The effect of calcineurin signaling inhibition on the treatment of endometriosis in rats

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CypA is one of the inflammatory marker which secreted various type of cell in the inflammatory condition. During the inflammation, CypA exacerbate inflammatory response by the activation of calcineurin signaling that increases the cytokine secretion and tissue degradation in inflammatory region. The aim of this study was to investigate the effect of calcineurin signaling inhibition on the treatment endometriosis in rats. Materials & Methods In the present study, 32 albino-wistar rat were used. All rats were divided into three groups as Cyclosporin A (n = 10), tacrolimus (n = 10) and control group (n = 12). Cyclosporin A group was administered intraperitoneal and tacrolimus group was administered intravenous, total 2 doses at intervals of 2 weeks respectively. All studies lasted for 8 weeks. Processed endometrial tissues will cut at the middle of the tissue samples and embedded into paraffin. Histological sections (5 µm) were stained with Ki-67, Bcl-2, Caspase-3 and VEGF. Results The size of endometriotic focus were 204.7 ± 153.4 mm 3 , 71.9 ± 85.4 mm 3 ve 30.6 ± 36.7 mm 3 in control, in CsA and in tacrolimus groups. Compared the control group the size of endometriotic focus was lower in CsA and tacrolimus group (p = 0.002). Microscopically, Ki-67 (p = 0.010) and VEGF (p = 0.007) immunoreactivity were lower in CsA and tacrolimus group than controls. Conclusion The inhibition of calcineurin signaling with CsA and tacrolimus treatment causes regression of endometriotic focus via decreases of endometriotic cell proliferation and angiogenesis in ectopic endometriotic tissue. Endometriosis Cyclophilin A Cyclosporin A Tacrolimus Rat Calcineurin Figures Figure 1 Figure 2 Figure 3 Introduction Endometriosis is defined as the presence and growth of endometrial-like tissue outside of the uterine cavity [ 1 ]. It is known to be a chronic inflammatory disease that frequently causes pelvic pain and female infertility [ 2 ]. Its prevalence is approximately 5%-10% in women of reproductive age worldwide. In the literature, several theories have been suggested to explain the development of endometriosis [ 3 , 4 ], although, the molecular and cellular mechanisms that underlie the condition are not yet fully understand. As a consequence, there is currently no consensus as to the most appropriate medical and/or surgical approaches (or combination of two) for the treatment of endometriosis [ 1 , 5 ]. Cyclophilin A (CypA) is a member of the immunophilin family of proteins that are secreted from various types of cells in response to inflammatory stimuli such as hypoxia, oxidative stress, and infection. Prior studies have demonstrated increased tissue expression and/or higher blood levels of CypA in patients with inflammatory diseases, including asthma, rheumatoid arthritis, cardiovascular diseases, and various cancers [ 6 – 12 ]. In the inflammatory condition, CypA plays a role in inflammatory cell migration and cytokine production. It also causes tissue destruction and cell invasion via the increased secretion of matrix metalloproteinases (MMPs) in the inflammatory areas [ 13 ]. These effects of CypA are mediated by the activation of calcineurin signaling [ 13 ]. Calcineurin, which is a Ca + 2 dependent serin/threonine protein kinase, plays an important role in relation to the inflammatory response and implantation [ 14 ]. This kinase also plays a central role in the adaptive immune response through the activation of the nuclear factor of activated T cells (NFAT) and the nuclear factor kappa beta (NF-κB) transcription factors in several cell types [ 15 ]. Cyclosporine A (CsA) and tacrolimus (FK506) are calcineurin inhibitors that work according to a similar mechanism. The pharmacological effects of both these drugs are based on the inhibition of calcineurin through binding to cyclophilin A, especially in immune effector cells [ 14 ]. In our previous study, we demonstrated the increased expression of CypA in both the eutopic and ectopic endometrial cells of patients with endometriosis [ 16 ]. Moreover, we found the CypA expression to be correlated with vascularity and endometrioma recurrence [ 16 ]. Prior cancer cell line and culture medium studies have shown that, the deficiency of CypA or CsA (or tacrolimus) treatment decrease tumor growth and increase the efficacy of chemotherapeutic agents in relation to the treatment of glioblastoma multiforme and endometrial cancer [ 17 , 18 ]. Thus, given the anti-inflammatory and immunomodulatory properties of CsA and tacrolimus, we investigated the added benefit of CsA and tacrolimus in terms of the treatment of endometriosis in an experimental rat model. Materials And Methods Ethical approval The study protocols was approved by Balikesir University animal research local ethics committee (Date: 25.06.2020, No:2020/4-14) before starting the study. This study was performed in Balikesir University Experimental Animals Laboratory. All of the experimental procedures and investigations were performed in complians with international guidelines on the ethical use of animals. Animals A total of thirty-six female non-pregnant wistar albino rats 10-12 weeks old, weighted 250-300 g were used for induction of the experimental endometriosis model. The all rats were housed at 20±2 o C room temperature during the adaptation and experimental procedure. The room with 12-hours light and 12-hours dark cycle with 50±10% humidity, the water and food add libitum. The animals were randomly classified into 3 groups (control group, cyclosporine A group, tacrolimus group). Before the surgical induction of endometriosis, the rats underwent daily vaginal lavages to detect the phase of estrus cycle. Vaginal secretions were examined under a light microscope to identify the estrus cycle by the dominancy of the anucleate cornified cells. Surgical procedure Experimental endometriosis was performed so that, the right uterine horn was removed, and a piece of the tissue was trimmed using microscissors. The removed endometrial tissue fragment was sutured into the abdominal wall of the same rat. One month after the first operation, second experiment was performed under anesthesia and the presence of endometrial foci was confirmed. During the procedure, endometriosis foci was measured and recorded. After the procedure, all rats were divided into three groups as Cyclosporin A (n=10), tacrolimus (n= 10) and control group (n=12). Cyclosporin A group was administered at 