Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis.

Lentinan (LNT) isolated from Lentinus edodes is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT on the mouse hepatocellular carcinoma (HCC) cell line Hepa1­6 and its possible mechanism. Mouse HCC apoptosis and its potential associated mechanism were then explored using in vitro and in vivo approaches. For in vitro approaches, the effect of LNT on the proliferation of Hepa1­6 cells was investigated by Cell Counting Kit­8 assay. Annexin V­FITC staining and flow cytometry were applied to explore HCC apoptosis. Western blotting was used to analyze related proteins, such as EGR1, phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (p­Akt), protein kinase B (Akt), B lymphocyte­2 (Bcl­2), Bcl2 family­associated X protein (Bax), etc. Cellular immunofluorescence staining was employed to assess the localization and expression of EGR1 and PTEN in nuclear and cytoplasmic fractions of Hepa1­6 cells. The association between EGR1 and PTEN was explored by EGR1 overexpression in cell lines. For in vivo methods, a mouse model of diethylnitrosamine (DEN)­induced primary liver cancer was established using C57BL/6 mice to investigate the inhibitory effect of LNT on liver cancer. Histopathology of liver tissue from mice was detected by hematoxylin­eosin staining and immunohistochemical assay. In vitro and in vivo results showed that LNT can inhibit the proliferation and promote the apoptosis of mouse HCC cells. Besides, LNT increased the expression of EGR1 in Hepa1­6 cells, which is translocated to the nucleus to function as a transcriptional factor. EGR1 then activates the expression of the tumor suppressor PTEN, thereby inhibiting the activation of the AKT signaling pathway. These data revealed a novel anti­tumor mechanism by which LNT can induce apoptosis to inhibit mouse HCC progression through the EGR1/PTEN/AKT axis. These results provide a scientific basis for the potential use of LNT in drug development and clinical applications associated with primary liver cancer.

Gene expression analysis in epileptic hippocampi reveals a promoter haplotype conferring reduced aldehyde dehydrogenase 5a1 expression and responsiveness.

Increasing evidence indicates the pathogenetic relevance of regulatory genomic motifs for variability in the manifestation of brain disorders. In this context, cis-regulatory effects of single nucleotide polymorphisms (SNPs) on gene expression can contribute to changing transcript levels of excitability-relevant molecules and episodic seizure manifestation in epilepsy. Biopsy specimens of patients undergoing epilepsy surgery for seizure relief provide unique insights into the impact of promoter SNPs on corresponding mRNA expression. Here, we have scrutinized whether two linked regulatory SNPs (rs2744575; 4779C > G and rs4646830; 4854C > G) located in the aldehyde dehydrogenase 5a1 (succinic semialdehyde dehydrogenase; ALDH5A1) gene promoter are associated with expression of corresponding mRNAs in epileptic hippocampi (n = 43). The minor ALDH5A1-GG haplotype associates with significantly lower ALDH5A1 transcript abundance. Complementary in vitro analyses in neural cell cultures confirm this difference and further reveal a significantly constricted range for the minor ALDH5A1 haplotype of promoter activity regulation through the key epileptogenesis transcription factor Egr1 (early growth response 1). The present data suggest systematic analyses in human hippocampal tissue as a useful approach to unravel the impact of epilepsy candidate SNPs on associated gene expression. Aberrant ALDH5A1 promoter regulation in functional terms can contribute to impaired γ-aminobutyric acid homeostasis and thereby network excitability and seizure propensity.

The anxiety and ethanol intake controlling GAL5.1 enhancer is epigenetically modulated by, and controls preference for, high-fat diet.

Excess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that environmentally modulated DNA methylation changes (5mC/5hmC) in regulatory regions of the genome that modulate mood and consumptive behaviours could contribute to susceptibility to these conditions. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. In contrast, we identified that allowing access of pregnant mothers to high-fat diet (> 60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection-based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli, such as EGR1 transcription factor expression and PKC agonism. Intriguingly, CRISPR-driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism or general appetite, significantly decreased intake of high-fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally influenced regulatory loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.

Chrysoeriol Prevents TNFα-Induced CYP19 Gene Expression via EGR-1 Downregulation in MCF7 Breast Cancer Cells.

