Resveratrol successfully treats experimental endometriosis through modulation of oxidative stress and lipid peroxidation

Endometriosis is a chronic disease with an unknown etiology, characterized with the presence of the endometrial gland and stroma out of the endometrial cavity.[] It is mainly a disease of the women of reproductive age and its estimated incidence is 10%.[] The current consensus is that endometriosis is a local pelvic inflammatory process with altered function of immune-related cells in the peritoneal environment. Supporting this concept are recent studies suggesting that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages that secrete various local products, such as growth factors, cytokines and possibly free oxygen radicals.[] A diverse group of agents is used in the medical treatment of endometriosis, however none of these agents have a marked effect upon the disease progress, thus, there is a need to explore new treatment modalities.[] The new treatment options, such as inhibitors of the arachidonic acid pathway, antioxidant vitamins, proteasome inhibitors (Bortezomib) and nuclear factor-kappa B (NF-kB) inhibitors (Dithiocarbamates) are being investigated for the treatment of endometriosis.[] Resveratrol (trans-3, 40, 5-trihydroxystilbene), a natural polyphenolic, non-flavonoid antioxidant, is a phytoalexin found in various food products with particularly high levels in grape skin (50-100 μg/g) and red wine (1.52 mg/l).[] Resveratrol has been shown to have significant anti-inflammatory, antioxidant and immunomodulatory properties.[] The anti-inflammatory effects of resveratrol have been demonstrated by suppression of production of reactive oxygen species (ROS) and inhibition of cyclooxygenase-2 (COX-2) expression and prostaglandin (PG) synthesis in various experimental models.[] On the basis of this background, as measuring of the oxidative stress parameters by biochemical methods and histopathological and immunohistochemical scores, we aimed to investigate the in vivo effects of resveratrol given in different doses on the development of endometriotic lesions in an experimental rat model.

This study was carried out in the Experimental Research Laboratory of the University, complying with the approval of the ethics committee and the guidelines for the care and use of experimental animals. Thirty-five adult female Wistar rats, each weighing between 300 and 350 g, were purchased from the University Animal Laboratory. All rats were examined by a veterinarian and determined to be in good health. The rats were housed in plastic cages, and they were kept under standard conditions: 12-h light and 12-h dark periods, 20°C constant temperatures, and a humidity range between 40% and 60%. The rats had free access to standard dry pellets ad libitum and tap water throughout the study. Before transplantation, all animals were hormonally synchronized in their four-day estrus phase to exclude differences in steroid synthesis, cell adhesion and growth, and thus, endometriosis development between the individual animals owing to hormonal variations. Synchronization was performed by administering two subcutaneous injections (55 mg/kg body weight estradiol) with a 24-h intermission, followed by one injection (7.5 mg/kg body weight progesterone) 20 h after the last estradiol injection.[] Daily vaginal smears of the rats were taken to establish the estrus cycle of each animal. Behavioral estrus occurred 4 h after the injection of progesterone. Vaginal smears were taken by cotton swab: The swab was inserted into the vagina and rotated 360° clockwise; then the swab was smeared onto a glass slide and fixed with ethanol. Smears were stained with the usual Papanicolau method and then evaluated by light microscopy by a cytopathologist who was unaware of the groups. The estrus cycle was determined as follows: Proestrus period (many centrally nucleolated epithelial cells), estrus period (cornified epithelial cells without nuclei), metestrus period (leukocytes, mucus and a few cornified cells), diestrus period (various epithelial cells, mucus and leukocytes). Rats were observed for at least two successive four-day estrus cycles. Endometriosis was induced surgically using the method described by Vernon and Wilson during estrus.[] Rats were divided randomly into two groups: Group A or the experimental group (n = 27) and Group B or the sham group (n = 8). All rats were anesthetized intraperitoneally (i.p.) with 20-30 mg/kg ketamine hydrochloride (Rotex, Germany). 