MicroRNAs are short non-coding RNAs that have been widely recognized as key mediators in the epigenetic control of gene expression and which are present in virtually all cells and tissues studied. These regulatory molecules are generated in multiple steps in a process called microRNA biogenesis. Distinct microRNA expression patterns during the different stages of oocyte and embryo development suggest important regulatory roles for these small RNAs. Moreover, studies antagonizing specific microRNAs and enzymes in microRNA biogenesis pathways have demonstrated that interference with normal miRNA function leads to infertility and is associated with some reproductive abnormalities. Endocrine disrupting chemicals such as Bisphenol A (BPA) are synthetic hormone mimics that have been found to negatively impact reproductive health. In addition to their direct effects on gene expression, these chemicals are widely implicated in the disruption of epigenetic pathways, including the expression and activity of miRNAs, thereby altering gene expression. In this review, the roles of microRNAs during mammalian oocyte and embryo development are outlined and the different mechanisms by which endocrine disruptors such as BPA interfere with these epigenetic regulators to cause reproductive problems is explored.
Nonalcoholic fatty liver disease (NAFLD) is a disorder of the liver found worldwide. The molecular mechanisms underlying NAFLD initiation and progression, however, remain poorly understood. In this study, fluorescence difference gel electrophoresis (DIGE) combined with mass spectrometry was performed to profile the intracellular processes in the rat liver at the proteome level when rats were fed a high-fat diet for 8 weeks. Dynamic changes of 27 protein spots were observed. Among them, upregulation of 14 spots and downregulation of 13 spots were observed during the eight weeks of the high fat diet-induction period. These spots were analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF), and ultimately 24 proteins were identified with more than 95% confidence. Gene ontology (GO) annotation indicated that these proteins were implicated in the metabolism of carbohydrates, lipids, and amino acids. Four proteins were validated by western blot. Further functional studies on these dynamically changing proteins may lead to a better understanding of the mechanisms of high fat diet-induced fatty liver disease.
Brain changes associated with risperidone, a dopamine-2/serotonin-2 receptor antagonist, have been documented in rats and humans, but not in nonhuman primates. This study characterized brain changes associated with risperidone in nonhuman primates. Rhesus monkeys were orally administered risperidone in a dose-escalation paradigm up to a maximum tolerated dose of 0.5 mg/kg/day for 3 weeks, or 3 months followed by a 3-month recovery period. Transient and fully reversible neurological signs consistent with risperidone pharmacology were observed. The results of a magnetic resonance imaging evaluation after 3 months of treatment and at the end of the 3-month recovery period showed no meaningful changes in the brain. There were no risperidone-related brain weight changes or gross findings. Histomorphological evaluation of brain sections stained with hematoxylin and eosin, ionized calcium binding adaptor molecule 1 (Iba1), and luxol fast blue/cresyl violet double staining showed no notable differences between control and risperidone groups. However, evaluation of the brain after glial fibrillary acidic protein (GFAP) immunohistochemical staining revealed increased staining in the cell bodies and processes of astrocytes in the putamen without apparent alterations in numbers or distribution. The increase in GFAP staining was present after 3 weeks and 3 months of treatment, but no increase in staining was observed after the 3-month recovery period, demonstrating the reversibility of this finding. The reversible increase in GFAP expression was likely an adaptive, non-adverse response of astrocytes, associated with the pharmacology of risperidone. These observations are valuable considerations in the nonclinical risk assessment of new drug candidates for psychiatric disorders.
Papillary renal cell carcinoma (PRCC) accounts for about 10 percent of all renal cell carcinomas, and the prognosis is poor for people with advanced disease. Interleukin-20 receptor subunit beta (IL20RB) is a single-pass type I membrane protein of the type II cytokine receptor family and is related to the pathogenesis of chronic inflammation and autoimmune diseases, including psoriasis, glaucoma, vitiligo, rheumatoid arthritis, and inflammatory bowel disease. However, little has been reported on IL20RB with respect to cancer, especially in PRCC. Thus, we performed this study to explore its biological characteristics in PRCC. Data from the TCGA database were used to analyze the expression and prognosis of IL20RB. qRT-PCR was used to detect the expression of IL20RB in PRCC cells in vitro. After knockdown of IL20RB with small interfering RNA (siRNA) technology, the proliferation, migration, and invasion of Ketr-3 cells and the expression of related proteins in the epithelial-mesenchymal transition (EMT) pathway were measured with Cell Counting Kit-8 (CCK-8), transwell, and western blot assays. The findings demonstrated that the expression of IL20RB was upregulated in both PRCC tissues and cells and that the high expression of IL20RB led to low overall survival (OS). Furthermore, after knockdown of IL20RB in vitro, the proliferation, migration, and invasion of Ketr-3 cells were reduced, and the expression of related proteins in the EMT pathway declined, suggesting that IL20RB plays a vital role in PRCC through the EMT pathway. These results reveal the biological significance of IL20RB in PRCC and provide new insight for future targeted drugs.
