Association of interleukin 1beta gene (+3953) polymorphism and severity of endometriosis in Turkish women

Endometriosis is regarded as a complex trait, in which genetic and environmental factors contribute to the disease phenotype. We investigated whether the interleukin (IL) 1beta (+3953) polymorphism is associated with the severity of endometriosis. Diagnosis of endometriosis was made on the basis of laparoscopic findings. Stage of endometriosis was determined according to the Revised American Fertility Society classification. 118 women were enrolled in the study. 78 women didnot have endometriosis, 6 women had stage I, 3 had stage II, 13 had stage III and 18 had stage IV endometriosis. Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP), and agarose gel electrophoresis techniques were used to determine the IL 1beta (+3953) genotype. Frequencies of the IL-1beta (+3953) genotypes in the control group were: CC, 0.397; TT, 0.115; CT, 0.487. Frequencies of the IL-1beta (+3953) genotypes in cases were: CC, 0.375; TT, 0.225; CT, 0.400. We found a 2.22 fold increase in TT genotype in the endometriosis group. However, the difference was not statistically significant (P > 0.05). We also observed an increase in the frequency of IL-1beta (+3953) T allele in the endometriosis group. However, the difference was not statistically significant. We also investigated the association between IL-1beta (+3953) polymorphism and the severity of endometriosis. The frequencies of CC+CT genotypes in stage I, III and IV endometriosis patients were 83.3, 84/6 and 72.2%, respectively; and TT genotypes were 16.7, 15.4 and 27.8%, respectively. We observed a statistically insignificant increase in TT genotype in stage IV endometriosis (P > 0.05). We suggest that IL-1beta (+3953) polymorphism is not associated with endometriosis in Turkish women.     

Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins

Sirtuins (Sir2-like proteins) are present in prokaryotes and eukaryotes. Here, two new human sirtuins (SIRT6 and SIRT7) are found to be similar to a particular subset of insect, nematode, plant, and protozoan sirtuins. Molecular phylogenetic analysis of 60 sirtuin conserved core domain sequences from a diverse array of organisms (including archaeans, bacteria, yeasts, plants, protozoans, and metazoans) shows that eukaryotic Sir2-like proteins group into four main branches designated here as classes I-IV. Prokaryotic sirtuins include members of classes II and III. A fifth class of sirtuin is present in gram positive bacteria and Thermotoga maritima. Saccharomyces cerevisiae has five class I sirtuins. Caenorhabditis elegans and Drosophila melanogaster have sirtuin genes from classes I, II, and IV. The seven human sirtuin genes include all four classes: SIRT1, SIRT2, and SIRT3 are class I, SIRT4 is class II, SIRT5 is class III, and SIRT6 and SIRT7 are class IV.     

Evaluation of serum CA 125 levels in patients with pelvic pain related to endometriosis

The aim of the study was to investigate the clinical value of the serum CA 125 level for diagnosing and determining the severity of endometriosis and pelvic pain associated with endometriosis. Eighty-six women who underwent operative laparoscopy were enrolled. Sixty-nine women with endometriosis and 17 without endometriosis participated in this study. In all of the patients, endometriosis was diagnosed and classified into stages according to the Revised American Fertility Society (R-AFS) classification. The mean serum CA 125 levels were determined in each patient. We also investigated the relationship between serum CA 125 concentration and the intensity of dysmenorrhea and dyspareunia in the study group. The mean serum CA 125 levels of women with endometriosis were higher than those of the control group (p<0.050). However, the mean serum CA 125 levels were higher in stage IV than in other stages of endometriosis according to the R-AFS classification. On the other hand, the percentage of patients with serum CA 125 levels >35 U/mL was elevated in the subgroups with severe dyspareunia and severe dysmenorrhea versus the asymptomatic subgroup but the differences had no statistical significance. In conclusion, CA 125 serum levels were related to endometriosis and R-AFS score in the evaluated patient series. No correlation was found between serum levels of CA 125 and pelvic pain in patients with endometriosis.     

