{"paper_id":"5201ea9e-bd3a-4aa3-9314-95f94c9ab154","body_text":"* Corresponding author: Windarti Windarti \n E-mail: windarti-2019@fk.unair.ac.id \n© 2023 by SPC (Sami Publishing Company) \n \nJournal of Medicinal and Chemical Sciences 6 (2023) 2952-2963 \n \n \nJournal homepage: http://www.jmchemsci.com/ \n \n \n \n \n \nOriginal Article \nCorrelation Between Vitamin D Administration and Reduced \nExpression of VEGF and MMP-9 in Abnormal Endometrial Stroma \n(Experimental in Mice Models with Endometriosis) \nWindarti Windarti*,1 \n , Ashon Sa’adi2 \n , Widjiati Widjiati3 \n  \n1Master of Reproductive Health, Airlangga University, Surabaya, Indonesia \n2Faculty of Medicine, Airlangga University, Airlangga, Indonesia \n3Faculty of Veterinary Medicine, Airlangga University, Airlangga, Indonesia \nA R T I C L E     I N F O  A B S T R A C T \nArticle history \nReceive: 2023-05-17 \nReceived in revised: 2023-07-09 \nAccepted: 2023-07-26 \nManuscript ID: JMCS-2306-2116 \nChecked for Plagiarism: Yes \nLanguage Editor:  \nDr. Fatima Ramezani \nEditor who approved publication:  \nDr. Mehrdad Hamidi  \nDOI:10.26655/JMCHEMSCI.2023.12.10 \n Background: Women with vitamin D deficiency are at higher risk of \nhaving endometriosis. Endometriosis is a benign gynecological disease that \noccurs in women of reproductive age with multifactorial etiopathogenesis \nwith high migratory and invasive potential. Invasion requires angiogenesis \nderived from vascularization mediated by VEGF and initiated by MMP. \nVitamin D acts on women reproduction in target gene regions by inhibiting \ncell proliferation in various cancer cells through induction of apoptosis and \nG0/G1 arrest, suppression of angiogenesis, and modulation of growth \nfactor receptor expression. \nThis study aims to prov e the role of graded vitamin D to decrease the \nexpression of VEGF and MMP -9 administered to endometriosis model \nmice. \nMethods: Experimental study with ethical due diligence certificate no. \n2.KE.144.12.2021 used 24 endom etriosis model mice divided into 4 \ngroups; 1 Control Group and 3 treatment groups that were administered \nvitamin D orally at a dose of 8 iu, 16 iu and 24 iu. The expression of VEGF \nand MMP-9 was assessed from the peritoneal tissue of mice in all groups. \nResults: It was found that there was a decreased average value of the \nexpression of VEGF and MMP -9 of treated endometriosis model mice \ncompared to that of untreated endometriosis model mice. The decrease in \nVEGF expression was not statistically significant, while the decrease in \nMMP-9 expressio n was statistically significant with a P value of 0.027. \nTherefore, there was a significant relationship between VEGF expression \nand MMP-9 expression with doses in the negative direction, the higher the \ndose, the lower the value of VEGF expression and MMP expression. \nConclusion: Vitamin D can suppress angiogenesis by reducing the \nexpression of VEGF and MMP -9 in endometriosis model mice at a dose of \n24 iu. \nK E Y W O R D S \nEndometriosis \nVitamin D \nVEGF \nMMP-9 \nReproductive health  \n \n \nG R A P H I C A L A B S T R A C T \n \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2953 | P a g e  \n \nIntroduction \nEndometriosis is a complex gynecological \ndisorder which occurs in women of reproductive \nage caused by a combination of several genetic \nand environmental factors characterized by a \nhigh migratory and invasive pote ntial such as \ntumor metastasis [1, 2]. The prevalence of \nendometriosis is 10 percent of women of \nreproductive age regardless of ethnicity and \nsocial status [3]. Various data on the prevalence \nof vitamin D deficiency which occurs in women of \nreproductive age in European, American and \nAsian countries vary from 42% -90% vitamin D : \nAn overview of vitamin D status and intake in \nEurope [4, 5], whereas the rate of vitamin D \ndeficiency in Indonesia reaches 63% [6]. The \nfeatures of women of reproductive age who have \nendometriosis are chronic type, thus affe cting \ntheir quality of life with the ma in symptoms of \npain and infertility [7], which interfere with \npsychological and social functions [8, 9]. \nIncreased activ ity of cytokines and steroid \nhormones in endometrial tissue increases Matrix \nMetalloproteinase-9 (MMP -9) expression and \nplays an important role in invasion and \nmetastasis [10]. Vascular Endothelial Growth \nFactor (VEGF) expression plays a role to form the \nvascular tissue cyc le by stimulating the \ndegradation of the extracellular matrix around \nendothelial cells [11]. MMP -9 is a group of \nproteolytic enzymes that degrade components of \nthe extracellular matrix in their progression [12].  \nVitamin D can suppress cyclooxygenase 2 (COX 2) \nexpression, thereby reducing levels of IL -6, TNF, \nand PG [13, 14]. Vitamin D can reduce the \ninflammatory response induced by IL-1β- or TNF-\nα so that the expression of MMP -2 and MMP9 \ndecreases through inhibition of nuclear factor -κB \n[15]. Vitamin D also reduces VEGF -A expression \nin the s troma of endometriosis lesions, and can \nreduce VEGF levels in diseases such as PCOS [16-\n18]. Research on nutrition and food groups that \ncorrelate with endometriosis is limited to \ndetermining potential of disease risk factor s in \nthe endometriosis pathogenesis to food groups \nand nutrition. Vitamins A, C, and E have been \nwidely researched to be correlated with \nendometriosis. However, the results show that \nthere is no statistically signific ant relationship, \nand only vitamin D shows significant results [13]. \nThe researchers are interested in conducting \nresearch on endometriosis model mice that are \nadministered vitamin D supplements at doses of \n8, 16, and 24 iu a decrease in the expression of \nMMP-9 and VEGF and the relationship of t he \ndecreased expression with the administered \ngraded doses.    \nMaterials and Methods \nExperimental animals \nThe experimental animals were obtained from \nthe Airlangga University Pusvetma. The research \nwas carried out at the Laboratory of Veterinary \nMedicine F aculty, Airlang ga University after \nobtaining a certificate of ethical due diligence no. \n2.KE.144.12.2021.  \nMaking endometriosis model mice \nAn endometriosis model mouse was mde by \ninjecting cyclosporin A on the mice, a nd then \nintraperitoneal injection of 0. 1 cc endometria l \nbiopsy tissue was carried out on them and on the \n5th day, the mice were administered \nintramuscular injection of estrogen with a dose of \n5.4 µg/mouse. After 14 days, endometriosis \nmodel mice were obtained. \nResearch design and stages \nThis type of research is true experimental using a \ncompletely randomized design posttest only \ncontrol group design. Using the randomization \ntechnique, the samples were categorized into 4 \ngroups, specifically the control grou p (C) which \nwas not given vitamin D, th e group treated  with \ndose I ( T1), dose II ( T2), and dose III ( T3). The \nneed for vitamin D was calculated according to \nthe Endocrine Society (21) and sampling was \ncarried out so that the mice in group T1 were \nadministered as  much as 8 iu vitamin D that \nequals to 0.2 cc; those in group T2 were \nadministered as much as16 iu Vitamin D that \nequals to 0.4 cc and those in group T3 received \n24 iu vitamin D equals to 0.6 cc. Vitamin D was \nadministered individually once daily. Tr eatment \nbegan on day 15 after the occur rence of  \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2954 | P a g e  \n \nendometriosis lesions by administering vitamin D \naccording to the dose per group. \nExamination on the expression of VEGF and MMP-9 \nSamples were taken from the peritoneal tissue of \nmice on day 37. The peritonea l tiss ue with \nendometriosis lesions was  fixed in 10% formalin \n[19, 20]. VEGF expression was analyzed using \nimmunohistochemical techniques. Staining was \ncarried out using DAB Kit with Coomasie brilliant \nblu counterstaining. MMP -9 expres sion an alysis \nwas performed by immunohistochemical staining \nusing MMP -9 polyclonal antibody. The sample \nwas evaluated using the Remmele scale index \n(Immuno Reactive Score/IRS) in a semi -\nquantitative manner. The assessment involved \nmultiplying the percentag e score  of \nimmunoreactive cells by the color intensity score \non these cells , as listed in Table 1 . For each \nsample, the data represented the average IRS \nvalue observed across ten different field of views \nusing the magnifications of 100x and 400x. \nResults and Discussion \nEffect of vitamin D on VEGF expression \nFigure 1  displays the impact of administering \nvitamin D supplements to mice with an \nendometriosis model. This figure shows that \nthere is VEGF expression in  cell tissue of \nendometriosis lesions taken from the peritoneum \nof endometriosis model mice. Accordingly, group \nC has stronger expression and it is clustered, \ncompared to group P1, P2, and P3 which have \nweaker expression and it is dispersed. The figure \non VEGF expression is in accordance with t he \ngraph on average values which tends to decrease \nin Figure 2. VEGF expression in the control group \nhad an average value of 5.6; the intervention \ngroup 1 with a dose of 8 iu had an average val ue \nof 5.5, intervention group 2 with a dose of 16 iu \nhad an average value of 4.3, and the lowest was \nthe intervention group 3 with a dose of 24 iu that \nhad an average value of 3.8. \nTable 1: Post Hoc Bonfferoni test of MMP-9 expression conducted on Endometriosis Model mice supplemented \nwith vitamin D \n Average value difference   P-value \nC vs. T1 1.67 0.213 \nC vs. T2 1.75 0.168 \nC vs. T3 2.37 0.027 \nT1 vs. T2 0.08 1.000 \nT1 vs. T3 0.70 1.000 \nT2 vs. T3 0.62 1.000 \nC: A group that serves as a control; T1: A group that receives an intervention with 8 iu of vitamin D; T2: A group that \nreceives an intervention with 16 iu of vitamin D; T3: A group that receives an intervention with 24 iu of vitamin D. \n \nFigure 1: VEGF expression on endometriosis model mice tissue shown by red arrows \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2955 | P a g e  \n \n \nFigure 2: Distribution of VEGF expression average values in endometriosis model mice \nBased on the graph, mice with an endometriosis \nmodel that received vitamin D supplementation \nat doses of 8 iu, 16 iu, and 24 iu showed \ndecreased V EGF expression compared to mice \nwith the endometriosis mo del without vita min D \nsupplementation. Among the different groups, \nthe mice receiving 24 iu of vitamin D \nsupplementation demonstrated the least amount \nof VEGF expression \nEffect of Vitamin D on MMP-9 Expression \nFigure 3 demonstrates the impact of vitamin D on \nthe MMP -9 expression. In addition , Figure 4 \ndisplays the MMP -9 expression in the tissue of \nmice with an endometriosis model. \nThe depicted graphs and figures illustrate the \neffects of vitamin D supplementation at doses of 8 \niu, 16 iu, and 24 iu on mice with an endometriosis \nmodel had lower MMP-9 expression compared to \nthat of endometriosis mod el mice that were not \nsupplemented with vitamin D and after the One -\nway ANOVA test w as carried out, P = 0.027 was \nobtained; thus at the real level we reject Ho. \nHence, there is a significant difference in the \naverage values of MMP -9 expression in the four \ngroups. These results are in line with Befferoni's \nPost Hoc analysis presented in Figure 4. \nThe association between varying doses of vitamin \nD supplementation and the variables of VEGF \nexpression and MMP -9 expression in lesions of \nmice with an endometriosis model was examined \nusing Spearman's Rho test, and the find ings are \npresented in Table 2. \nBased on the results of the Spearman's Rho test \non VEGF expression with graded dose of vitamin \nD, p -value of 0.022 and MMP -9 of 0.007 with \ngraded doses of vitamin D are obtai ned. Based on \nthese results, it can be concluded that there is a \nsignificant relat ionship between the two \nvariables. Whereas, judging from the correlation \ncoefficient, it is shown that the two variables have \na moderate correlation and negative direction, a s \nshown in the following scatter plot graph  (Figure \n5). \nThe scatter plot graph depicted above exhibits a \ndistinct pattern wherein the data points align in a \nstraight line, extending from the lower right \ncorner to the upper left corner. This pattern \nindicates a linear and negative correlation \nbetween the dosage of  vitamin D and the \nexpression levels of VEGF and MMP -9. In simpler \nterms, as the dosage  of vitamin D increases, the \nexpression of VEGF and MMP -9 decreases, and \nvice versa. \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2956 | P a g e  \n \n \nFigure 3: Graph on MMP-9 expression value distribution on endometriosis model mice Control group (C) zero \ndose. Intervention Group 1 (T1) with a dose of vitamin D 8 iu, Intervention Group 2 (T2) with a dose of vitamin D \n16 iu, and Intervention Group 3 (T3)  with a dose of vitamin D 24 iu \n \nFigure 4: Images on MMP-9 expression at endometriosis model mice tissue shown by red arrows \nTable  2. The relationship on vitamin D supplementation  at  various doses with the expression of  VEGF and \nMMP-9 at  Endometriosis Mice Model \nNo. Relationship Correlation coefisient P-value \n1 VEGF and Dose -0.467* 0.022 \n2 MMP and Dose -0.539** 0.007 \nNotes: Relationship on vitamin D supplementation at various doses with the expression of VEGF and MMP-9 \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2957 | P a g e  \n \nFigure 5: Scatter plot graph on e xpression of  VEGF dan MMP -9 at Endometriosis Model mice supplemented \nwith vitamin D \nIn this study, a heterologous endometriosis \ninduction method was employed to create the \nendometriosis model. The process involved the \ninjection of human endomet rial tissue into the \nperitoneal cavity of mice. The levels of VEGF and \nMMP-9 expression were assessed in the resulting \nendometriosis lesions. This method provides \nseveral benefits for investigating the initial \nphases of the disease, encompassing factors li ke \nangiogenesis, abnormal cell death (apoptosis), \nproliferation of endometrial cel ls, and \ninflammation [21]. \nFurthermore, this approach closely mimics the \npathophysiology of retrograde menstruation in \nhumans, making it a valuable  tool for studying \nendometriosis lesions in humans [22]. According \nto Sampson's theory o f retrograde menstruation, \nendometriosis develops when shed endometrial \ntissue during menstruation migrates to the \ncommon attachment sites on the peritoneal wall, \ninvades the  extracellular matrix (ECM), and \nproliferates to form endometriotic lesions. \nEndometriosis is characterized by the presence of \neutopic and ectopic endometrial stromal cells \n(ESC), which exhibit invasive, adhesive, and \nproliferative properties. Women diagno sed with \nendometriosis exhibit decreased apoptosis and \nelevated expression levels of vascular endothelial \ngrowth factor (VEGF), urokinase plasminogen \nactivator, and matrix metalloproteinase -3 (MMP-\n3) in their endometrial tissues [23]. VEGF is a \nheparin-binding glycoprotein  secreted in the \nform of a homodimer (45 kDa). Heparin interacts \nwith VEGF through the formation of the Heparin -\nVEGF complex which leads to changes in \nmolecular conformation so that VEGF becomes \nmore stable, and is resistant to inactivation and \nhas a longe r half -life. The formation of the \nHeparin-VEGF complex also leads to an increase \nin the affinity of VEGF receptors that exist on the \ncell surface so that intracellular signals are \nformed as a means of proliferation to activate. \nUnder normal circumstances, VEGF is expressed \nto varying degrees by different tissues, including \nthe brain, ki dney, liver, and spleen. Exposure to \nhypoxic conditions induces rapid expression of \nVEGF. In contrast, under the conditions of normal \noxygen levels (normoxia), VEGF expressio n \ndecreases and stabilizes [24]. The results of the \nstudy on VEGF expression of endometriosis \nmodel mice supplemented with various doses of \nVitamin D showed a decreasing trend when \ncompared to that of endometriosis mice that \nwere not supplemented with vitamin D. These \nfindings indicate that the vitamin D receptor \n(VDR) is consistently expressed in all types of \nendometrial cells, and specifically in stromal \ncells. The endometrium , being responsive to \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2958 | P a g e  \n \nvitamin D, possesses the capacity to transform \n25(OH) vitamin D3 into its active state . These \ndiscoveries provide support for the involvement \nof vitamin  D in the reproductive hormonal \nprocesses [16]. Research has exposed that \nvitamin D plays a role in inhibiting cell growth in \nvarious cancer cells. This effect is achieved \nthrough the induction of apoptosis (cell death) \nand G0/G1 arrest, as well as through the \nsuppression of angiogenesis (the formation of \nnew blood vessels) and the modulation of \nexpression of growth factor receptors.  