F-Box and Leucine-Rich Repeat Protein 20 (FBXL20), Negatively Regulated by microRNA (miR)-195-5p, Accelerates the Malignant Progression of Ovarian Cancer.

Ovarian cancer (OC) is one of the most common cancers among women, characterized by various histological subtypes. Here, we aimed to investigate the biological function of F-box and leucine-rich repeat protein 20 (FBXL20) in the malignant phenotype of OC cells and its related mechanism. The expression of FBXL20 in OC tissue and normal tissue samples was analyzed through the GEPIA database. Quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) and Western blot were employed to detect the expression of miR-195-5p and FBXL20 in OC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) experiment and flow cytometry were applied to detect cell proliferation, cell cycle and apoptosis. Bioinformatics analysis and dual-luciferase reporter gene experiments were adopted to predict and validate the targeting relationship between miR-195-5p and FBXL20 mRNA 3'-untranslated region (3'UTR). Correlation between the expressions of miR-195-5p and FBXL20 mRNA was analyzed by Pearson correlation analysis. FBXL20 expression was upregulated in OC, and its high expression level was significantly associated with higher International Federation of Gynecology and Obstetrics (FIGO) stage and poor tumor differentiation. Functionally, overexpression of FBXL20 promoted proliferation, inhibited apoptosis and accelerated the cell cycle in OC cells in comparison to control group, and knockdown of FBXL20 exerted the opposite effects. Mechanistically, miR-195-5p directly targeted FBXL20 and negatively regulated its expression. Pearson correlation analysis indicated that miR-195-5p was negatively correlated with FBXL20 mRNA expression. In addition, overexpression of miR-195-5p reversed the above biological functions of FBXL20 in OC cells. FBXL20, negatively regulated by miR-195-5p, accelerates the proliferation and cell cycle progression of OC cells, and inhibits cell apoptosis, which might act as a prospective prognostic biomarker and a promising therapeutic target for OC.

Activity and structure of methanogenic microbial communities in sediments of cascade hydropower reservoirs, Southwest China.

Freshwater reservoirs are an important source of the greenhouse gas methane (CH4). However, little is known about the activity and structure of microbial communities involved in methanogenic decomposition of sediment organic matter (SOM) in cascade hydropower reservoirs. In this study, we targeted on sediments of three cascade reservoirs in Wujiang River, Southwest China. Our results showed that the content of sediment organic carbon (SOC) was between 3% and 11%, and it's positively correlated with both C/N ratio and recalcitrant organic carbon content of SOM. Meanwhile, SOC content was positively correlated with CH4 production rates but had no significant correlation with total CO2 production rates of the sediments, when rates were normalized to sediment volume. Resultantly, the sediment anaerobic decomposition rates hardly significantly increase along with the SOC content. These results suggested that the terrestrial organic matter accumulated after damming stimulated CH4 production from the reservoir sediments even though its decomposition rate was limited. Meantime, high throughput sequencing of 16S rRNA genes indicated that not only the hydrogenotrophic and acetoclastic, but also the methylotrophic methanogens (Methanomassiliicoccus) are abundant in the reservoir sediments. Moreover, metagenomic sequencing also suggested that methylotrophic methanogenesis are potentially important in the sediment of cascade reservoirs. Finally, the hydraulic residence time of the reservoir could be the key controlling factor of the structures of bacterial and archaeal communities as well as the CH4 production rates of the reservoir sediments.

Oxidative status of cardinal ligament in pelvic organ prolapse.