UVA influenced the SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in human skin primary fibroblasts

Both SIRT1 and UVA radiation are involved in cellular damage processes such as apoptosis, senescence and ageing. MicroRNAs (miRNAs) have been reported to be closely related to UV radiation, as well as to SIRT1. In this study, we investigated the connections among SIRT1, UVA and miRNA in human skin primary fibroblasts. Our results showed that UVA altered the protein level of SIRT1 in a time point–dependent manner. Using miRNA microarray, bioinformatics analysis, we found that knocking down SIRT1 could cause up‐regulation of miR‐27a‐5p and the latter could down‐regulate SMAD2, and these results were verified by qRT‐PCR or Western blot. Furthermore, UVA radiation (5 J/cm²), knocking down SIRT1 or overexpression of miR‐27a‐5p led to increased expression of MMP1, and decreased expressions of COL1 and BCL2. We also found additive impacts on MMP1, COL1 and BCL2 under the combination of UVA radiation + Sirtinol (SIRT1 inhibitor), or UVA radiation + miR‐27a‐5p mimic. SIRT1 activator resveratrol could reverse damage changes caused by UVA radiation. Besides, absent of SIRT1 or overexpression of miR‐27a‐5p increased cell apoptosis and induced cell arrest in G2/M phase. Taken together, these results demonstrated that UVA could influence a novel SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in skin primary fibroblasts, and may provide potential therapeutic targets for UVA‐induced skin damage.     

Cytopathology of peritoneal endometriosis caused by ruptured ovarian cysts

The cytopathologic features of two cases of peritoneal endometriosis (secondary to ruptured ovarian endometrial cysts) are described. Both patients presented with abdominal distension and tenderness and were clinically thought to have an abdominal tumor. Preoperative cytologic examination of peritoneal fluids gave a diagnosis of endometriosis in both cases. The endometrial tissue was present in the smears as honeycombs, syncytia and tight clusters of both epithelial and stromal cells. Subsequent surgery confirmed the cytodiagnosis in both cases. These cases emphasize the need to include endometriosis in the differential diagnosis of peritoneal effusions, especially in women.     

Delphinidin induces antiproliferation and apoptosis of endometrial cells by regulating cytosolic calcium levels and mitochondrial membrane potential depolarization

Endometriosis is a benign gynecological disease of women of reproductive ages, wherein endometrial cells grow ectopically, decreasing their quality of life due to chronic pelvic pain and severe dysmenorrhea. Although surgery and hormone therapies are gold standards for treating endometriosis, side effects are common and the recurrence rate is nearly 50%. Recent studies are exploring phytochemicals as pharmacological adjuvants for treating endometriosis. Delphinidin is an anthocyanin with anti-inflammatory, antioxidative, and anticancerous properties. In this study, delphinidin showed antiproliferative and apoptotic effects on human endometrial cells. Additionally, treatment with delphinidin decreased the mitochondrial membrane potential and increased cytosolic calcium levels in VK2/E6E7 and End1/E6E7 cells. Delphinidin decreased the phosphorylation of proliferative signaling molecules, including ERK1/2, AKT, P70S6K, and S6, while increasing the phosphorylation of P38 MAPK and P90RSK. These results imply that delphinidin is a novel therapeutic agent for treating and managing endometriosis, and has fewer side effects.     

Extracellular matrix-dependent regulation of Fas ligand expression in human endometrial stromal cells.

Interaction between endometrial stromal cells and extracellular matrix (ECM) components has a crucial role in the development of endometriosis. Endometrial stromal cells attach to the mesothelial surface of peritoneum by means of integrins during their initial implantation and growth in endometriosis. Similarly, interaction between integrin and the extracellular matrix is also crucial for the remodeling of the endometrium during early pregnancy. We hypothesized that adhesion of endometrial stromal cells to the extracellular matrix could suppress the immunologic reaction to implanting endometrial cells by inducing the expression of Fas ligand (FasL), a mediator of the apoptotic pathway. Western blot analysis of human endometrial stromal cells plated onto fibronectin, laminin, and collagen IV revealed higher levels of FasL protein expression compared with endometrial stromal cells that plated to BSA-coated plates (control). Immunocytochemistry results from endometrial stromal cells plated to extracellular matrix proteins demonstrated a similar up-regulation of FasL expression. Eutopic endometrial stromal cells from women with endometriosis demonstrated higher FasL expression on control plates and those coated with extracellular matrix proteins compared with those from women without endometriosis. Disruption of actin cytoskeleton in endometrial stromal cells by treatment with cytochalasin D blocked the increase of FasL protein expression that occurred in response to adhesion to the extracellular matrix. These results suggest that attachment of endometrial stromal cells during retrograde menstruation to a new environment such as peritoneum with increased expression of laminin, fibronectin, and collagen IV could lead to an increase in FasL expression. Induction of FasL expression by adhesion of endometrial stromal cells to the extracellular matrix may take part in the development of a relative immunotolerance by inducing apoptosis of cytotoxic T lymphocytes, which will allow further development of ectopic implants.     