Estrogen overproduction is closely associated with the development of estrogen receptor-positive breast cancer. Aromatase, encoded by the cytochrome P450 19 (CYP19) gene, regulates estrogen biosynthesis. This study aimed to identify active flavones that inhibit CYP19 expression and to explore the underlying mechanisms. CYP19 expression was evaluated using reverse transcription PCR, quantitative real-time PCR, and immunoblot analysis. The role of transcription factor early growth response gene 1 (EGR-1) in CYP19 expression was assessed using the short-hairpin RNA (shRNA)-mediated knockdown of EGR-1 expression in estrogen receptor-positive MCF-7 breast cancer cells. We screened 39 flavonoids containing 26 flavones and 13 flavanones using the EGR1 promoter reporter activity assay and observed that chrysoeriol exerted the highest inhibitory activity on tumor necrosis factor alpha (TNFα)-induced EGR-1 expression. We further characterized and demonstrated that chrysoeriol inhibits TNFα-induced CYP19 expression through inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated EGR-1 expression. Chrysoeriol may be beneficial as a dietary supplement for the prevention of estrogen receptor-positive breast cancer, or as a chemotherapeutic adjuvant in the treatment of this condition.

Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Time course of photo-induced Egr-1 expression in the hypothalamus of a seasonally breeding songbird.

Many seasonally-breeding species use daylength to time reproduction. Light-induced release of progonadal hormones involves a complex cascade of responses both inside and outside the brain. In this study, we used induction of early growth response 1 (Egr-1), the protein product of an immediate early gene, to evaluate the time course of such responses in male white-throated sparrows (Zonotrichia albicollis) exposed to a single long day. Induction of Egr-1 in the pars tuberalis began ∼11 h after dawn. This response was followed ∼6 h later by dramatic induction in the tuberal hypothalamus, including in the ependymal cells lining the third ventricle. At approximately the same time, Egr-1 was induced in dopaminergic and vasoactive intestinal peptide neurons in the tuberal hypothalamus and in dopaminergic neurons of the premammillary nucleus. We noted no induction in gonadotropin-releasing hormone (GnRH) neurons until 2 h after dawn the following morning. Overall, our results indicate that Egr-1 responses in GnRH neurons occur rather late during photostimulation, compared with responses in other cell populations, and that such induction may reflect new synthesis related to GnRH depletion rather than stimulation by light cues.

Low temperature induces von-willebrand factor expression via increased early growth response 1 transcriptional activity in splenic sinusoidal endothelial cells.

von Willebrand factor (vWF) is a large plasma glycoprotein that plays an important role in hemostasis by forming molecular bridges with platelets following vascular injury. Previously, we reported that hypothermia enhanced vWF production in the spleen, which resulted in the activation of the platelet pool in a hypothermia-induced murine model. However, the mechanisms that regulate vWF expression under hypothermic conditions remain unclear. In this study, we focused on vWF expression under hypothermic conditions in splenic endothelial cell culture. Human splenic endothelial cells (HSEC) were incubated at 20 °C for 1 h. Total RNA was extracted from the cells, and cDNA microarray gene expression analysis was performed. Genes that may be associated with vWF expression in low temperature culture conditions were then selected for further analysis. Gene expression analysis showed that low temperature conditions increased the expression of FOS and EGR1. We then hypothesized that these factors upregulate vWF mRNA expression in HSEC. The transcriptional inhibitors of EGR1 significantly inhibited vWF mRNA expression in HSEC cultured at a low temperature. Our analysis revealed that low temperatures enhance the gene expression of EGR1, which transcriptionally increases vWF expression. This acute-phase reaction may play an important role in platelet activation in the spleen during hypothermia.

Active fraction of Polyrhachis vicina Rogers (AFPR) suppressed breast cancer growth and progression via regulating EGR1/lncRNA-NKILA/NF-κB axis.

Breast cancer (BC) is a major contributor of cancer-associated mortality in women. It is essential to find new therapeutic targets and drugs. Polyrhachis vicina Rogers is one of the Traditional Chinese Medicine (TCM). Our previous studies have shown an active fraction of Polyrhachis vicina Rogers (AFPR) has significant anti-inflammatory activity, suggesting its anti-cancer effect. Here, we aimed to explore the inhibitory effects of AFPR on BC and reveal its mechanism. The effects of AFPR on BC were examined by cell proliferation assay, wound healing assay, invasion assay and xenograft assay. Microarray sequencing, qRT-PCR, Western blot, chromatin immunoprecipitation assay and luciferase reporter assay were performed to investigate the regulation of AFPR on related genes and underlying mechanisms. As a result, AFPR suppressed BC cell growth, migration and invasion and inhibited tumor growth. LncRNA NKILA was most prominently upregulated in AFPR-treated MCF7 cells. AFPR inactivated NF-κB signaling pathway via regulating NKILA. Furthermore, AFPR regulated the expression of NKILA by inhibiting its transcript suppressor EGR1. This study firstly indicated that AFPR was a potential inhibitor of BC development via regulating EGR1/NKILA/NF-κB axis.

