Consumption of Soya Isoflavones Improved Polycystic Ovary Syndrome (PCOS)-Associated Metabolic Disorders in a Rat Model.

Polycystic ovary syndrome (PCOS) is associated with increased risks for certain metabolic disorders such as insulin resistance, non-alcoholic fatty liver disease (NAFLD), and suppressed ovarian follicular development. This study aimed to examine whether soya isoflavones (ISF) mitigate these PCOS-associated metabolic disorders in a rat model. Weanling Sprague-Dawley female rats were randomly divided into 6 groups and were treated with either 0 or 83 µg/day dihydrotestosterone (DHT) to induce PCOS and fed diets containing 0, 0.5, or 1g ISF/kg diet for 8 weeks. DHT treatment increased food intake, body weight gain (BWG, p<0.001), percentage of primordial follicles (60% vs 50.9%, p<0.05), and accumulation of lipid droplets in the livers. It also elevated serum total cholesterol (TC), free cholesterol (FC), triglycerides, non-esterified fatty acids (NEFA), and leptin, and hepatic TC and NEFA. Additionally, DHT treatment reduced the percentage of primary follicles (13.8% vs 30.2%, p<0.05), ovary weight, and length (p<0.001), as well as insulin sensitivity (p<0.01) compared to the Control. ISF intake at 1g/kg reduced BWG, serum TC, FC, NEFA, leptin, and hepatic triglycerides and DHT-induced insulin resistance (p<0.01). ISF intake at both levels decreased DHT-induced lipid droplet accumulation in the livers, and changes in the percentages of primordial and primary follicles. Dietary soya ISF alleviated DHT-induced BWG, insulin resistance, and hepatic lipid droplet accumulation, as well as suppressed ovarian follicular development. This suggests that consumption of soya foods or ISF supplements may be beneficial for the individuals with PCOS, mitigating the associated metabolic disorders such as diabetes and NAFLD.

Lack of Annexin A1 Exacerbates Inflammatory Response in Acute Endometritis Model.

Annexin A1 (AnxA1) is a glucocorticoid-inducible protein and an important endogenous modulator of inflammation. However, its effect in the endometrial microenvironment is poorly explained. This study aimed to evaluate the role of endogenous AnxA1 in an endometritis mouse model induced by lipopolysaccharide (LPS). Female C57BL/6 wild-type (WT) and AnxA1-/- mice were divided into two groups: SHAM and LPS. To induce endometritis, mice received a vaginal infusion of 50 µL of LPS (1 mg/mL) dissolved in phosphate-buffered saline. After 24 h, the mice were euthanized, and blood and uteri samples were collected. The endometrium inflammatory scores were significantly increased in the LPS-treated group. AnxA1-/- mice from the LPS group demonstrated a significant increase in the number of degranulated mast cell levels compared to AnxA1-/- SHAM mice. The Western blotting analysis revealed that a lack of AnxA1 promoted the upregulation of NLRP3 and pro-IL-1ß in the acute endometritis animal model compared to WT LPS animals. LPS-induced endometritis increased the number of blood peripheral leukocytes in both WT and AnxA1-/- mice compared with SHAM group mice (p < 0.001). AnxA1-/- mice also showed increased plasma levels of IL-1ß (p < 0.01), IL-6, IL-10, IL-17, and TNF-α (p < 0.05) following LPS-induced endometritis. In conclusion, a lack of endogenous AnxA1 exacerbated the inflammatory response in an endometritis model via NLRP3 dysregulation, increased uterine mast cell activation, and plasma pro-inflammatory cytokine release.

Galectin-3 is a key hepatoprotective molecule against the deleterious effect of cisplatin.