Pelvic organ prolapse (POP) is a common and distressing health problem in adult women, but the pathophysiological mechanism is yet to be fully elucidated. Previous studies have indicated that oxidative stress may be associated with POP. Thus, the aim of the present study was to investigate the oxidative status of pelvic supportive tissue in POP and further demonstrate that oxidative stress is associated with the pathogenesis of POP. A total of 60 samples were collected from females undergoing hysterectomy for POP or cervical intraepithelial neoplasia (CIN). This included 16 females with POP II, 24 females with POP III-IV (according to the POP-Q system) and 20 females with CIN II-III as the control group. Immunohistochemistry was utilized to measure the expression of oxidative biomarkers, 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxynonenal (4-HNE). Major antioxidative enzymes, mitochondrial superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx1) were measured through reverse transcription-quantitative polymerase chain reaction, western blotting and enzyme activity assays. The results demonstrated that in the cardinal ligament, the expression of 8-OHdG and 4-HNE was higher in the POP III-IV group compared with the POP II group and control group. The MnSOD and GPx1 protein level and enzyme activity were lower in the POP III-IV group compared with the POP II or the control group, while the mRNA expression level of MnSOD and GPx1 was increased. In conclusion, oxidative damage is increased in the pelvic supportive ligament of female patients with POP and the antioxidative defense capacity is decreased. These results support previous findings that oxidative stress is involved in the pathogenesis of POP.

The role of GPX1 in the pathogenesis of female pelvic organ prolapse.

Gestation and delivery can increase intra-abdominal pressure, which are well-known risk factors for Pelvic Organ Prolapse (POP). But the pathogenesis mechanism of POP remains unclear. Our previous research showed that the expression of glutathione peroxidase type 1 (GPX1) decreased in pelvic floor ligaments from POP patients, implying that oxidative stress (OS) may be related to POP. The aim of this study was to figure out the role of GPx1 regulation in the pathogenesis of POP. Women (>45 years) who received hysterectomy surgery were enrolled in this research, identified by screening. We applied mechanical strain of 0µ, 5333 µ to GPX1-overexpressing human uterosacral ligament fibroblasts (hUSLFs) isolated from menopausal women without POP respectively for 4 hours, in order to investigate the changes of cell apoptosis, oxidative status and ECM metabolism when cytomechanics model loaded on GPX1-overexpressing hUSLFs. Comparing with the non-transfection and mock-vehicle groups, we found that GPX1 not only protects hUSLFs from cell apoptosis, oxidative damage, but also improves the remodeling of ECM induced by mechanical stimulation. These results suggested that mechanical strain caused abnormalities of ECM metabolism via OS pathway in hUSLFs, which was involved in the pathogenesis of POP, and that GPx1 played a significant role in regulating mechanical strain induced POP.

Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse.

Mechanical loading on pelvic supports contributes to pelvic organ prolapse (POP). However, the underlying mechanisms remain to be elucidated. Our previous study identified that mechanical strain induced oxidative stress (OS) and promoted apoptosis and senescence in pelvic support fibroblasts. The aim of the present study is to investigate the molecular signaling pathway linking mechanical force with POP. Using a four­point bending device, human uterosacral ligament fibroblasts (hUSLF) were exposed to mechanical tensile strain at a frequency of 0.3 Hz and intensity of 5333 µÎµ, in the presence or absence of LY294002. The applied mechanical strain on hUSLF resulted in apoptosis and senescence, and decreased expression of procollagen type I α1. Mechanical strain activated phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling and resulted in downregulated expression of glutathione peroxidase 1 and Mn­superoxide dismutase, and accumulation of intracellular reactive oxygen species. These effects were blocked by administration of LY294002. Furthermore, it was demonstrated that PI3K/Akt was activated in the uterosacral ligaments of POP patients, and that OS was increased and collagen type I production reduced. The results from the present study suggest that mechanical strain promotes apoptosis and senescence, and reduces collagen type I production via activation of PI3K/Akt-mediated OS signaling pathway in hUSLF. This process may be involved in the pathogenesis of POP as it results in relaxation and dysfunction of pelvic supports.

Collagen metabolic disorder induced by oxidative stress in human uterosacral ligament­derived fibroblasts: A possible pathophysiological mechanism in pelvic organ prolapse.

