Deletion of proapoptotic Puma selectively protects hematopoietic stem and progenitor cells against high-dose radiation.

Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here, we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively induced by irradiation in primitive hematopoietic cells, and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy.

Tumor-specific expression of microRNA-26a suppresses human hepatocellular carcinoma growth via cyclin-dependent and -independent pathways.

MicroRNA-26a (miR-26a) is a tumor suppressor that is reduced in hepatocellular carcinoma (HCC). Increasing evidence indicates that the liver is a hormone-responsive organ like the breast. The purpose of this study was to investigate whether miR-26a, regulated by a human α-fetoprotein (hAFP) and human telomerase reverse transcriptase (hTERT) dual promoter, could be specifically expressed in liver tumor cells to suppress their growth and to clarify whether estrogen receptor-α (ERα) is regulated by miR-26a and involved in the HCC process. Our data show that miR-26a expression driven by a hAFP-TERT dual promoter was tumor-specific and decreased the viability of tumor cells by regulating ERα, progesterone receptor (PR) and P53 except for cyclin D2 or cyclin E2 in vitro and in vivo. Our data also show that estradiol (E2) promotes the growth of liver cancer cells similar to breast cancer cells partly via the E2-ERα pathway and that miR-26a significantly down regulates ERα and prevents the stimulation of hepatoma cell growth by E2. These data suggest that ERα, which is regulated by miR-26a, is important for liver tumor cell growth. Moreover, hAFP-TERT dual promoter-mediated miR-26a expression could specifically exert potential antitumor activity and provide a novel targeting approach for cancer therapy.

[Effects of Chinese herbal medicine Neiyi Recipe-medicated serum on angiopoiesis of endometriosis in the chick chorioallantoic membrane model].

OBJECTIVE: To compare angiopoiesis ability of eutopic and ectopic endometrial tissue isolated from women with endometriosis and endometrium isolated from women without endometriosis (control), and to explore the inhibitory effects of medicated serum of Neiyi Recipe, a compound traditional Chinese herbal medicine. METHODS: Chick chorioallantoic membrane (CAM) model of endometriosis was established by transplanting endometrium onto CAM. The CAMs were then hatched with blank serum or medicated serum of danazol or Neiyi Recipe, which were prepared in rats by orally administering. The sizes of the transplanted tissue and new vessels around the transplanted tissue were measured. Expression of vascular endothelial growth factor (VEGF) was detected by immunohistochemical method. RESULTS: There was no difference in the sizes of transplanted tissue among CAM models of ectopic and eutopic endometrial tissue isolated from women with endometriosis or control (P>0.05), and more new vessels were found around the ectopic and eutopic endometrial tissue than the endometrial tissue of control (P<0.05). Compared with the controls, the size of the transplanted tissue and positive area of new vessels were significantly inhibited by Neiyi Recipe-medicated serum (P<0.01, P<0.05), and similar changes happened in the danazol groups, except for the size of transplanted tissue from ectopic endometrial tissue (P>0.05). Expression of VEGF was significantly higher in eutopic and ectopic endometrial tissue than in the control (P<0.01); the level of VEGF obviously reduced in the Neiyi Recipe and danazol groups (P<0.01), but no significant difference was detected between them. CONCLUSION: Endometrium from women with endometriosis stimulates the formation of new vessels by increase the expression of VEGF. Neiyi Recipe-medicated serum significantly decreases the expression of VEGF in eutopic and ectopic endometrial tissues and thus restrains the formation of new vessels, reduces the blood supply and inhibits growth of ectopic endometrial tissue, which are similar to danazol, but has greater efficacy in suppressing the expression of VEGF.

Androgen Excess Induced Mitochondrial Abnormality in Ovarian Granulosa Cells in a Rat Model of Polycystic Ovary Syndrome.

