Hylocereus Polyrhizus Peel Ethanol Extract - The Potential Effect to Tumor Necrosis Factor - A, Macrophage, and Matrix Metalloproteinase - 9 in Endometriosis Mice

On endometriosis, macrophage and TNF -α was found in higher concentration. Tumor Necrosis Factor-α activates NF-κB pathway and increase the expression of MMP-9’s gen. NF-κB pathway can be blocked by Hylocereus polyrhizus peel. The objective of this study was to know the effect of Hylocereus polyrhizus peel ethanol extract at dose 0,25; 0,5 mg/gram BW/day; and 1 mg/gram on TNF-α concentration, number of macrophage, and MMP-9 expression on mouse model of endometriosis. This study was a laboratory experimental research. Thirty female mice were used as samples and divided into 5 groups: 1 positive control, 1 negative control, and 3 treatment groups. Positive control and treatment groups were induced as model of endometrios is for 14 days. The next 14 days, Na - CMC 0,5% was given to both control groups, while Hylocereus polyrhizus peel ethanol extract dose 0,25; 0,5 mg/gram BW/day; and 1 mg/gram were given to treatment groups orally. Peritoneum fluid and endometriosis lesion were examined. There were significantly differences on TNF -α concentration (p=0.021), number of macrophage (p=0.00), and MMP -9 expression (p=0.002) among control groups and treatment groups. TNF -α concentrations were higher on treatment groups. Hylocereus polyrhizus peel ethanol extract can be used as alternative therapy for attenuating the alteration of macrophage and MMP-9 in endometriosis disease. Experimental article (J Int Dent Med Res 2017; 10(3): pp. 1070-1073)

Hylocereus polyrhizus peel ethanol extract, endometriosis, TNF -α, macrophage, MMP-9. Received date: 05 August 2017 Accept date: 11 September 2017

Endometriosis is defined as the presence of endometriosis -like tissue outside the uterus, which induces a chronic inflammatory reaction. 1 The prevalence of endometriosis in the population is difficult to determine but estimates between 2 to 10% within the female population while 50% in infertile women. Most women with endometriosis experience painful symptoms thus infertility. 2 Management of endometriosis can be divided into empirical treatments and surgery. However, the recurrence rate after treatment is higher, about 35% in mild endometriosis and 74% in severe endometriosis. 3 The high recurrence rate due to endometriosis is a progressive disease and needed long-time treatment.4

