inflammatory area HIF -1 alpha \nexpression among treatments. Positive immunoreactive cells are \nshown by inflammatory cells, endothelial cells, and fibroblasts \n(arrows). It is evident in this study that HIF expression \n(chromogen brown color) of the group that received hyperbaric \noxygen therapy (B) is much lower than that of the pretest control \ngroup (A) and the group that did not received hyperbaric oxygen \ntherapy (C) (Immunohistochemistry staining, 1000 times \nmagnification; Nikon H600L microscope; 300 megapixels DS \nFi2 camera).   \n \nA significant HIF-1 alpha expression difference was \nfound between the three groups (p= 0.000). The difference \nwas found between Group 1 and Group 2 (p= 0.000) and \nbetween Group 2 and Group 3 (p= 0.036). The HIF -1 \nalpha expression comparison between Groups is \ndescribed in the following table and graph. \n \nTable 1. Expression HIF-1 alpha comparison between group \nGroup HIF-1 alpha expression p \n1 8,25 ± 1,25  \n \n0,000 2 1,60 ± 1,43 \n3 3,42 ± 0,98 \n \n3.1.2 Endometriosis tissue size \n \nAll mice on all groups were healthy on the day after the \nmodeling process is completed. Surgery was conducted \non mice in Group 1 on the day after the modeling process \nis completed. All mice in Group 1 developed \nendometriosis with the appearance of plaque and hyper \nvascularization. The result suggested that the \nendometriosis modeling process was carried out \nsuccessfully and other groups could p roceed with the \nstudy. \nSurgery was conducted on mice in Group 2 and 3 on \nthe eleventh day after the surgery on Group 1. \nEndometriosis tissue size of Group 2 was found smaller \nthan that of Group 1 and 3 as seen in figure 1.  \n \n2\nE3S Web of Conferences 151, 01003 (2020) https://doi.org/10.1051/e3sconf/202015101003\n1st ICVAES 2019\n\n \n \n \n \n \nFigure 2. Peritoneun of endometriosis mice. Endometriosis \nlesions were found in the form of plaque growth (yellow arrows) \nand hypervascularization (blue arrows). There was a decrease in \nthe area of endometriosis lesions in the group given HBO (B) \ncompared to the pretest control group (A) and the group that did \nnot receive hyperbaric oxygen (C) therapy. \n \nA significant endometriosis tissue size difference \nwas found among the three groups (p= 0.002). the \ndifference was found between Group 1 and Group 2 (p= \n0.013) and between Group 2 and Group 3 (p= 0.027). The \nendometriosis tissue size comparison between Gro ups is \ndescribed in the following table and graph. \n \nTable 2. Endometriosis tissue size comparison between groups. \nGroup Endometriosis Tissue Size \n(mm2) \np \n1 207,09 ± 91,25  \n \n0,002 2 49,36 ± 48,94 \n3 209,53 ± 190,44 \nA significant difference was found between mice \nwith HBO and those without HBO. Endometriosis tissue \nsize on Group 2 was nearly ten times smaller than that of \nGroup 1 and was five times smaller than that of Group 3. \n3.2 Discussion \n \nThe pretest control group (Group 1) is the Group that was \ndesigned to identify the endometriosis tissue before HBO \ntherapy. HIF-1 alpha expression was found highest in this \ngroup. This finding indicates that endometriosis develops \nin hypoxic conditions. This conditio n was predicted to \ninduce a series of regulations that would allow the \nendometriosis tissue to continue its development.  \nExpression of HIF-1 alpha on the group that \nreceived HBO therapy on 30 -minutes 2.4 ATA dose 3 \ntimes daily for 10 days was found signif icantly lower. \nThis finding indicates that HBO therapy on 30 -minutes \n2.4 ATA dose 3 times daily for 10 days increases cellular \noxygenation which corrects the tissue hypoxic condition. \nThe increase in oxygen level automatically develops \nnormoxic – or even h yperoxic – condition. Under the \nnormoxic and hyperoxic conditions, HIF -1 will be \nhydroxylated on specific prolyl residual due to prolyl \nhydroxylase (PHD) enzyme activation. Under this \ncondition, von Hippel Lindau protein (pVHL) will form a \ncomplex that wil l activate E3 ubiquitin ligation that will \nform ubiquitinylate HIF-1 alpha; this process degrades the \nHIF-1 alpha.[8]  \nThe mice group that did not receive HBO therapy \n(Group 3) was treated in a chamber filled with free air \nwithout extra pressure. HIF -1 alp ha expression on this \ngroup was found lower than that of the pretest control \ngroup; however, it was higher than that of Group 2. The \npossible cause of this finding was the endometriosis tissue \ndevelopment that had to reach its optimum level in 14 \ndays afte r modeling (maturation stage). As it has been \nknown, the endometriosis tissue did not spontaneously \ndevelop in the modeled mice; instead, the development \nwas stimulated by implantation followed by hormonal and \nimmunosuppressant administration. When the mod eling \nprocess was estimated to be complete on the fourteenth \nday, Group 3 no longer had the triggering factor needed \nfor the development of endometriosis tissue.  This \nsituation caused the absence of a growth signal which \nleads to HIF -1 alpha not be expres sed. This finding \nconfirms the result of a previous study that found a lower \nNFkβ expression on the HBO therapy group compared \nwith the control group. The decrease of NFkβ expression \nis estimated to cause the absence of growth signal which \nleads to the dec rease of HIF -1 alpha expression. This \nhappens because HIF -1 alpha regulation is strongly \ninfluenced by NFkβ activity.[13]    \nThe presence of HIF -1 alpha is estimated to \ninfluence the increase of endometriosis tissue size. This is \nproved by the decrease of endometriosis tissue size due to \nthe decrease of HIF -1 alpha expression. This is because \nHIF-1 alpha has an important role in various mechanisms \nthat form the endometriosis tissue. The decrease of HIF-1 \nalpha expression will activate DUSP -2 which inhibits \nERK phosphorylation; this will prevent the formation of \nthe COX -2 enzyme which would be necessary for the \nformation of prostaglandin E2 (PGE2) .[14,15] As a result, \nthe modulation of endometriosis tissue development \nprocesses such as steroidogenesis, cell p roliferation, \nangiogenesis, and immune cell suppression does not \noccur. Moreover, the low PGE2 level will prevent the \nfeedback loop effect on HIF-1 alpha.[2] \nAccording to the discussion above, it can be \ndeduced that HBO has an important role in decreasing or \nin increasing the HIF -1 alpha expression. HBO dose of \n30-minutes 2.4 ATA 3 times daily for 10 days  was \nsignificantly effective in decreasing HIF -1 alpha \nexpression on endometriosis tissue of model mice. The \ndecrease of HIF-1 alpha expression is a strategic condition \nin the management of endometriosis. This confirms \nseveral previous studies that found that HIF -1 alpha was \nsubcellular protein necessary for cell proliferation on \nendometriosis tissue. \n \n4 Conclusion \n \nHyperbaric Oxygen (HBO) dose of 30-minutes 2.4 ATA \n3 times daily for 10 days  can decrease endometri osis \ntissue size by decreasing HIF-1 alpha expression. \n \nAcknowledgments \n \nThe authors would like to thank Balai Penelitian dan \nPengembangan Kesehatan (BALITBANGKES) \nDepartemen Kesehatan RI  for financial support and  \nLembaga Kesehatan Kelautan (LAKESLA) RSAL \n3\nE3S Web of Conferences 151, 01003 (2020) https://doi.org/10.1051/e3sconf/202015101003\n1st ICVAES 2019\n\n \n \nRamelan Surabaya for the opportunity to use the animal \nhyperbaric chamber. The authors would also like to thank \nMrs. Widjiati, DVM, M.Sc, PhD, Mr. Djoko Legowo \nDVM, M.Sc and team for the technical support on this \nstudy. \n \nReferences \n \n1. Tariverdian N, Theoharides TC, Siedentopf F,  et al .: \nNeuroendocrine- Immune Disequilibrium and \nEndometriosis: an Interdisciplinary Approach.  