{"paper_id":"18c5fb67-f461-4a65-afac-9f6bbd0842e7","body_text":"Journal of International Dental and Medical Research ISSN 1309-100X   H.Polyrhizus Peel Ethanol Extract toward Endometriosis  \nhttp://www.jidmr.com                                                                                                                                        Anindya Hapsari, and et al  \n  \n \n \n  Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017  \n \n \nPage 1070  \n \nHylocereus Polyrhizus Peel Ethanol Extract- the Potential Effect to Tumor Necrosis Factor-Α,  \nMacrophage, and Matrix Metalloproteinase-9 in Endometriosis Mice  \n  \nAnindya Hapsari1, Hendy Hendarto2, Widjiati3*  \n  \n1. Faculty of Sport Science. Universitas Negeri Malang,Malang, Indonesia and Posgraduate student of Reproductive Health Science, \nFaculty of Medicine Universitas Airlangga Surabaya-Indonesia.  \n2. Department of Obstetry and Gynecology, Faculty of Medicine Universitas Airlangga. Surabaya-Indonesia.  \n3. Department of Embriology, Faculty of Veterinary Medicine Universitas Airlangga. Surabaya-Indonesia.  \n  \nAbstract  \n      On endometriosis, macrophage and TNF -α was found in higher concentration. Tumor Necrosis \nFactor-α activates NF-κB pathway and increase the expression of MMP-9’s gen. NF-κB pathway can \nbe blocked by Hylocereus polyrhizus peel.   \n      The objective of this study was to know the effect of Hylocereus polyrhizus peel ethanol extract \nat dose 0,25; 0,5 mg/gram BW/day; and 1 mg/gram on TNF-α concentration, number of macrophage, \nand MMP-9 expression on mouse model of endometriosis.   \n      This study was a laboratory experimental research. Thirty female mice were used as samples and \ndivided into 5 groups: 1 positive control, 1 negative control, and 3 treatment groups. Positive control \nand treatment groups were induced as model of endometrios is for 14 days. The next 14 days, Na -\nCMC 0,5% was given to both control groups, while Hylocereus polyrhizus peel ethanol extract dose \n0,25; 0,5 mg/gram BW/day; and 1 mg/gram were given to treatment groups orally. Peritoneum fluid \nand endometriosis lesion were examined.   \n      There were significantly differences on TNF -α concentration (p=0.021), number of macrophage \n(p=0.00), and MMP -9 expression (p=0.002) among control groups and treatment groups. TNF -α \nconcentrations were higher on treatment groups.   \n      Hylocereus polyrhizus peel ethanol extract can be used as alternative therapy for attenuating the \nalteration of macrophage and MMP-9 in endometriosis disease.  \nExperimental article (J Int Dent Med Res 2017; 10(3): pp. 1070-1073)  \n      Keywords: Hylocereus polyrhizus peel ethanol extract, endometriosis, TNF -α, macrophage, \nMMP-9.  \n      Received date: 05 August 2017                                         Accept date:  11 September 2017       \n  \n  Introduction  \n  \nEndometriosis is defined as the presence \nof endometriosis -like tissue outside the uterus, \nwhich induces a chronic inflammatory reaction. 1 \nThe prevalence of endometriosis in the population \nis difficult to determine but estimates between 2 to \n10% within the female population while 50% in \ninfertile women. Most women with endometriosis \nexperience painful symptoms thus infertility. 2 \nManagement of endometriosis can be divided into \nempirical treatments and surgery. However, the \nrecurrence rate after treatment is  \nhigher, about 35% in mild endometriosis and 74% \nin severe endometriosis. 3 The high recurrence \nrate due to endometriosis is a progressive disease \nand needed long-time treatment.4  \n  \nMaterials and methods  \n  \nThis study has been received approval \nethical clea rance letter of animal subjects from \nFaculty of Veterinary Medicine Universitas \nAirlangga with number 710 -KE. This study was a \nlaboratory experimental research. Thirty female  \nmice (Mus musculus), aged 2-3 months, weighing \n20-25 gram, were used as samples.  \n\n  \nJournal of International Dental and Medical Research ISSN 1309-100X   H.Polyrhizus Peel Ethanol Extract toward Endometriosis  \nhttp://www.jidmr.com                                                                                                                                        Anindya Hapsari, and et al  \n  \n \n \n  Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017  \n \n \nPage 1071  \n \n*Corresponding author:  \nDr. Widjiati, M.Sc., DVM.,  This study was conducted in Faculty of Department of Embriology, Faculty of Veterinary Medicine, \nVeterinary Medicine Universitas Airlangga from Universitas Airlangga, Surabaya. Indonesia.  June to July 2017.    \nE-mail:  widjiati@fkh.unair.ac.id; anindya.hapsari.fik@um.ac.id  \n  After adaptation for a week, female mice then \ndivided into 5 groups, which are: positive  \ncontrol group (K1), negative control group (K2), \ntreatment group with Hylocereus polyrhizus  peel \nethanol extract dose 0,25 mg/gram  BW/day (K3), \ntreatment group with Hylocereus polyrhizus  peel \nethanol extract dose 0,5 mg/gram  BW/day (K4), \nand treatment group with Hylocereus polyrhizus  \npeel ethanol extract dose 1 mg/gram/day (K5). \nPositive contro l and treatment groups were \ninduced as model of endometriosis by this \nfollowing steps: 1) injection of cyclosporin A (0,2 \nml/mice) intramuscular on day one to make mice \nin immunodefficiency state. Cyclosporin A was \npurchased from Sandimmun (North Ryde,  \nAustralia); 2) injection of ethinyl estradiol 20.000 \nIU (0,1 ml/mice) on day one and five; 3) injection \nof implant tissue (0,1 ml/mice) in the peritoneal \ncavity on day one. Implant tissue is derived from \nthe myometrium and endometrium of gynecologic \nbenign t umor patient who underwent surgery \nprocedure and didn’t use hormonal contraception \nat least for last 3 months before surgery. The \nanimal model was observed for 14 days to be the \nmice model of endometriosis.  \nStarting from 15 th day, Na -CMC 0,5% \n(Clorogreen Gemilang™, Bandung, Indonesia) \nwas given to both control groups as placebo. \nHylocereus polyrhizus peel ethanol extract dose \n0,25 mg/gram  BW/day was given to group K3, \nHylocereus polyrhizus peel ethanol extract dose \n0,5 mg/gram  BW/day was given to group K4, and \nHylocereus polyrhizus peel ethanol extract dose 1 \nmg/gram/day was given to group K5. These \nplacebo and extract was administered orally (0,2 \nml/25 mg/gram  BW/day) for 14 days with an oral \ngavage.  \nAt the end of experiment, mice were \nanesthetized with ketamin (Ketamin Hydrochloride \nPfizer®, New Jersey, USA) and acepromazine \n(Castran®,Venray, Holland). Peritoneal fluid \nsamples were obtained by peritoneum puncture to \nexamine TNF -α concentration using Enzyme \nLinked Immunosorbent Assay kit (Elabscience™, \nWuhan, China), then endometriosis lesions in \nperitoneum cavity were collected. Formalin -fixed, \nparaffin-embedded tissue sections from \nendometriosis lesion were tested by \nimmunohistochemistry to see MMP-9 expressions \n(Bioss Antibody Incorporation™, Massachu ssets, \nUSA).  The other tissue sections from \nendometriosis lesion were tested by Hematoxillin \nEosin (HE) staining (DAKO™, California,USA) to \ncount macrophages on the lesions. The number of \nmacrophages from 5 field -view then categorized \nby Klopfleisch scoring system. This score criterias \nare: 0 if there was no macrophage infiltration, 1 if \nthere were less than 10 macrophage infiltration, 2 \nif there were 11 -50 macrophage infiltration, 3 if \nthere were 51-100 macrophage infiltration, and 4 if \nthere were more th an 100 macrophage \ninfiltration.13  \nMatrix Metalloproteinase -9 expressions \nwere analyzed using modified Remmele and \nStegnes semiquantitative scoring system (1986). \nThis Immuno