This study has been received approval ethical clea rance letter of animal subjects from Faculty of Veterinary Medicine Universitas Airlangga with number 710 -KE. This study was a laboratory experimental research. Thirty female mice (Mus musculus), aged 2-3 months, weighing 20-25 gram, were used as samples. Journal of International Dental and Medical Research ISSN 1309-100X H.Polyrhizus Peel Ethanol Extract toward Endometriosis http://www.jidmr.com Anindya Hapsari, and et al Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017 Page 1071 *Corresponding author: Dr. Widjiati, M.Sc., DVM., This study was conducted in Faculty of Department of Embriology, Faculty of Veterinary Medicine, Veterinary Medicine Universitas Airlangga from Universitas Airlangga, Surabaya. Indonesia. June to July 2017. E-mail: [email protected]; [email protected] After adaptation for a week, female mice then divided into 5 groups, which are: positive control group (K1), negative control group (K2), treatment group with Hylocereus polyrhizus peel ethanol extract dose 0,25 mg/gram BW/day (K3), treatment group with Hylocereus polyrhizus peel ethanol extract dose 0,5 mg/gram BW/day (K4), and treatment group with Hylocereus polyrhizus peel ethanol extract dose 1 mg/gram/day (K5). Positive contro l and treatment groups were induced as model of endometriosis by this following steps: 1) injection of cyclosporin A (0,2 ml/mice) intramuscular on day one to make mice in immunodefficiency state. Cyclosporin A was purchased from Sandimmun (North Ryde, Australia); 2) injection of ethinyl estradiol 20.000 IU (0,1 ml/mice) on day one and five; 3) injection of implant tissue (0,1 ml/mice) in the peritoneal cavity on day one. Implant tissue is derived from the myometrium and endometrium of gynecologic benign t umor patient who underwent surgery procedure and didn’t use hormonal contraception at least for last 3 months before surgery. The animal model was observed for 14 days to be the mice model of endometriosis. Starting from 15 th day, Na -CMC 0,5% (Clorogreen Gemilang™, Bandung, Indonesia) was given to both control groups as placebo. Hylocereus polyrhizus peel ethanol extract dose 0,25 mg/gram BW/day was given to group K3, Hylocereus polyrhizus peel ethanol extract dose 0,5 mg/gram BW/day was given to group K4, and Hylocereus polyrhizus peel ethanol extract dose 1 mg/gram/day was given to group K5. These placebo and extract was administered orally (0,2 ml/25 mg/gram BW/day) for 14 days with an oral gavage. At the end of experiment, mice were anesthetized with ketamin (Ketamin Hydrochloride Pfizer®, New Jersey, USA) and acepromazine (Castran®,Venray, Holland). Peritoneal fluid samples were obtained by peritoneum puncture to examine TNF -α concentration using Enzyme Linked Immunosorbent Assay kit (Elabscience™, Wuhan, China), then endometriosis lesions in peritoneum cavity were collected. Formalin -fixed, paraffin-embedded tissue sections from endometriosis lesion were tested by immunohistochemistry to see MMP-9 expressions (Bioss Antibody Incorporation™, Massachu ssets, USA). The other tissue sections from endometriosis lesion were tested by Hematoxillin Eosin (HE) staining (DAKO™, California,USA) to count macrophages on the lesions. The number of macrophages from 5 field -view then categorized by Klopfleisch scoring system. This score criterias are: 0 if there was no macrophage infiltration, 1 if there were less than 10 macrophage infiltration, 2 if there were 11 -50 macrophage infiltration, 3 if there were 51-100 macrophage infiltration, and 4 if there were more th an 100 macrophage infiltration.13 Matrix Metalloproteinase -9 expressions were analyzed using modified Remmele and Stegnes semiquantitative scoring system (1986). This Immuno Reactive Score (IRS) is a result from multiplication between immunoreactive cells procentage (A) and colour intensity score on immunoreactive cells (B). Datas were collected from 5 field-view. The immunoreactive cells score citerias (A) are: 0 if there was no positive cell, 1 if procentage of positive cells was less than 10%, 2 if proc entage of positive cells was 11 -50%, 3 if procentage of positive cells was 51 -80%, and 4 if procentage of positive cells was more than 80%. While colour intensity score (B) criterias are: 0 if there was no colour reaction, 1 if the intensity of colour is low, 2 if the intensity of colour is average, and 3 if the intensity of colour is high.14 Shapiro Wilk test was used to know the normality of data and Levene test was used to know the homogenity of data. If distribution and homogenity of data were normal (p>0.05), One Way Anova test was conducted, followed with posthoc Bonferroni, but, if the data distribution or homogenity wasn’t normal, Kruskall -Wallis test followed with Mann Whitney test, were conducted. Analysis was performed with IBM SPSS Statistics versions 24.00 (New York, USA). Journal of International Dental and Medical Research ISSN 1309-100X H.Polyrhizus Peel Ethanol Extract toward Endometriosis http://www.jidmr.com Anindya Hapsari, and et al Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017 Page 1072

Result showed that there were mean differences among K1, K2, K3, K4, and K5 groups, as seen on table 1. Table 1. TNF-α Concentration on Mice Model Endometriosis AmongzControl Groups and Treatment Groups. The level of TNF -α concentration was higher in the endometriosis model groups (K2, K3, K4, and K5) compared to the negative control group (K1). All doses of Hylocereus polyrhizus peel ethanol extract also have higher level of TNF- α concentration compared to K1. Statistical analysis was conducted by One Way Anova test with p=0.021, that means there was significantly differences among groups. Posthoc Bonferroni test showed that there were significantly differences between K1 and K4.

