Abstract
Objective:
Endometriosis is an estrogen-dependent chronic inflammatory disease observed in reproductive period. The aim of the present study is to assess the efficacy of colchicine, widely used to treat many inflammatory diseases, in an experimental rat endometriosis model.
Study Design:
Experimental endometriosis was constituted with implantation of autogenous endometrial tissue. Rats were divided randomly into 2 groups as colchicine group (n = 8) and control group (n =8). Although oral 0.1 mg/kg colchicine was administered 4 weeks to the colchicine group, the same amount of saline solution was administered to the control group. Before and after 30 days of treatment period, peritoneal and tissue tumor necrosis factor α (TNF-α), the volumes and histopathological properties of the implants were evaluated.
Results:
Although the implant volume decreased significantly in the colchicine group (89.2 ± 13.4 mm3 to 35.2 ± 4.5 mm3, P < .05), the implant volume increased in the control group (85.1 ± 14.2 mm3 to 110.3 ± 10.5 mm3, P < .05). When compared to the control group, the colchicine group had significantly lower histopathologic sores (1.4 ± 0.2 vs 2.6 ± 0.4, P < .001). Although peritoneal fluid TNF-α levels were significantly decreased in the colchicine group (45.2 ± 5.3 pg/mL vs 12.1 ± 5.2 pg/mL, P < .001), the peritoneal fluid TNF-α levels were significantly increased in the control group after the treatment (44.2 ± 3.5 pg/mL vs 61.3 ± 12.2 pg/mL; P < .001). Tissue TNF-α levels were significantly lower in the colchicine group when compared to the control group (45.4 ± 8.6 pg/mL vs 71.3 ± 11.2 pg/mL; P < .001).
Conclusion:
Colchicine resulted in regression of endometrial implant volumes in experimental rat endometriosis model and decreased peritoneal and tissue TNF-α levels.
Introduction
Endometriosis is defined as the presence of endometrial gland and stroma in extrauterine sites. 1 Although ectopic endometrial focuses are generally localized in pelvis, they could be present anywhere in the body. Endometriosis is an estrogen-dependent, benign, and chronic disease that is commonly seen during active reproductive period. 2,3 It might be asymptomatic but also it might cause chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility symptoms. 4 As endometriosis might be asymptomatic, its incidence in general population is not exactly known but it is estimated as 10%. 5 Its incidence increases to 30% to 50% in patients who have infertility. 6,7 Symptom-targeted treatment modalities such as surgery, medical treatment, and assisted reproductive technology are being applied in the management of endometriosis. 8,9 The disease could reoccur or ovarian functions might be affected negatively following these methods. Although the necessity of nonhormonal medical treatment has been suggested in prevention and treatment of endometriosis and associated symptoms, there is no such treatment option yet. 10
Up to the present, the pathophysiology of endometriosis has not been clarified exactly but retrograde menstruation, changed immunity, coelemic metaplasia, and metastatic spread were supported. 11 –14 The role of changed cellular and humoral immunity in development of endometriosis has been demonstrated. 15 Tumor necrosis factor α (TNF-α) secreted from active macrophages modulates the secretion of other cytokines and plays a crucial role in pathogenesis of endometriosis. 16
Colchicine is an alkaloid extracted from the plant of Colchicum autumnale, and for long years colchicine is being used in treatment of inflammatory diseases such as gout, pseudo-gout, Behcet syndrome, and familial Mediterranean fever. 17,18 Although the exact mechanism of anti-inflammatory effect of colchicine is not known, it provides cytokine modulation and decreases TNF-α-mediated immune response. 19,20
The aim of the present study is to investigate the efficacy of colchicine, which is used in treatment of many diseases due to its anti-inflammatory properties, in experimental rat endometriosis model.
Materials and Methods
Rats
The study was performed after obtaining ethical committee approval from Mustafa Kemal University Animal Laboratory Ethical Committee. Twenty Wistar albino adult female rats with weight of 200 to 250 g were included in the study. Rats were caged individually in a controlled environment with 12-hour light–dark cycles, at 20°C to 22°C room temperature with 50% to 55% relative humidity and were fed ad libitum. For adaptation to the environment, they were waited for 10 days (acclimatize). By vaginal smears, it was determined that the animals have normal estrous cycles. All operations were performed in the rats at estrous phase. In all procedures that were applied to the animals, local ethical committee laboratory rules and rules of Guidelines for the Care and Use of Laboratory Animals of the US National Institutes of Health (Washington, District of Coumbia) were obeyed.