5mg/kg intraperitoneal and tacrolimus group was administered at 0,3mg/kg intravenous, total 2 doses at intervals of 2 weeks respectively. Saline infusion was given at 50 mg/kg with the same dose intervals by subcutaneous injection in control group. All studies lasted for 8 weeks. At the end of the treatment period all rats was sacrificed and eutopic and ectopic endometrial tissue were removed. Tissue evaluation After 28 days of medication in experimental group, the rats were euthanized wiht ketamine 10% and xylazine 2%, and a laparotomy was done. Ectopic endometrial tissue was isolated and measured three dimentions (lenght x width x height) using a digital meter. The macroscopic volume of the tissue was calculated with the prolated elipsoid formula which was descripted elsewhere [19]. For histopathological and immunohistochemical evaluation, all endometriotic tissue samples were fixed in 10% formalin and then with phosphate buffered saline overnight, and stored in 70% EtOH until processing. Processed endometrial tissues will cut at the middle of the tissue samples and embedded into paraffin. Histological sections (5 μm) were stained with Ki-67 (anti-human Ki-67 Antibody DAKO, California, USA), Bcl-2 (Bcl-2 Antibody (C-2): sc-7382, Santa Cruz Biotechnology, California, USA), Caspase-3 (Anti-Caspase-3 Antibody, Rabbit Polyclonal, Sino Biological, Houston, USA) and VEGF (Anti-VEGF183/VEGFA antibody, Sino Biological, Pennsylvania, USA). IHC examination A 5 µm thick endometriotic tissue sections were obtained and stained in hematoxylin eosin. All samples were visualized under light microscopy (Olympus BX48, Tokyo, Japan) and evaluated according to semi-quantitative method described previously (Nis Elements Advantages Research Microscope Imaging Software; Nikon Instruments Europe BV, Amsterdam, Netherlands). All histopathological and immunohistochemical evaluation was done same histopathologist and during the evaluation the histopathologist was blinded. Assessment of Ki-67, Bcl-2, Caspase-3 and VEGF Immunoreactivity The expression of Ki-67, Bcl-2, Caspase-3 and VEGF in the all tissue samples was compared according to IHC staining intensity in all tissue samples. Ki-67 and Caspase-3 immunoreactivity were predominantly localized in the nucleus of endometriotic cells. In contrast, the Bcl-2 immunoreactivity was predominantly localized in intracytoplasmic and cell membrane surface in the same cells. The degree of positive staining for Ki-67 and Caspase-3 was calculated as the ratio of positively stained endometriotic cells to total endometriotic cells at × 400 magnification in the selected field. Any nuclear staining was regarded as positive. A semiquantitative scoring system was used to evaluate the degree of Bcl-2 and VEGF immunoreactivity. Brown-color staining in the cell membrane surface of the endometriotic cells revealed the presence of Bcl-2 expression. Brown-color staining in the cytoplasm of the cells revealed the presence of VEGF expression. Degree of positive staining of Bcl-2 and VEGF was scored as 0, negative; 1, weak positive; 2, moderate positive; 3, strongly positive. Statistical analysis All the statistical analyses were performed using MedCalc statistical package for medical sciences (MedCalc Inc., Belgium) version 19.2. All data were presented as mean±SD or proportion as appropriate. The parametric data were compared between study groups using one-way analysis of variance (ANOVA) with post-hoc tests for subgroup analyses. Non-parametric data were compared using Kruskal Wallis one-way analysis of variance. Independent t test was used to compare the parametric variables between two study groups. A two-sided p-value of less than 0.05 was considered statistically significant. Results A total of 36 rats was randomly divided into the 3 study group, 4 of the rats were exculuded from the study for anestesia complications. Finally, 32 rats were included the study and all of them were divided as control (n=12), Cyclosporine A (n = 12) and tacrolimus (n = 10). There was no significant differences between the baseline characteristics of the groups (Table 1). After the treatment the size of endometriotic focus were 204.7±153.4 mm 3 in controls, 71.9±85.4 mm 3 in cyclosporine A and 30.6±36.7 mm 3 in tacrolimus groups. Compared the control group the macroscopic sizes of endometriotic focus were lower in both Cyclosporin A and tacrolimus group (p=0.002). Microscopically Ki-67 and VEGF immunoreactivity were lower in Cyclosporin A and tacrolimus groups than controls (p=0.010 and p=0.007, respectively). Moreover, Bcl-2 immunoreactivity was lower in both Cyclosporine A and tacrolimus groups, however this differences did not reach statistical significance (p = 0.056). There was no significant differences in Caspase-3 immunoreactivity between the groups (p = 0.178). Our subgroup analysis was demonstrated that there was no significant differences in Ki-67, Bcl-2, Caspase-3 and VEGF immunoreactivity between cyclosporine A and tacrolimus groups. Table 1: Pre-treatment and post-treatment endometriotic implant volume of the groups and immunohistochemical evaluation results. Control Group (n = 12) Tacrolimus Group (n = 10) Cyclosporin A Group (n = 10) P Values* Pre-treatment endometriotic implant volume 165.3±129.1 213.6±166.8 214.8±193.9 0.716 Post-treatment endometriotic implant volume 204.7±153.4 a 30.6±36.7 71.9±85.4 0.002 Ki-67, mean ± SD 1.8±0.7 a 1.1±0.5 1.0±0.6 0.010 Bcl-2 mean±SD 1.8±0.7 1.2±0.8 1.1±0.7 0.056 Caspase-3 mean±SD 1.6±0.7 2.1±0.9 2.2±0.9 0.178 VEGF mean ± SD 1.9±0.7 b 1.1±0.6 1.0±0.8 0.007 * ANOVA Bcl-2 (B-cell lymphoma 2) VEGF (Vascular Endothelial Growth Factor) a: Group 1 is statistically different from Groups 2 and 3 (p< 0.05) b: Group 1 is statistically different from Groups 2 and 3 (p< 0.05) Discussion In this experimental study, we observed that CsA and tacrolimus treatment were equally capable of significantly reducing the size of endometriosis. We also found that they decreased the histological grade (as demonstrated by the Ki-67 and VEGF expression) in endometriotic implants in rats when compared with the control group. To the best of our knowledge, this is the first study to evaluate the effects of CsA and tacrolimus mediated calcineurin signaling inhibition with regard to the treatment of endometriosis. CsA and tacrolimus are two well-known immunosuppressive drugs with the similar therapeutic mechanisms. Both drugs work by binding to CypA and inhibiting calcineurin signaling. Importantly, calcineurin signaling plays a central role in the adaptive immune response and the