Background: Androgen excess could profoundly lead to follicular dysplasia or atresia, and finally result in polycystic ovary syndrome (PCOS); however, the exact mechanism remains to be fully elucidated. Methods: PCOS model rats were induced by dehydroepiandrosterone, and their fertility was assessed. The ovarian granulosa cells (GCs) from matured follicles of PCOS model rats were collected and identified by immunofluorescence. The mitochondrial ultrastructure was observed by transmission electron microscope and the mitochondrial function was determined by detecting the adenosine triphosphate (ATP) content and mtDNA copy number. Besides, the expressions of respiratory chain complexes and ATP synthases in relation to mitochondrial function were analyzed. Results: The PCOS model rats were successfully induced, and their reproductive outcomes were obviously adverse. The GCs layer of the ovarian was apparently cut down and the mitochondrial ultrastructure of ovarian GCs was distinctly destroyed. The ATP content and mtDNA copy number of ovarian GCs in PCOS model rats were greatly reduced, and the expressions of NDUFB8 and ATP5j were significantly down-regulated without obvious deletion of mtDNA 4834-bp. Conclusions: Androgen excess could damage mitochondrial ultrastructure and function of GCs in rat ovary by down-regulating expression of NDUFB8 and ATP5j in PCOS.

Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity.

Endometriosis (EMs) occurs in approximately 50% of women with infertility. The main causes of EMs-related infertility are follicle dysplasia and reduced oocyte quality. Iron overload occurs in ovarian follicular fluid (FF) of patients with EMs, and this condition is associated with oocyte maturation disorder. However, the underlying molecular mechanism remains largely unknown. In the present study, we identified the mechanism underlying ferroptosis in ovarian granulosa cells and oocyte maturation failure in EMs based on a retrospective review of in vitro fertilization/intracytoplasmic sperm injection-frozen embryo transfer outcomes in infertile patients with EMs. Mouse granulosa cells were treated with EMs-related infertile patients' follicular fluid (EMFF) in vitro. Western blot analysis, quantitative polymerase chain reaction, fluorescence staining, and transmission electron microscopy were used to assess granulosa cells ferroptosis. The effects of exosomes were examined by nanoparticle tracking analysis, RNA-seq, and Western blot analysis. Finally, the therapeutic values of vitamin E and iron chelator (deferoxamine mesylate) in vivo were evaluated in an EMs-related infertility model. Patients with ovarian EMs experienced poorer oocyte fertility than patients with non-ovarian EMs. We observed that EMFF with iron overload-induced granulosa cell ferroptosis in vitro and in vivo. Mechanically, nuclear receptor coactivator four-dependent ferritinophagy was involved in this process. Notably, granulosa cells undergoing ferroptosis further suppressed oocyte maturation by releasing exosomes from granulosa cells. In therapeutic studies, vitamin E and iron chelators effectively alleviated EMs-related infertility models. Our study indicates a novel mechanism through which EMFF with iron overload induces ferroptosis of granulosa cells and oocyte dysmaturity in EMs-related infertility, providing a potential therapeutic strategy for EMs-related infertility.

Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3 and increasing MYL-9 expression.