showed that there were mean differences among K1, K2, K3, K4, and K5 groups, as seen on table 2. The numbers of macrophage were decreased on treatment groups. This reduction was linear with the increasing of Hylocereus polyrhizus peel ethanol extract doses. Statistical analysis was conducted by Kruskall Wallis test with p=0.000, that means there was at least 1 significantly differences between 2 groups. Mann Whitney test showed that there were significantly differences between: K1 and K2, K1 and K3, K1 and K4, K2 and K3, K2 and K4, K2 and K5, and K3 and K5. Table 2. Number of Macrophage on Mice Model Endometriosis Among Control Groups and Treatment Groups. Table 3. Matrix Metalloproteinase -9 on Mice Model Endometriosis Among Control Groups and Treatment Groups. Figure 1. The macrophage inflitration. Massive macrophage infiltration in positive control group (K2) compared with another groups (Hematoxillin Eosin, magnification 400x, Nikon H600L microscope from Nikon Instrumen Inc™, New York, USA). Journal of International Dental and Medical Research ISSN 1309-100X H.Polyrhizus Peel Ethanol Extract toward Endometriosis http://www.jidmr.com Anindya Hapsari, and et al Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017 Page 1073 Figure 2. The MMP -9 expression (blue arrow). MMP-9 expression in positive control group (K2) was the strongest among another groups (immunohitochemistry, magnification 1000x, Nikon H600L microscope from Nikon Instrumen Inc™, New York, USA).

showed th at there were mean differences among K1, K2, K3, K4, and K5 groups, as seen on table 3, figure 1, 2. The MMP -9 expressions were decreased on treatment groups. This reduction was linear with the increasing of Hylocereus polyrhizus peel ethanol extract’s d oses. Statistical analysis was conducted by Kruskall Wallis test with p=0.002, that means there was at least 1 significantly differences between 2 groups. Mann Whitney test showed that there were significantly differences between: K1 and K2, K1 and K3, K2 and K3, K2 and K4, K2 and K5, and K3 and K4.

The result of this study showed that Hylocereus polyrhizus peel ethanol extract was able to reduce the number of macrophage and MMP-9 expression on mice model endometriosis. But, the extract wasn’t able to reduce TNF -α concentration.10 Hylocereus polyrhizus peel ethanol extract which contain betalain, were given to mice model endometriosis to inhibit NFκB pathway, which is activated by macrophage and endometriosis cells when t hese cells bind to their receptors.15,16 By blocking NF -κB pathway, it was supposed that the genes expression of TNF -α, MMP-9, and MCP -1 reduced. This study result presented that the secretion of TNF -α by macrophage wasn’t reduced. This may because the production of TNF-α by macrophage isn’t only through NF -κB pathway 16,17. There is possibility that TNF -α can be produced through MAPK (mitogen-activated protein kinase) pathway. 19,20 So that, although the extract inhibited NF -κB pathway, the production of TN Fα was still high through MAPK pathway. This study also presented that MMP-9 and number of macrophage were significantly greater in the positive control group compared to other groups. These increased levels of MMP -9 in mice model endometriosis were significantly reduced by 0,5 mg/gram BW/day and 1 mg/gram administration of Hylocereus polyrhizus peel ethanol extract to those comparable to the negative control group. Administration of Hylocereus polyrhizus peel ethanol extract dose 1 mg/gram also can red uce the number of macrophage to those comparable to the negative control group.

The conclusion of this study was Hylocereus polyrhizus peel ethanol extract can be used as alternative therapy for attenuating the alteration of macrophage and MMP-9 in endometriosis disease.

The authors wish to thank Djoko Legowo, DVM, M.Sc for help with the interpretation of histology. Declaration of Interest The authors declare no conflict of interest.