Reactive Score (IRS) is a result from \nmultiplication between immunoreactive cells  \nprocentage (A) and colour intensity score on \nimmunoreactive cells (B). Datas were collected \nfrom 5 field-view. The immunoreactive cells score \nciterias (A) are: 0 if there was no positive cell, 1 if \nprocentage of positive cells was less than 10%, 2 \nif proc entage of positive cells was 11 -50%, 3 if \nprocentage of positive cells was 51 -80%, and 4 if \nprocentage of positive cells was more than 80%. \nWhile colour intensity score (B) criterias are: 0 if \nthere was no colour reaction, 1 if the intensity of \ncolour is low, 2 if the intensity of colour is average, \nand 3 if the intensity of colour is high.14   \nShapiro Wilk test was used to know the \nnormality of data and Levene test was used to \nknow the homogenity of data. If distribution and \nhomogenity of data were normal (p>0.05), One \nWay Anova test was conducted, followed with \nposthoc Bonferroni, but, if the data distribution or \nhomogenity wasn’t normal, Kruskall -Wallis test \nfollowed with Mann Whitney test, were conducted. \nAnalysis was performed with IBM SPSS  \nStatistics versions 24.00 (New York, USA).  \n  \n\n  \nJournal of International Dental and Medical Research ISSN 1309-100X   H.Polyrhizus Peel Ethanol Extract toward Endometriosis  \nhttp://www.jidmr.com                                                                                                                                        Anindya Hapsari, and et al  \n  \n \n \n  Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017  \n \n \nPage 1072  \n \nResults  \n  \nResult showed that there were  mean \ndifferences among K1, K2, K3, K4, and K5 groups, \nas seen on table 1.   \n  \n  \nTable 1.  TNF-α Concentration on Mice Model \nEndometriosis AmongzControl Groups and \nTreatment Groups.  \n  \nThe level of TNF -α concentration was \nhigher in the endometriosis model groups (K2, K3, \nK4, and K5) compared to the negative control \ngroup (K1). All doses of Hylocereus polyrhizus  \npeel ethanol extract also have higher level of TNF-\nα concentration compared to K1.  \nStatistical analysis was conducted by One \nWay Anova test with p=0.021, that means there \nwas significantly differences among groups. \nPosthoc Bonferroni test showed that there were \nsignificantly differences between K1 and K4.  \nResult showed that there were  mean \ndifferences among K1, K2, K3, K4, and K5 groups, \nas seen on table 2. The numbers of macrophage \nwere decreased on treatment groups. This \nreduction was linear with the increasing of \nHylocereus polyrhizus peel ethanol extract doses. \nStatistical analysis was conducted by Kruskall \nWallis test with p=0.000, that means there was at \nleast 1 significantly differences between 2 groups. \nMann Whitney test showed that there were \nsignificantly differences between: K1 and K2, K1 \nand K3, K1 and K4, K2 and K3, K2 and K4, K2 and \nK5, and K3 and K5.  \n  \n  \nTable 2. Number of Macrophage on Mice Model \nEndometriosis Among Control Groups and \nTreatment Groups.  \n  \n  \nTable 3.  Matrix Metalloproteinase -9 on Mice \nModel Endometriosis Among Control Groups and \nTreatment Groups.  \n  \n  \nFigure 1.  The macrophage inflitration. Massive \nmacrophage infiltration in positive control group \n(K2) compared with another groups (Hematoxillin \nEosin, magnification 400x, Nikon H600L \nmicroscope from Nikon Instrumen Inc™, New \nYork, USA).  \n  \n\n\n  \nJournal of International Dental and Medical Research ISSN 1309-100X   H.Polyrhizus Peel Ethanol Extract toward Endometriosis  \nhttp://www.jidmr.com                                                                                                                                        Anindya Hapsari, and et al  \n  \n \n \n  Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017  \n \n \nPage 1073  \n \n  \nFigure 2.  