BACKGROUND: Abnormal proliferation, apoptosis, migration and contraction of airway smooth muscle (ASM) cells in airway remodeling in asthma are basically excessive repair responses to a network of inflammatory mediators such as PDGF, but the mechanisms of such responses remain unclear. Nogo-B, a member of the reticulum family 4(RTN4), is known to play a key role in arteriogenesis and tissue repair. Further studies are needed to elucidate the role of Nogo-B in airway smooth muscle abnormalities. METHODS: A mouse model of chronic asthma was established by repeated OVA inhalation and subjected to Nogo-B expression analysis using immunohistochemistry and Western Blotting. Then, primary human bronchial smooth muscle cells (HBSMCs) were cultured in vitro and a siRNA interference was performed to knockdown the expression of Nogo-B in the cells. The effects of Nogo-B inhibition on PDGF-induced HBSMCs proliferation, migration and contraction were evaluated. Finally, a proteomic analysis was conducted to unveil the underlying mechanisms responsible for the function of Nogo-B. RESULTS: Total Nogo-B expression was approximately 3.08-fold lower in chronic asthmatic mice compared to naïve mice, which was obvious in the smooth muscle layer of the airways. Interference of Nogo-B expression by siRNA resulted nearly 96% reduction in mRNA in cultured HBSMCs. In addition, knockdown of Nogo-B using specific siRNA significantly decreased PDGF-induced migration of HBSMCs by 2.3-fold, and increased the cellular contraction by 16% compared to negative controls, but had limited effects on PDGF-induced proliferation. Furthermore, using proteomic analysis, we demonstrate that the expression of actin related protein 2/3 complex subunit 5 (ARPC 2/3) decreased and, myosin regulatory light chain 9 isoform a (MYL-9) increased after Nogo-B knockdown. CONCLUSIONS: These data define a novel role for Nogo-B in airway remodeling in chronic asthma. Endogenous Nogo-B, which may exert its effects through ARPC 2/3 and MYL-9, is necessary for the migration and contraction of airway smooth muscle cells.

Upregulation of programmed death-1 on T cells and programmed death ligand-1 on monocytes in septic shock patients.

INTRODUCTION: Studies on the role of programmed death-1(PD-1) and its main ligand (PD-L1) during experimental models of sepsis have shown that the PD-1/PD-L1 pathway plays a pathologic role in altering microbial clearance, the innate inflammatory response and accelerated apoptosis in sepsis. However, the expression of PD-1 and PD-L1 and their role during the development of immune suppression in septic patients have not been elucidated. The present study was designed to determine whether the expression of PD-1 and PD-L1 is upregulated in septic shock patients and to explore the role of this pathway in sepsis-induced immunosuppression. METHODS: Nineteen septic shock patients and 22 sex-matched and age-matched healthy controls were prospectively enrolled. Apoptosis in lymphocyte subpopulations and PD-1/PD-L1 expression on peripheral T cells, B cells and monocytes were measured using flow cytometry. Apoptosis of T cells induced by TNFα or T-cell receptor ligation in vitro and effects of anti-PD-L1 antibody administration were measured by flow cytometry. CD14+ monocytes of septic shock patients were purified and incubated with either lipopolysaccharide, anti-PD-L1 antibody, isotype antibody, or a combination of lipopolysaccharide and anti-PD-L1 antibody or isotype antibody. Supernatants were harvested to examine production of cytokines by ELISA. RESULTS: Compared with healthy controls, septic shock induced a marked increase in apoptosis as detected by the annexin-V binding and active caspase-3 on CD4+ T cells, CD8+ T cells and CD19+ B cells. Expression of PD-1 on T cells and of PD-L1 on monocytes was dramatically upregulated in septic shock patients. PD-1/PD-L1 pathway blockade in vitro with anti-PD-L1 antibody decreased apoptosis of T cells induced by TNFα or T-cell receptor ligation. Meanwhile, this blockade potentiated the lipopolysaccharide-induced TNFα and IL-6 production and decreased IL-10 production by monocytes in vitro. CONCLUSIONS: The expression of PD-1 on T cells and PD-L1 on monocytes was upregulated in septic shock patients. The PD-1/PD-L1 pathway might play an essential role in sepsis-induced immunosuppression.

The Mechanism Exploration of Follicular Fluids on Granulose Cell Apoptosis in Endometriosis-Associated Infertility.