Regulation of SIRT1 determines initial step of endometrial receptivity by controlling E-cadherin expression

Sirtuin 1 (SIRT1), originally found as a class III histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. We examined the role of SIRT1 in the regulation of uterine receptivity using Ishikawa and RL95-2 endometrial carcinoma cell lines. Exogenous expression of SIRT1 significantly enhanced E-cadherin expression, while small interfering RNA-mediated depletion of endogenous SIRT1 resulted in a significant reduction of E-cadherin expression. A SIRT1 activator resveratrol elevated E-cadherin expression in a dose dependent manner, while SIRT1 repressors nicotinamide and sirtinol exhibited a dose dependent reduction of E-cadherin expression. We also showed that both forced expression of SIRT1 and activation of SIRT1 promote E-cadherin-driven reporter gene constructs, and SIRT1 is localized at E-cadherin promoter containing E-box elements in Ishikawa cells. Using an in vitro model of embryo implantation, we demonstrate that exogenous expression of SIRT1 and stimulation of SIRT1 activity resulted in the Ishikawa cell line becoming receptive to JAR cell spheroid attachment. Furthermore, resveratrol enhanced E-cadherin and Glycodelin protein expression at sites of intercellular contact, suggesting an additive role of resveratrol in promoting implantation. The initial step of human reproduction depends on the capacity of an embryo to attach and implant into the endometrial wall, and these results revealed the novel mechanism that activation and increased expression of SIRT1 play an important role in uterine receptivity.     

Inhibition of SIRT6 in prostate cancer reduces cell viability and increases sensitivity to chemotherapeutics

SI RT6 is an important histone modifying protein that regulates DNA repair, telomere maintenance, energy metabolism, and target gene expression. Recently SIRT6 has been identifi ed as a tumor suppressor and is downregulated in certain cancer types, but not in other cancers. From deposited gene profi ling studies we found that SIRT6 was overexpressed in prostate tumors, compared with normal or paratumor prostate tissues. Tissue microarray studies confi rmed the higher levels of SIRT6 in both prostate tumor tissues and prostate cancer cells than in their normal counterparts. Knockdown of SIRT6 in human prostate cancer cells led to sub-G1 phase arrest of cell cycle, increased apoptosis, elevated DNA damage level and decrease in BCL2 gene expression. Moreover, SIRT6-deficiency reduced cell viability and enhanced chemotherapeutics sensitivity. Taken together, this study provides the fi rst evidence of SIRT6 overexpression in human prostate cancer, and SIRT6 regulation could be exploited for prostate cancer therapy.     

The role of DJ-1 in the pathogenesis of endometriosis

Background

Endometriosis is an estrogen-dependent disease causing pelvic pain and infertility in 10% of reproductive-aged women. Despite a long history of the disease the pathogenesis of endometriosis is poorly understood. It is known that the expression of several proteins is either up or down regulated during endometriosis, but their precise role remains to be determined. DJ-1 is one such protein that is upregulated in eutopic endometrium of women having endometriosis suggesting that DJ-1 may be involved in the pathogenesis of endometriosis.