AIMS: Evaluate the role of galectin-3 in the liver using an acute model of cisplatin-induced toxicity. MATERIAL AND METHODS: Modified citrus pectin (MCP) treatment was used to inhibit galectin-3. Rats were distributed into four groups: SHAM, CIS, MCP and MCP + CIS. On days 1-7, animals were treated by oral gavage with 100 mg/kg/day of MCP (MCP and MCP + CIS groups). On days 8, 9 and 10, animals received intraperitoneal injection of 10 mg/kg/day of cisplatin (CIS and MCP + CIS groups) or saline (SHAM and MCP groups). KEY FINDINGS: Cisplatin administration caused a marked increase in hepatic leukocyte influx and liver degeneration, and promoted reactive oxygen species production and STAT3 activation in hepatocytes. Plasma levels of cytokines (IL-6, IL-10), and hepatic toxicity biomarkers (hepatic arginase 1, α-glutathione S-transferase, sorbitol dehydrogenase) were also elevated. Decreased galectin-3 levels in the livers of animals in the MCP + CIS group were also associated with increased hepatic levels of malondialdehyde and mitochondrial respiratory complex I. Animals in the MCP + CIS group also exhibited increased plasma levels of IL-1ß, TNF-α, and aspartate transaminase 1. Furthermore, MCP therapy efficiently antagonized hepatic galectin-9 in liver, but not galectin-1, the latter of which was increased. SIGNIFICANCE: Reduction of the endogenous levels of galectin-3 in hepatocytes favors the process of cell death and increases oxidative stress in the acute model of cisplatin-induced toxicity.

Histomorphometric analysis of the endometrium in an ectopic model of endometriosis in mice.

Objective: Evaluate histomorphometry of ectopic and eutopic endometrial tissues in receptor mice. Method: Eighteen female Balb/c were divided into 3 groups, 6 animals each: GI Control, no procedure; GII - Sham, animals that had the same procedures as GIII without receiving the ectopic endometrial implant. Instead, they received saline solution; GIII - endometriosis model, animals had surgical intervention with an ectopic endometrial implant. GI and GIII mice were treated with 17ß-estradiol, 100 µg/kg each. All animals were euthanized to collect uterine horns, which were fixed in 4% paraformaldehyde, embedded in paraffin, stained with Hematoxilin and Eosin and submitted to histomorphometric analyzes. Data underwent one-way ANOVA followed by Tukey's test. Results: Local tissue growth, showing important lesions and adhesions, as well as dark cysts were noticed. In GIII group, there was an increase in number of blood vessels and glands (GIII ≥ GI and GIII p > .001). Thickening of the GIII endometrial epithelial was also evident (GIII ≥ GI and GIII. p > .001). We also noticed an increase in the number of eosinophils (GIII (GIII ≥ GI and GIII. p > .001). Conclusion: Easy to perform model, capable of reproducing morphological endometriosis characteristics. From our findings, there was an increase of endometrial thickness as well as an increase in the eosinophils population.

Isoflavone Protects the Renal Tissue of Diabetic Ovariectomized Rats via PPARγ.

Diabetes associated with post-menopause is related to a worse condition of kidney disease. Taking into consideration that this disorder may be regulated by estrogenic mediators, we evaluated the renal protective effect of isoflavone. We investigated the role of the PPARγ in the pathogenesis of the disease. For this study, we used diabetic female rats in a postmenopausal model through ovariectomy. The animals were treated with isoflavone or 17ß-estradiol. A dosage was administered to bring on blood glycemia, and through immunohistochemistry, we evaluated the immunoreactivity of PPARγ in the endometrium and renal tissue. We analyzed the immunoreactivity of renal injury molecule KIM-1 and the collagen and glycogen densities in the kidney. Through bioinformatics analysis, we observed PPARγ and COL1A1 gene expression under the influence of different glucose doses. In particular, we observed that isoflavone and 17ß-estradiol regulate blood glycemia. Renal injury was inhibited by isoflavone, observed by a reduction in KIM-1, along with glycogen accumulation. These benefits of isoflavone may be associated with PPARγ overexpression in the kidneys and endometrium of diabetic ovariectomized rats.

Chondroitin Sulfate Protects the Liver in an Experimental Model of Extra-Hepatic Cholestasis Induced by Common Bile Duct Ligation.