activation of calcineurin is dependent CypA that is on the calcineurin receptore. CypA mediates the action of these immunosuppressant drugs via peptidyl-prolyl cis-trans isomerases (PPIases) [20,21]. Once CsA and tacrolimus bind to their respective proteins, PPIase activity is abolished [20]. The effect of CsA and tacrolimus become active by forming complexes with the PPIases in the body. These complexes then bind to calcineurin and inhibit its phosphatase activity. The inhibitory effects of these drugs in relation to calcineurin work by competitively binding to and inhibiting Ca +2 and calmodulin-dependent phosphatase calcineurin [22]. According to the findings of the present study, the inhibition of calcineurin signaling by means of CsA and tacrolimus treatment causes the regression of endometriotic tissue, leading to decreased cell proliferation and angiogenesis in the endometriotic tissue. In the inflammatory condition, the secretion of CypA exacerbates the inflammatory response in many different benign and malign diseases, such as atherosclerosis, asthma, rheumatoid arthritis, diabetes mellitus, and lung and endometrial cancer [6–12]. In our previous study, we clearly demonstrated that women with endometriosis exhibited increased expression of CypA in both their eutopic and ectopic endometrial tissues when compared with control subjects. Moreover, CypA expression has been found to be correlated with the vascularity of endometriotic tissue [16]. In the present study, we showed that the inhibition of CypA-mediated calcineurin signaling caused the regression of the experimental endometriotic tissues in rats. Endometriosis is characterized by the presence and growth of the functional endometrial glands and/or stromal tissue in the abnormal region outside of the uterine cavity. The condition has a multifunctional etiology and prior studies have demonstrated that various inflammatory and immunological biomarkers play an important role in its pathogenesis. The expression of proinflammatory cytokines and chemokines is increased in response to the presence of ectopic endometriotic tissue. Previous studies have shown that the expression of IL-1β, TNF- α, IL-6, IL-8 and VEGF is increased in the blood samples, pelvic peritoneum and endometriotic tissue samples of patients with endometriosis [23,24]. Similarly, prior studies have demonstrated that the activation of calcineurin signaling causes the upregulation of proinflammatory cytokine and chemokine including TNF- α, IL-6 by the activation of NF-κB [25]. In addition, blocking of calcineurin signaling by the calcineurin inhibitors decreases the TNF-α and IL-6 [25]. According to the present results, the CsA and tacrolimus-mediated inhibition of calcineurin signaling may lead to a decrease in proinflammatory cytokines and chemokine production. This hypothese should be checked in the future studies. Prior studies have clearly shown that calcineurin acts through the activation of the NF-κB and NFAT transcription factors. More specifically, the activation of the calcineurin/NFAT signaling pathway by angiotensin II increases the endometrial stromal cell proliferation and blastocyst implantation via the induction of cyclooxygenase-2 gen expression in rats [26]. On the other hand, the activation of NF-κB in macrophages releases proinflammatory cytokines in endometriotic cells, which promotes cytokine production in ectopic endometriotic cells, thereby causing an increase in the synthesis of antiapoptotic factors, preventing apoptosis in endometrial cells, and contributing to endometriotic cell survival [27]. In a recent study that sought to investigate the pathway leading to the activation of NF-κB, the authors found that MAPKs, ERK1/2, JNK, and p38 were all activated by CypA treatment. However, only the ERK inhibitor PD98059 was able to decrease the activation of NF-κB and attenuate the MMP-9 activity induced by CypA [28]. Additionally, previous animal model studies have shown that, CsA inhibits endometrial stromal cell proliferation via abolishing calcineurin/NFAT dephosphorylation and translocation to the nucleus [26]. Recent cancer cell line and culture medium studies have shown that the overexpression of CypA associated with cancer cells proliferation, cell cycles progression, cell invasion, cell migration and inhibition of cell apoptosis [8,29,30]. Interestingly, presence of CypA causes resistance to chemotherapeutic drugs. Some recent studies have demonstrated that medical treatments with anti-cancer agents such as 5-aza-2-deoxycytidine, celecoxib, 5-fluorouracil, and paclitaxel are related to decreases in CypA expression within cancer cells [18,31–33]. However, the knockdown of CypA or CsA has been shown to increase the chemotherapeutic effect of cisplatin and paclitaxel in the treatment of glioblastoma multiforme and endometrial cancer [17,18]. On the other hand, it is known that CsA and tacrolimus have immunosuppresive effect with the adverse reactions incluiding nephrotoxicity, hepatotoxicity, and electrolyte imbalance in the body. We recommended that non-immunosuppresive calcineurin signal inhibitors might be more effective for treatment of endometriosis. Main limitations, this study was conducted in experimental rat model and we couldn’t evaluate cytokine and chemokine levels in both blood and endometriotic tissue. Conclusion The inhibition of CypA mediated calcineurin signaling with using CsA and tacrolimus treatment cause decreases of the cell proliferation and angiogenesis in the endometriotic tissue of rats. Declarations Acknowledgment: The study was supported by Scientific Investigations Foundation of Balikesir University (Project number: BAP.2018/145). This study was produced from the thesis of residency graduation of Cagla Bahar Bulbul, MD. Financial Disclosure: The authors have no connection to any companies or products mentioned in this article. Conflicts of interest: The authors declare there is no conflict of interest. Ethics approval: The study protocols was approved by Balikesir University animal research local ethics committee (Date: 25.06.2020, No:2020/4-14) before starting the study. This study was performed in Balikesir University Experimental Animals Laboratory. All of the experimental procedures and investigations were performed in complians with international guidelines on the ethical use of animals. Consent to participate: Not applicable Consent for publication: Not applicable Availability of data and material (data transparency): All data of this study was repositoried by corresponding author. If requested data is available. References Taylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. The Lancet. 