The MMP -9 expression (blue arrow). \nMMP-9 expression in positive control group (K2) \nwas the strongest among another groups \n(immunohitochemistry, magnification 1000x, \nNikon H600L microscope from Nikon Instrumen \nInc™, New York, USA).   \n  \nResult showed th at there were  mean \ndifferences among K1, K2, K3, K4, and K5 groups, \nas seen on table 3, figure 1, 2.   \nThe MMP -9 expressions were decreased \non treatment groups. This reduction was linear \nwith the increasing of Hylocereus polyrhizus peel \nethanol extract’s d oses. Statistical analysis was \nconducted by Kruskall Wallis test with p=0.002, \nthat means there was at least 1 significantly \ndifferences between 2 groups. Mann Whitney test \nshowed that there were significantly differences \nbetween: K1 and K2, K1 and K3, K2 and K3, K2 \nand K4, K2 and K5, and K3 and K4.  \n  \nDiscussion   \n  \nThe result of this study showed that \nHylocereus polyrhizus  peel ethanol extract was \nable to reduce the number of macrophage and \nMMP-9 expression on mice model endometriosis.  \nBut, the extract wasn’t able to reduce TNF -α \nconcentration.10 Hylocereus polyrhizus  peel \nethanol extract which contain betalain, were given \nto mice model endometriosis to inhibit NFκB \npathway, which is activated by macrophage and \nendometriosis cells when t hese cells bind to their \nreceptors.15,16  \nBy blocking NF -κB pathway, it was \nsupposed that the genes expression of TNF -α, \nMMP-9, and MCP -1 reduced. This study result \npresented that the secretion of TNF -α by \nmacrophage wasn’t reduced. This may because \nthe production of TNF-α by macrophage isn’t only \nthrough NF -κB pathway 16,17. There is possibility \nthat TNF -α can be produced through MAPK \n(mitogen-activated protein kinase) pathway. 19,20 \nSo that, although the extract inhibited NF -κB \npathway, the production of TN Fα was still high \nthrough MAPK pathway.   \nThis study also presented that MMP-9 and \nnumber of macrophage were significantly greater \nin the positive control group compared to other \ngroups. These increased levels of MMP -9 in mice \nmodel endometriosis were significantly reduced by \n0,5 mg/gram  BW/day and 1 mg/gram \nadministration of Hylocereus polyrhizus  peel \nethanol extract to those comparable to the \nnegative control group. Administration of  \nHylocereus polyrhizus peel ethanol extract dose 1 \nmg/gram also can red uce the number of \nmacrophage to those comparable to the negative \ncontrol group.  \n  \nConclusions   \n  \nThe  conclusion  of  this  study \n was Hylocereus polyrhizus peel ethanol extract \ncan be used as alternative therapy for attenuating \nthe alteration  of  macrophage  and \n MMP-9  in endometriosis disease.  \n  \nAcknowledgements   \n  \nThe authors wish to thank Djoko Legowo, \nDVM, M.Sc for help with the interpretation of \nhistology.  \n Declaration of \nInterest   \n  \nThe authors declare no conflict of interest.  \n    \n  References  \n  \n1. Management of Women with Endometriosis Guideline of the \nEuropean Society of Human Reproduction and Embriology.  \nEuropean Society of Human Reproduction and Embriology. \nESHRE Endometriosis Guideline Development Group. 2013.  \n2. Hendarto H. Endometriosis From Theory Aspects To Clinical \nManagement. Surabaya:Airlangga University Press. 2015.  \n3. Speroff L, Fritz MA. Endometriosis in Clinical Gynecologic \nEndocrinology and Infertility 7 th Edition. Philadelphia:Lippincott \nWilliam and Wilkins. 2005.  \n4. Annas JY, Hendarto H, Widjiati. The Effect of Curcumin in Various \nDoses to Endometriosis Progressivity in Mice Model \nEndometrios. Obstetry and Gynaecologic Magazine.  \n2014:22(3):118-8.  \n5. Oepomo TD. Tumor Necrosis Factor -α Concentration on \nPeritoneal Fluid of Endometriosis Patients. Biotechnology. \n2006;2(2):54-4.  \n6. 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