Methodology and Principal Findings

The role of DJ-1 in the pathogenesis of endometriosis was investigated. For this purpose the influence of DJ-1 on endometrial cell survival, attachment, proliferation, migration, and invasion either by overexpressing DJ-1 in normal endometrial cells or by knocking down DJ-1 expression in endometriotic cells using siRNA was investigated. The results indicated that DJ-1 protects endometrial cells from oxidative stress mediated apoptosis. Overexpression of DJ-1 in normal endometrial epithelial cells increases the adhesion on collagen type IV. However, no significant difference was observed incase of stromal cells. It was further demonstrated that DJ-1 regulates cell proliferation, migration, and invasion in normal endometrial and endometriotic epithelial cells whereas in the case of normal endometrial and endometriotic stromal cells, it regulates cell proliferation and invasion but not migration. Furthermore, the present study also indicated that DJ-1 regulates these cellular processes by modulating PI3K/Akt pathway by interacting and negatively regulating PTEN.

Conclusions

Abnormally high levels of DJ-1 expression may be involved in endometriosis, possibly by stimulating endometrial cell survival, proliferation, migration, and invasion.     

Markers of Collagen Remodeling Detect Clinically Significant Fibrosis in Chronic Hepatitis C Patients

Background and Aim

Detection of advanced fibrosis (Metavir F≥3) is important to identify patients with a high urgency of antiviral treatments vs. those whose treatment could be deferred (F≤2). The aim was to assess the diagnostic value of novel serological extracellular matrix protein fragments as potential biomarkers for clinically significant and advanced fibrosis.

Methods

Specific protein fragments of matrix metalloprotease degraded type I, III, IV and VI collagen (C1M, C3M, C4M, C6M) and type III and IV collagen formation (Pro-C3 and P4NP7S) were assessed in plasma from 403 chronic hepatitis C patients by specific ELISAs. Patients were stratified according to Metavir Fibrosis stage; F0 (n = 46), F1 (n = 161), F2 (n = 95), F3 (n = 44) and F4 (n = 33) based on liver biopsy.

Results

Pro-C3 was significantly elevated in patients with significant fibrosis (≥F2) compared to F0-F1 (p<0.05), while the markers C3M, C4M, C6M and P4NP7S were significantly elevated in patients with advanced fibrosis (≥F3) compared to F0-F2 (p<0.05). C1M showed no difference between fibrosis stages. Using Receiver Operating Characteristics analysis, the best marker for detecting ≥F2 and ≥F3 was Pro-C3 with AUC = 0.75 and AUC = 0.86. Combination of Pro-C3 and C4M with age, BMI and gender in a multiple ordered logistic regression model improved the diagnostic value for detecting ≥F2 and ≥F3 with AUC = 0.80 and AUC = 0.88.

Conclusion

The Pro-C3 protein fragment provided clinically relevant diagnostic accuracy as a single marker of liver fibrosis. A model combining Pro-C3 and C4M along with patient’s age, body mass index and gender increased the diagnostic power for identifying clinically significant fibrosis.     

Hyperprolactinemia and luteal insufficiency in infertile patients with mild and minimal endometriosis.

The objective of the present paper was to assess the presence of hormonal alterations in infertile women with stage I or II endometriosis (Group III, n = 20) compared to fertile women without endometriosis (Group I, n = 14) and to fertile women with endometriosis (Group II, n = 7). Serum levels of FSH, LH, estradiol, TSH, and PRL were measured between days 1 and 5 of the early follicular phase; in the luteal phase, three serum samples were collected for progesterone measurement, and endometrial biopsies were performed. Serum estradiol levels were lower (p = 0.035) in infertile patients with endometriosis than in fertile patients without endometriosis. Six infertile patients with endometriosis presented prolactin levels above 20 ng/ml. This was not observed in the other groups. Luteal insufficiency was more frequent in infertile patients with endometriosis (78.9%) than in fertile patients with (42.9%) or without endometriosis (0%). In a multiple logistic regression analysis, only the presence of endometriosis and infertility was significantly associated with luteal insufficiency. The serum levels of LH, FSH, and TSH were not significantly different among the groups. Luteal insufficiency and altered prolactin secretion were associated with endometriosis, and could be important mechanisms causing infertility in this group of patients.     