Pelvic organ prolapse (POP) is a global health problem, for which the pathophysiological mechanism remains to be fully elucidated. The loss of extracellular matrix protein has been considered to be the most important molecular basis facilitating the development of POP. Oxidative stress (OS) is a well­recognized mechanism involved in fiber metabolic disorders. The present study aimed to clarify whether OS exists in the uterosacral ligament (USL) with POP, and to investigate the precise role of OS in collagen metabolism in human USL fibroblasts (hUSLFs). In the present study, 8­hydroxyguanosine (8­OHdG) and 4 hydroxynonenal (4­HNE), as oxidative biomarkers, were examined by immunohistochemistry to evaluate oxidative injury in USL sections in POP (n=20) and non­POP (n=20) groups. The primary cultured hUSLFs were treated with exogenous H2O2 to establish an original OS cell model, in which the expression levels of collagen, type 1, α1 (COL1A1), matrix metalloproteinase (MMP)­2, tissue inhibitor of metalloproteinase (TIMP)­2 and transforming growth factor (TGF)­ß1 were evaluated by western blot and reverse transcription­quantitative polymerase chain reaction analyses. The results showed that the expression levels of 8­OHdG and 4­HNE in the POP group were significantly higher, compared with those in the control group. Collagen metabolism was regulated by H2O2 exposure in a concentration­dependent manner, in which lower concentrations of H2O2 (0.1­0.2 mM) stimulated the anabolism of COL1A1, whereas a higher concentration (0.4 mM) promoted catabolism. The expression levels of MMP­2, TIMP­2 and TGF­ß1 exhibited corresponding changes with the OS levels. These results suggested that OS may be involved in the pathophysiology of POP by contributing to collagen metabolic disorder in a severity­dependent manner in hUSLFs, possibly through the regulation of MMPs, TIMPs and TGF­ß1 indirectly.

Oxidative damage to human parametrial ligament fibroblasts induced by mechanical stress.

The aim of the present study was to explore the underlying mechanisms of the roles of mechanical factors in the pathogenesis of pelvic organ prolapse (POP). The experiments were performed on fibroblasts derived from uterosacral ligaments and cardinal ligaments of patients who received total hysterectomy due to benign disease excluding POP. Fibroblasts were cultured after collagenase digestion and identified by morphological observation and immunocytochemical methods. A four­point bending device was used to subject fibroblasts at passage 4­6 to strains of 0, 1,333 µ (1 mm), 2,666 µ (2 mm) or 5,333 µ (4 mm) at a frequency of 0.1 Hz for 4 h. Intracellular reactive oxygen species (ROS) were quantified using the fluorescent probe 2',7'­dichlorodihydrofluorescein diacetate. Changes in the mitochondrial membrane potential were verified using the fluorescent dye JC­1, and apoptosis was detected using Annexin V/propidium iodide staining and flow cytometric analysis. Mechanical strain changed the morphology and adherence ability of parametrial ligament fibroblasts. Furthermore, the production of ROS was significantly increased and the mitochondrial membrane potential obviously declined with the enhancement of mechanical stress loading. In addition, the apoptotic rate of fibroblasts subjected to high mechanical strain was significantly increased compared with that in fibroblast under low­intensity strain. In conclusion, the present study showed that mechanical strain enhanced intracellular ROS levels, decreased the mitochondrial membrane potential and increased the apoptotic rate in human parametrial ligament fibroblasts, which may contribute to POP.

Transvaginal genital fistula repair with insertion of Foley catheter via fistula tract.

AIM: Genital fistula is one of the most devastating injuries in women. Despite advances in medical care, it continues to be a distressing problem, and the success rate of repair surgery is still limited. We herein describe our experience with the surgical approach using Foley catheter to repair genital fistula after gynecological surgery. METHODS: We retrospectively reviewed 29 patients who had received genital fistula repair surgery with Foley catheter between October 2011 and December 2013. Based on traditional transvaginal genital fistula repair surgery, we inserted a Foley catheter into the bladder or intestine through the fistula opening. As a result, the fistula opening could be tracked, which allows for a clear view to improve fistula repair. All 29 patients were followed up at 1, 4, and 12 weeks postoperatively. RESULTS: Of the 29 patients, 28 had successful surgical outcome (96.55% success rate). The mean operative time was 85 ± 8.1 min. The mean blood loss was 109 ± 23.4 mL. No intraoperative complications were observed. The mean postoperative hospitalization time was 10 ± 2.8 days. The follow-up rate was 100%. CONCLUSIONS: Repair of transvaginal genital fistula using Foley catheter had a high success rate, short operative time, minimal blood loss, low morbidity and short hospital stay. Therefore, this approach is minimally invasive and effective.