OBJECTIVE: To explore the mechanisms of follicular fluids (FFs) on granulose cell (GC) apoptosis in endometriosis-associated infertility. MATERIALS AND METHODS: 60 infertile women were enrolled. The FFs from 30 endometriosis-associated infertility (EI) patients were collected and processed by ELISA hormone assay and proteomic profiling. The ovary GCs collected from 30 tubal-associated infertility (TI) patients were cultured in follicular fluids of endometriosis-associated infertility patients (EI-FFs), and the apoptosis mechanisms were explored by flow cytometry assay, real-time PCR, Western blotting, and protein-protein interaction (PPI) network analysis. RESULTS: Our results showed that the expression of 22 specific proteins was significantly different in the FFs from EI and TI patients, and the level of testosterone and anti-Müllerian hormone was not obviously different between the two groups. EI-FFs could accelerate the apoptosis process of granulose cells of tubal-associated infertility patients (TI-GCs) by regulating the expression of 5 apoptosis-related proteins including BCL2, BAX, CASP3, CASP9, and TP53. The correlation of these 22 specific proteins and 5 apoptosis-related proteins was analyzed by PPI, and 5 protein biomarkers (INS, CXCL10, ICAM1, WIF1, and TNFRSF13C) and 5 signaling pathways (cytokine-cytokine receptor interaction, apoptosis, regulation of actin cytoskeleton, MAPK, and p53 signaling pathway) were predicted. CONCLUSION: This research clarified the effect and explored the mechanisms of EI-FFs on the apoptosis of TI-GCs and indicated the protein biomarkers and signaling pathways for further study.

Aggregative Perivascular Tumor Cell Growth Pattern of Primary Central Nervous System Lymphomas Is Associated with Hypoxia-Related Endoplasmic Reticulum Stress.

Primary central nervous system lymphomas (PCNSLs) often present a unique histopathological feature of aggregative perivascular tumor cells (APVT). Our previous studies showed that patients of PCNSL with APVTs exhibited poor long-term outcomes and increased expression of the endoplasmic reticulum stress (ERS) factor X-box-binding protein (XBP1). However, very little is known about molecular mechanism of the APVT formation in PCNSLs. The aim of this study is to determine if hypoxia-induced ERS is related to the APVT formation in PCNSLs. In this study, cell culture was used to observe the interplay between diffuse large B cell lymphoma (DLBCL) tumor cells and human brain microvascular endothelial cells (HBMECs) in different oxygen conditions. The expression of XBP1, CXCR and CD44 was manipulated by molecular cloning and siRNA technology. Mouse in vivo experiments and clinical studies were conducted to confirm our hypothesis. Our results showed that activated B-cell type-DLBCL cells easily migrated and invaded, and expressed high levels of XBP1 and stromal molecules CXCR4 and CD44 during hypoxia-induced ERS and dithiothreitol unfolded protein response (UPR). The gene upregulation (using overexpression vector) and downregulation (siRNA gene knock-out) in cultured cells and in mouse models further confirmed a close relation of the expression of XBP1, CXCR4, and CD44 with APVT formation, which is coincided with our clinical observation that increased expression of XBP1, CXCR4, and CD44 in the APVT cells in PCNSLs were associated with poor clinical outcomes. The results suggest that hypoxia-induced ERS and UPR might be associated with APVTs formation in PCNSL and its poor clinical outcomes. The results will help us better understand the progression of PCNSL with APVTs feature in daily pathological work and could be valuable for future target treatment of PCNSLs.

p53-insensitive PUMA down-regulation is essential in the early phase of liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: Liver regeneration after partial hepatectomy involve proliferation and apoptosis of hepatocytes. PUMA, the well-known proapoptotic member of the Bcl-2 family, can respond to distinct stimuli. This study explores the role of PUMA and its relationship with other Bcl-2 family members in this process. METHODS: The expression patterns of PUMA and its related proteins were investigated in livers after 70% hepatectomies. The contributions of PUMA to liver regeneration were assessed by manipulating its expression levels using adenovirus vectors. The differences in PUMA expression levels in human normal livers and hepatitis, as well as hepatoma tissues were characterised. RESULTS: During the first 72h after hepatectomy, PUMA was highly down-regulated transcriptionally, while the levels of p53, Slug, Bax, and Bcl-X(L) proteins increased continuously. Highly induced expression of PUMA in the liver by Ad-PUMA caused lethal fulminant hepatitis 48h after treatment. Slightly induced expression was enough to impair liver regeneration, with an elevation of post-hepatectomy mortality, an increase of apoptosis, a decrease of proliferation, an up-regulation of Bax levels, an induction of inflammatory chemokines (KC and macrophage inflammatory protein-2), and an increase in the neutrophil infiltration relative to the control. In contrast to the results from the regenerating liver, PUMA expression showed an increased trend in human hepatitis and hepatoma tissues. CONCLUSIONS: Sharply p53-insensitive down-regulation of PUMA, coupled with Bcl-X(L) up-regulation, may play a cytoprotective role in liver regeneration after hepatectomy. Furthermore, the increased expression of PUMA in hepatitis and hepatoma may indicate misregulation of the apoptotic network in these diseases.