These \nfindings highlight the significance of vitamin D in \ncontrolling cellular processes related to cancer \ndevelopment [25]. VEGF expression of the \ntreatment group supplemented with vitamin D at \nvarious doses, sequentially from the highest to \nthe lowest was shown in group T3, T2, T1, and C, \nin the T3 group, which received a 24 iu  dose of \nvitamin D supplementation, the VEGF expression \nin the lesions was found to be the lowest. \nDalbandi's study additionally demonstrated that \ntreatment with vitamin D enhanced the \nattachment of endometrial stromal cells (ESC) \nfrom various sources to t he extracellular matrix \n(ECM). In contrast, it was observed that the VDR \nagonist, elocalcitol, reduced the capacity of \nendometrial cells in mice to bind to collagen.  \nExcess VEGF in abnormal endometrial stroma:  \n(1) VEGF stimulates the formation of new bloo d \nvessels, so excess VEGF in abnormal endometrial \nstroma can cause an increase in blood vessels in \nthe tissue , (2) VEGF can also stimulate the \nproliferation of endothelial cells (cells lining \nblood vessels), which can contribute to the \nabnormal growth and expansion of endometrial \nstroma, (3) VEGF can increase vascular \npermeability, which can facilitate the movement \nof cells and substances into tissues, including \ncells involved in inflammatory or disease \nprocesses [26]. VEGF deficiency in abnormal \nendometrial stroma:  (1) Deficiency of VEGF can \ninhibit the formation of new blood vessels \nneeded to supply nutrients and oxygen to tissues. \nThis can lead to disturbances in growth and \nabnormal endometrial s troma function  and (2) \nVEGF also plays a role in the wound healing \nprocess. VEGF deficiency can inhibit the healing \nprocess in damaged or abnormal endometrial \nstroma [27]. Vitamin D exerts its effects at an \noptimal concentration dose of 10 -7 M, which is \ncrucial for cellular maintenance and corresponds \nto the physiological levels of reproducti ve \nhormones [16]. The expression level of VEGF i s \nalso influenced by the presence of inflammatory \ncytokines and growth hormone , including \nEpidermal Growth Factor (EGF), Interleukin -1β, \nplatelet derived growth factor (PDGF), tumor \nnecrosis factor -α (TNF -α), and transforming \ngrowth factor -β (TGF -1β)[24]. MMP normally \nregulates macrophage activity by degrading the \nextracellular matrix of cells to b e phagocytosed \n[28]. Excess MMP -9 in abnormal endometrial \nstroma: \n(1) MMP-9 is an enzyme involved in extracellular \nmatrix degradation. MMP -9 excess may lead to \nincreased extracellular matrix degradation in \nabnormal endometrial stroma, which may \ncontribute to structural changes and tissue \nsoftness, (2) MMP-9 can facili tate the invasion of \ncells into healthy tissue. Excess MMP -9 in \nabnormal endometrial stroma can increase the \nability of cells to spread beyond their normal area \nand invade surrounding tissue  [29]. MMP-9 \ndeficiency in abnormal endometrial stroma:  (1) \nDeficiency of MMP -9 may interfere with the \nextracellular matrix remodeling process required \nfor normal changes in the endometrial stroma \nduring the menstrual cycle. This can contribute to \nabnormal stromal development or interference in \nthe tissue regeneration process  and (2) MMP -9 \ndeficiency can inhibit the ability of cells to move \nand spread to new areas. This may affect the \nability of the abnormal endometrial stroma to \ninvade surro unding tissue or particip ate in \nproliferation and healing processes [30]. Changes \nin the expression of MMP family members in the \nendometrium with endometriosis may represent \na key mechan ism which relates to the invasive \npotential of endometrial reflux to have this \ndisease [31]. Regulation of MMP expression in the \nendometrium is mediated by steroids that \nspecifically influence the formation of ectopic \nlesions. Increased MMP expression in \nendometriosis occurs due to a decrease in the \nprogesterone action and a n increase in \nproinflammatory cytokine exposure, which