|Year : 2018 | Volume
| Issue : 6 | Page : 261-265
Inhibitory effect of genistein on MMP-2 and MMP-9 expression through suppressing NF-κB activity in peritoneum of murine model of endometriosis
Dwi Yuliawati1, Karyono Mintaroem2, Sutrisno Sutrisno3
1 STIKES Karya Husada Kediri, East Java, Indonesia
2 Laboratory of Pathology, Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia
3 Division of Fertility, Endocrinology and Reproduction, Department of Obstetrics and Gynecology, Saiful Anwar Hospital, Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia
|Date of Submission||21-Jun-2018|
|Date of Decision||18-Jul-2018|
|Date of Acceptance||10-Sep-2018|
|Date of Web Publication||30-Nov-2018|
STIKES Karya Husada Kediri, East Java
Source of Support: None, Conflict of Interest: None
Objective: To analyze the inhibitory effect of genistein on matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) through inhibition of nuclear factor-kappa B (NF-κB) activation. Methods: A total of 36 female mice were divided into six groups (n=6 in each group): control group (untreated mice), endometriosis mice group, and endometriosis mice groups administered with genistein at different doses (1.30 mg/day; 1.95 mg/day; 2.60 mg/day; and 3.25 mg/day). The genistein treatment was performed for 14 d. The expressions of NF-κB, MMP-2 and MMP-9 on the endometriosis lesions were analyzed by the immunohistochemical technique. Results: The activity of NF-κB in the endometriosis group increased significantly than that of the control group (P<0.05). The expression of MMP-2 or MMP-9 in the endometriosis group increased significantly than that of the control group (P<0.05). Administration of genistein at different doses was capable of significantly reducing the activity of NF-κB compared to that of the endometriosis group (P<0.05), reaching the level comparable to that of the control group at the third highest dose (P>0.05). The four doses of genistein administration were capable of significantly reducing the expression of MMP-2 compared to that of the endometriosis group (P<0.05), reaching an expression comparable to that of the control group for the highest dose (P>0.05). Administration of genistein at different doses was capable of significantly reducing the expression of MMP-9 compared to that of the endometriosis group (P<0.05), reaching the level comparable to that of the control group at the highest dose (P>0.05). Conclusions: Genistein suppresses the expression of MMP-2 and MMP-9 through suppressing NF-κB activity in the peritoneum of murine model of endometriosis.
Keywords: Peritoneum, Metalloproteinase, Genistein, Endometriosis, Isoflavones
|How to cite this article:|
Yuliawati D, Mintaroem K, Sutrisno S. Inhibitory effect of genistein on MMP-2 and MMP-9 expression through suppressing NF-κB activity in peritoneum of murine model of endometriosis. Asian Pac J Reprod 2018;7:261-5
|How to cite this URL:|
Yuliawati D, Mintaroem K, Sutrisno S. Inhibitory effect of genistein on MMP-2 and MMP-9 expression through suppressing NF-κB activity in peritoneum of murine model of endometriosis. Asian Pac J Reprod [serial online] 2018 [cited 2022 Jan 26];7:261-5. Available from: http://www.apjr.net/text.asp?2018/7/6/261/246344
| 1. Introduction|| |
Endometriosis is a chronic and heterogeneous disease with the presence of the stroma and functional endometrial glands outside the uterine cavity, and it is often found in the peritoneum, ovary, or rectovaginal septum,. The etiology of most endometriosis is unknown. The incidence of endometriosis in women of reproductive age is 6%-11%, with the highest incidence at 25-35 years old,. The main symptoms of endometriosis are pelvic pain and infertility, but it is also asymptomatic.
Despite the histologically benign lesion, endometriosis lesions also exhibit malignant cancer behaviors, including local invasion and aggressive spread to distant organs. This behavior is due to the ability of endometriosis lesions to form an estrogenic environment for proliferation of cells and stimulation of proinflammatory cytokines,. Two important proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β, activate the transcription factors—nuclear factor-kappa B (NF-κB) and activator protein-1. Furthermore, these transcription factors induce gene transcription and code for products, and one of them is the matrix metalloproteinases (MMPs), including MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9. The MMPs trigger the degradation and remodeling of the extracellular matrix of peritoneal surfaces and other organs.
The growth and development of endometriosis require the activation of gelatinase-type MMPs (MMP-2/gelatinase A and MMP-9/ gelatinase B). Previous findings have demonstrated an increased expression and activity of MMP-2 and MMP-9 in the stages of organ development, and a variety of pathological conditions involving tissue remodeling. The expression of MMP-2 and MMP-9 is found to be higher in women with endometriosis than that in controls which modulates by the activation of NF-κB,,. The stimulator for this activity is tumor necrosis factor-α and interleukin-1β.