PD-L1 blockade improves survival in experimental sepsis by inhibiting lymphocyte apoptosis and reversing monocyte dysfunction.

INTRODUCTION: Lymphocyte apoptosis and monocyte dysfunction play a pivotal role in sepsis-induced immunosuppression. Programmed death-1 (PD1) and its ligand programmed death ligand-1 (PD-L1) exert inhibitory function by regulating the balance among T cell activation, tolerance, and immunopathology. PD-1 deficiency or blockade has been shown to improve survival in murine sepsis. However, PD-L1 and PD-1 differ in their expression patterns and the role of PD-L1 in sepsis-induced immunosuppression is still unknown. METHODS: Sepsis was induced in adult C57BL/6 male mice via cecal ligation and puncture (CLP). The expression of PD-1 and PD-L1 expression on peripheral T cells, B cells and monocytes were measured 24 hours after CLP or sham surgery. Additionally, the effects of anti-PD-L1 antibody on lymphocyte number, apoptosis of spleen and thymus, activities of caspase-8 and caspase-9, cytokine production, bacterial clearance, and survival were determined. RESULTS: Expression of PD-1 on T cells, B cells and monocytes and PD-L1 on B cells and monocytes were up-regulated in septic animals compared to sham-operated controls. PD-L1 blockade significantly improved survival of CLP mice. Anti-PD-L1 antibody administration prevented sepsis-induced depletion of lymphocytes, increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production, decreased IL-10 production, and enhanced bacterial clearance. CONCLUSIONS: PD-L1 blockade exerts a protective effect on sepsis at least partly by inhibiting lymphocyte apoptosis and reversing monocyte dysfunction. Anti-PD-L1 antibody administration may be a promising therapeutic strategy for sepsis-induced immunosuppression.

Correlation of contractile function recovery with acetylcholine receptor changes in a rat muscle flap model.

The aim of this study was to investigate the correlation between contractile function recovery and changes of acetylcholine receptors (AChR) in a transferred muscle flap following reinnervation. Orthotopic transfer of the gracilis muscle flap with repair of its nerve was performed bilaterally in 48 rats. The rats were randomly divided into six experimental groups based on the time intervals for assessments (1, 4, 5, 10, 20, and 30 weeks). Sixteen gracilis muscle samples from eight rats without surgery were used as the controls. In each group, muscle contractile force and weight were measured (n = 16). The AChR numbers (n = 8) and subunits (epsilon and gamma) mRNA (n = 8) were examined using [(125)I]-alpha-bungarotoxin and fluorescent quantitative-PCR. The results showed the AChR numbers in the muscle flap increased from 4 to 20 weeks after reinnervation and correlated with recovery of the tetanic contraction force. However, correlation between the increase of AChR number with the specific tension (peak contractile force normalized to wet muscle weight) was only found from 4 to 10 weeks postoperatively. The expression of gamma-subunit mRNA increased at the early period after flap transfer and then decreased rapidly, whereas the epsilon-subunit mRNA recovered gradually since fourth week postoperatively. A small amount of gamma-subunit mRNA could still be detected at 30 weeks after surgery. In conclusion, following reinnervation of the transferred muscle flap, the contractile functional recovery is partially correlated to increase of the AChRepsilon. Our findings may provide evidence for further study of improving muscle function in functional reconstruction by targeting the AChR.