Silicosis is a serious occupational disease characterized by pulmonary fibrosis, and its mechanism and progression have not been fully elucidated yet. In this study, silicosis models of rat were established by a one-time dusting method, and the rats were sacrificed after 30, 60, and 120 days (herein referred to as the 30, 60, and 120 days groups, respectively). The rats without dust exposure were used as the control. The lungs were removed to observe pathological changes using hematoxylin and eosin and Masson’s trichrome staining and transmission electron microscopy, and the degree of collagen type I and III deposition in the lung was evaluated by enzyme‐linked immunosorbent assay. The levels of malondialdehyde and superoxide dismutase were measured by spectrophotometry, and the expression levels of fibrosis-related genes (transforming growth factor beta 1, type I collagen, type III collagen) were assessed by real-time quantitative polymerase chain reaction. The results suggested that the rats in the model groups exhibited obvious collagen fibrosis and that the severity of the lung injury increased as the time after exposure to SiO2 increased. There was a significant response to lung inflammation in the model rats, especially in the 30 days group. The degree of lipid peroxidation in bronchoalveolar lavage fluid cells and lung tissues in experiment group rats significantly increased. Among the three fibrosis-related genes, transforming growth factor beta 1was elevated in both bronchoalveolar lavage fluid cells and lung tissues of the experiment group rats, while collagen type I and III were only elevated in lung tissues. Hence, we concluded that as silicosis progressed, inflammation, fibrosis, and the expression of fibrosis-related genes showed different time-dependent changes and that a number of causal relationships existed among them.
To clarify difference in the responses on the reprogramming of metabolism toward carcinogenesis between genotoxic and non-genotoxic hepatocarcinogens in the liver, rats were repeatedly administered genotoxic hepatocarcinogens (N-nitrosodiethylamine, aflatoxin B1, N-nitrosopyrrolidine, or carbadox) or non-genotoxic hepatocarcinogens (carbon tetrachloride, thioacetamide, or methapyrilene hydrochloride) for 28, 84, or 90 days. Non-genotoxic hepatocarcinogens revealed transcript expression changes suggestive of suppressed mitochondrial oxidative phosphorylation (OXPHOS) after 28 days and increased glutathione S-transferase placental form-positive (GST-P+) foci downregulating adenosine triphosphate (ATP) synthase subunit beta, mitochondrial precursor (ATPB), compared with genotoxic hepatocarcinogens after 84 or 90 days, suggesting that non-genotoxic hepatocarcinogens are prone to suppress OXPHOS from the early stage of treatment, which is in contrast to genotoxic hepatocarcinogens. Both genotoxic and non-genotoxic hepatocarcinogens upregulated glycolytic enzyme genes and increased cellular membrane solute carrier family 2, facilitated glucose transporter member 1 (GLUT1) expression in GST-P+ foci for up to 90 days, suggesting induction of a metabolic shift from OXPHOS to glycolysis at early hepatocarcinogenesis by hepatocarcinogens unrelated to genotoxic potential. Non-genotoxic hepatocarcinogens increased c-MYC+ cells after 28 days and downregulated Tp53 after 84 or 90 days, suggesting a commitment to enhanced metabolic shift and cell proliferation. Genotoxic hepatocarcinogens also enhanced c-MYC activation-related metabolic shift until 84 or 90 days. In addition, both genotoxic and non-genotoxic hepatocarcinogens upregulated glutaminolysis-related Slc1a5 or Gls, or both, after 28 days and induced liver cell foci immunoreactive for neutral amino acid transporter B(0) (SLC1A5) in the subpopulation of GST-P+ foci after 84 or 90 days, suggesting glutaminolysis-mediated facilitation of cell proliferation toward hepatocarcinogenesis. These results suggest differential responses between genotoxic and non-genotoxic hepatocarcinogens on reprogramming of energy metabolic pathways toward carcinogenesis in liver cells from the early stage of hepatocarcinogen treatment.
