Kaempferol Alleviates Hepatic Injury in Nonalcoholic Steatohepatitis (NASH) by Suppressing Neutrophil-Mediated NLRP3-ASC/TMS1-Caspase 3 Signaling.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a significant hepatic condition that has gained worldwide attention. Kaempferol (Kae), renowned for its diverse biological activities, including anti-inflammatory, antioxidant, anti-aging, and cardio-protective properties, has emerged as a potential therapeutic candidate for non-alcoholic steatohepatitis (NASH). Despite its promising therapeutic potential, the precise underlying mechanism of Kae's beneficial effects in NASH remains unclear. Therefore, this study aims to clarify the mechanism by conducting comprehensive in vivo and in vitro experiments. RESULTS: In this study, a murine model of non-alcoholic steatohepatitis (NASH) was established by feeding C57BL/6 female mice a high-fat diet for 12 weeks. Kaempferol (Kae) was investigated for its ability to modulate systemic inflammatory responses and lipid metabolism in this model (20 mg/kg per day). Notably, Kae significantly reduced the expression of NLRP3-ASC/TMS1-Caspase 3, a crucial mediator of liver tissue inflammation. Additionally, in a HepG2 cell model induced with palmitic acid/oleic acid (PA/OA) to mimic NASH conditions, Kae demonstrated the capacity to decrease lipid droplet accumulation and downregulate the expression of NLRP3-ASC/TMS1-Caspase 3 (20 µM and the final concentration to 20 nM). These findings suggest that Kae may hold therapeutic potential in the treatment of NASH by targeting inflammatory and metabolic pathways. CONCLUSIONS: These findings suggest that kaempferol holds potential as a promising therapeutic intervention for ameliorating non-alcoholic fatty liver disease (NAFLD).

Delivering umbilical cord mesenchymal stem cell exosomes through hydrogel ameliorates vaginal atrophy in ovariectomized rats.

BACKGROUND: Menopausal and postmenopausal women often experience vaginal atrophy due to estrogen deficiency. Mesenchymal stem cell exosomes have emerged as potential therapeutic agents, capable of promoting tissue regeneration and repair. OBJECTIVE: This study aimed to explore the benefits of exosomes on VK2 cells and the therapeutic effect of topical exosomal hydrogel on atrophic vaginas. METHODS: Exosomes were extracted using the high-speed centrifugation method, and their effects on VK2 cell proliferation, migration, and differentiation were observed through co-culture. The menopause model was induced by ovariectomy in rats, followed by the injection of exosome-loaded hydrogel into their vaginas. The treatment's effectiveness was evaluated by measuring vaginal epithelium thickness using HE staining, and assessing vaginal mucosa proliferation and lamina propria angiogenesis using Ki67 and anti-CD31 staining, respectively. RESULTS: Exosomes significantly promoted VK2 cell proliferation and migration, but had no significant effect on differentiation. The exosome hydrogel increased the expression of Ki67 and CD31, leading to a significant improvement in epithelial thickness. CONCLUSIONS: UcMSC- ex can stimulate the proliferation and migration of VK2 cells, but do not appear to promote differentiation. Topical application of exosome hydrogel enhances vaginal epithelium thickness to a certain degree, offering a promising non-hormonal therapeutic strategy to alleviate vaginal atrophy in postmenopausal women.

Effect of Vitamin D on the Proliferation and Barrier of Atrophic Vaginal Epithelial Cells.

Atrophic vaginitis is very common in postmenopausal women due to declining estrogen levels. Vitamin D plays an important role in promoting epithelial cell proliferation, migration and adhesion. We established a rat model of ovariectomy (OVX) induced atrophic vaginitis with the aim of investigating the effects of Vitamin D supplementation on the vaginal epithelial barrier. The results showed that ovariectomised rats had significantly higher vaginal pH, reduced Lactobacillus, significantly lower uterine and vaginal weights, and lower vaginal epithelial PCNA, occludin, and E-cadherin mRNA expression compared with sham-operated rats. Vitamin D supplementation could reduce the vaginal pH, promote the proliferation and keratinization of vaginal epithelial cells, enhance the expression of PCNA mRNA in vaginal tissues, and improve the vaginal and uterine atrophy. Vitamin D can also increase the expression of E-cadherin and occludin proteins in vaginal tissues, maintain the integrity of the vaginal epithelium, increase the number of Lactobacillus, and reduce pathogenic bacterial infections. In vitro experiments demonstrated that 1,25(OH)2D3 could promote the proliferation and migration of VK2/E6E7 vaginal epithelial cells and increase the expression of E-cadherin protein. In conclusion, we demonstrated that Vitamin D can regulate the expression of vaginal epithelial tight junction proteins, promotes cell proliferation, and improves vaginal atrophy due to estrogen deficiency.