Transferrin Insufficiency and Iron Overload in Follicular Fluid Contribute to Oocyte Dysmaturity in Infertile Women With Advanced Endometriosis.

Purpose: To screen out specific protein with different concentration in follicular fluid from advanced endometriosis and determine its direct effect on mouse oocytes matured in vitro. Methods: FF samples were obtained from 25 patients (EMS group, n = 15; control group, n = 10) to screen the differential proteins by using iTRAQ Labeling and 2D LC-MS. Transferrin (TRF) in was found significantly decreased in EMS group, which was verified using ELISA in enlarged FF samples (EMS group, n = 31; control group, n = 27). The contents of ferric ion in FFs were detected by ELISA and TRF saturations were calculated in two groups. Germinal vesicle (GV) oocytes of mouse were maturated in vitro interfered with the FFs in five groups, whose concentrations of TRF were modulated, and maturation in vitro rates were compared among groups. Results: The reduced concentration of TRF with three analogs and increased concentration of ferric ion were found in the FF of the EMS group (p < 0.05). The numerical values of TSAT was 54.8% in EMS group, indicating iron overload in the FF. The EMS-FF showed significantly decreased maturation in vitro rate (p < 0.05) of mouse oocytes, which was improved with the supplementation of TRF, compared with the control-FF. The effect was blocked by the TRF antibody (p < 0.05). Conclusions: Being aware of the relatively small sample size, our results possibly suggest that TRF insufficiency and iron overload in FF from advanced EMS contribute to oocytes dysmaturity, which may be a cause of EMS-related infertility.

Correlation Between Fecal Metabolomics and Gut Microbiota in Obesity and Polycystic Ovary Syndrome.

Objective: This study aimed to explore the relationship between the fecal metabolites and gut microbiota in obese patients with PCOS and provide a new strategy to elucidate the pathological mechanism of obesity and PCOS. Methods: The fecal samples of obese patients with PCOS (n = 18) and obese women without PCOS (n = 15) were analyzed by 16S rRNA gene sequencing and untargeted metabolomics. The peripheral venous blood of all subjects was collected to detect serum sex hormones. The association among fecal metabolites, gut microbiota, and serum sex hormones was analyzed with the R language. Results: A total of 122 named differential fecal metabolites and 18 enrichment KEGG pathways were obtained between the groups. Seven fecal metabolites can be used as characteristic metabolites, including DHEA sulfate. The richness and diversity of gut microbiota in the obese PCOS group were lower than those in the control group. Lachnoclostridium, Fusobacterium, Coprococcus_2, and Tyzzerela 4 were the characteristic genera of the obese patients with PCOS. Serum T level significantly and positively correlated with the abundance of fecal DHEA sulfate (p < 0.05), and serum DHEAS level significantly and negatively correlated with the abundance of fecal teasterone (p < 0.05). Conclusion: Specific fecal metabolites may be used as characteristic metabolites for obese patients with PCOS. The closely relationship among gut microbiota, fecal metabolites, and serum sex hormones may play a role in the related changes caused by hyperandrogenemia.

Characteristic gut microbiota and predicted metabolic functions in women with PCOS.

Polycystic ovary syndrome (PCOS) is a chronic endocrine and metabolic disease. Gut microbiota is closely related to many chronic diseases. In this study, we conducted a cross-sectional study and recruited 30 obese (OG) and 30 non-obese (NG) women with PCOS, 30 healthy women (NC) and 11 healthy but obese women (OC) as controls to investigate the characteristic gut microbiota and its metabolic functions in obese and non-obese patients with PCOS. The blood and non-menstrual faecal samples of all the participants were collected and analysed. As a result, the Hirsutism score, LH/FSH and serum T level in NG and OG both increased significantly compared with their controls (P < 0.05). High-throughput 16S rRNA gene sequencing revealed that the abundance and diversity of the gut microbiota changed in patients with PCOS. The linear discriminant analysis (LDA) indicated that Lactococcus was the characteristic gut microbiota in NG, while Coprococcus_2 in OG. Correlation heatmap analysis revealed that the sex hormones and insulin levels in human serum were closely related to the changes in the gut microbiota of NG and OG. Functional prediction analysis demonstrated that the citrate cycle pathway enriched both in NG and OG, and other 12 gut bacterial metabolic pathways enriched in NG. This study highlighted significant differences in the gut microbiota and predictive functions of obese and non-obese women with PCOS, thereby providing insights into the role and function of the gut microbiota that may contribute to the occurrence and development of PCOS in obese and non-obese women.

Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS.

High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and enzyme-linked immunosorbent assays. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique, and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, Baicalin decreased the testosterone concentrations in a dose-and time-dependent manner in NCI-H295R cells. GEP revealed that 3ß-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity, and decreased the expression of HSD3B2 in ovarian. In summary , our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.

Follicle-stimulating hormone-induced activation of Gata4 contributes in the up-regulation of Cyp19 expression in rat granulosa cells.

Several studies have suggested that the transcription factor GATA4 plays an important role in ovarian function. This study evaluated the effects of GATA4 on the regulation of the Cyp19 gene in primary rat granulosa cells under basal conditions and in response to stimulation by FSH. A significant increase in GATA4 mRNA, protein, and DNA binding activity was observed in rats treated with pregnant mare serum gonadotropin, a hormone that binds to the FSH receptors, and in granulosa cells incubated with FSH. Enrichment of the Cyp19 promoter was observed in granulosa cells treated with FSH after chromatin precipitation with an anti-GATA4 antibody. Mutation of the GATA binding site on the Cyp19 promoter and inhibition of GATA4 expression with specific small interfering RNA significantly reduced FSH-enhanced Cyp19 expression, whereas overexpression of GATA4 increased Cyp19 promoter activity. A synergistic effect observed between GATA4 overexpression and FSH treatment in Cyp19 expression was abolished by mutating Ser105 in the GATA4 protein or by pretreating granulosa cells with a protein kinase A inhibitor. Inhibition of phosphatidylinositol-dependent kinase (PI3-K)/casein kinase 2 or ERK1/2 attenuated GATA4/FSH synergism, whereas the simultaneous blockade of PI3-K/casein kinase 2 and ERK1/2 activity eliminated Cyp19 stimulation. Finally, we demonstrated that FSH increases GATA4 phosphorylation and that GATA4 activation requires the activation of multiple kinases, including ERK1/2, PI3-K, and protein kinase A. These findings demonstrate that GATA4 contributes in the regulation of Cyp19 expression in the rat ovary and provide the first evidence that FSH regulates GATA4 activity.

Regulation of aromatase P450 expression by puerarin in endometrial cell line RL95-2.

OBJECTIVE: To study the effects of puerarin on the aromatase P450 (P450(arom)) mRNA expression and the effects of low-dose puerarin on transcription factors of the P450(arom) gene (P II) 5'-flanking region. METHODS: The effects of puerarin on the P450(arom) mRNA expression were determined by real-time polymerase chain reaction (RT-PCR). The 5'-flanking region was amplified by PCR using human genomic cDNA as a template. By means of the restriction sites and sequence confirmation, the PCR product was cloned into reporter vector. Series of sequential deletion reporter constructs were transiently transfected into RL95-2 cells which were treated with or without puerarin. Luciferase activity was measured by Dual-Luciferase Reporter Assay System and Luminoskan Ascent luminometer. Furthermore, by using web-based search program, the most possible cis-acting elements and transcription factors were evaluated. RESULTS: The data demonstrated that low-dose puerarin treatment could decrease P450(arom) expression at mRNA level compared to dimethyl sulphoxide (DMSO) treatment (P<0.01), and puerarin (10(-7)mol/L) had a time-course effect on P450(arom) mRNA expression, which reached the bottom at 12h (P<0.01). Cells transfected with the -763/+8 bp constructs showed decrease in relative luciferase activity after puerarin (10(-7)mol/L) treatment compared to DMSO treatment (P<0.05), indicating an essential regulatory site between -763 bp and -543 bp responsible for the transcription suppression by puerarin. Furthermore, the most possible transcription factors, which turned out to be AP-1(c-jun/c-fos) at -410/-401 bp were also evaluated. The activity of exogenous AP-1 was reduced after 12 hours of puerarin treatment (P<0.05). The inhibition of c-jun mRNA also showed a time-course effect, which bottomed out at 12h in parallel with that of P450(arom) (P<0.01). The protein level of c-jun was also down-regulated by puerarin (10(-7)mol/L) treatment at 12h. CONCLUSION: The suppression of P450(arom) expression and activity may be associated with the down-regulation of transcription factor AP-1/c-jun. This partially explains the mechanisms whereby puerarin treats endometriosis.