The morphological effects of β-naphthoflavone (β-NF) on placental development in pregnant rats were examined. β-NF, administered to pregnant rats intraperitoneally at 15 mg/kg bw from gestation day (GD) 9 to GD 14, had no effect on maternal body weight gain, mortality, or clinical sign. In the β-NF-exposed rats, intrauterine growth retardation (IUGR) rates increased on GDs 17 and 21, although there was no effect on fetal mortality rate, fetal or placental weight, or external fetal abnormality. Histopathologically, β-NF induced apoptosis and inhibition of cell proliferation of the trophoblastic septa in the labyrinth zone, resulting in its poor development. In the basal zone, β-NF induced spongiotrophoblast apoptosis and delayed glycogen islet regression, resulting in their cystic degeneration. β-NF-induced CYP1A1 expression was detected in the endothelial cells of the fetal capillaries in the labyrinth zone and in the endothelial cells of the spiral arteries in the metrial gland, but not in any trophoblasts. This indicates that CYP1A1 is inducible in the endothelial cells of the fetal capillaries in the labyrinth zone, and that these cells have an important role in metabolizing CYP1A1 inducers crossing the placental barrier.
Herein, we describe the case of a 6-year-old female ferret that died within a few days of the onset of anorexia and reduced spontaneous locomotor activity. Necropsy revealed a dark red abdominal mass of unknown origin between the right lobes of the pancreas and the proximal jejunum, with massive blood retention in the peritoneal cavity. Histopathologically, spindle-shaped or sometimes polygonal tumor cells were proliferating with irregularly shaped vascular spaces containing blood components and surrounding-tissue infiltration. In some areas, tumor cells formed distinctly dilated blood vessel-like structures. Immunohistochemically, most of the tumor cells were strongly positive for CD31, but factor VIII-related antigen immunoreactivity was confined to the area with dilated blood vessel-like structures. Based on these findings, the tumor was diagnosed as an abdominal hemangiosarcoma. Abdominal hemangiosarcoma excluding cases of the liver and spleen are rare in ferrets.
Spontaneous nonneoplastic proliferative lesions of the cardiac hemangioendothelium are extremely rare in humans and animals. Here, we describe a spontaneous hemangioendothelial cell hyperplasia in the heart of a 9-week-old male ICR mouse. The lesion was observed focally in the interventricular septum, with no compression of the surrounding tissues. In the lesion, a single layer of hemangioendothelial cells that had a polygonal shape with enlarged nuclei and plump cytoplasm closely lined surrounding widened capillary vascular spaces and cardiac muscles. There was little cellular atypia, and there were no multilayered endothelial cells. Immunohistochemical staining revealed that these cells were partly positive for factor VIII and CD31, hemangioendothelial cell markers, and negative for Ki-67. These features were consistent with those in aged female B6C3F1 mice in the only report in mice of spontaneous cardiac hemangioendothelial cell hyperplasia. Therefore, this is the first report of spontaneous hemangioendothelial cell hyperplasia in the heart of a young mouse.
Aplasia of the uterine horn caused by developmental defects has been reported in several species but has not been reported in RccHan:WIST rats. We encountered spontaneous segmental aplasia of a right uterine horn in two RccHan:WIST rats and detailed its pathological characteristics. The right uterine horn of both rats had similar gross and histological appearances. At necropsy, there was segmental loss of the tissues corresponding to normal right uterine horn, which consisted of a fibrous band connected to the uterine cervix. A cystic structure with clear and colorless fluid was observed in the cranial segment of the right uterine horn close to the right oviduct. The cystic structure was thought to be a partially developed tissue to the right uterine horn. The cystic structure seemed to be derived from the right uterine horn. Histologically, a single layer of cuboidal epithelium lined the luminal surface of the cystic structure, the endometrium was thin, and no uterine glands were observed. The fibrous band was composed of α-SMA positive smooth muscle cells, connective tissue, and blood vessels, but cytokeratin AE1/AE3 positive epithelium and uterine endometrium were absent. Based on these gross findings and histological features, segmental aplasia of a uterine horn was diagnosed. To our knowledge, these cases of segmental aplasia of a uterine horn are the first ones described in RccHan:WIST rats.