The relationship between pelvic floor functions and vaginal microbiota in 6-8 weeks postpartum women.

The impairment of pelvic floor muscle functions and Lactobacillus-deficient vaginal microbiota is common in postpartum women. However, few studies have explored the correlation between pelvic floor muscle functions and vaginal microbiota. Given this research gap, our study aims to investigate any potential association between these two conditions of postpartum women (6-8 weeks after childbirth). A total of 230 women who required postpartum pelvic floor function examination at Peking University International Hospital from December 2021 to April 2022 were enrolled in this study. The collected questionnaire information included progestational weight, body mass index (BMI), weight gain during pregnancy, neonatal weight, delivery type, multiparity, postpartum time, and urinary incontinence (UI). A total of 187 samples of vaginal secretions were collected, and the vaginal microbiota was detected by 16S rRNA sequence analysis. Finally, 183 samples were analyzed in the trial. All individuals were divided into two groups according to the results of pelvic floor muscle assessment to explore the difference between the incidence of postpartum urinary incontinence and vaginal microbiota. We found that the prevalence of UI was higher in the group with weakened pelvic floor muscles. Vaginal delivery, overweight, age, neonatal weight, and weight gain during pregnancy were all risk factors for postpartum urinary incontinence. The vaginal microbiome was no longer Lactobacillus dominant of most postpartum women (91.8%), while the diversity of microbiota increased. The Lactobacillus-deficient community, commonly labeled as community state type (CST) IV, was sub-divided into four communities. The abundance of vaginal Lactobacillus decreased in the group with compromised pelvic muscle functions, while the species richness and diversity increased significantly. In conclusion, the decreased pelvic floor muscle functions of postpartum women 6-8 weeks after delivery may disrupt the balance of vaginal microbiota, and the restoration of pelvic floor functions may contribute to a healthy and balanced vaginal microbiota.

The impact of pelvic floor electrical stimulation on vaginal microbiota and immunity.

Pelvic floor electrical stimulation (ES) is an effective treatment for pelvic floor dysfunction. However, the impact of ES on vaginal microbiota and local inflammatory response is yet poorly understood. Therefore, we designed a longitudinal study to investigate the impact of ES on vaginal microbiota and cytokines. A total of 170 participants were recruited into the study at Peking University International Hospital, Beijing, China, from December 2021 to April 2022. They were divided into two groups concerning the follow-up: long-term cohort (n = 147) following up to seven treatment sessions and short-term cohort (n = 23) following up to 7 h after a 30-min treatment. Paired vaginal discharge samples were collected from 134 individuals. Vaginal microbiota was characterized by 16S rRNA sequencing, and local cytokines concentrations were detected by the cytometric bead array method. A significant increase in the relative abundance of Lactobacillus spp. was observed after ES treatment (P < 0.001). In addition, L. crispatus (P = 0.012) and L. gasseri (P = 0.011) also increased significantly. Reduced microbial diversity was observed in the vaginal microbiota after the treatment. In the long-term cohort, a significant downregulation of IFN-γ, IL-2, IL-4, IL-10, IL-17A, and TNF-α was compared with baseline. However, the short-term cohort presented with an elevated IL-6 level at 7 h after the treatment. In conclusion, this study suggested that transvaginal electrical stimulation might help to restore and maintain a healthy vaginal microbiota dominated by Lactobacillus, reducing the risk of vaginal inflammation.

Identification of kaempferol as an OSX upregulator by network pharmacology-based analysis of qianggu Capsule for osteoporosis.