Effect of Mongolian warm acupuncture on the gene expression profile of rats with insomnia.

BACKGROUND: The mechanism of Mongolian warm acupuncture (MWA) for the treatment of insomnia has not been previously reported. OBJECTIVE: To investigate the effect of MWA on gene expression profile in the p-chlorophenylalanine (PCPA)-induced rat model of insomnia. METHODS: A rat model of insomnia was established and the animals were divided into five groups: control, PCPA (untreated), PCPA+estazolam, PCPA+MA (manual acupuncture), and PCPA+MWA. The rats were euthanased at 7 days after treatment, and hypothalamic tissue was harvested to extract total RNA for the analysis of gene expression profile. Micro-array and Partek Genomics Suite analysis system were used to analyse differential expression of genes between groups. Furthermore, ingenuity pathways analysis was used to analyse the main regulators. RESULTS: After treatment, in rats with improved sleep, micro-array data from the follow-up phase compared with baseline showed that MWA down-regulated 11 genes compared with the control group and 16 genes compared with the PCPA group. Six genes were selected following the micro-array detection to perform quantitative polymerase chain reaction (qPCR) verification, and the results showed that the coincidence rate was up to 90%, which verified the reliability of the microarray results. Compared with the PCPA group, transcription levels of Egr 1, Btg2 and BDNF in the PCPA+MWA group were up-regulated (P<0.05). CONCLUSION: In combination, the findings of this study suggests that MWA is efficacious at improving sleep in an experimental rat model of insomnia.

The lncRNA MIR2052HG regulates ERα levels and aromatase inhibitor resistance through LMTK3 by recruiting EGR1.

BACKGROUND: Our previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG's mechanism of action. METHODS: RNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines. RESULTS: MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. CONCLUSIONS: Our findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Early growth response-1 regulates acetylcholinesterase and its relation with the course of Alzheimer's disease.

Our previous studies showed that the transcription factor early growth response-1 (EGR1) may play a role in keeping the brain cholinergic function intact in the preclinical stages of Alzheimer's disease (AD). In order to elucidate the mechanisms involved, we first performed data mining on our previous microarray study on postmortem human prefrontal cortex (PFC) for the changes in the expression of EGR1 and acetylcholinesterase (AChE) and the relationship between them during the course of AD. The study contained 49 patients, ranging from non-demented controls (Braak stage 0) to late AD patients (Braak stage VI). We found EGR1-mRNA was high in early AD and decreased in late AD stages, while AChE-mRNA was stable in preclinical AD and slightly decreased in late AD stages. A significant positive correlation was found between the mRNA levels of these two molecules. In addition, we studied the relationship between EGR1 and AChE mRNA levels in the frontal cortex of 3-12-months old triple-transgenic AD (3xTg-AD) mice. EGR1- and AChE-mRNA were lower in 3xTg-AD mice compared with wild-type (WT) mice. A significant positive correlation between these two molecules was present in the entire group and in each age group of either WT or 3xTg-AD mice. Subsequently, AChE expression was determined following up- or down-regulating EGR1 in cell lines and the EGR1 levels were found to regulate AChE at both the mRNA and protein levels. Dual-luciferase assay and electrophoretic mobility shift assay in the EGR1-overexpressing cells were performed to determine the functionally effective binding sites of the EGR1 on the AChE gene promoter. We conclude that the EGR1 can upregulate AChE expression by a direct effect on its gene promoter, which may contribute significantly to the changes in cholinergic function in the course of AD. The 3xTg-AD mouse model only reflects later stage AD.

The MEK/ERK/CREB signaling pathway is involved in atrazine induced hippocampal neurotoxicity in Sprague Dawley rats.