Currently, no drug successfully cures endometriosis completely and in some cases it causes side effects of long-term use. Certain drugs can even lead to delays in conception and have not been shown to have fertility effects after treatment. Therefore, an ideal drug is currently developed with the aim of regression of the disease and its symptoms without the negative effects associated with hypoestrogenic conditions. The drug is an anti-estrogenic selective estrogen receptor modulator.
Genistein is an isoflavone capable of acting as selective estrogen receptor modulators, which serves as a pure antagonist when acting via estrogen receptor alpha, but it can serve as a partial agonist when acting via estrogen receptor beta. With regard to the structural similarities of 17β-estradiol and genistein and to endometriosis with estrogenic environmental conditions to maintain cellular viability, genistein can be a pure antagonist. Previous studies have shown that genistein is capable of inhibiting the activation of NF-κB. Therefore, the purpose of the present study was to analyze the inhibitory effect of genistein on MMP-2 and MMP-9 through inhibition of NF-κB activation.
| 2. Materials and methods|| |
Mice (Mus musculus) were obtained from the Laboratory of Embryology, Faculty of Veterinary Medicine, Airlangga University (FKH Unair) Surabaya, East Java, Indonesia. Mice selected for the present study were healthy female mice, with 2-3 months old, 20-30 g in weight, having fine hairs and glowing eyes, and having no limp and scars. Mice were kept in plastic cages of 45.0 cm × 35.5 cm × 14.5 cm in size with a wire-mesh cage cover. The base of the cages was covered with sawdust of 0.5-1.0 cm in thickness and was changed every three days. Room light was carefully controlled, with 12 h of light (06:00 to 18:00 West Indonesia Time) and 12 h of dark (18:00 to 6:00 West Indonesia Time). Room temperature ranged 27 °C to 28 °C. The mice were fed ad libitum twice a day with Guyofeed chicken pellets containing 12% protein.
A total of 36 mice were divided into six groups (6 animals per group): control group (untreated mice), endometriosis mice group (without genistein treatment) and endometriosis mice groups administered with genistein at different doses (1.30 mg/day; 1.95 mg/day; 2.60 mg/day; 3.25 mg/day).
2.2. Generation of murine model of endometriosis
Endometrial and myometrial tissues were taken from adenomyotic patients who were subjected to a surgery at Syaiful Anwar Hospital Malang. Tissues were removed after the patient’s signature of the informed consent. It was subsequently stored in a phosphate buffer saline -containing Falcon tube. Furthermore, tissues were cut into small pieces of 0.5 cm in thickness, washed with phosphate buffer saline, and centrifuged twice at 3 000 rpm at a temperature of 4 °C for 10 min. The implant tissues were then intraperitoneally injected into the mice subjected to the immunodeficient condition by administration of cyclosporin A and to hyperestrogenic condition by injection of ethinyl estradiol. The development of endometriosis was established by means of direct evaluation of the peritoneum of mice (Mus musculus) model of endometriosis, and the expression of estrogen receptor alpha and estrogen receptor beta in the endometriosis lesions of the murine peritoneal tissue was measured,.
Dosage of genistein was determined based on previous studies, which was converted for administration in mice. The genistein used was that of genistein trademark manufactured by Bioworld, America, CAS 446-72-0, Catalog 40000007-2. Genistein purity was > 95%. Genistein was powder-shaped, purified from soybeans. Genistein was administered for 14 d by dissolving it in sesame oil to achieve the desired dose,.
2.4. Collection of peritoneal tissues
Upon completion of treatment, mice were anesthetized and dissected for collection of peritoneal tissues. Prior to dissection, they were anesthetized with ketamine. Peritoneal tissue samples were stored at −80 °C until analysis.
2.5. Expression of NF-κB
This expression was analyzed on the endometriosis lesions of the peritoneal tissue using immunohistochemical technique. The antibody anti NF-κβ 65 was used (produced by Bioss Inc.; bs-0465R). The Remmele index using the immuno reactive score was the product of multiplication between the percentage of immunoreactive cells and color intensity score of the immunoreactive cells.
2.6. Expression of MMP-2
Expression of MMP-2 was the expression of MMP-2 on the endometriosis lesions of the peritoneal tissues in the murine model of endometriosis using the immunohistochemical technique. The antibody anti MMP-2 was used (produced by Bioss Inc.; bs-0412R). Expression of MMP-2 was assessed semi-quantitatively using the same technique as that of the expression of NF-κB .