2021;397:839–52. Macer ML, Taylor HS. Endometriosis and Infertility: A Review of the Pathogenesis and Treatment of Endometriosis-associated Infertility. Obstet Gynecol Clin North Am. 2012;39:535–49. Sampson JA. 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Lou J, Fatima N, Xiao Z, Stauffer S, Smythers G, Greenwald P, et al. Proteomic profiling identifies cyclooxygenase-2-independent global proteomic changes by celecoxib in colorectal cancer cells. Cancer Epidemiol Biomark Prev Publ Am Assoc Cancer Res Cosponsored Am Soc Prev Oncol. 2006;15:1598–606. Wong CS-C, Wong VW-K, Chan CM-L, Ma BB-Y, Hui EP, Wong MC-K, et al. Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Oncol Rep. 2008;20:89–98. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1930806","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":128674520,"identity":"a3ea722e-e8fc-431f-892e-f29bba111ed0","order_by":0,"name":"Cagla Bahar Bulbul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDCCAyCigCEBREkwMNgAKcbGA4S1GMC1pIG0NJCk5TBCEBfgu3344ccvBnZ5/LObD974uee83dr2w0BbamyicWmRPJdmLC1jkFwscedYsmXPs9vJ284kArUcS8ttwKHF4AwPg7SEAXNiw40cMwmeA7eTzQ4AtTA2HManhfm3hEF94vwb+d8k/xw4l2x2/iFBLWySHwwOJ264kcMmzXPggJ3ZDQK2SJ5hM7NmMDieuPFGmrG1zIHkBLMbQFsS8PiF7wzz45s/KqoT591IfnjzzQE7e7Pz6Q8ffKixwakFBJh5kDiJYJUJeJSDAOMPJI49AcWjYBSMglEwAgEA4uJpxwyJMf4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4128-499X","institution":"Balikesir Ataturk State Hospital","correspondingAuthor":true,"prefix":"","firstName":"Cagla","middleName":"Bahar","lastName":"Bulbul","suffix":""},{"id":128674521,"identity":"85fd6a04-a535-4bf8-b3ea-f522898eb58c","order_by":1,"name":"Gulay Turan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gulay","middleName":"","lastName":"Turan","suffix":""},{"id":128674522,"identity":"a1f357da-93c4-41dd-aed5-f18b5cbc404c","order_by":2,"name":"Ceyda Sancakli Usta","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ceyda","middleName":"Sancakli","lastName":"Usta","suffix":""},{"id":128674523,"identity":"fbe64055-9364-48ea-a509-40a02b530258","order_by":3,"name":"Ozgur Bulmus","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ozgur","middleName":"","lastName":"Bulmus","suffix":""},{"id":128674524,"identity":"0124e19a-19b8-444a-8d4d-6bbda71c4822","order_by":4,"name":"Akin Usta","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Akin","middleName":"","lastName":"Usta","suffix":""}],"badges":[],"createdAt":"2022-08-04 20:06:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1930806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1930806/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25327098,"identity":"5f9d1c62-6576-4622-aaad-01e7c706d702","added_by":"auto","created_at":"2022-08-17 15:16:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":804179,"visible":true,"origin":"","legend":"\u003cp\u003eA; Strong immunoreactivity of Ki-67 in the control group, B; Weak immunoreactivity of Ki-67 in cyclosporine A group\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1930806/v1/d600f9c0eeb904564d8317ca.png"},{"id":25327563,"identity":"e6ae81de-7e68-408c-935c-6d6ae7756e10","added_by":"auto","created_at":"2022-08-17 15:21:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":641668,"visible":true,"origin":"","legend":"\u003cp\u003eA; Strong immunoreactivity of Bcl-2 in the control group, B; Weak immunoreactivity of Bcl-2 in cyclosporine A group\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1930806/v1/781618399ec26b80b9e038b6.png"},{"id":25327099,"identity":"8cca3533-a49a-47a6-b042-0a94ecf2885c","added_by":"auto","created_at":"2022-08-17 15:16:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":660064,"visible":true,"origin":"","legend":"\u003cp\u003eA; Strong immunoreactivity of VEGF in the control group, B; Weak immunoreactivity of VEGF in cyclosporine A group\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1930806/v1/18b639fb947e5c1e0085f123.png"},{"id":25866592,"identity":"5b753f1b-e9fe-4262-9e2a-c82f92232e66","added_by":"auto","created_at":"2022-08-30 20:57:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2137855,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1930806/v1/742d33b5-31ee-4411-a4af-64570c85b381.pdf"}],"financialInterests":"","formattedTitle":"The effect of calcineurin signaling inhibition on the treatment of endometriosis in rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is defined as the presence and growth of endometrial-like tissue outside of the uterine cavity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is known to be a chronic inflammatory disease that frequently causes pelvic pain and female infertility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its prevalence is approximately 5%-10% in women of reproductive age worldwide.\u003c/p\u003e \u003cp\u003eIn the literature, several theories have been suggested to explain the development of endometriosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], although, the molecular and cellular mechanisms that underlie the condition are not yet fully understand. As a consequence, there is currently no consensus as to the most appropriate medical and/or surgical approaches (or combination of two) for the treatment of endometriosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCyclophilin A (CypA) is a member of the immunophilin family of proteins that are secreted from various types of cells in response to inflammatory stimuli such as hypoxia, oxidative stress, and infection. Prior studies have demonstrated increased tissue expression and/or higher blood levels of CypA in patients with inflammatory diseases, including asthma, rheumatoid arthritis, cardiovascular diseases, and various cancers [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the inflammatory condition, CypA plays a role in inflammatory cell migration and cytokine production. It also causes tissue destruction and cell invasion via the increased secretion of matrix metalloproteinases (MMPs) in the inflammatory areas [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These effects of CypA are mediated by the activation of calcineurin signaling [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Calcineurin, which is a Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e dependent serin/threonine protein kinase, plays an important role in relation to the inflammatory response and implantation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This kinase also plays a central role in the adaptive immune response through the activation of