The aim of this study was to elucidate the renal lesions of leptin receptor-deficient medaka showing hyperglycemia and hypoinsulinemia and to evaluate the usefulness of the medaka as a model of diabetic nephropathy. Leptin receptor-deficient medaka at 20 and 30 weeks of age showed hyperglycemia and hypoinsulinemia; they also showed a higher level of plasma creatinine than the control medaka. Histopathologically, dilation of glomerular capillary lumina and of afferent/efferent arterioles was observed in leptin receptor-deficient medaka at 20 weeks of age, and then glomerular enlargement with cell proliferation and matrix expansion, formation of fibrin cap-like lesions, glomerular atrophy with Bowman’s capsule dilation, and renal tubule dilation were observed at 30 weeks of age. These histopathological characteristics of leptin receptor-deficient medaka were similar to the characteristics of kidney lesions of human and rodent models of type II diabetes mellitus, making leptin receptor-deficient medaka a useful model of diabetic nephropathy.
Circulating peroxiredoxin-4 (Prx4) is suggested as a prognosis marker as well as a regulator of many diseases. We aimed to examine 1) whether Prx4 is secreted from the liver in an animal model of sepsis and 2) effects of GYY4137, a hydrogen sulfide donor molecule, on septic liver injury as well as the hepatic secretion of Prx4. Rats (Wistar, male, 6 weeks old) were administered lipopolysaccharide (LPS, 15 mg/kg body weight, i.p.) with or without pre-administration of GYY4137 (50 mg/kg body weight, i.p.) and sacrificed 24 h after LPS administration. Hematoxylin-eosin and Elastica Masson-Goldner stains were used to evaluate hepatic injuries. Cytokine expression levels were determined by qPCR, and the levels of Prx4 in the serum and liver were determined by immunoblotting. Hepatocytes were isolated from rat liver, and the levels of Prx4 in the medium as well as the cells were determined 24 h after the administrations of LPS (1 µg/ml), tumor necrosis factor-α (TNFα, 50 ng/ml), or interleukin-1β (IL-1β, 10 ng/ml), with or without GYY4137 (300 µM). Hepatic inflammation and damage in LPS-administered rats were suppressed by GYY4137. An increase in plasma Prx4 level caused by LPS was observed, but the increase was attenuated by pre-administration of GYY4137. Prx4 was secreted from isolated hepatocytes after stimulation with LPS, TNFα, or IL-1β. GYY4137 attenuated the IL-1β-induced Prx4 secretion from hepatocytes. Secretion from hepatocytes is likely involved in the increase in circulating Prx4 during sepsis. GYY4137 attenuates not only hepatic injury but also Prx4 secretion.
Polyhexamethylene guanidine hydrochloride (PHMG-HCl), an antimicrobial additive in humidifier disinfectants, was associated with the pulmonary disease outbreak in South Korea. However, PHMG-mediated oxidative stress has only been studied in vitro. Here, we evaluated PHMG-induced oxidative stress in the lungs of rats exposed to PHMG-HCl. Male F344 rats were exposed to different concentrations of PHMG-HCl for 13-weeks via whole-body inhalation. Histopathological examination of the exposed rats showed the presence of lung lesions, including alveolar/interstitial fibrosis with inflammatory cell infiltration, bronchioalveolar hyperplasia, bronchiolar/alveolar squamous metaplasia, bronchial/bronchiolar epithelial detachment, and alveolar hemorrhage. Immunohistochemical analysis showed that 4-hydroxynonenal (4-HNE) was expressed in the bronchiolar epithelium, mainly in Clara cells and macrophages of the fibrotic tissue. The number of 4-HNE-positive cells increased significantly in a dose-dependent manner. This is the first in vivo study to report PHMG-induced oxidative stress. Our study provides clues to elucidate the mechanisms underlying PHMG-induced damage in patients affected by humidifier disinfectants.
Digital pathology, including image analysis and automatic diagnosis of pathological tissue, has been developed remarkably. HALO is an image analysis platform specialized for the study of pathological tissues, which enables tissue segmentation by using artificial intelligence. In this study, we used HALO to quantify various histopathological changes and findings that were difficult to analyze using conventional image processing software. Using the tissue classifier module, the morphological features of degeneration/necrosis of the hepatocytes and muscle fibers, bile duct in the liver, basophilic tubules and hyaline casts in the kidney, cortex in the thymus, and red pulp, white pulp, and marginal zone in the spleen were learned and separated, and areas of interest were quantified. Furthermore, using the cytonuclear module and vacuole module in combination with the tissue classifier module, the number of erythroblasts in the red pulp of the spleen and each area of acinar cells in the parotid gland were quantified. The results of quantitative analysis were correlated with the histopathological grades evaluated by pathologists. By using artificial intelligence and other functions of HALO, we recognized morphological features, analyzed histopathological changes, and quantified the histopathological grades of various findings. The analysis of histopathological changes using HALO is expected to support pathology evaluations.