MicroRNA‐488 inhibits endometrial glandular epithelial cell proliferation,migration, and invasion in endometriosis mice via Wnt by inhibiting FZD7

Endometriosis is a chronic inflammatory syndrome and nearly 6%‐10% of women are affected by it during the reproductive period. Previous studies have proved that microRNAs (miRNAs) are implicated in the pathogenesis of ovarian endometriosis. In this study, we aimed to investigate that restored miR‐488 would effectively inhibit the development of endometriosis. The microarray‐based data analysis was performed to screen endometriosis‐related differentially expressed genes (DEGs). The mouse model in endometriosis syndrome was established by being subcutaneously injected with Estradiol benzoate, and the ectopic endometrial tissues and normal endometrial tissues were collected. Additionally, the endometrial glandular epithelial cells were extracted from the endometrial glandular epithelial tissues from normal and endometriosis mice. In order to examine the role of miR‐488 in mice with endometriosis, we measured miR‐488 expression and expression levels of Frizzled‐7 (FZD7), cyclinD1, β‐catenin, and c‐Myc in vivo and in vitro. Finally, we detected the effect of miR‐488 on cell proliferation, apoptosis, migration and invasion in vitro. FZD7 was upregulated in human endometriosis. The data showed higher expression levels of FZD7, β‐catenin, c‐Myc and cyclinD1, and lower miR‐488 expression in mouse endometrial tissues. FZD7 was the target gene of miR‐488. Furthermore, elevated miR‐488 in isolated mouse endometrial glandular endometrial cells inhibited FZD7, the translocation of β‐catenin to nucleus, the activation of Wnt pathway, and the cell proliferation, migration and invasion. Collectively, these findings indicated that up‐regulated miR‐488 may reduce the proliferation, migration and invasion of endometrial glandular epithelial cells through inhibiting the activation of Wnt pathway by down‐regulating FZD7.     

Atractylenolide III ameliorates Non-Alcoholic Fatty Liver Disease by activating Hepatic Adiponectin Receptor 1-Mediated AMPK Pathway

Background: Nonalcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic liver diseases worldwide. At present, there are no effective pharmacological therapies for NAFLD except lifestyle intervention-mediated weight loss. Atractylenolide III (ATL III), the major bioactive component found in Atractylode smacrocephala Koidz, has been shown to exert anti-oxidant, anti-tumor, anti-allergic response, anti-bacterial effects and cognitive protection. Here we investigate the therapeutic potential and underlying mechanisms of ATL III for the treatment of NAFLD.Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) and treated with ATL III. Lipid accumulation was analyzed by Oil Red O staining in liver tissues and free fatty acids (FFAs)-treated hepatocytes. AMP-activated protein (AMPK) and sirtuin 1(SIRT1) signaling pathways were inhibited by Compound C and EX527 in vitro, respectively. Small-interfering RNA (siRNA) was used to knockdown adiponectin receptor 1 (AdipoR1) expression in HepG2 cells.Results: ATL III treatment ameliorated liver injury and hepatic lipid accumulation in the HFD-induced NAFLD mouse model as demonstrated by that ATL III administration significantly reduced serum levels of alanine aminotransferase, glutamic oxaloacetic transaminase, triglycerides, total cholesterol and low-density lipoprotein. Furthermore, treatment with ATL III alleviated hepatic oxidative stress, inflammation and fibrosis in the HFD feeding model. To study the underlying mechanisms, we performed Computer Aided Design assay and found that open-formed AdipoR1 and adiponectin receptor 2 were the potential receptors targeted by ATL III. Interestingly, HFD feeding or FFAs treatment only reduced hepatic AdipoR1 expression, while such reduction was abolished by ATL III administration. In addition, in vitro treatment with ATL III activated the AdipoR1 downstream AMPK /SIRT1 signaling pathway and reduced lipid deposition in HepG2 cells, which was diminished by silencing AdipoR1. Finally, inhibition of AMPK or SIRT1, the AdipoR1 downstream signaling, abolished the protective effects of ATL III on lipid deposition and oxidative stress in FFAs-treated HepG2 cells.Conclusion: Our findings suggest that ATL III is a therapeutic drug for the treatment of NAFLD and such protective effect is mediated by activating hepatic AdipoR1-mediated AMPK/SIRT1 signaling pathway.     