the nuclear factor of activated T cells (NFAT) and the nuclear factor kappa beta (NF-κB) transcription factors in several cell types [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Cyclosporine A (CsA) and tacrolimus (FK506) are calcineurin inhibitors that work according to a similar mechanism. The pharmacological effects of both these drugs are based on the inhibition of calcineurin through binding to cyclophilin A, especially in immune effector cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our previous study, we demonstrated the increased expression of CypA in both the eutopic and ectopic endometrial cells of patients with endometriosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, we found the CypA expression to be correlated with vascularity and endometrioma recurrence [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Prior cancer cell line and culture medium studies have shown that, the deficiency of CypA or CsA (or tacrolimus) treatment decrease tumor growth and increase the efficacy of chemotherapeutic agents in relation to the treatment of glioblastoma multiforme and endometrial cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, given the anti-inflammatory and immunomodulatory properties of CsA and tacrolimus, we investigated the added benefit of CsA and tacrolimus in terms of the treatment of endometriosis in an experimental rat model.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocols was approved by Balikesir University animal research local ethics committee (Date: 25.06.2020, No:2020/4-14) before starting the study. This study was performed in Balikesir University Experimental Animals Laboratory. All of the experimental procedures and investigations were performed in complians with international guidelines on the ethical use of animals. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of thirty-six female non-pregnant wistar albino rats 10-12 weeks old, weighted 250-300 g were used for induction of the experimental endometriosis model. The all rats were housed at 20\u0026plusmn;2\u003csup\u003eo\u003c/sup\u003eC room temperature during the adaptation and experimental procedure. The room with 12-hours light and 12-hours dark cycle with 50\u0026plusmn;10% humidity, the water and food add libitum. The animals were randomly classified into 3 groups (control group, cyclosporine A group, tacrolimus group). Before the surgical induction of endometriosis, the rats underwent daily vaginal lavages to detect the phase of estrus cycle. Vaginal secretions were examined under a light microscope to identify the estrus cycle by the dominancy of the anucleate cornified cells. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurgical procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental endometriosis was performed so that, the right uterine horn was removed, and a piece of the tissue was trimmed using microscissors. The removed endometrial tissue fragment was sutured into the abdominal wall of the same rat. One month after the first operation, second experiment was performed under anesthesia and the presence of endometrial foci was confirmed. During the procedure, endometriosis foci was measured and recorded. After the procedure, all rats were divided into three groups as Cyclosporin A (n=10), tacrolimus (n= 10) and control group (n=12). Cyclosporin A group was administered at 5mg/kg intraperitoneal and tacrolimus group was administered at 0,3mg/kg intravenous, total 2 doses at intervals of 2 weeks respectively. Saline infusion was given at 50 mg/kg with the same dose intervals by subcutaneous injection in control group. All studies lasted for 8 weeks. At the end of the treatment period all rats was sacrificed and eutopic and ectopic endometrial tissue were removed. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 28 days of medication in experimental group, the rats were euthanized wiht ketamine 10% and xylazine 2%, and a laparotomy was done. Ectopic endometrial tissue was isolated and measured three dimentions (lenght x width x height) using a digital meter. The macroscopic volume of the tissue was calculated with the prolated elipsoid formula which was descripted elsewhere [19]. For histopathological and immunohistochemical evaluation, all endometriotic tissue samples were fixed in 10% formalin and then with phosphate buffered saline overnight, and stored in 70% EtOH until processing. Processed endometrial tissues will cut at the middle of the tissue samples and embedded into paraffin. Histological sections (5 \u0026mu;m) were stained with Ki-67 (anti-human Ki-67 Antibody DAKO, California, USA), Bcl-2 (Bcl-2 Antibody (C-2): sc-7382, Santa Cruz Biotechnology, California, USA), Caspase-3 (Anti-Caspase-3 Antibody, Rabbit Polyclonal, Sino Biological, Houston, USA) and VEGF (Anti-VEGF183/VEGFA antibody, Sino Biological, Pennsylvania, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 5 \u0026micro;m thick endometriotic tissue sections were obtained and stained in hematoxylin eosin. All samples were visualized under light microscopy (Olympus BX48, Tokyo, Japan) and evaluated according to semi-quantitative method described previously (Nis Elements Advantages Research Microscope Imaging Software; Nikon Instruments Europe BV, Amsterdam, Netherlands). All histopathological and immunohistochemical evaluation was done same histopathologist and during the evaluation the histopathologist was blinded. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Ki-67, Bcl-2, Caspase-3 and VEGF Immunoreactivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of Ki-67, Bcl-2, Caspase-3 and VEGF in the all tissue samples was compared according to IHC staining intensity in all tissue samples.\u0026nbsp;Ki-67 and Caspase-3 immunoreactivity were predominantly localized in the nucleus of endometriotic cells. In contrast, the Bcl-2 immunoreactivity was predominantly localized in intracytoplasmic and cell membrane surface in the same cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe degree of positive staining for Ki-67 and\u0026nbsp;Caspase-3\u0026nbsp;was calculated as the ratio of positively stained endometriotic cells to total endometriotic cells at \u0026times;\u0026thinsp;400 magnification in the selected field. Any nuclear staining was regarded as positive.\u003c/p\u003e\n\u003cp\u003eA semiquantitative scoring system was used to evaluate the degree of Bcl-2 and VEGF immunoreactivity. Brown-color staining in the cell membrane surface of the endometriotic cells revealed the presence of Bcl-2 expression. Brown-color staining in the cytoplasm of the cells revealed the presence of VEGF expression. Degree of positive staining of Bcl-2 and VEGF was scored as 0, negative; 1, weak positive; 2, moderate positive; 3, strongly positive. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the statistical analyses were performed using MedCalc statistical package for medical sciences (MedCalc Inc., Belgium) version 19.2. All data were presented as mean\u0026plusmn;SD or proportion as appropriate. The parametric data were compared between study groups using one-way analysis of variance (ANOVA) with post-hoc tests for subgroup analyses. Non-parametric data were compared using Kruskal Wallis one-way analysis of variance. Independent t test was used to compare the parametric variables between two study groups. A two-sided p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 36 rats was randomly divided into the 3 study group, 4 of the rats were exculuded from the study for anestesia complications. Finally, 32 rats were included the study and all of them were divided as control (n=12), Cyclosporine A (n = 12) and tacrolimus (n = 10). There was no significant differences between the baseline characteristics of the groups (Table 1). After the treatment the size of endometriotic focus were 204.7\u0026plusmn;153.4 mm\u003csup\u003e3\u003c/sup\u003e in controls, 71.9\u0026plusmn;85.4 mm\u003csup\u003e3\u003c/sup\u003e in cyclosporine A and 30.6\u0026plusmn;36.7 mm\u003csup\u003e3\u003c/sup\u003e in tacrolimus groups. Compared the control group the macroscopic sizes of endometriotic focus were lower in both Cyclosporin A and tacrolimus group (p=0.002). Microscopically Ki-67 and VEGF immunoreactivity were lower in Cyclosporin A and tacrolimus groups than controls (p=0.010 and p=0.007, respectively). \u0026nbsp; Moreover, Bcl-2 immunoreactivity was lower in both Cyclosporine A and tacrolimus groups, however this differences did not reach statistical significance (p = 0.056). There was no significant differences in Caspase-3 immunoreactivity between the groups (p = 0.178). Our subgroup analysis was demonstrated that there was no significant differences in Ki-67, Bcl-2, Caspase-3 and VEGF immunoreactivity between cyclosporine A and tacrolimus groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1: Pre-treatment and post-treatment endometriotic implant volume of the groups and immunohistochemical evaluation results.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.71153846153846%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.07051282051282%\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.275641025641026%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTacrolimus Group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.51923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCyclosporin A Group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.423076923076923%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP Values*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.71153846153846%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePre-treatment endometriotic implant volume\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.07051282051282%\"\u003e\n \u003cp\u003e165.3\u0026plusmn;129.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.275641025641026%\"\u003e\n \u003cp\u003e213.6\u0026plusmn;166.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.51923076923077%\"\u003e\n \u003cp\u003e214.8\u0026plusmn;193.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.423076923076923%\"\u003e\n \u003cp\u003e0.716\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.71153846153846%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePost-treatment endometriotic implant volume\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.07051282051282%\"\u003e\n \u003cp\u003e\u003cstrong\u003e204.7\u0026plusmn;153.4\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.275641025641026%\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.6\u0026plusmn;36.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.51923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003e71.9\u0026plusmn;85.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.423076923076923%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.71153846153846%\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi-67, mean\u003c/strong\u003e\u0026plusmn;\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.07051282051282%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.8\u0026plusmn;0.7\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.275641025641026%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.1\u0026plusmn;0.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.51923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.0\u0026plusmn;0.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.423076923076923%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.010\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.71153846153846%\"\u003e\n \u003cp\u003eBcl-2 \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.07051282051282%\"\u003e\n \u003cp\u003e1.8\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.275641025641026%\"\u003e\n \u003cp\u003e1.2\u0026plusmn;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.51923076923077%\"\u003e\n \u003cp\u003e1.1\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.423076923076923%\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.71153846153846%\"\u003e\n \u003cp\u003eCaspase-3 \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.07051282051282%\"\u003e\n \u003cp\u003e1.6\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.275641025641026%\"\u003e\n \u003cp\u003e2.1\u0026plusmn;0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.51923076923077%\"\u003e\n \u003cp\u003e2.2\u0026plusmn;0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.423076923076923%\"\u003e\n \u003cp\u003e0.178\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.71153846153846%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVEGF \u0026nbsp;mean\u003c/strong\u003e\u0026plusmn;\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.07051282051282%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.9\u0026plusmn;0.7\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.275641025641026%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.1\u0026plusmn;0.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.51923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.0\u0026plusmn;0.