Semin \nImmunopathol. 2007; 29: 193-210. \n2. Hsiao KY, Lin SC,Wu MH, Tsai SJ:  Pathological \nfunctions of hypoxia in endometriosis .  Frontiers in \nBioscience, Elite. 2015; 7 : 352-366. \n3. Ren X, He YL, Pan SL et al . :Expression of hypoxia -\ninducible factor -1alpha in endometriosis . Nan Fang Yi \nKe Da Xue Xue Bao. 2007; 27(4):538-540. \n4. Wu MH, Chen KF, Lin SC, et al. : Aberrant expression \nof leptin in human endometriotic stromal cells is \ninduced by elevated levels of hypoxia inducible factor-1 \nalpha. Am J Pathol. 2007; 170(2): 590-598. \n5. Wu MH, Lu CW, Chang FM, et al. : Estrogen receptor \nexpression affected by hypoxia inducible factor -1α in \nstromal cells from patients with endometriosis . Taiwan \nJ Obstet Gynecol. 2012; 51(1): 50-54. \n6. Goteri G, Lucarini G, Zizzi A, et al .: Proangiogenetic \nmolecules, hypoxia -inducible factor -1alpha and nitric \noxide synthase isoforms in ovarian endometriotic cysts. \nVirchows Arch. 2010; 456(6):703-710. \n7. Imesch P, Samartzis EP, Schneider M, et al. : Inhibition of \ntranscription, expression, and secretion of the vascular \nepithelial growth factor in human epithelial \nendometriotic cells by romidepsin.  Fertil Steril .  2011 ; \n95(5):1579-1583. \n8. Mulawarmanti D : Profil cAMP, NFkB, iNOS, dan \nMMP3 Jaringan Period ontal Tikus Winstar Pada \nKondisi Hiperglikemia yang Terpapar Hiperoksia \nHiperbarik. Dissertation. Program Pasca Sarjana \nUniversitas Airlangga Surabaya. 2008. \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n9. Vika SP, Hendarto H, Suhartono DS, et al . : Pengaruh \nPemberian Siklosporin A Sebagai Penurun Jumlah \nLimfosit serum Terhadap Terjadinya Implan \nEndometriosis Pada Mencit.  Available in Pengaruh \nSuplementasi Kurkumin Terhadap Maturasit Oosit dan \nViabilitas Embrio Hasil Fertilisasi In Vitro Pada Mencit \nModel Endometriosis. Laporan Penelitian Un ggulan \nStrategis Nasional. 2010; 18. \n10. Hendarto H, Widjiati, Sudiana IK, et al . : Khasiat \nkurkumin terhadap Perubahan ekspresi VEGF, GDF -\n9, Hyaluronan untuk Perbaikan Terapi dan Hasil \nFertilisasi Pada Endometriosis.  Laporan Penelitian \nUnggulan Strategis Nasional. 2009; 17.  \n11. Sudiana IK  : Teknologi Ilmu Jaringan dan \nImunohistokimia Ed.1. Sagung Seto. 2005; 1-51. \n12. Novak M, Madej JA, Dziegeil P: Intensity of Cox 2 \nexpression in Cell of Soft Tissue Fibrosarcomas in Dog \nAs Related to Grade of Tumor malignation. Bull Vet inst \nPulawy. 2007; 51: 275-279.  \n13. Jung YJ, Isaacs JS, Lee S, et al. : IL-1beta-mediated up-\nregulation of HIF -1alpha via an NFkappaB/COX -2 \npathway identifies HIF -1 as a critical link between \ninflammation and oncogenesis . FASEB J . \n2003 ;17(14):2115-2117. \n14. Kaidi A, Qualtrough D,  Williams AC, et al . : Direct \nTranscriptional Up-regulation of Cyclooxygenase -2 by \nHypoxia-Inducible Factor (HIF)-1 Promotes Colorectal \nTumor Cell Survival and Enhances HIF -1 \nTranscriptional Activity during Hypoxia . Cancer Res . \n2006 ; 66 (13): 6683-6692. \n15. Wu MH, Lin SC, Hsiao KY  :  Hypoxia-inhibited dual - \nspecificity phosphatase -2 expression in endometriotic \ncells regulates cyclooxygenase -2 expression . J Pathol . \n2011; 225(3):390-400. \n4\nE3S Web of Conferences 151, 01003 (2020) https://doi.org/10.1051/e3sconf/202015101003\n1st ICVAES 2019","source_license":"CC0","license_restricted":false}