1. Management of Women with Endometriosis Guideline of the European Society of Human Reproduction and Embriology. European Society of Human Reproduction and Embriology. ESHRE Endometriosis Guideline Development Group. 2013. 2. Hendarto H. Endometriosis From Theory Aspects To Clinical Management. Surabaya:Airlangga University Press. 2015. 3. Speroff L, Fritz MA. Endometriosis in Clinical Gynecologic Endocrinology and Infertility 7 th Edition. Philadelphia:Lippincott William and Wilkins. 2005. 4. Annas JY, Hendarto H, Widjiati. The Effect of Curcumin in Various Doses to Endometriosis Progressivity in Mice Model Endometrios. Obstetry and Gynaecologic Magazine. 2014:22(3):118-8. 5. Oepomo TD. Tumor Necrosis Factor -α Concentration on Peritoneal Fluid of Endometriosis Patients. Biotechnology. 2006;2(2):54-4. 6. Yeniel AO, Erbas O, Ergenoglu, AM, “et al.” Effect Of Oxytocin Treatment On Explant Size, Plasma, and Peritoneal Levels of Journal of International Dental and Medical Research ISSN 1309-100X H.Polyrhizus Peel Ethanol Extract toward Endometriosis http://www.jidmr.com Anindya Hapsari, and et al Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017 Page 1074 MCP-1, VEGF, TNF -α, and Histopathological Parameters In A Rat Endometriosis Model. European Journal of Obstetrics and Gynecology and Reproductive Biology. 2014;175:134-6. 7. Grund EM, Kagan D, Tran CA, “et al.” Tumor Necrosis Factor -α Regulates Inflammatory and Mesenchymal Responses via Mitogen-Activated Protein Kinase, p38, and Nuclear Factor-κB in Human Endometriotic Epithelial Cells. Molecular Pharmacology. 2008;73(5):1394-11. 8. Liu H, Wang J, Wang H, “et al.” Correlation Between Matrix Metalloproteinase-9 and Endometriosi s. International Jornal Clinical Pathology. 2015;8(10):13399-6. 9. Gonzalez-Ramos R, Defrere S., Devoto L. Nuclear FactorKappaB: A Main Regulator Of Inflammation and Cell Survival In Endometriosis Pathophysiology. Fertility and Sterility. 2012;98(3):520-8. 10. Sarasmita MA, Laksmiani NPL. The Cytotoxicity Effect of Hylocereus polyrhizus Peel Ethanol Extract on Carcinoma Mammae Cells Invitro and Insilico. Udayana Pharmacy Journal. 2015;4(2):91-7. 11. Nurliyana SZ, Suleiman IMK, Aisyah MR, Rahim KR. Antioxydant Study Of Pulp And Peels Of Dragon Fruits : A Comparative Study. International Food Research Journal. 2010;17:367-9. 12. Wybraniec S, Nowak-Wydra B, Mitka K. Minor Betalain in Fruit of Hylocereus species. J Phytochem. 2007;68:251-9. 13. Klopfleisch R. Multiparametri c and semiquantitative scoring systems for the evaluation of mouse model histopathology - a systematic review. Veterinary Research. 2013;9:123. 14. Nowak M, Janusz M, Dziegiel P. Intensity of COX2 Expression in Cells of Soft Tissue Fibrosarcomas in Dogs as Re lated to Grade of Tumour Malignancy. Bull vet Ins Pulawy. 2007;5:275- 5. 15. Martinez RM, Longhi -Balbinot DT, Zarpelon AC, “et al.” Antiinflammatory Activity of Betalain -Rich Dye of Beta vulgaris: Effect on Edema, Leucocyte Recruitment, Superoxide Anion, and Cytokine Production. Archives of Pharmacal Research. 2014;38:494-11. 16. Capobianco A, Rovere -Querini P. Endometriosis, A Disease Of The Macrophage. Frontiers in Immunology. 2013;4(9):1-14. 17. Bruggen T, Nijenhuis S, Raaij E, Verhoef J, Sweder B, Asbeck. Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Monocytes Involves the Raf - 1/MEK1MEK2/ERK1-ERK2 Pathway. Infection and Immunity. 1999;67(8):3824-6. 18. Dumitru CD, Ceci JD, Tsatsanis C, “et al.” TNF -α Induction by LPS Is Regulated Posttranscriptionally via a Tpl2/ERKDependent Pathway. Cell. 2000;103:1071-13. 19. Setyadewi W., Oki A.S., Sunariani J. Moderate Intensity Physical Exercise Effect on PMN and Macrophage Expression in Rattus Norvegicus Post Tooth Extraction. Journal of International Dental and Medical Research. 2016;9(1):364-367. 20. Ismaili B., and Bokonjic D. Short-Term Low-Level Laser Therapy Attenuates Inflammation and Production of Interleukin1, but Elevates the Level of Matrix Metalloproteina se 9 in Chronic Periodontitis. Journal of International Dental and Medical Research. 2014;7(1)7-13.