2.7. Expression of MMP-9
Expression of MMP-9 was the expression of MMP-9 on the endometriosis lesions of the peritoneal tissues of the murine model of endometriosis using the immunohistochemical technique. The antibody anti MMP-9 was used (produced by Bioss Inc.; bs-0397R). Expression of MMP-9 was assessed semi-quantitatively using the same technique as that of the expression of NF-κB .
The present study passed the ethics review (No. 275/EC/ KEPK/07/2016) and was approved by the Health Research Ethics Committee, Faculty of Medicine, Brawijaya University, Malang, Indonesia (July 26, 2016).
2.9. Statistical analysis
The expression of NF-κB, MMP-2 and MMP-9 was shown in mean ± standard deviation (mean ± SD). Differences among treatment groups were analyzed using one-way analysis of variance test by means of SPSS 17.0 statistical package software. A post-hoc test was conducted in the event that the analysis of variance test found a within-group significance. A P<0.05 was a statistically significant difference.
| 3. Results|| |
[Figure 1] showed the activity of NF-κB on the peritoneal tissues of different groups. The activity of NF-κB was higher significantly in the endometriosis group [(4.55±0.53)%] compared to the control group [(0.00±0.00)%] (P<0.05). Administration of genistein at different doses significantly reduced the activity of NF-κB compared to that of the endometriosis group (P<0.05), reaching the level comparable to that of the control group at the third highest dose [(0.00 ± 0.00)%] (P>0.05).
|Figure 1: NF-κBexpression in peritoneal tissue of various research groups.|
Note: Data are presented in mean ±SD; aP<0.05 compared to control group; bP<0.05 compared to endometriosis group.
Click here to view
[Figure 2] showed the expression of MMP-2 in peritoneal tissue of different groups. MMP-2 expression significantly increased in the endometriosis group [(5.00 ± 0.77)%] campared to the control group [(0.00±0.00)%] (P<0.05). The four doses of genistein administration significantly reduced the expression of MMP-2 compared to that of the endometriosis group (P<0.05), reaching an expression comparable to that of the control group at the highest dose [(0.45 ± 0.38)%] (P>0.05).
|Figure 2: MMP-2 expression in peritoneal tissue of various research groups.|
Note: Data are presented in mean ± SD; aP<0.05 compared to control group; bP<0.05 compared to endometriosis group; cP<0.05 compared to endometriosis group received first dose of genistein; dP<0.05 compared to endometriosis group received second dose of genistein.
Click here to view
[Figure 3] showed the expression of MMP-9 in peritoneal tissue of different groups. MMP-9 expression significantly increased in the endometriosis group [(4.50±0.38)%] compared to that of the control group [(0.00±0.00)%] (P<0.05). Administration of genistein at different doses significantly decreased the expression of MMP-9 compared to that of the endometriosis group (P<0.05), reaching levels comparable to that of the control group at the highest dose [(0.75±0.41)%] (P>0.05).
|Figure 3: MMP-9 expression in peritoneal tissue of various research groups.|
Note: Data are presented in mean ± SD; aP<0.05 compared to control group; bP<0.05 compared to endometriosis group.
Click here to view
| 4. Discussion|| |
The present study showed that the activity of NF-κB and the expression of MMP-2 and MMP-9 in the endometriosis group significantly increased than those of the control groups. This indicates that the translated protein products, namely MMP-2 and MMP-9, were upregulated at least via the increased activity of the transcription factor NF-κB . The estrogen induces transcription factors that modulate the activity of major pro-inflammatory cytokines. This finding is consistent with that of previous studies that the activation of NF-κB is increased on endometriosis lesions which involve proliferation, apoptosis, adhesion, invasion, and angiogenesis in endometriosis,. With regard to the peritoneum, NF-κB acts via regulation of the expression of MMPs in the invasion and adhesion of endometriosis cells on the peritoneal surface. A previous study has shown that MMP-2 is expressed in the endometrial graft in the peritoneum. Other studies have also found expression of MMP-2 and MMP-9 in peritoneal endometriosis. Materials that can suppress the production of MMP-2 and MMP-9 will inhibit the adhesion and migration of endometriosis cells in the peritoneum.