During liver fibrogenesis, there is an imbalance between regeneration and wound healing. The current treatment is the withdrawal of the causing agent; thus, investigation of new and effective treatments is important. Studies have highlighted the action of chondroitin sulfate (CS) in different cells; thus, our aim was to analyze its effect on an experimental model of bile duct ligation (BDL). Adult Wistar rats were subjected to BDL and treated with CS for 7, 14, 21, or 28 days intraperitoneally. We performed histomorphometric analyses on Picrosirius-stained liver sections. Cell death was analyzed according to caspase-3 and cathepsin B activity and using a TUNEL assay. Regeneration was evaluated using PCNA immunohistochemistry. BDL led to increased collagen content with corresponding decreased liver parenchyma. CS treatment reduced total collagen and increased parenchyma content after 21 and 28 days. The treatment also promoted changes in the hepatic collagen type III/I ratio. Furthermore, it was observed that CS treatment reduced caspase-3 activity and the percentage of TUNEL-positive cells after 14 days and cathepsin B activity only after 28 days. The regeneration increased after 14, 21, and 28 days of CS treatment. In conclusion, our study showed a promising hepatoprotective action of CS in fibrogenesis induced by BDL.

Disclosing the involvement of proteases in an eczema murine animal model: Perspectives for protease inhibitor-based therapies.

Eczema is a skin condition characterized by itchy and inflammatory patches. The accumulation of neutrophils and the imbalance between enzymes and their inhibitors appears to be related to this condition. We proposed a neutrophil elastase (NE)-based eczema model in mice in order to verify histopathological features as well as the expression and activity of proteases and inhibitors. Mice skins were topically administered with human NE (0-2 pmol/cm2) for 24-168 h. It was observed thickening of epidermis, parakeratosis, spongiosis and leukocyte infiltration. Also, NE-treated skins presented high activity of epidermal kallikreins 5 and 7, and cathepsin B on synthetic substrates, and expression evaluated by RT-qPCR. The proteolytic activity was inhibited by soybean trypsin inhibitor, CA074 and Caesalpinia echinata kallikrein inhibitor (CeKI). The topic application of CeKI reversed eczema phenotype in NE-treated skins. Elafin expression was shown to be increased in NE-treated skins. These results suggest that the NE may trigger morphological and biochemical changes in skin similar to those observed in eczematous diseases. In addition to the establishment of this in vivo model, this work opens perspectives for the use of protease inhibitor-based drugs for the management of this skin condition.

Effects of estrogen deficiency followed by streptozotocin-induced diabetes on periodontal tissues of female rats.

Although both estrogen deficiency and diabetes contribute to periodontal tissue deterioration, the combined effects of these conditions on periodontium is unknown. Thus, we analyzed the combined effects of ovariectomy followed by streptozotocin (STZ)-induced diabetes on periodontal tissues of rats. Twenty adult rats were ovariectomized (OVX) or SHAM-operated (SHAM). After 3 weeks, the rats received an intraperitoneal injection of STZ (60 mg/kg/body weight) to induce diabetes or vehicle (blank) solution. The groups were assigned as follows (n = 5): SHAM-vehicle (SHAM), OVX-vehicle (OVX), SHAM + STZ (SHAM-Di), and OVX + STZ (OVX-Di). Seven weeks post-diabetes induction, the rats were euthanized. Blood samples were collected for glucose measurements and maxillae were processed for paraffin embedding. Sections stained with hematoxylin/eosin, Masson's trichrome, and picrosirius-red were used for alveolar bone loss and collagen fiber analysis in the lamina propria. Immunohistochemistry was performed for runt-related transcription factor 2 (Runx2), matrix metalloproteinase 9 (MMP-9), and tryptase detection. Alveolar bone loss and fewer collagen fibers were observed in the OVX-Di group, collagen fibers with irregular organization, and MMP-9 immunoreactivity were more evident in diabetic groups, and MMP-9-positive osteoclasts on alveolar bone surface were noticed in all groups. The OVX-Di group showed lower Runx2 immunoreactivity (osteoblast formation marker), and more tryptase-positive cells (mast cell marker) in the alveolar bone marrow. Our results indicate that estrogen depletion, followed by STZ-induced diabetes, promotes periodontal tissue deterioration that is more evident than both interventions applied alone. Furthermore, our results points to a possible participation of bone-derived mast cells in this process.