Atrazine (ATR) is a commonly used artificial synthetic herbicide world-wide, which has been implicated as a potential threat to human health. Previous studies have demonstrated that exposure to ATR affects hippocampus-dependent learning and memory in rodents, but the exact molecular mechanism remains to be elucidated. In this study, we investigated the effect of ATR on the hippocampus of postnatal day 35 male Sprague Dawley (SD) rats administered doses of either 10 or 100 mg/kg body weight (BW)/day of ATR for a period of 30 days. A Morris water maze (MWM) test revealed that ATR treatment impaired memory performance in the spatial probe test, especially amongst the high-dose group. Moreover, analysis by electron microscopy showed that hippocampal neuron ultrastructure in the dentate gyrus (DG) and cornu ammonis 1 (CA1) sub-regions was impaired in the ATR-treated groups. Finally, a downregulation in the mRNA and protein expression levels of members of the MEK/ERK/CREB pathway and downstream factors brain-derived neurotrophic factor (BDNF) and Zif268 was observed in hippocampal tissue following ATR treatment. Taken together, these results suggest that developmental exposure to ATR is able to induce functional and morphological lesions in the hippocampus of SD rats, and that the MEK/ERK/CREB signaling pathway may be involved in this process.

Influence of the Klotho/FGF23/Egr1 signaling pathway on calcium-phosphorus metabolism in diabetic nephropathy and the intervention of Shenyuan granules.

This study aimed to explore the effects of Shenyuan granules on the Klotho/FGFR23/Egr1 signaling pathway and calcium-phosphorus metabolism in diabetic mice models with impairment of renal function. Streptozotocin-induced diabetic nephropathy (DN) mice models were randomly divided into three groups: Shenyuan granules group (n=10), model control group (n=10), and blank control group (n=10). Corresponding drugs were given by gavage for 8 weeks. Blood glucose and serum creatinine (SCr), urea nitrogen (BUN), calcium (Ca), phosphorus (P) and mLAB were detected before and after administration. Moreover, RT-qPCR was performed to detect the expression of CYP24 and CYP27 mRNA in kidney tissue. Blood FGF23 was detected by ELISA. Western-blot and immunohistochemistry were performed to detect the expressions of Klotho, FGFR1, Egr1, CYP24, CYP27, ERK1/2 and p-ERK1/2. Compared with the blank control group, in the model control group serum FGF23,P, SCr and 24-hour proteinuria levels increased (P<0.05), serum Ca significantly decreased (P<0.05), expressionss of Egr1, CYP24, CYP27 and p-ERK1/2 were up-regulated (P<0.05), and the expressions of Klotho and FGFR1 were down-regulated (P<0.05). After treatment, compared with the model control group, in the Shenyuan granule group serum FGF23, P, SCr levels decreased (P<0.05), serum Ca increased (P<0.05), expressions of Egr-1, CYP24, CYP27 and p-ERK1/2 were down-regulated (P<0.05), and the expressions of Klotho and FGFR1 were up-regulated (P<0.05). Shenyuan granules may partly intervene in the expressions of CYP24 and CYP27 through the Klotho/FGF23/Egr1 signaling pathway, thereby improving calcium and phosphorus metabolism and alleviating renal injury in diabetic nephropathy.

Inflammation-Induced Epithelial-to-Mesenchymal Transition and GM-CSF Treatment Stimulate Mesenteric Mesothelial Cells to Transdifferentiate into Macrophages.

In our previous work, we showed that during inflammation-induced epithelial-to-mesenchymal transition (EMT), mesenteric mesothelial cells express ED1 (pan-macrophage marker), indicating that they are transformed into macrophage-like cells. In this paper, we provide additional evidences about this transition by following the phagocytic activity and the TNFα production of mesenteric mesothelial cells during inflammation. Upon injection of India ink particles or fluorescent-labeled bioparticles (pHrodo) into the peritoneal cavity of rats pretreated with Freund's adjuvant, we found that mesothelial cells efficiently engulfed these particles. A similar increase of internalization could be observed by mesothelial cells in GM-CSF pretreated primary mesenteric culture. Since macrophages are the major producers of tumor necrosis factor, TNFα, we investigated expression level of TNFα during inflammation-induced EMT and found that TNFα was indeed expressed in these cells, reaching the highest level at the 5th day of inflammation. Since TNFα is one of the target genes of early growth response (EGR1) transcription factor, playing important role in monocyte-macrophage differentiation, expression of EGR1 in mesothelial cells was also investigated by Western blot and immunocytochemistry. While mesothelial cells did not express EGR1, a marked increase was observed in mesothelial cells by the time of inflammation. Parallel to this, nuclear translocation of EGR1 was shown by immunocytochemistry at the day 5 of inflammation. Caveolin-1 level was high and ERK1/2 became phosphorylated as the inflammation proceeded showing a slight decrease when the regeneration started. Our present data support the idea that under special stimuli, mesenteric mesothelial cells are able to transdifferentiate into macrophages, and this transition is regulated by the caveolin-1/ERK1/2/EGR1 signaling pathway.