The present study showed that administration of genistein at different doses was capable of significantly reducing the activity of NF-κB compared to that of the endometriosis group, reaching the level comparable to that of the control group at the third highest dose (2.60 mg/day). This suggests that genistein is capable of suppressing the activation of NF-κB on the peritoneum of the murine model of endometriosis. This finding is consistent with the previous one that genistein is capable of inhibiting the translocation of NF-κB and expression of genes modulated by NF-κB. Genistein inhibits the DNA binding of NF-κB through blocking the phosphorylation of the protein inhibitor Iκβα, preventing the translocation of NF-κB. Furthermore, inhibition of this activation suppresses expression of the proteins MMP-2 and MMP-9. The present study demonstrated that all the doses of genistein were capable of significantly decreasing the expression of MMP-2 and MMP-9 compared to that of the endometriosis group. The expression of MMP-2 comparable to that of the control group was achieved at the highest dose (3.25 mg/day), while that of MMP-9 was achieved at the highest dose. This suggests that genistein is capable of suppressing the expression of MMP-2 and MMP-9 at least through suppressing the activity of NF-κ B. The differences in the optimal dose of genistein, which can return to the basal value, are thought to be caused by the presence of other regulators of transcription factors in the expression of MMP-2, namely signal transducer and activator of transcription-1 (STAT-1) and STAT-3. Genistein is an activation inhibitor of NF-κB and STAT-1. Meanwhile, regulation of MMP-9 involves NF-κβ/p65, activator protein-1/c-jun, transcription factor E2F2, Smad2, STAT-1 and STAT-3. In addition, genistein can also act by means of bonding to estrogen receptors,.
In the focus of endometriosis, the effects of genistein on blood flow and vascularization are inconsistent. Genistein can suppress angiogenesis in endometriosis tissue through down-regulation of vascular endothelial growth factor and hypoxia inducible factor-1α. In another study analyzing vascularization and blood flow, it was found that genistein could suppress vascularization (assessed by microvillar density) but did not affect blood flow in endometriosis graft. In this study, differences in genistein effects in MMP-2 and MMP-9 expression suppression still require continued research to assess tissue remodeling anatomy.
The strength of this study is the analysis of MMP-2 and MMP-9 which simultaneously perform as a product of NF-κB transcription factor. The weakness of this study is that no other regulatory factors are assessed (especially STAT signals) and no anatomical measurements of tissue remodeling are performed. This will be the target for further study.
In conclusion, genistein suppresses the expression of MMP-2 and MMP-9 through suppressing NF-κB activity in the peritoneum of a murine model of endometriosis.
Conflict of interest statement
All authors hereby state that there is no conflict of interest.
| References|| |
Riccio LGC, Santulli P, Marcellin L, Abrao MS, Batteux F, Chapron C. Immunology of endometriosis. Best Practice Res Clin Obstet Gynecol
Burghaus S, Fehm T, Fasching PA, Blum S, Renner SK, Baier F, et al. The International Endometriosis Evaluation Program (IEEP Study)–A systematic study for physicians, researchers and patients. Geburtshilfe Frauenheilkd
Zondervan KT, Rahmioglu N, Morris AP, Nyholt DR, Montgomery GW, Becker CM, et al. Beyond endometriosis genome-wide association study: From genomics to phenomics to the patient. Semin Reprod Med
Williams KE, Miroshnychenko O, Johansen EB, Niles RK, Sundaram R, Kannan K, et al. Urine, peritoneal fluid and omental fat proteomes of reproductive age women: Endometriosis – related changes and associations with endocrine disrupting chemicals. J Proteom
Asghari S, Valizadeh A, Aghebati-Maleki L, Nouri M, Yousefi M. Endometriosis: Perspectives, lights, and shadow of etiology. Biomed Pharmacother
Wu M, Hsiao K, Tsai S. Endometriosis and possible inflammation markers. Gynecol Minimally Invasive Ther
Liu Y, Qin X, Lu X. Crocin improves endometriosis by inhibiting cell proliferation and the release of inflammatory factors. Biomed Pharmacother
Patel BG, Lenk EE, Lebovic DI, Shu Y, Yu J, Taylor RN. Pathogenesis of endometriosis: Interaction between endocrine and inflammatory pathways. Best Practice Res Clin Obstet Gynecol