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.423076923076923%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* ANOVA\u003c/p\u003e\n \u003cp\u003eBcl-2 (B-cell lymphoma 2)\u003c/p\u003e\n \u003cp\u003eVEGF (Vascular Endothelial Growth Factor)\u003c/p\u003e\n\u003cp\u003ea: Group 1 is statistically different from Groups 2 and 3 (p\u0026lt; 0.05)\u003c/p\u003e\n \u003cp\u003eb: Group 1 is statistically different from Groups 2 and 3 (p\u0026lt; 0.05)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this experimental study, we observed that CsA and tacrolimus treatment were equally capable of significantly reducing the size of endometriosis. We also found that they decreased the histological grade (as demonstrated by the Ki-67 and VEGF expression) in endometriotic implants in rats when compared with the control group. To the best of our knowledge, this is the first study to evaluate the effects of CsA and tacrolimus mediated calcineurin signaling inhibition with regard to the treatment of endometriosis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCsA and tacrolimus are two well-known immunosuppressive drugs with the similar therapeutic mechanisms. Both drugs work by binding to CypA and inhibiting calcineurin signaling. Importantly, calcineurin signaling plays a central role in the adaptive immune response and the activation of calcineurin is dependent CypA that is on the calcineurin receptore. CypA mediates the action of these immunosuppressant drugs via peptidyl-prolyl cis-trans isomerases (PPIases)\u0026nbsp;[20,21]. Once CsA and tacrolimus bind to their respective proteins, PPIase activity is abolished\u0026nbsp;[20]. The effect of CsA and tacrolimus become active by forming complexes with the PPIases in the body. These complexes then bind to calcineurin and inhibit its phosphatase activity. The inhibitory effects of these drugs in relation to calcineurin work by competitively binding to and inhibiting Ca\u003csup\u003e+2\u003c/sup\u003e and calmodulin-dependent phosphatase calcineurin [22]. According to the findings of the present study, the inhibition of calcineurin signaling by means of CsA and tacrolimus treatment causes the regression of endometriotic tissue, leading to decreased cell proliferation and angiogenesis in the endometriotic tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the inflammatory condition, the secretion of CypA exacerbates the inflammatory response in many different benign and malign diseases, such as atherosclerosis, asthma, rheumatoid arthritis, diabetes mellitus, and lung and endometrial cancer [6\u0026ndash;12]. In our previous study, we clearly demonstrated that women with endometriosis exhibited increased expression of CypA in both their eutopic and ectopic endometrial tissues when compared with control subjects. Moreover, CypA expression has been found to be correlated with the vascularity of endometriotic tissue [16]. In the present study, we showed that the inhibition of CypA-mediated calcineurin signaling caused the regression of the experimental endometriotic tissues in rats. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEndometriosis is characterized by the presence and growth of the functional endometrial glands and/or stromal tissue in the abnormal region outside of the uterine cavity. \u0026nbsp;The condition has a multifunctional etiology and prior studies have demonstrated that various inflammatory and immunological biomarkers play an important role in its pathogenesis.\u003c/p\u003e\n\u003cp\u003eThe expression of proinflammatory cytokines and chemokines is increased in response to the presence of ectopic endometriotic tissue. Previous studies have shown that the expression of IL-1\u0026beta;, TNF- \u0026alpha;, IL-6, IL-8 and VEGF is increased in the blood samples, pelvic peritoneum and endometriotic tissue samples of patients with endometriosis [23,24]. Similarly, prior studies have demonstrated that the activation of calcineurin signaling causes the upregulation of proinflammatory cytokine and chemokine including TNF- \u0026alpha;, IL-6 by the activation of NF-\u0026kappa;B [25]. In addition, blocking of calcineurin signaling by the calcineurin inhibitors decreases the TNF-\u0026alpha; and IL-6 [25]. According to the present results, the CsA and tacrolimus-mediated inhibition of calcineurin signaling may lead to a decrease in proinflammatory cytokines and chemokine production. This hypothese should be checked in the future studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrior studies have clearly shown that calcineurin acts through the activation of the NF-\u0026kappa;B and NFAT transcription factors.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMore specifically, the activation of the calcineurin/NFAT signaling pathway by angiotensin II increases the endometrial stromal cell proliferation and blastocyst implantation via the induction of cyclooxygenase-2 gen expression in rats [26]. On the other hand, the activation of NF-\u0026kappa;B in macrophages releases proinflammatory cytokines in endometriotic cells, which promotes cytokine production in ectopic endometriotic cells, thereby causing an increase in the synthesis of antiapoptotic factors, preventing apoptosis in endometrial cells, and contributing to endometriotic cell survival [27]. In a recent study that sought to investigate the pathway leading to the activation of NF-\u0026kappa;B, the authors found that MAPKs, ERK1/2, JNK, and p38 were all activated by CypA treatment. However, only the ERK inhibitor PD98059 was able to decrease the activation of NF-\u0026kappa;B and attenuate the MMP-9 activity induced by CypA [28]. Additionally, previous animal model studies have shown that, CsA inhibits endometrial stromal cell proliferation via abolishing calcineurin/NFAT dephosphorylation and translocation to the nucleus [26]. Recent cancer cell line and culture medium studies have shown that the overexpression of CypA associated with cancer cells proliferation, cell cycles progression, cell invasion, cell migration and inhibition of cell apoptosis [8,29,30]. Interestingly, presence of CypA causes resistance to chemotherapeutic drugs. \u0026nbsp;Some recent studies have demonstrated that medical treatments with anti-cancer agents such as 5-aza-2-deoxycytidine, celecoxib, 5-fluorouracil, and paclitaxel are related to decreases in CypA expression within cancer cells [18,31\u0026ndash;33]. However, the knockdown of CypA or CsA has been shown to increase the chemotherapeutic effect of cisplatin and paclitaxel in the treatment of glioblastoma multiforme and endometrial cancer [17,18].