Osteoporosis is the most common metabolic disease of skeleton with reduced bone density and weaker bone. Qianggu Capsule as a traditional chinese medicine has been widely used to treat osteoporosis. The potential pharmacological mechanism of its active ingredient Gusuibu is not well understood. The purpose of this work is to analyze the anti-osteoporosis function of Gusuibu based on network pharmacology, and further explore the potential mechanism of Qianggu Capsule. The active compounds and their corresponding targets of Gusuibu were obtained from TCMSP, TCMID, and BATMAN-TCM databases. Potential therapeutic targets for osteoporosis were obtained through DisGeNET, TTD, GeneCards, MalaCards, CTD, and OMIM databases. The overlapping targets of Gusuibu and osteoporosis were obtained. GO and KEGG pathway enrichment analysis were performed. The "Gusuibu-active compounds-target genes-osteoporosis" network and protein-protein interaction (PPI) network were constructed, and the top hub genes were screened by using the plug-in CytoHubba. Molecular docking was used to verify the binding activity of hub genes and key compounds. We identified 21 active compounds and 140 potential therapeutic targets that may be related to Gusuibu and 10 hub genes (AKT1, IL6, JUN, TNF, MAPK3, VEGFA, EGFR, MAPK1, CASP3, PTGS2). Molecular docking analysis demonstrated that four key active small molecules in Gusuibu (including Luteolin, Naringenin, Kaempferol, and Beta-sitosterol) have excellent binding affinity to the target proteins encoded by the top 10 hub genes. Our new findings indicated that one key active compound kaempferol activated the expression of osteoblast specific transcription factor OSX through JNK kinase pathway.

Metformin With or Without Clomiphene Citrate Versus Laparoscopic Ovarian Drilling With or Without Clomiphene Citrate to Treat Patients With Clomiphene Citrate-Resistant Polycystic Ovary Syndrome: A Systematic Review and Meta-Analysis.

Introduction: Which is optimal to treat clomiphene citrate-resistant polycystic ovary syndrome (CCR-PCOS) with LOD or metformin remains a problem. There are three inconsistent or even contradictory views. Objectives: The present meta-analysis aimed to evaluate the effectiveness and safety of Metformin with or without CC and to compare them with LOD with or without CC (Met/Met-CC vs. LOD/LOD-CC) in women with CCR-PCOS who also have anovulation. Data source: The PubMed, Cochrane, and Embase databases were searched to identify relevant studies reported between 1 Jan 1966 and 31 Aug 2019; the search was updated on 17 May 2022. Study eligibility criteria: We included randomized controlled trials (RCTs) of CCR-PCOS that had considered Met/Met-CC and LOD/LOD-CC as the exposure variables and fertility as the main outcome variable. Study appraisal and synthesis methods: We assessed study quality using the Cochrane risk-of-bias tool. The primary effectiveness outcome was live birth/ongoing pregnancy rate and the primary safety outcome was miscarriage rate. A fixed-effect meta-analysis was performed. The robustness of the results was assessed using sensitivity analyses. Meta-regression and subgroup analysis were performed to examine the reasons for heterogeneity. Publication bias was examined using the funnel plot, Egger linear regression, and Begg rank correlation tests. The quality of this meta-analysis was estimated according to the GRADE approach. This meta-analysis has been registered in PROSPERO (CRD42021240156). Results: Among 71 potentially relevant studies, we included five RCTs in our meta-analysis. We found no difference in effectiveness between Met-CC and LOD in terms of live birth/ongoing pregnancy (RR = 1.02, 95% CI: 0.87-1.21, z = 0.28; p = 0.780), and miscarriage rates (RR = 0.79, 95% CI: 0.46-1.36, z = 0.86; p = 0.390). I2 tests results revealed moderate or no heterogeneity (I2 = 51.4%, p = 0.083; I2= 0.0%; p = 0.952). Sensitivity analysis confirmed the robustness of the results. Funnel plot, Egger linear regression, and Begg rank correlation tests implied no publication bias (p > 0.05). LOD was more expensive than Met (€1050 vs. €50.16). The evidence quality was moderate. Conclusion: There is no evidence on the difference in the outcomes between the two interventions regarding ovulation, pregnancy, and live birth. As LOD is an invasive procedure and carries inherent risks, the use of Met/Met-CC should be the second-line treatment for women with CCR-PCOS. Systematic Review Registration: identifier CRD42021240156.

Vaginal Microbiota Changes Caused by HPV Infection in Chinese Women.

Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases. After studying 602 unvaccinated Chinese women using 16S rRNA to detect cervical-vaginal microecology, we analyzed the relationship between HPV infection and vaginal microecology including 20 HPV types. In Chinese women, L. gasseri-dominated and L. jensenii-dominated clusters were significantly absence. Microbial alpha diversity was significantly higher in HPV-infected and cervical intraepithelial neoplasia (CIN)-diagnosed groups than in healthy control group. Certain bacteria were associated with HPV infection and CIN, including Streptococcus, Prevotella, Chlamydia, Bifidobacterium, Ralstonia, and Aerococcus. With the development of disease, the proportions of community state type III (CST-III) and CST-IV-B gradually increased, whereas the proportions of CST-I and CST-IV-A gradually decreased. In addition, age was an influential factor for HPV infection. With aging, the probability of HPV infection and the proportion of CST-IV-B increase. In conclusion, our study was a large cross-sectional study that evaluated the relationship between vaginal microbiota and HPV infection, and brought essential comparable data.

A cross-sectional analysis about bacterial vaginosis, high-risk human papillomavirus infection, and cervical intraepithelial neoplasia in Chinese women.

Bacterial vaginosis (BV) is a genital infection that frequently presents in women infected with human papillomavirus (HPV), but the correlation between BV, HPV and cervical intraepithelial neoplasia (CIN) development is still elusive. We organized a cross-sectional analysis which enrolled 624 participants and obtained 423 samples of vaginal secretions from them, including 193 HPV-negative samples and 230 HR-HPV-positive samples. We used 16S rRNA sequencing to measure the vaginal microbiota diversity in women with different BV, HPV and CIN status, and then calculated risk factors for CIN by logistic regression. We found that the diversity of vaginal microbiota was significantly increased after BV, HPV and BV-infected CIN group. The Observed species and Chao1 index of H.C group showed little difference with normal group, while its Shannon index was considerable higher than normal group. L. iners enriched in HPV infection group compared with others significantly. BV (OR = 0.358; 95% CI = 0.195-0.656; P < .05) and HR-HPV infection (OR = 0.016; 95% CI = 0.004-0.072; P < .001) were risk factors for CIN. In conclusion, we consider BV as a risk factor for CIN. The enrichment of L. iners under HPV infection state may contribute to maintenance of vaginal dysbiosis, and BV infection could facilitate the disturb.

Mooring Stone-Like Arg114 Pulls Diverse Bulged Peptides: First Insight into African Swine Fever Virus-Derived T Cell Epitopes Presented by Swine Major Histocompatibility Complex Class I.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating pig disease threatening the global pork industry. However, currently, no commercial vaccines are available. During the pig immune response, major histocompatibility complex class I (MHC-I) molecules select viral peptide epitopes and present them to host cytotoxic T lymphocytes, thereby playing critical roles in eliminating viral infections. Here, we screened peptides derived from ASFV and determined the molecular basis of ASFV-derived peptides presented by the swine leukocyte antigen 1*0101 (SLA-1*0101). We found that peptide binding in SLA-1*0101 differs from the traditional mammalian binding patterns. Unlike the typical B and F pockets used by the common MHC-I molecule, SLA-1*0101 uses the D and F pockets as major peptide anchor pockets. Furthermore, the conformationally stable Arg114 residue located in the peptide-binding groove (PBG) was highly selective for the peptides. Arg114 draws negatively charged residues at positions P5 to P7 of the peptides, which led to multiple bulged conformations of different peptides binding to SLA-1*0101 and creating diversity for T cell receptor (TCR) docking. Thus, the solid Arg114 residue acts as a "mooring stone" and pulls the peptides into the PBG of SLA-1*0101. Notably, the T cell recognition and activation of p72-derived peptides were verified by SLA-1*0101 tetramer-based flow cytometry in peripheral blood mononuclear cells (PBMCs) of the donor pigs. These results refresh our understanding of MHC-I molecular anchor peptides and provide new insights into vaccine development for the prevention and control of ASF. IMPORTANCE The spread of African swine fever virus (ASFV) has caused enormous losses to the pork industry worldwide. Here, a series of ASFV-derived peptides were identified, which could bind to swine leukocyte antigen 1*0101 (SLA-1*0101), a prevalent SLA allele among Yorkshire pigs. The crystal structure of four ASFV-derived peptides and one foot-and-mouth disease virus (FMDV)-derived peptide complexed with SLA-1*0101 revealed an unusual peptide anchoring mode of SLA-1*0101 with D and F pockets as anchoring pockets. Negatively charged residues are preferred within the middle portion of SLA-1*0101-binding peptides. Notably, we determined an unexpected role of Arg114 of SLA-1*0101 as a "mooring stone" which pulls the peptide anchoring into the PBG in diverse "M"- or "n"-shaped conformation. Furthermore, T cells from donor pigs could activate through the recognition of ASFV-derived peptides. Our study sheds light on the uncommon presentation of ASFV peptides by swine MHC-I and benefits the development of ASF vaccines.