Rats were weighed before the operation and intramuscular ketamine hydrochloride (50 mg/kg Ketalar; Eczacıbasi, İstanbul, Turkey) and xylazine hydrochloride (10 mg/kg Rompun; Bayer Türk İlac Ltd, İstanbul, Turkey) were used for anesthesia. Endometriosis was induced surgically using the method described by Vernon and Wilson during estrous phase. 21 After perp and drep of the skin, a midline incision was made to enter the abdominal cavity. A 0.5 × 0.5 × 0.1-cm piece excised by micro scissors from the right uterine horn was attached to the peritoneum only on the right side of the ventral abdominal wall close to an artery via the surgical autotransplantation technique using 5/0 vicryl polyglactin 910 (Ethicon Ltd, Sommerville, New Jersey). Serosal side of the uterus was attached directly to the peritoneum as endometrial side was always facing the abdominal cavity. 22 The rats were individually caged after the operation and were left for a recovery period. After 4 weeks, their daily vaginal smears were monitored, and a second laparotomy was performed in their estrus phase to determine the attachment and viability of endometrial implants. Of the 20 experimental rats, 4 did not develop any signs of endometriosis, and therefore, these were excluded from the study. Rats were divided randomly into 2 groups: group 1 (colchicine) or the experimental group (n = 8) and group 2 or the control group (n =8). The pretreatment implant volumes in each group were calculated by measuring their dimensions (length, width, and height, in millimeters). For volume calculations, the ellipsoid volume formula (0.52 × length × width × height) was used. Abdomen was washed with saline solution (%0.9 NaCl), and peritoneal fluid was taken and immediately frozen at −80°C and stored. Following the second laparotomy colchicine (Sigma Chemical Co, St Louis, Missouri), 0.1 mg/kg was given orally for 4 weeks to the first group. The same amount of saline was given by gavage to the second group. After 4 weeks of colchicine and placebo treatment, the rats were weighed and third laparotomy was performed. Abdomen was washed with saline solution and peritoneal fluid was taken. The sizes of endometriotic implants were measured again, and volume was calculated. Endometriotic implants were excised, and rats were killed under ketamine anesthesia. Some of the endometriotic implants were fixed in formalin 10% for histological examination and some were stored at −80°C for biochemical analysis.
Histological Evaluation
Tissue samples were fixed in 10% buffered formalin solution for 24 hours. After fixation, a routine tissue-processing procedure was performed, and the sampled tissues were embedded in paraffin. Paraffin wax blocks were cut in 5-μm thickness. Prepared sections were stained with hematoxylin and eosin. Five hematoxylin and eosin sections were examined per autograft. Slides were examined under a light microscope. The pathologist assessing the treatment effects was blinded to the treatment groups.
The histologic diagnosis was based on the morphologic identification of endometrial glandular tissue and stroma: glands and stroma of the endometrial type, with epithelial lining and luminal formation. The persistence of epithelial cells in endometrial autografts was evaluated semiquantitatively. The pathologic evaluation of the uterine autografts was performed according to a previously published method as follows: a well-preserved epithelial layer = score 3, a moderately preserved epithelium with leukocyte infiltrate = score 2, a poorly preserved epithelium = score 1, and no epithelium = score 0. 23
Biochemical Analysis
Peritoneal fluid and tissue TNF-α levels were measured using the enzyme-linked immunosorbent assay according to manufacturer’s instructions (Wuhan EIAab Science Co, Ltd, China). The TNF-α concentration were presented as pg/mL protein.
Statistics
Continuous variables were expressed as mean ± standard deviation. The differences between numeric variables were evaluated by Kruskal-Wallis test, and Mann-Whitney U test was utilized for post hoc analysis. In all statistical analyses, P < .05 was recognized as statistically significant. We conducted our statistical analyses with SPSS 20.0 (SPSS Inc, Chicago, Illinois) package program.
Results
No statistically significant change was observed in rat weights before and after treatment in colchicine (230.4 ± 11.2 g and 221.3 ± 9.8 g, respectively, P > .05) and control groups (234.2 ± 9.4 g and 231.3 ± 9.23 g, respectively, P > .05). There was no statistically significant difference in pretreatment implant volumes between the colchicine and the treatment groups (89.2 ± 13.4 mm3 and 85.1 ± 14.2 mm3, respectively, P > .05; Table 1). Posttreatment implant volumes in the colchicine group were significantly less than the control group (35.2 ± 4.5 mm3 and 110.3 ± 10.5 mm3, respectively; Figure 1).
Comparison of Implant Volumes, Histopathologic Scores, and Tissue TNF-α Levels Between Colchicine and Control Groups.
Abbreviations: NS, not significant; TNF-α, tumor necrosis factor α.
Implant volumes before and after treatment.
Although the posttreatment implant volumes significantly decreased according to the pretreatment sizes in the colchicine group (89.2 ± 13.4 mm3 vs 35.2 ± 4.5 mm3, P < .05), it was increased in the control group (85.1 ± 14.2 mm3 vs 110.3 ± 10.5 mm3, P < .05; Figure 2). Histopathological scoring values in the colchicine group were significantly lower than the control group (1.4 ± 0.2 vs 2.6 ± 0.4, P < .001; Figure 3).
Histopathologic pictures of endometriotic implants in control and colchicine groups.
Histological scores of experimental and control groups.
Tissue TNF-α levels in the colchicine group were significantly lower than the control group (45.4 ± 8.6 pg/mL vs 71.3 ± 11.2 pg/mL; P < .001; Figure 4). The TNF-α levels in peritoneal fluid after treatment significantly decreased when compared with the pretreatment levels in the colchicine group (45.2 ± 5.3 pg/mL vs 12.1 ± 5.2 pg/mL P < .001; Figure 5). Whereas in the control group, TNF-α levels in peritoneal fluid after the treatment significantly increased when compared with the pretreatment levels (44.2 ± 3.5 pg/mL vs 61.3 ± 12.2 pg/mL P < .001; Figure 6 and Table 2).