Beneficial Effects of Isoflavones in the Kidney of Obese Rats Are Mediated by PPAR-Gamma Expression.

Several studies have demonstrated an important association between altered lipid metabolism and the development of kidney injury because of a high-fat diet. Fructose is also closely associated with renal injury. We opted for a combination of fructose and saturated fats in a diet (DH) that is a model known to induce renal damage in order to evaluate whether soy isoflavones could have promising use in the treatment of renal alterations. After two months of ingestion, there was an expansion of visceral fat, which was associated with long-term metabolic disorders, such as sustained hyperglycemia, insulin resistance, polyuria, dyslipidemia, and hypertension. Additionally, we found a decrease in renal blood flow and an increase in renal vascular resistance. Biochemical markers of chronic kidney disease were detected; there was an infiltration of inflammatory cells with an elevated expression of proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß), the activation of the renin-angiotensin system, and oxidative/nitrosative stress. Notably, in rats exposed to the DH diet for 120 days, the concomitant treatment with isoflavones after 60 days was able to revert metabolic parameters, renal alterations, and oxidative/nitrosative stress. The beneficial effects of isoflavones in the kidney of the obese rats were found to be mediated by expression of peroxisome proliferator-activated receptor gamma (PPAR-γ).

The influence of phytoestrogens or estrogens on the proliferation of the rat endocervical mucosa.

OBJECTIVE: Although estrogen therapy is widely used against post-menopausal symptoms, it can present adverse effects, including endometrial cancer. Soy isoflavones are considered a possible alternative to estrogen therapy. However, there are still concerns whether isoflavones exert trophic effects on the uterine cervix. To evaluate the histomorphometric and immunohistochemical alterations in the uterine cervix of ovariectomized rats treated with soy isoflavones (Iso). METHODS: Fifteen adult Wistar rats were ovariectomized (Ovx) and divided into three groups: Group I (Ovx), administered with vehicle solution; Group II (OVX-Iso), administered with concentrated extract of Iso (150 mg/kg) by gavage; and Group III (OVX-E2), treated with 17ß-estradiol (10 µg/kg), subcutaneously. After 30 days of treatments, the uterine cervix was fixed in 10% formaldehyde and processed for paraffin-embedding. Sections were stained with Hematoxylin and eosin for morphological and morphometric studies or subjected to immunohistochemistry for detections of Ki-67 and vascular endothelial growth factor-A (Vegf-A). The data obtained were subjected to statistical analysis (p ≤ 0.05). RESULTS: We noted an atrophic uterine cervix in GI, whereas it was more voluminous in GII and even more voluminous in GIII. The thickness of the cervical mucosa was significantly higher in GIII, as compared to GI and GII. The cell proliferation (Ki-67) was significantly elevated in the estradiol and isoflavones treated groups, whereas Vegf-A immunoexpression was significantly higher in GIII, as compared to groups GII and GI. CONCLUSIONS: Soy isoflavones cause less trophic and proliferative effects in the uterine cervix of rats as compared to estrogen.

Heparin Attenuates Visceral Apoptosis in a Swine Model of Hemorrhagic Shock and Reperfusion Injury.