Mining and Functional Verification of Calcitonin Genes from the Genome Database.

BACKGROUND: With more countries in the world entering elderly society, osteoporosis is a common disease among the elderly, especially middle-aged and elderly women. Although calcitonin is an effective drug used to treat osteoporosis in clinical practice, it also exists such problems as high cost, short half-life, and high immunogenicity. Therefore, to explore more efficient calcitonin has important clinical significance. OBJECTIVE: Given the emergence of new-generation gene sequencing, numerous genome sequences of marine species have been revealed. This study aimed to identify new, highly active Calcitonins (CTs) from the gene database. METHODS: Candidate CT sequences were obtained by BLAST and analyzed. The evolutionary tree of these sequences was constructed using the Neighbor-Joining method of MEGA 7 software. Secondary structures were analyzed by Circular Dichroism (CD). The biological activities of CTs were estimated using the standard of the rat hypocalcemic activity assay in vivo. The half-life and immunogenicity of CT sequences were determined by ELISA. The physicochemical properties of peptides were analyzed with ProtParam and HeliQuest. RESULTS: A total of 64 candidate CT gene and amino acid sequences from different species were obtained by BLAST using the salmon CT (sCT) sequence as the query sequence. These sequences were clustered to 27 different CT polypeptide sequences, and then the evolutionary tree was constructed. A total of 13 sequences were selected for chemical synthesis and activity assay. Results showed that although their secondary structures were similar, four types of candidate CTs had 30% higher activities than sCT, three other types had similar activities to sCT, and the remaining four types had much lower activities than sCT. Among the three designed CTs, the activities of CT-01 and CT-02 were at least 50% higher than those of sCT. Furthermore, all three CT sequences had a similar half-life to sCT and lower immunogenicity. CONCLUSION: CTs from Monodelphis domestica, Gallus gallus, Ornithorhynchus anatinus, and Carassius auratus had high activities. The exploration and mining of the marine-life genome database can be extremely valuable considering broad application prospect.

Targeted therapy and immunotherapy for platinum-refractory advanced ovarian adenosquamous carcinoma: a case report.

BACKGROUND: Ovarian adenosquamous carcinoma is an extremely rare type of ovarian histology. Platinum-refractory disease is also uncommon, but can be fatal because of the lack of available treatment options. To date, there is no study or case report on platinum-refractory ovarian adenosquamous carcinoma or its relevant treatment. CASE PRESENTATION: Herein, we report the case of a 38-year-old Chinese woman with platinum-refractory advanced ovarian adenosquamous carcinoma who received clinical benefit from poly adenosine diphosphate ([ADP] ribose) polymerase and programmed death-1 inhibitors after failure of prior multiline chemotherapies and antiangiogenic agents. The targeted therapy and immunotherapy-controlled disease deterioration and improved performance status. Thus far, the patient has survived longer than 15 months, and she is taking nivolumab as maintenance treatment. CONCLUSION: Targeted therapy and immunotherapy may be options for rare categories of ovarian cancer, but this warrants more clinical evidence of efficacy and toxicity.