Tissue tumor necrosis factor α (TNF-α) levels in experimental and control groups.
Peritoneal tumor necrosis factor α (TNF-α) levels in experimental group before and after treatment.
Peritoneal tumor necrosis factor α (TNF-α) levels in control group before and after treatment.
Peritoneal TNF-α Levels Before and After Treatment in Groups.
Abbreviation: TNF-α, tumor necrosis factor α.
Discussion
The present study results revealed that colchicine has regressed significantly endometriotic implant sizes and it also decreased histopathological scoring values when compared with the control group. Furthermore, TNF-α levels in endometrial implant tissue were found to be significantly decreased in the colchicine group when compared with the control group. When compared with the pretreatment values, peritoneal TNF-α levels were found to be significantly decreased after colchicine treatment. In the present study, for the first time in the literature, colchicine, an anti-inflammatory drug, has macroscopically decreased endometrial implants and provided improvement in histopathological and biochemical values in rat endometriosis model.
Endometriosis is accepted as a chronic inflammatory disease. 24 Activated macrophages, inflammatory cytokines, chemokines, and prostaglandins are increased in peritoneal fluid of endometriosis. 25 Decreased cytotoxic T cells and natural killer cell activity are observed in peritoneal fluid of the patients with endometriosis. 26 Increased levels of interleukin (IL) 1, IL-6, IL-8, IL-18, and TNF-α were detected during the inflammatory process of endometriosis. 27,28 Activated macrophages, endometriotic lesions, and peritoneal mesothelial cells secrete TNF-α. 16 The TNF-α is a cytokine with a key role in many inflammatory processes as well as in endometriosis, and its primary function is to initiate a cascade of cytokines and other inflammatory factors. 23 A relation between TNF-α levels and the endometriosis severity has been established. 29 In the current study, we assessed TNF-α levels that have important role in the pathogenesis of endometriosis. We observed that TNF-α levels of endometriosis tissue in the colchicine group were significantly lower than the control group. Besides, posttreatment peritoneal TNF-α level was found to be decreased significantly in the colchicine group according to the pretreatment levels. However, an increase was observed in peritoneal TNF-α levels in the control group.
For many years, colchicine has been used in treatment of inflammatory diseases such as gout, pseudo-gout, Behcet syndrome, and familial Mediterranean fever. 17,18 Colchicine binds tubulin and forms colchicine–tubulin complex. 30 This complex causes modulation in microtubule dynamics which plays role in cell division, signal transduction, gene expression, migration, and secretion. 31 It is supposed that most of the anti-inflammatory effect of colchicine occurs by disturbing microtubule functions. Colchicine inhibits rolling, adhesion, and cytokine secretory functions of leukocytes. 20 Besides, colchicine decreases the chemotactic response of neutrophils to leukotriene B4 and IL-8 and TNF-α-mediated immune response in macrophages. 32 According to the results of our experiment, we propose that the main effect of colchicine on experimental rat endometriosis model is its inhibition of TNF-α mediated immune response.
To the best of our knowledge, this is the first study investigating the effects of colchicine in the treatment of endometriosis in a rat experimental model, but there may be some limitations to perform this treatment in a similar manner in humans. As the mechanism of anti-inflammatory effect of colchicine isn’t known exactly, in the present study, only TNF-α levels were evaluated and other cytokines were not assessed. One of the disadvantages of our study is that rat’s estrogen levels are much lower than that in humans, and there may be some limitations while extrapolation of the results from this model to humans. 33 Also, the immunologic properties of species are different, and this may change the efficacy of the treatment in humans. In addition, the colchicine dosage administered in the present study might be accepted as a limitation. The equivalent human dose that was used for the rats (0.1 mg/kg/d) in the present study would be very high in humans. Moreover, the rats’ ovaries and eutopic endometrium were not assessed for any detrimental effect from the colchicine in our study, which might be subject of the future studies.
Up to the present, many immunomodulator drugs such as imiquimod, levamizole, and etanercept were tried in treatment of rat endometriosis models. 23,34,35 As a result of these treatments, a decrease in endometrial implant volume has been achieved, but in all studies, it was emphasized that there is need of less toxic treatment modalities. It is also shown that TNF-α blocker drugs may cause infection and cancer by disturbing the host defense mechanism. 36 Colchicine is currently being used confidently in many autoimmune diseases. Besides, there is no increase in anomaly risk due to colchicine in pregnancy that might develop during treatment. 37
In conclusion, the findings of the present study showed that endometriosis regressed in size and also peritoneal and serum TNF-α levels of endometriotic implants were decreased in the group treated with colchicine in rats. When compared with TNF-α blocker drugs, colchicine is promising in treatment of endometriosis as it has few side effects and as it is used in many inflammatory diseases nowadays. There is need of detailed and clinical data to investigate the effects of colchicine on endometriosis.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.