BACKGROUND: The use of unfractionated heparin in hypovolemic shock, aortic clamping, and visceral reperfusion is still not established, despite evidence of inhibition of early cell damage. This study investigated the potential protective effect of unfractionated heparin on hepatic and renal apoptosis in a porcine ischemia and reperfusion model. METHODS: Twenty-one male swine (Sus scrofa) were divided into 3 groups: sham (n = 5), heparin (n = 8), and nonheparin (n = 8). The heparin and nonheparin groups underwent hypovolemic shock for 30 min, supraceliac aortic clamping for 1 h and reperfusion for 3 h. Unfractionated heparin 200 mg/kg was administered to the heparin group during aortic clamping. Hemodynamic and laboratory parameters were monitored, including aminotransferase and serum urea. Histological lesion scores were applied to hematoxylin and eosin-stained liver and kidney sections. Apoptosis quantification was performed by caspase-3 immunohistochemistry. RESULTS: The proposed model caused a severe cardiocirculatory disturbance in the heparin and nonheparin groups, observed by the carotid-femoral pressure gradient and lactic acidosis. There was no significant difference in hemodynamic and laboratory parameters between these two groups. The mean values of liver and renal histological lesion scores did not present any significant differences. Caspase-3 immunoexpression was lower in the heparin than the nonheparin group for both liver and kidney. CONCLUSIONS: Attenuation of liver and kidney cell apoptosis in pigs undergoing systemic heparinization suggests a potential use for heparin in modulating cell death under critical hemodynamic conditions.

Evidence that Melatonin Increases Inhibin Beta-A and Follistatin Gene Expression in Ovaries of Pinealectomized Rats.

Melatonin plays an important role in the regulation of ovarian function including oocyte maturation in different mammalian species. Many studies indicate that melatonin has an impact on the ovarian function of a variety of ovarian cells. However, the information on the exact mechanism and involved hormones is low. To evaluate inhibin beta-A (INHBA) and follistatin (FST) expression in the ovaries of pinealectomized rats treated with melatonin, thirty adult female Wistar rats were randomized into three groups of ten animals each: group 1 (GSh), sham-operated controls receiving vehicle; group 2 (GPx), pinealectomized animals receiving vehicle; and group 3 (GPxMe), pinealectomized animals receiving replacement melatonin (1.0 mg/kg body weight. It was assumed that each animal drank 6.5 ± 1.2 ml per night and weighs approximately 300 g.) for 60 consecutive days. The ovaries were collected for mRNA abundance and protein of INHBA and FST by qRT-PCR and immunohistochemical analyses, respectively. Treatment with melatonin resulted in the upregulation of INHBA and FST genes in the ovarian tissue of the melatonin-treated animals (GPxMe), when compared with GPx. These findings were then confirmed by analyzing the expression of protein by immunohistochemical analyses, which revealed higher immunoreactivity of INHBA and FST in GPxMe animals in the follicular cells compared with GSh and GPx rats. Melatonin increases the expression of INHBA and FST in the ovaries of pinealectomized female rats.

The influence of phytoestrogens or estrogens on the proliferation of the rat endocervical mucosa

SUMMARY INTRODUCTION Although estrogen therapy is widely used against post-menopausal symptoms, it can present adverse effects, including endometrial cancer. Soy isoflavones are considered a possible alternative to estrogen therapy. However, there are still concerns whether isoflavones exert trophic effects on the uterine cervix. OBJECTIVES To evaluate the histomorphometric and immunohistochemical alterations in the uterine cervix of ovariectomized rats treated with soy isoflavones (Iso). METHODS Fifteen adult Wistar rats were ovariectomized (Ovx) and divided into three groups: Group I (Ovx), administered with vehicle solution; Group II (OVX-Iso), administered with concentrated extract of Iso (150 mg/kg) by gavage; and Group III (OVX-E2), treated with 17β-estradiol (10 µg/kg), subcutaneously. After 30 days of treatments, the uterine cervix was fixed in 10% formaldehyde and processed for paraffin-embedding. Sections were stained with Hematoxylin and eosin for morphological and morphometric studies or subjected to immunohistochemistry for detections of Ki-67 and vascular endothelial growth factor-A (Vegf-A). The data obtained were subjected to statistical analysis (p ≤ 0.05). RESULTS We noted an atrophic uterine cervix in GI, whereas it was more voluminous in GII and even more voluminous in GIII. The thickness of the cervical mucosa was significantly higher in GIII, as compared to GI and GII. The cell proliferation (Ki-67) was significantly elevated in the estradiol and isoflavones treated groups, whereas Vegf-A immunoexpression was significantly higher in GIII, as compared to groups GII and GI. CONCLUSIONS Soy isoflavones cause less trophic and proliferative effects in the uterine cervix of rats as compared to estrogen.