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 auto-transplantation technique. For the sham group (n = 8), 4-0 nylon sutures, with or without fat tissues, were attached to the peritoneum, except in the auto-transplantation of endometriotic implants. The rats were individually caged after the operation and were left for a recovery period. After three weeks, their daily vaginal smears were monitored, and a second laparatomy was performed in their estrus phase to determine the attachment and viability of endometrial implants. Of the 27 experimental rats, three did not develop any signs of endometriosis, and therefore, these were excluded from the study. In the sham group, the fat tissues showed no growth. 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 (P/6 × length × width × height) was used.[] The 24 rats with endometriosis were randomized (using random number tables) into three groups: (1) Control group (n = 8), (2) low dose resveratrol group (n = 8) and (3) high dose resveratrol group (n = 8). In the first group, a daily dose of 0.2 ml dimethyl sulfoxide (DMSO, Sigma Aldrich, Saint-Quentin-Fallavier, France), the vehicle of resveratrol, was injected intraperitoneally (i.p.) and this group was served as control. The rats in Group 2 (low dose group) were then treated i.p. resveratrol with a dose of 1 mg/kg/day. High dose resveratrol (10 mg/kg/day) was administered i.p. to rats in Group 3. The drug doses were chosen based on previous biological studies related to the resveratrol in experimental rat models.[] The treatment was continued for 7 consecutive days at approximately the same time each day. A third laparotomy was performed while the rats were fixed in the supine position at the end of the treatment. The volumes of the endometriotic implants were measured again with the same method by the same researchers who were blinded to the groups. All biochemical determinations were performed on the tissue and serum obtained after centrifugation using spectrophotometric methods. In order to evaluate the prooxidant-antioxidant balance, we determined the free radicals production by measuring of lipid peroxidation (malondialdehyde, MDA), and activity of some enzymatic antioxidants (superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GSH-Px). All tissues were homogenized in ice-cold 140 mM KCI at 16,000 rpm for 2 min using a homogenizer (IKA Ultra-Turrax T25 basic homogenizer, Germany). MDA and protein analyses were measured at this homogenate stage. The homogenate was then centrifuged at 5000 × g for 60 min to remove debris. The clear upper supernatant fluid was taken and CAT, GSH-Px activities and protein concentration was carried out in this stage. The supernatant solution was extracted with an equal volume of an ethanol/chloroform mixture (5:3, volume per volume [v/v]). After centrifugation at 5000 × g for 30 min, the clear upper layer (the ethanol phase) was taken and used in the SOD activity and protein assays. All preparation procedures were performed at 4°C. Total (Cu-Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al.[] SOD activity was also expressed as units per milligram protein and U/ml in tissue and serum respectively. CAT (EC 1.11.1.6) activity was determined according to Aebi's method.[] Tissue CAT activity was expressed as k/g protein. GSH-Px activity was measured by the method of Pagia and Valentine.[] GSH-Px activity was also expressed as units per gram protein and U/L in the tissue and serum respectively. The tissue and serum MDA was determined by a method based on the reaction with thiobarbituric acid (TBA) at 90-100°C.[] In the TBA test reaction, MDA or MDA-like substances and TBA react with the production of a pink pigment having an absorption maximum at 532 nm. The reaction was performed at pH 2-3 at 90°C for 15 min. The sample was mixed with two volumes of cold 10% (w/v) trichloroacetic acid to precipitate protein. The precipitate was pelleted by centrifugation, and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm. The results were expressed according to a standard graphic which was prepared from a standard solution (1, 1, 3, 3-tetramethoxypropane). The endometrial implants were then excised and processed for histological and immunohistochemical studies. Formalin-fixed specimens were embedded in paraffin, cut into 5-mm-thick sections, and stained with hematoxylin and eosin. The sections were also stained for proliferating cell nuclear antigen (PCNA) immunohistochemistry. The histological diagnosis of endometriosis was based on the morphological identification of endometrial glandular tissue (GT) and stromal tissue (ST). All proliferating nuclei in the stromal, glandular and endometrial epithelial cells were stained with mouse monoclonal antibody against PCNA (Clone PC10; Sigma Corp., Missouri, USA). PCNA is a nuclear protein that has its peak expression during the S-phase of the cell cycle, and it has been used previously to identify proliferating cells. Monoclonal anti-PCNA clone PC10 from mouse ascites fluid was diluted to 1/1000 and applied to 5-mm paraffin sections deparaffinized in xylene using the labeled streptavidin-biotin method. For PCNA immunostaining, human tonsil tissue served as the positive control. Negative controls (primary antibody omitted) were routinely performed on adjacent serial sections. Histological slides were evaluated for ST, GT and PCNA immunoreactivity under light microscopy. A semi-quantitative grading system that was previously described by the same research team was used to score the degree of histological change of stromal, glandular and luminal epithelial cells.[] The scalings were conducted to determine the degree of PCNA immunoreactivity of implant cells (0, no PCNA-positive cells per high-power field (×40); 1 (mild), between 1 and 10 PCNA-positive cells; 2 (moderate), between 10 and 20 PCNA-positive cells; and 3 (severe), >20 PCNA-positive cells). They were also conducted to determine the degree of GT (0, no secretory glands per high-power field (×40); 1 (mild), between 1 and 5 secretory glands; 2 (moderate), between 6 and 10 secretory glands; and 3 (severe), >10 secretory glands). Finally, scalings were conducted to determine the degree of ST (0, no ST; 1 (mild), 25% ST; 2 (moderate), 25-50% ST; and 3 (severe), >50% ST). Statistical analyses were performed using the SPSS for Windows version 15.0 program. Normality for continuous variables in the groups was determined by the Shapiro-Wilk test. The normally distributed variables (P > 0,05) were compared by a one-way ANOVA test. The variables with non-normal distributions were compared by the Kruskal-Wallis H test. For multiple comparisons of the groups, the Tukey test and the Conover test were used where appropriate. A paired sample t-test was used to compare the pre-treatment and post-treatment implant volumes within each group. A value of P < 0.05 was considered statistically significant.

The pre-treatment volumes of endometriotic implants were similar between the groups and demonstrated in Table 1 (P = 0.384). Post-treatment implant volumes were significantly lower in Group 2 and Group 3 (P = 0.004) than in the Control group. In addition, post-treatment volumes of implants in Group 2 were not significantly different than those of rats in Group 3 (P > 0.05). When the pre- and post-treatment volumes of endometriotic implants were compared in the same group, it was found that the post-treatment volumes decreased significantly in Group 2 (P < 0.001) and Group 3 (P = 0.002) groups. Nevertheless, in the Control group, the post-treatment volumes were significantly increased (P = 0.008). Serum and tissue levels of antioxydant enzymes and MDA were presented in Table 2. Treatment with resveratrol resulted a significant and dose-dependent increase in activities of SOD and GSH-Px in serum and tissue of the rats (P < 0.05). Similarly, serum and tissue MDA levels were significantly higher in Group 3 than that of Group 1 (P = 0.009 and P = 0.002, respectively). In low-dose resveratrol group, serum and tissue activity of MDA was lower than Control group, although the difference was not statistically different (P = 0.144 and P = 0.210, respectively). The proliferative indices and immunostaining scores of the groups are presented in Table 3. The histological scores for both GT and ST were found to be significantly lower in Group 2 and Group 3 than in the Control groups (P < 0.001). Glandular and stromal cells of the endometriotic implants demonstrated higher histochemical staining for PCNA in the Control group than those in the Group 2 and Group 3 (P < 0.001). The immunohistochemical detection of PCNA revealed the proliferation of many stromal, glandular and endothelial cells in the implants of control animals. In contrast, endometriotic implants of rats in Group 2 and Group 3 developed only a few proliferating cells, which were mainly localized within the endometrial stroma [Figure 1].