Macrophage Migration Inhibitory Factor Is Involved in Ectopic Endometrial Tissue Growth and Peritoneal-Endometrial Tissue Interaction In Vivo: A Plausible Link to Endometriosis Development

Pelvic inflammation is a hallmark of endometriosis pathogenesis and a major cause of the disease''s symptoms. Abnormal immune and inflammatory changes may not only contribute to endometriosis-major symptoms, but also contribute to ectopic endometrial tissue growth and endometriosis development. A major pro-inflammatory factors found elevated in peritoneal fluid of women with endometriosis and to be overexpressed in peritoneal fluid macrophages and active, highly vascularized and early stage endometriotic lesions, macrophage migration inhibitory factor (MIF) appeared to induce angiogenic and inflammatory and estrogen producing phenotypes in endometriotic cells in vitro and to be a possible therapeutic target in vivo. Using a mouse model where MIF-knock out (KO) mice received intra-peritoneal injection of endometrial tissue from MIF-KO or syngeneic wild type (WT) mice and vice versa, our current study revealed that MIF genetic depletion resulted in a marked reduction ectopic endometrial tissue growth, a disrupted tissue structure and a significant down regulation of the expression of major inflammatory (cyclooxygenease-2), cell adhesion (αv and β3 integrins), survival (B-cell lymphoma-2) and angiogenic (vascular endothelial cell growth) factorsrelevant to endometriosis pathogenesis, whereas MIF add-back to MIF-KO mice significantly restored endometriosis-like lesions number and size. Interestingly, cross-experiments revealed that MIF presence in both endometrial and peritoneal host tissues is required for ectopic endometrial tissue growth and pointed to its involvement in endometrial-peritoneal interactions. This study provides compelling evidence for the role of MIF in endometriosis development and its possible interest for a targeted treatment of endometriosis.     

Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by integrin-dependent cell adhesion.

Shed menstrual endometrium is viable and has the ability to implant and grow in women, who eventually develop endometriosis. Many of the cell-to-cell or cell-to-extracellular matrix (ECM) connections are mediated by integrins. Monocyte chemotactic protein (MCP)-1, a potent chemotactic factor produced in many cell types, is elevated in the peritoneal fluid of women with endometriosis. In this study, we investigated whether endometrial stromal cell (ESC) adhesion itself induces the expression of MCP-1 and whether this process is integrin mediated. ESC were plated on Petri dishes and 24-well plates coated with fibronectin, laminin, collagen IV, poly-L-lysine, or mouse anti-human integrin beta(1) and beta(2) monoclonal antibodies. Adherence of ESC to various ECM substrates, except for poly-L-lysine, a non-integrin-dependent adhesion matrix, induced the expression of MCP-1 at both mRNA and protein levels. Engagement of beta(1)-containing integrins was associated with ESC adhesion and resulted in up-regulation of MCP-1 gene expression and protein secretion. Disruption of the actin cytoskeleton by treating ESC with cytochalasin D completely blocked the increase of MCP-1 induced in response to integrin activation. These findings indicate a novel mechanism of MCP-1 regulation. Cell adhesion to ECM is an important event that leads to stimulation of MCP-1 expression, and this process is mediated by integrins.     

Functional aspects of peritoneal macrophages in endometriosis of women

Peritoneal fluid was collected in the periovulatory phase of the cycle from 25 women undergoing laparoscopy. Endometriosis was diagnosed in 13 patients (AFS score 1, N = 9; AFS score 2, N = 4) and 12 patients without endometriosis served as controls. In endometriosis patients the total peritoneal fluid cell number and cell concentration was significantly higher than in controls, indicating peritoneal irritation by endometrial implants. Peritoneal fluid macrophages in patients with endometriosis showed significantly increased erythrophagocytosis and lower chemiluminescence than in controls, suggesting an advanced differentiation of the macrophages in endometriosis patients. The macrophages in this stage of differentiation may interfere with gametes and embryos and thus contribute to endometriosis-associated subfertility.