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, it is known that CsA and tacrolimus have immunosuppresive effect with the adverse reactions incluiding nephrotoxicity, hepatotoxicity, and electrolyte imbalance in the body. We recommended that non-immunosuppresive calcineurin signal inhibitors might be more effective for treatment of endometriosis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMain limitations, this study was conducted in experimental rat model and we couldn\u0026rsquo;t evaluate cytokine and chemokine levels in both blood and endometriotic tissue.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe inhibition of CypA mediated calcineurin signaling with using CsA and tacrolimus treatment cause decreases of the cell proliferation and angiogenesis in the endometriotic tissue of rats.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003eThe study was supported by Scientific Investigations Foundation of Balikesir University (Project number: BAP.2018/145). This study was produced from the thesis of residency graduation of Cagla Bahar Bulbul, MD. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial Disclosure:\u003c/strong\u003e The authors have no connection to any companies or products mentioned in this article.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare there is no conflict of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThe study protocols was approved by Balikesir University animal research local ethics committee (Date: 25.06.2020, No:2020/4-14) before starting the study. This study was performed in Balikesir University Experimental Animals Laboratory. All of the experimental procedures and investigations were performed in complians with international guidelines on the ethical use of animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material (data transparency):\u0026nbsp;\u003c/strong\u003eAll data of this study was repositoried by corresponding author. If requested data is available.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTaylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. The Lancet. 2021;397:839\u0026ndash;52. \u003c/li\u003e\n\u003cli\u003eMacer ML, Taylor HS. 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Calcineurin, Calcium-Dependent Serine-Threonine Phosphatase Activation by Prion Peptide 106\u0026ndash;126 Enhances Nuclear Factor-\u0026kappa;B-Linked Proinflammatory Response through Autophagy Pathway. ACS Chem Neurosci. American Chemical Society; 2021;12:3277\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eAbraham F, Sacerdoti F, Le\u0026oacute;n RD, Gentile T, Canellada A. Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells. PLOS ONE. Public Library of Science; 2012;7:e37750. \u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez-Ramos R, Van Langendonckt A, Defr\u0026egrave;re S, Lousse J-C, Colette S, Devoto L, et al. Involvement of the nuclear factor-\u0026kappa;B pathway in the pathogenesis of endometriosis. Fertil Steril. 2010;94:1985\u0026ndash;94. \u003c/li\u003e\n\u003cli\u003eJin Z-G, Lungu AO, Xie L, Wang M, Wong C, Berk BC. 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Proteomic profiling identifies cyclooxygenase-2-independent global proteomic changes by celecoxib in colorectal cancer cells. Cancer Epidemiol Biomark Prev Publ Am Assoc Cancer Res Cosponsored Am Soc Prev Oncol. 2006;15:1598\u0026ndash;606. \u003c/li\u003e\n\u003cli\u003eWong CS-C, Wong VW-K, Chan CM-L, Ma BB-Y, Hui EP, Wong MC-K, et al. Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Oncol Rep. 2008;20:89\u0026ndash;98. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, Cyclophilin A, Cyclosporin A, Tacrolimus, Rat, Calcineurin","lastPublishedDoi":"10.21203/rs.3.rs-1930806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1930806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe molecular and cellular mechanism underlying endometriosis is still under investigation. CypA is one of the inflammatory marker which secreted various type of cell in the inflammatory condition. During the inflammation, CypA exacerbate inflammatory response by the activation of calcineurin signaling that increases the cytokine secretion and tissue degradation in inflammatory region. The aim of this study was to investigate the effect of calcineurin signaling inhibition on the treatment endometriosis in rats.\u003c/p\u003e\u003ch2\u003eMaterials \u0026amp; Methods\u003c/h2\u003e \u003cp\u003eIn the present study, 32 albino-wistar rat were used. All rats were divided into three groups as Cyclosporin A (n\u0026thinsp;=\u0026thinsp;10), tacrolimus (n\u0026thinsp;=\u0026thinsp;10) and control group (n\u0026thinsp;=\u0026thinsp;12). Cyclosporin A group was administered intraperitoneal and tacrolimus group was administered intravenous, total 2 doses at intervals of 2 weeks respectively. All studies lasted for 8 weeks. Processed endometrial tissues will cut at the middle of the tissue samples and embedded into paraffin. Histological sections (5 \u0026micro;m) were stained with Ki-67, Bcl-2, Caspase-3 and VEGF.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe size of endometriotic focus were 204.7\u0026thinsp;\u0026plusmn;\u0026thinsp;153.4 mm\u003csup\u003e3\u003c/sup\u003e, 71.9\u0026thinsp;\u0026plusmn;\u0026thinsp;85.4 mm\u003csup\u003e3\u003c/sup\u003e ve 30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;36.7 mm\u003csup\u003e3\u003c/sup\u003e in control, in CsA and in tacrolimus groups. Compared the control group the size of endometriotic focus was lower in CsA and tacrolimus group (p\u0026thinsp;=\u0026thinsp;0.002). Microscopically, Ki-67 (p\u0026thinsp;=\u0026thinsp;0.010) and VEGF (p\u0026thinsp;=\u0026thinsp;0.007) immunoreactivity were lower in CsA and tacrolimus group than controls.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe inhibition of calcineurin signaling with CsA and tacrolimus treatment causes regression of endometriotic focus via decreases of endometriotic cell proliferation and angiogenesis in ectopic endometriotic tissue.\u003c/p\u003e","manuscriptTitle":"The effect of calcineurin signaling inhibition on the treatment of endometriosis in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-17 15:16:46","doi":"10.21203/rs.3.rs-1930806/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6c9ceef7-d75f-4e7b-96e0-011b675df18d","owner":[],"postedDate":"August 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-30T20:57:35+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-17 15:16:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1930806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1930806","identity":"rs-1930806","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}