Strict Assembly Restriction of Peptides from Rabbit Hemorrhagic Disease Virus Presented by Rabbit Major Histocompatibility Complex Class I Molecule RLA-A1.

Rabbits are pivotal domestic animals for both the economy and as an animal model for human diseases. A large number of rabbits have been infected by rabbit hemorrhagic disease virus (RHDV) in natural and artificial pandemics in the past. Differences in presentation of antigenic peptides by polymorphic major histocompatibility complex (MHC) molecules to T-cell receptors (TCR) on T lymphocytes are associated with viral clearance in mammals. Here, we screened and identified a series of peptides derived from RHDV binding to the rabbit MHC class I molecule, RLA-A1. The small, hydrophobic B and F pockets of RLA-A1 capture a peptide motif analogous to that recognized by human class I molecule HLA-A*0201, with more restricted aliphatic anchors at P2 and PΩ positions. Moreover, the rabbit molecule is characterized by an uncommon residue combination of Gly53, Val55, and Glu56, making the 310 helix and the loop between the 310 and α1 helices closer to the α2 helix. A wider A pocket in RLA-A1 can induce a special conformation of the P1 anchor and may play a pivotal role in peptide assembly and TCR recognition. Our study broadens the knowledge of T-cell immunity in domestic animals and also provides useful insights for vaccine development to prevent infectious diseases in rabbits.IMPORTANCE We screened rabbit MHC class I RLA-A1-restricted peptides from the capsid protein VP60 of rabbit hemorrhagic disease virus (RHDV) and determined the structures of RLA-A1 complexed with three peptides, VP60-1, VP60-2, and VP60-10. From the structures, we found that the peptide binding motifs of RLA-A1 are extremely constraining. Thus, there is a generally restricted peptide selection for RLA-A1 compared to that for human HLA-A*0201. In addition, uncommon residues Gly53, Val55, and Glu56 of RLA-A1 are located between the 310 helix and α1 helix, which makes the steric position of the 310 helix in RLA-A1 much closer to the α2 helix than that found in other mammalian MHC class I molecules. This special conformation between the 310 helix and α1 helix plays a pivotal role in rabbit MHC class I assembly. Our results provide new insights into MHC class I molecule assembly and peptide presentation of domestic mammals. Furthermore, these data also broaden our knowledge on T-cell immunity in rabbits and may also provide useful information for vaccine development to prevent infectious diseases in rabbits.

Retraction Note: DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA.

An amendment to this paper has been published and can be accessed via the original article.

DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA.

BACKGROUND: Detection of DNA methylome at single-base resolution is a significant challenge but promises to shed considerable light on human disease etiology. Current technologies could not detect DNA methylation genome-wide at single-base resolution with small amount of sequencing data and could not avoid detecting the methylation of repetitive elements which are considered as "junk DNA". METHODS: In this study, we have developed a novel DNA methylome profiling technology named MB-seq with its ability to identify genome-wide 5mC and quantify DNA methylation levels by introduced an assistant adapter AluI-linker This linker can be ligated to sonicated DNA and then be digested after the bisulfite treatment and amplification, which has no effect of MeDIP enrichment. Because many researchers are interested in investigating the methylation of functional regions such as promoters and gene bodies, we have also developed a novel alternative method named MRB-seq, which can be used to investigate the DNA methylation of functional regions by removing the repeats with Cot-1 DNA. RESULTS: In this study, we have developed MB-seq, a novel DNA methylome profiling technology combining MeDIP-seq with bisulfite conversion, which can precisely detect the 5mC sites and determine their DNA methylation level at single-base resolution in a cost-effective way. In addition, we have developed a new alternative method, MRB-seq (MeDIP-repetitive elements removal-bisulfite sequencing), which interrogates 5mCs in functional regions by depleting nearly half of repeat fragments enriched by MeDIP. Comparing MB-seq and MRB-seq to whole-genome BS-seq using the same batch of DNA from YH peripheral blood mononuclear cells. We found that the sequencing data of MB-seq and MRB-seq almost reaches saturation after generating 7-8 Gbp data, whereas BS-seq requires about 100 Gbp data to achieve the same effect. In comparison to MeDIP-seq and BS-seq, MB-seq offers several key advantages, including single-base resolution, discriminating the methylated sites within a CpG and non-CpG pattern and overcoming the false positive of MeDIP-seq due to the non-specific binding of 5-methylcytidine antibody to genomic fragments. CONCLUSION: Our novel developed method MB-seq can accelerate the decoding process of DNA methylation mechanism in human diseases because it requires 7-8 Gbp data to measure human methylome with enough coverage and sequencing depth, affording it a direct and practical application in the study of multiple samples. In addition, we have also provided a novel alternative MRB-seq method, which removes most repetitive sequences and allows researchers to genome-wide characterize DNA methylation of functional regions.