[Effect of mechanical stress on human parametrial ligament fibroblasts apoptosis].

OBJECTIVE: To explore the possible mechanism of mechanical factors in the pathogenesis of pelvic organ prolapse (POP). METHODS: The experiment material were uterosacral ligament and cardinal ligament- derived fibroblast from 10 patients who received total hysterectomy due to benign disease except POP. Fibroblast were cultured after collagenase digestion and identified by morphology and immocytochemical methods. Fibroblasts of 4-8 generations were stretched by four-point bending system for 0µ (0 mm), 1 333 µ (1 mm), 2 666 µ (2 mm), 5 333 µ (4 mm) strain, using 0 mm strain as the control group. Parameters was set to a frequency of 0.1 Hz, 4 hours. Cell apoptosis was tested by flow cytometry. RESULTS: Mechanical strain increased cell apoptosis [0 µ: (7.4 ± 1.5)%, 1 333 µ: (8.7 ± 2.2)%, 2 666 µ: (19.4 ± 3.4)%, 5333 µ: (50.9 ± 6.6)%, respectively]. Cell apoptosis rate was significantly higher under strong mechanical strain (2 666 µ and 5 333 µ versus 0 µ, all P < 0.05). CONCLUSION: Increased mechanical stress loading on human parametrial ligament fibroblasts could raise the rate of apoptosis.

Clinical efficacy of add-back therapy in treatment of endometriosis: a meta-analysis.

OBJECTIVE: A meta-analysis was conducted to determine the effectiveness of using gonadotropin-releasing hormone analogues (GnRH-a), both with and without hormonal add-back therapy, for the management of endometriosis. METHODS: Cochrane library, Ovid (Embase) and Pubmed databases were searched between the years 1998 and 2013 for published, prospective, randomised controlled trials (RCT) that assessed the effectiveness of "add-back" therapy for EMs treatment. The meta-analysis was performed using RevMan V5.0. The main outcome measures were as follows: lumbar spine bone mineral density (BMD) immediately after treatment and after 6 months of follow-up; femoral neck BMD; serum estradiol levels; changes in the Kupperman index score; the pelvic pain score, including dysmenorrhoea and dyspareunia; and pelvic tenderness. RESULTS: A total of 13 RCT, including 945 participants, were identified. The evidence suggested that "add-back" therapy was more effective for symptom relief than GnRH-a alone. BMD was significantly different when comparing "add-back" therapy to GnRH-a alone, both immediately after treatment and at 6 months. The "add-back" therapy increased serum oestrogen and did not reduce the efficacy of GnRH-a for treating dysmenorrhoea and dyspareunia. A variety of add-back regimens had a same effect for the treatment of endometriosis. CONCLUSIONS: "Add-back" therapy, based on the GnRH-a dose, does not reduce the efficacy of using GNRH-a for the management of endometriosis. "Add-back" therapy reduced the occurrence of side effects that can occur with GnRH-a therapy alone, such as osteoporosis and menopausal syndrome. There were no statistically significant differences when comparing the effectiveness of a variety of "add-back" regimens to each other.

Analgesic and uterine relaxant effects of isoliquiritigenin, a flavone from Glycyrrhiza glabra.