leads \nto an increase in the ability to build ectopic \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2959 | P a g e  \n \ngrowths in endometriosis [31]. This study \nsignificantly showed that the endometriosis \nmodel mice that were supplemented with vitamin \nD had lower MMP -9 expression compared to that \nof the endomet riosis model mice that were not \nsupplemented with vitamin D. Among the \ndifferent groups, the administration of vitamin D \nat a dosage of 24 iu exhibited the lowest MMP -9 \nexpression in the lesions when compared to the \nother groups. The results of this study \ncorroborate the research of Garcia -Gomes et al. , \nwhere MMP -9 had decreased expression (mRNA \nand protein) and enzyme activity, caused by the \npresence of prostaglandins (PGE2) in the \nperitoneal fluid [32]. In addition, PGE2 activity \ntakes place by the EP2/EP4 (recep tor for MMPs) \nwhich depends on PKA pathway. Another study \nreported that inactivation of phagocytosis by \nPGE2 via CD36 inhibition is mediated by EP -2 \n[28]. With the decrease in MMP -9 expression, the \nability to develop invasive ectopic growth also \ndoes not occur [31]. Vitamin D affects \nangiogenesis, changes cell ad hesion, migration, \nand re duces cancer cell invasion. Vitamin D not \nonly reduces expression and secretion of \nmetalloproteinase (MMP) 2 and 9, but also \ndecreases cathepsin K activity, increases tissue \ninhibitory MMP1 (TIMP1) and regulates various \ncomponents of the plasminogen activa tor system \n[33]. Based on the results of the dose correlation \ntest for VEGF expression in this study, it was \nconcluded that there was a significant \nrelationship between VEGF expression and dose, \nwhile the correlation coefficient showed that the \ntwo variables had a nega tive \ncorrelation/relationship. Decreased expression of \nVEGF gene after treatment with Vitamin D is a \npossible beneficial effect of this hormone in \npatients with endometriosis. In a recent study by \nYildirim et al., it was observed that endometriosis \nmice tr eated with vitamin D exhi bited lower \nVEGF immunoreactivity compared to the control \ngroup. Decreased expression of VEGF (Vascular \nEndothelial Growth Factor)  and MMP -9 (Matrix \nMetalloproteinase-9) in abnormal endometrial \nstroma can be correlated with the giv en dose of \nvitamin D beca use vitamin D has a role in \nregulating gene expression and enzyme activity. \nSeveral factors that can explain this correlation \nare as follows: \n(1) Genetic regulation: Vitamin D can interact \nwith vitamin D receptors (VDR) present on target \ncells, including e ndometrial stromal cells. VDR \nactivation by vitamin D will affect gene \nexpression, including genes involved in the \nproduction of VEGF and MMP -9. High doses of \nvitamin D can produce different gene regulatory \neffects than low doses o r vitamin D deficiency  \n[34]. \n(2) Immunomodulatory effect: Vitamin D also has \nan immunomodulatory effect, which can affect \nthe activity of immune system cells, including \nendometrial stromal cells. VEGF and MMP -9 can \nbe produced by immune cells, including \nmacrophages and endometrial stromal cells. High \ndoses of vitamin D can alter the immune \nresponse and inhibit the production of VEGF and \nMMP-9 by these cells.  \n(3) Inhibition of angiogenesis: Studies have \nshown that vitamin D can inhibit the process of \nangiogenesis by inhibiting the VEGF expression. \nHigh dos es of vitamin D can ampli fy this \ninhibitory effect, potentially reducing VEGF \nexpression in the abnormal endometrial stroma. \n(4) Inhibition of enzyme activity: Vitamin D can \naffect enzyme activity, including MMP -9 enzyme \nactivity. High doses of vitamin D c an inhibit the \nactivity of this enzyme, which can reduce MMP -9 \nexpression in the abnormal endometrial stroma  \n[35]. \nConversely, according to Dalbandi et al. , vitamin \nD did not have an impact on the proliferation of \nendometrial stro mal cells (ESC) in any of the \ngroups studied in the absence of fibronectin. \nThese findings emphasize the potential \ntherapeutic use of vitamin D3 in preventing the \ndissemination of endometriosis. Consistent with \nDalbandi's findings, Liu et al.  provided evid ence \nthat the signaling process facilitated by \nextracellular fibronectin played a pivotal role in \nthe proliferative reaction of thyroid cancer cells \nto vitamin D. In another study by Dalbandi et al., \nit was found that vitamin D administration led to \na decr ease in VEGF expression in endometrial \nepithelial cells (EESC) at 18 and 48 hours after \ntreatment, depending on the specific time points. \nHowever, no significant effects of vitamin D were \n\nWindarti W., et al. / J. Med. Chem. Sci. 2023, 6(12) 2952-2963 \n2960 | P a g e  \n \nobserved in other cell groups across all time \nintervals [16]. While the conclusion of the \ncorrelative test results f or MMP -9 expression at \nvarious doses in this study was significantly \nstrong in the medium category and in the \nnegative direction, the higher the d ose, the lower \nthe MMP-9 value. \nIn a pilot study conducted b y Dalbandi, it showed \nthat Vitamin D exerts its effects that depends on a \ndose. Predetermined optimal concentration of \n10-7 M Vitamin D for cell maintenance equals to \nphysiological levels of reproductive hormones. \nAccording to [16], vitamin D exhibits a biphasic \neffect on cell proliferation, where it su ppresses \ncell growth at concentrations higher than 10-8 M. \nIn their study, [10] discovered that they could \nregulate the levels of VEGF, MMP -9, and tissue \ninhibitor of metalloproteinase-2 (TIMP-2) in mice \nwith an endometriosis model, it was possible to \ninduce apoptosis in en dometriotic lesions.  \nElevated 1α -hydroxylase in pa tients with \nendometriosis leads to local production of the \nactive form of vitamin D, 1.25 (OH)2D3 that \nreduces endometriosis cell growth. The high \nexpression of key vitamin D enzymes in the \nendometrium of patients with endometr iosis \nunderlines the potentia l for local autocrine or \nparacrine responses rather than the classic \nendocrine effects of vitamin D [35]. \nThis sug gests that Vitamin D has the ability to \ninhibit the gro wth of ectopic endometrial cells. \n[15] provided evidence to support the notion that \nvitamin D hampers the nf -κB activation, as a \nresult, there was a decrease in the quantity and \nDNA replication of abnormal endometrial cells. \nFurthermore, the administration of vitamin D to \nthe mice with the end ometriosis model caused a \ndecline in the expression of VEGF -A, VEGF, and \nMMP-9 in the abnormal endometrial stroma, \neffectively suppressing th e angiogenic potential \nassociated with endometriosis [25],[16]. \nDelbandi et al.  (2016) also demonstrated the \npositive effects of vit amin D on endometriosis \nstromal cells by decreasing the rates of invasion \nand proliferation of the abnormal endometrial \ncells. \nMoreover, [36] concluded that vitamin D played a \nrole in regressing the extent of endometrios is \nlesions through the angiogenesis modulation, this \nwas accomplished by reducing the VEGF \nexpression and inhibiting the MMP-9 activity. \nConclusion \nThe finding of this research provid e support for \nthe pat hogenesis theory of endometriosis and \nconfirm the fi ndings of Vitamin D to treat \ntargeted mediators and molecules related to \ninflammation in the endometriosis treatment. \nThe decreased expression of VEGF and MMP -9 \nsignificantly correlated with dose, and wit h \nhigher dose of Vitamin D, the expression of VEGF \nand MMP-9 in the abnormal endometrial stroma \nwas diminished more significantly so that the \ncapacity for endometriosis angiogenesis in \nendometriosis stromal cells decreased its level of \ninvasion and proliferation. \nDisclosure Statement \nNo potential conflict o f interest was reported by \nthe authors. \nFunding \nThis research did not receive any spe cific grant \nfrom funding agencies in the public, commercial, \nor not-for-profit sectors. \nAuthors' Contributions \nAll authors contributed to data analysis, drafting, \nand revi sing of the paper and agreed to be \nresponsible for all the aspects of this work. \nORCID \nWindarti Windarti \nhttps://orcid.org/0000-0002-6264-2418 \n Ashon Sa’adi \nhttps://orcid.org/0000-0003-2682-3139 \nWidjiati Widjiati \nhttps://orcid.org/0000-0002-8376-1176 \nReferences \n[1]. 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