Harmine suppresses the proliferation and migration of human ovarian cancer cells through inhibiting ERK/CREB pathway.

Ovarian cancer is the most lethal gynaecological cancer and the sixth most common cause of cancer related death among Western women. Recent studies show that harmine, a small-molecular ß-carboline alkaloid present in medicinal plants, displayed obvious anticancer effects in several cancer cells. However, the effect of harmine on ovarian cancer is not well understood. In the present study, the effect of harmine on the cell proliferation and migration of ovarian cancer SKOV-3 cells and the underlying mechanism were investigated. Our results indicated that harmine significantly suppressed the proliferation of SKOV-3 cells in a dose-dependent manner. Interestingly, it also inhibited the epidermal growth factor (EGF)-induced proliferation of SKOV-3 cells. Moreover, the migration of SKOV-3 cells was markedly inhibited by harmine treatment. Further study showed that harmine inhibited not only the basal phosphorylation level of extra-cellular signal-regulated kinase 1/2 (ERK1/2) and cyclic adenosine monophosphate response element-binding protein (CREB) but also EGF-induced ERK1/2 and CREB phosphorylation. Finally, harmine significantly suppressed the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP) family MMP-2, and MMP-9. In conclusion, our data revealed that harmine inhibited the proliferation and migration of SKOV-3 cells, which might be mediated by ERK/CREB pathway. These findings elucidate that harmine may act as a potential therapeutic drug for ovarian cancer treatment.

Ovarian Damages Produced by Aerosolized Fine Particulate Matter (PM2.5) Pollution in Mice: Possible Protective Medications and Mechanisms.

BACKGROUND: Ambient aerosol fine particulate matter (PM2.5) is associated with male reproductive toxicity in experiments and may have adverse effects in the female. However, studies evaluating the protective effects and precise mechanisms of aspirin, Vitamin C, Vitamin E, or ozone against toxic effects of PM2.5are sparse. This study was conducted to investigate the possible protective effects and mechanisms of aspirin, Vitamin C, Vitamin E, or ozone on fertility in female mice treated with PM2.5. METHODS: Eighty-four ICR mice were divided into six groups: control group, PM2.5group, PM2.5 + aspirin group, PM2.5 + Vitamin C group, PM2.5 + Vitamin E group, and PM2.5 + ozone group. PM2.5was given by intratracheal instillation every 2 days for 3 weeks. Aspirin, Vitamin C, and Vitamin E were given once a day by oral gavage for 3 weeks, and ozone was administered by intraperitoneal injection once a day for 3 weeks. The levels of anti-Müllerian hormone (AMH), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured using enzyme-linked immunosorbent assay. Western blotting analysis was used to analyze the expressions of Bcl-2, Bax, and caspase-3 in ovaries. Changes in histological structure were examined by light microscope and electron microscopy was used to detect ultramicrostructure. RESULTS: The results demonstrated that PM2.5 decreased AMH levels (P < 0.001); however, aspirin (P < 0.001), Vitamin C (P < 0.001), Vitamin E (P = 0.001), and ozone (P = 0.002) alleviated the decrease. Changes of IL-6, TNF-α, 8-OHdG, Bax/Bcl-2, and caspase-3 in PM2.5group were increased compared to control group (P < 0.001), while in PM2.5 + aspirin, PM2.5 + Vitamin C, PM2.5 + Vitamin E, and PM2.5 + ozone groups, they were statistically decreased compared to PM2.5group (P < 0.001 or P< 0.05). CONCLUSIONS: PM2.5cause the damage of ovaries, and aspirin, Vitamin C, Vitamin E, and ozone antagonizes the damage. The protective mechanism is probably due to its ability to blunt the inflammatory and oxidative stress caused by PM2.5, which subsequently suppressing the expression of apoptotic regulatory protein and reducing the incidence of ovary apoptosis.