Vercellini P, Buggio L, Somigliana E. Role of medical therapy in management of deep rectovaginal endometriosis. Fertil Steril
Yang H, Liu J, Fan Y, Guo Q, Ge L, Yu N, et al. Associations between various possible promoter polymorphisms of MMPs genes and endometriosis risk: A meta-analysis. Eur J Obstet Gynecol Reprod Biol
Barisic A, Pavlic SD, Ostojic S, Pereza N. Matrix metalloproteinase and tissue inhibitors of metalloproteinases gene polymorphisms in disorders that influence fertility and pregnancy complications: A systematic review and meta-analysis. Gene
Kim SH, Park MJ. Effects of phytoestrogen on sexual development. Korean J Pediatr
Lin ML, Lu YC, Chung JG, Wang SG, Lin HT, Kang SE, et al. Down-regulation of MMP-2 through the p38 MAPK-NF-kappaB-dependent pathway by aloe-emodin leads to inhibition of nasopharyngeal carcinoma cell invasion. Mol Carcinogen
Aznaurova YB, Zhumataev MB, Roberts TK, Aliper AM, Zhavoronkov AA. Molecular aspect of development and regulation of endometriosis. Reprod Biol Endocrinol
Yan W, Fan W, Chen C, Wu Y, Fan Z, Chen J, et al. IL-15 up-regulates the MMP-9 expression levels and induces inflammatory infiltration of macrophages in polymyositis through regulating the NF-kB pathway. Gene
Elnashar A. Emerging treatment of endometriosis. Middle East Fertil Soc J
Ganai AA, Farooqi H. Bioactivity of genistein: A review of in vitro
and in vivo
stuides. Biomed Pharmacother
Mazumder MAR, Hongsprabhas P. Genistein as antioxidant and antibrowning agents in in vivo
and in vitro:
A review. Biomed Pharmacother
Amelia SWN, Yueniwati Y, Sutrisno S. The expression of vascular endothelial growth factor is affected by hypoxia inducible factor-1α in peritoneum of endometriosis mice treated with genistein. Middle East Fertil Soc J
Sutrisno S, Wulandari RRCL, Sulistyowati DWW, Wulandari RF, Wahyuni ES, Yueniwati Y, et al. Effect of genistein on proinflammatory cytokines and estrogen receptor-β in mice model of endometriosis. Asian Pac J Reprod
Yavuz E, Oktem M, Esinler I, Toru SA. Genistein causes regression of endometriotic implants in the rat model. Fertil Steril
Nowak M, Janusz M, Dziegiel P. Intensity of COX-2 expression in cells of soft tissue fibrosarcomas in dogs as related to grade of tumour malignancy. Bull Vet Inst Pulawy
Ramos RG, Rocco J, Rojas C, Sovino H, Poch A, Kohen P, et al. Physiologic activation of nuclear factor kappa b in the endometrium during the menstrual cycle is altered in endometriosis patient. Fertil Steril
Zhou B, Rao L, Peng Y, Wang Y, Qie M, Zhang Z, et al. A functional promoter polymorphism in NFKB1 increases susceptibility to endometriosis. DNA Cell Biol
Arablou T, Kolahdouz-Mohammadi R. Curcumin and endometriosis: Review on potential roles and molecular mechanisms. Biomed Pharmacother
Weigel MT, Kramer J, Schem C, Wenners A, Alkatout I, Jonat W, et al. Differential expression of MMP-2, MMP-9 and PCNA in endometriosis and endometrial carcinoma. Eur J Obstet Gynecol Reprod Biol
Kim JH, Yang YI, Ahn JY, Lee JG, Lee KT, Choi JH. Deer (Cervus elaphus)
antler extract suppresses adhesion and migration of endometriotic cells and regulates MMP-2 and MMP-9 expression. J Ethnopharmacol
Emmerson E, Campbell L, Ashcroft GS, Hardma MJ. The phytoestrogen genistein promotes wound healing by multiple independent mechanisms. Mol Cell Endocrinol
Sutrisno S, Aprina H, Simanungkalit HM, Andriyani A, Barlianto W, Sujuti H, et al. Genistein modulates the estrogen receptor and suppresses angiogenesis and inflammation in the murine model of peritoneal endometriosis. J Trad Complement Med
Laschke MW, Schwender C, Vollmar B, Menger MD. Genistein does not affect vascularization and blood perfusion of endometriotic lesions and ovarian follicles in dorsal skinfold chambers of Syrian golden hamsters. Reprod Sci
[Figure 1], [Figure 2], [Figure 3]
|This article has been cited by|
||G Protein-Coupled Estrogen Receptor 1 (GPER) as a Novel Target for Schizophrenia Drug Treatment
| ||Danielle S MacÍdo,Lia Lira Olivier Sanders,Raimunda das Candeias,Cyntia de Freitas Montenegro,David Freitas de Lucena,Adriano Josť Maia Chaves Filho,Mary V Seeman,Aline Santos Monte |
| ||Schizophrenia Bulletin Open. 2020; 1(1) |
|[Pubmed] | [DOI]|
||Isoflavone Ameliorated Oxidative Stress and Vascular Damages in Nicotine-Administrated Mice
| ||Wanida Sukketsiri,Kiyoto Hoshino,Hirona Kugo,Tomomi Nakamura,Tsukasa Sasoh,Tatsuya Moriyama,Nobuhiro Zaima |
| ||Journal of Oleo Science. 2019; 68(12): 1241 |
|[Pubmed] | [DOI]|