Shaoyao-gancao-tang, a Chinese medicinal formula consisting of peony and licorice has been used for the treatment of dysmenorrhea for thousands of years. The purpose of the present study was to demonstrate the analgesic and uterine relaxant effects of isoliquiritigenin (ISL), a flavonoid isolated from the roots of Glycyrrhiza glabra (a type of licorice). In vitro, isoliquiritigenin caused concentration-dependent inhibition of spontaneous contraction of isolated rat uterus and the contraction induced by various types of stimulants, such as acetylcholine (Ach, 10 mM), KCl (40 mM) and oxytocin (1 mU/mL). The uterine contractile response to cumulative concentrations of CaCl2 was blocked by 0.1 and 1 mM of isoliquiritigenin. The isoliquiritigenin-induced relaxation was partly inhibited by the nitric oxide synthase (NOS) inhibitor Nv-nitro-L-arginine methylester (L-NAME, 100 mM) and the COX-1/COX-2 inhibitor indomethacin (10mM). In vivo, isoliquiritigenin could cause a significant reduction in the acetic acid-induced writhing response and hot-plate test at the high dose. These results indicate that isoliquiritigenin, a flavonoid isolated from the roots of Glycyrrhiza glabra, not only has a spasmolytic effect on uterine contraction, which is in relation to Ca²âº channels, NOS and COX, but also an effective activity in reducing pain.

The anti-HSV-2 effect of alumen: In vitro and in vivo experimental studies.

This study investigated the anti-HSV-2 effect of alumen through in vitro and in vivo experiments. Viable cell counting was employed to assess the toxicity of alumen on Vero cells. The inhibition rate of HSV-2 was defined as the cytopathic effect (CPE) of the cells infected with the virus. Alumen suppositories of different concentrations were vaginally applied to the guinea pigs which were then infected with HSV-2 via a vaginal route. The clinical symptoms were observed and the local virus titer calculated. The results showed that alumen had an in vitro anti-HSV-2 effect by means of antiviral duplication, direct killing of the virus, and antiviral adsorption. Alumen suppositories of different concentrations could reduce or completely inhibit HSV-2 infection in guinea pigs. It was concluded that alumen had an in vitro anti-HSV-2 effect through multiple approaches and it could suppress in vivo vaginal HSV-2 infection of guinea pig to some extent.

The Anti-HSV-2 Effect of Alumen: In Vitro and In Vivo Experimental Studies / 华中科技大学学报（医学）（英德文版）

This study investigated the anti-HSV-2 effect of alumen through in vitro and in vivo experiments.Viable cell counting was employed to assess the toxicity of alumen on Vero cells.The inhibition rate of HSV-2 was defined as the cytopathic effect (CPE) of the cells infected with the virus.Alumen suppositories of different concentrations were vaginally applied to the guinea pigs which were then infected with HSV-2 via a vaginal route.The clinical symptoms were observed and the local virus titer calculated.The results showed that alumen had an in vitro anti-HSV-2 effect by means of antiviral duplication,direct killing of the virus,and antiviral adsorption.Alumen suppositories of different concentrations could reduce or completely inhibit HSV-2 infection in guinea pigs.It was concluded that alumen had an in vitro anti-HSV-2 effect through multiple approaches and it could suppress in vivo vaginal HSV-2 infection of guinea pig to some extent.

TSLC1 gene silencing in cutaneous melanoma.

Tumor suppressor in lung cancer 1 (TSLC1) is a tumor suppressor gene that encodes a member of the immunoglobulin superfamily, which is involved in the progression of some types of cancer. Several studies have shown that loss of TSLC1 expression is strongly correlated to methylation of the gene promoter, thus leading to poor prognosis in these cancers. However, the role of TSLC1 in cutaneous melanoma (CM) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of TSLC1 inactivation in CM. The expression and promoter methylation of TSLC1 were analyzed in 120 CMs. TSLC1 expression was examined by immunohistochemistry, whereas its methylation status was determined by methylation-specific PCR. TSLC1 expression was lost in 84 of 120 (70%) CMs; 36 (30%) CMs were scored as positive for TSLC1 protein expression. The TSLC1 promoter was methylated in 58 (48.33%) of 120 CMs. The incidence of the loss of expression and methylation of TSLC1 significantly increased as the tumor stage advanced (P=0.032 and 0.0021, respectively). Furthermore, in CM, disease-related survival was significantly shorter in patients with tumors losing TSLC1 or harboring methylated TSLC1 (P=0.0003 and 0.0329, respectively). The epigenetic silencing of TSLC1 through methylation is an important event in the pathogenesis of CM, and TSLC1 provides an indicator for poor prognosis.