The results from the present study have clearly showed that resveratrol, a natural compound with profound free radical scavenger activity, has therapeutic potential in the treatment of endometriosis, a chronic disease with unclear etiology. The observed therapeutic efficacy in the current study was dose-dependent and related with an increase of endogenous antioxidant capacity and therefore suppression of oxidative damage which has been considered as a promoting factor in the pathophysiology of endometriosis. It has been hypothesized that peritoneal macrophage activation is a pivotal step in the initiation and progression of endometriosis.[] In addition, increased production of free oxygen radicals by peritoneal fluid macrophages, with increased lipid peroxidation in patients with endometriosis, has been known for a long time.[] Liu et al. have found that a marked suppression of peritoneal fluid antioxidant levels and elevated oxidized lipoproteins in the peritoneal microenvironment of patients with endometriosis.[] Similarly, Szczepanska et al. reported that women with endometriosis had significantly lower levels of SOD and GSH-Px in peritoneal fluid compared to fertile control women.[] Furthermore, Shanti et al. found similar results in a study comparing women with endometriosis to women having tubal ligation, in which endometriosis was associated with significantly higher levels of lipid peroxide-modified rabbit serum albumin, malondialdehyde-modified low-density lipoprotein, and oxidized low-density lipoprotein as measured in serum compared to tubal ligation; however, no differences were detected in the peritoneal fluid.[] Interestingly, Ota et al. suggested that expression of xanthine oxidase, an enzyme that generates free oxygen radicals, in ectopic and eutopic endometrium remained high throughout the menstrual cycle in women with endometriosis; in contrast, cyclic variations in its expression were seen in controls.[] On the other hand, several contradictory results have also been published and to date, there is no clear consensus about the role of oxidative stress in the pathophysiology of endometriosis.[] In accordance with the results of previous studies supporting an imbalance in oxidant-antioxidant activity in endometriotic patients, in the present study, we demonstrated an increased oxidative stress in rats with peritoneal endometriosis and that resveratrol treatment have provided a significant regression of endometriotic implants together with activation of endogenous antioxidant capacity. According to the best of our knowledge, this is the first time such an observation has been reported in an in vivo experimental model. Elevated concentrations of oxidized low-density lipoproteins are discussed in various studies, mostly supporting the promoter effects of such oxidized lipoproteins on the development of endometriosis. Murphy et al. found increased oxidation of low-density lipoprotein in patients with pelvic endometriosis and increased concentrations of oxidized low-density lipoproteins in the peritoneal fluid of women in whom the disease was developing.[] Higher levels of lysophosphatidyl choline, another indicator of lipoprotein peroxidation, were found in the peritoneal fluid of patients with endometriosis as well.[] In their prospective controlled study, Mier-Cabrera et al. investigated whether antioxidant vitamins may have an effect on the concentration of MDA in peripheral blood and in the pregnancy rate of women with endometriosis. They found serum levels of MDA in women with endometriosis was significantly lowered after 6 months of Vitamin C and Vitamin E treatment; the pregnancy rate, however, did not improve during or after the intervention.[] In the present study, we found a significant increase of serum and tissue levels of MDA in control rats, which represents increased lipid peroxidation in peritoneal implants and plasma; this increase was suppressed by treatment with resveratrol at a dose level of 10 mg/kg/day. Therefore, we speculate that as a potent antioxidant, resveratrol supplementation in women with endometriosis can have protective effect on the peritoneal microenvironment that could certainly contribute positive impacts on fertilization and embryo development. There are several limitations to the present study. This is an experimental study with small number of rats per group. The small size issue was due to our ethical concern regarding to conform to the ′principle of reduction′ in animal experiments; however, much larger numbers would be needed to clarify minor biochemical, histological and immunohistochemical differences between the groups. The second limitation was that we used a subjective scoring system to evaluate the histological changes of the implants. Using an image-analysis program that allows an objective/automated interpretation of histological changes would be more accurate in order to make a clear conclusion; however, this program was not available in our current conditions. In conclusion, the possible role of oxidative stress in the pathophysiology of endometriosis provides us another point of view for the treatment of the disease. Evidences from the current study demonstrated successful treatment of experimental endometriosis can be accomplished by resveratrol through increased antioxidant capacity and decreased lipid peroxidation that could provide insights into the new endometriosis treatment modalities, which is an area of ongoing research. These preliminary information suggests that resveratrol as a common and cost-saving product that is available as dietary supplements can be used as an alternative option in the medical treatment of endometriosis.

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Mier-Cabrera J, Genera-García M, De la Jara-Díaz J, Perichart-Perera O, Vadillo-Ortega F, Hernández-Guerrero C. Effect of vitamins C and E supplementation on peripheral oxidative stress markers and pregnancy rate in women with endometriosis Int J Gynaecol Obstet. 2008;100:252–6 Source of Support: Nil. Conflict of Interest: None declared.