RESUMO INTRODUÇÃO Embora a terapia estrogênica seja amplamente utilizada contra sintomas pós-menopausais, ela pode apresentar efeitos adversos, incluindo câncer de mama e endometrial. Assim, as isoflavonas da soja são consideradas uma alternativa possível à terapia estrogênica. No entanto, ainda há controvérsias se estes compostos exercem efeitos tróficos significativos no colo do útero. OBJETIVOS Avaliar as alterações histomorfométricas e imuno-histoquímicas no colo do útero de ratas ovariectomizadas tratadas com isoflavonas da soja (iso). MÉTODOS Quinze ratas Wistar adultas foram ovariectomizadas bilateralmente (Ovx) e separadas em três grupos: Grupo I (Ovx) - veículo (propilenoglicol); Grupo II (Ovx-Iso) - receberam extrato concentrado de Iso (150 mg/kg) e Grupo III (Ovx-E2) - tratado com 17β-estradiol (10 µg/kg); as soluções foram administradas via gavagem por 30 dias consecutivos. Posteriormente, os colos uterinos foram retirados, fixados em formaldeído a 10% tamponado e processados para inclusão em parafina. Cortes (4 µm) foram coradas com hematoxilina e eosina para estudo morfológico e morfométricos, enquanto outros foram submetidos à imuno-histoquímica para detecção de Ki-67 e do fator de crescimento endotelial vascular-A (Vegf-A). Os dados obtidos foram submetidos à análise estatística (p≤0,05). RESULTADOS Observamos a presença de colo uterino atrófico no GI (Ovx), sendo este mais volumoso no GII (Ovx+Iso) e ainda mais volumoso no GIII (Ovx+E2). A espessura da mucosa cervical foi significativamente maior no GIII (Ovx-E2), em comparação ao GI (Ovx) e ao GII (Ovx-Iso). A proliferação celular (Ki-67) foi significativamente mais elevada nos grupos tratados com estradiol e isoflavonas, enquanto a imunoexpressão de Vegf-A foi significativamente maior no GIII (Ovx-E2), em comparação ao GII (Ovx-Iso) e ao GI (Ovx-E2). CONCLUSÕES As isoflavonas da soja causam menos efeitos tróficos e proliferativos no colo do útero de ratas em comparação ao estrogênio.

Melatonin may prevent or reverse polycystic ovary syndrome in rats.

OBJECTIVE: To evaluate the ovarian effects of melatonin (Mel) in a rat model of polycystic-ovary-syndrome (PCOS) before and after permanent estrus induction. METHODS: Thirty-two adult-female rats with regular estrous cycle were equally divided into four groups: 1) GCtrl - at estrous phase. 2) GPCOS - at permanent-estrous phase. 3) GMel1 - treated for 60 days with Mel (0.4 mg/Kg) during permanent estrus induction and 4) GMel2 - rats with PCOS and treated for 60 days with Mel. After that, the animals were euthanized, and the ovaries were removed and processed for paraffin embedding. Sections were stained with H.E. for histomorphometry or subjected to immunohistochemistry for Ki-67 and cleaved caspase-3 (Casp-3) detections. RESULTS: The GPCOS showed lack of corpus luteum and several ovarian cysts, as well as interstitial-like cells. The presence of corpus luteum and a significant increase in primary and antral follicles were observed in Mel-treated groups, which also showed a decrease in the number of ovarian cysts and in the area occupied by interstitial-like cells. These results were more evident in GMel1. The percentage of Ki-67-positive cells was significantly higher in the Mel-treated groups, mainly in the GMel2, as compared to GPCOS. On the other hand, the percentage of Casp-3-positive cells was significantly lower in granulosa cells of GMel1, whereas it was significantly higher in the interstitial-like cells of GMel2, in comparison to GPCOS. CONCLUSION: Melatonin administration prevents the permanent estrus state in the PCOS rat model. This effect is more efficient when melatonin is administered before permanent estrus induction.