Down-regulation of Gli transcription factor leads to the inhibition of migration and invasion of ovarian cancer cells via integrin ß4-mediated FAK signaling.

BACKGROUND: Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including ovarian cancer. Therefore, chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for ovarian cancer. RESULTS: In this study, we show that activation of Hh signaling promoted cellular migration and invasion, whereas blockade of Hh signaling with GANT61 suppressed cellular migration and invasion in ovarian cancer cells. After treatment with GANT61, cDNA microarray analyses revealed changes in many genes such as Integrin ß4 subunit (ITGB4), focal adhesion kinase (FAK), etc. Furthermore, ITGB4 expression was up-regulated by Sonic Hedgehog (Shh) ligand and down-regulated by Hh signaling inhibitor. The Shh-mediated ovarian cell migration and invasion was blocked by neutralizing antibodies to ITGB4. In addition, phosphorylations of FAK were increased by Shh and decreased by Hh signaling inhibitor. Inhibition of Gli1 expression using siRNA mimicked the effects of GANT61 treatment, supporting the specificity of GANT61. Further investigations showed that activation of FAK was required for Shh-mediated cell migration and invasion. Finally, we found that down-regulation of Gli reduced the expression of ITGB4 and the phosphorylated FAK, resulting in the inhibition of tumor growth in vivo. CONCLUSIONS: The Hh signaling pathway induces cell migration and invasion through ITGB4-mediated activation of FAK in ovarian cancer. Our findings suggest that the diminishment of crosstalk between phosphorylated FAK and ITGB4 due to the down-regulation of Gli family transcription factors might play a pivotal role for inhibiting ovarian cancer progression.

Hedgehog signaling pathway and ovarian cancer.

Epithelial ovarian carcinoma (EOC) is the most common form of ovarian malignancies and the most lethal gynecologic malignancy in the United States. To date, in spite of treatment to it with the extensive surgical debulking and chemotherapy, the prognosis of EOC remains dismal. Recently, it has become increasingly clear that in many instances, the signaling and molecular players that control development are the same, and when inappropriately regulated, drive tumorigenesis and cancer development. Here, we discuss the possible involvement of Hedgehog (Hh) pathway in the cellular regulation and development of cancer in the ovaries. Using the in vitro and in vivo assays developed has facilitated the dissection of the mechanisms behind Hh-driven ovarian cancers formation and growth. Based on recent studies, we propose that the inhibition of Hh signaling may interfere with spheroid-like structures in ovarian cancers. The components of the Hh signaling may provide novel drug targets, which could be explored as crucial combinatorial strategies for the treatment of ovarian cancers.

Effect of WFDC 2 silencing on the proliferation, motility and invasion of human serous ovarian cancer cells in vitro.

OBJECTIVE: To investigate effect and possible mechanisms of silencing human WFDC2 (HE4) gene on biological behavior changes as cell proliferation, apoptosis, movement and invasion of human serous ovarian cancer cell line SKOV3. METHODS: Lentiviral WFDC2 gene sequence of small interfering siRNA was stablely transfected into SKOV3 identified by Q-PCR and western-blot. Obtained SKOV3 stable strains with silenced HE4 were measured by proliferation, apoptosis, migration, and invasion. RESULTS: Gene sequencing showed that the oligonucleotides were successfully inserted into the expected site. After silencing HE4 in the SKOV3, proliferation was significantly inhibited (P<0.05). G(0)/G(1) phase was arrested by the cell cycle (P<0.01) and capacity of the migration and invasion decreased significantly (P<0.01). Slight early apoptosis ratio and no change of late apoptosis were found without change of Caspase-3 or Bcl-2 protein. Proteins involved in ERK pathway as phosphorylated protein as p-EGFR, p-ERK decreased and protease protein involved in tissue remodeling as matrix metalloproteinases MMP-9, MMP-2 and cathepsin B decreased compared with control group. CONCLUSIONS: HE4 gene plays an important role in regulating proliferation, apoptosis, migration, invasion of serous ovarian cancer cells by ERK pathway and protease system. Its role in apoptosis needs to be further explored, and it may be a potential target for serous ovarian cancer.