Restoring Effects of Natural Anti-Oxidant Quercetin on Cellular Senescent Human Dermal Fibroblasts.

The oxidative damage initiated by reactive oxygen species (ROS) is a major contributor to the functional decline and disability that characterizes aging. The anti-oxidant flavonoid, quercetin, is a plant polyphenol that may be beneficial for retarding the aging process. We examined the restoring properties of quercetin on human dermal fibroblasts (HDFs). Quercetin directly reduced either intracellular or extracellular ROS levels in aged HDFs. To find the aging-related target genes by quercetin, microarray analysis was performed and two up-regulated genes LPL and KCNE2 were identified. Silencing LPL increased the expression levels of senescence proteins such as p16INK4A and p53 and silencing KCNE2 reversed gene expressions of EGR1 and p-ERK in quercetin-treated aged HDFs. Silencing of LPL and KCNE2 decreased the expression levels of anti-oxidant enzymes such as superoxide dismutase and catalase. Also, the mitochondrial dysfunction in aged HDFs was ameliorated by quercetin treatment. Taken together, these results suggest that quercetin has restoring effect on the cellular senescence by down-regulation of senescence activities and up-regulation of the gene expressions of anti-oxidant enzymes in aged HDFs.

Bu-shen-zhu-yun decoction promotes synthesis and secretion of FSHß and LHß in anterior pituitary cells in vitro.

Luteal phase defects (LPD) are an important etiology of infertility which has increased in recent years. Studies have shown that bu-shen-zhu-yun decoction (BSZY-D) can lower the expression of estrogen receptor and progesterone receptor, in rats endometrium of embryonic implantation period, which upregulated by mifepristone, and improve uterine receptivity. The aim of present study was to determine the effect of BSZY-D on the synthesis and secretion of gonadotropic hormones in the anterior pituitary cells of rats. Rats were treated with saline (control) or BSZY-D two times/day for three estrous cycles by gavage. The cerebrospinal fluid (CSF) were collected for further cell treatment. The components in BSZY-D, serum and CSF were analysed by High Performance Liquid Chromatography (HPLC). Cells were either pretreated with normal CSF or BSZY-D/CSF before being stimulated with or without cetrorelix. The mRNA and proteins levels of receptors, hormones, and transcription factors were detected by RT-PCR, western blot analysis and immunostaining. We show that non-toxic concentrations of cetrorelix, a GnRH antagonist, can reduce the mRNA and protein levels of GnRHR, LH, and FSH. This effect could be reversed by the addition of BSZY-D/CSF. We also show decreased mRNA and protein expression of transcription factors, such as CREB, and Egr-1 and secretory vescicles, including SNAP-25 and Munc-18 upon treatment with cetrorelix could be reversed post co-treatment with BSZY-D/CSF. These results indicate that BSZY-D/CSF treatment led to increased levels of GnRHR, transcription factors, and secretory vesicles leading to increased secretion of FSH and LH. Thus, BSZY-D presents a promising candidate to treat luteal phase defects and infertility.

Sinomenine inhibits fibroblast-like synoviocyte proliferation by regulating α7nAChR expression via ERK/Egr-1 pathway.

Fibroblast like synoviocyte (FLS) is a crucial in the pathogenesis of rheumatoid arthritis (RA), and involved in inflammation and joint destruction. Sinomenine (SIN), an alkaloid derived from the plant Sinomenium acutum, has anti-inflammatory and analgesic effect and been used for RA treatment in China. Alpha 7 nicotinic acetylcholine receptors (α7nAChR), as the key receptor in cholinergic anti-inflammatory pathway (CAP) to inhibit inflammation, has been detected in RA patients synovium, but its role is still unclear. Here we investigated the association between the aggressive proliferation of FLS and α7nAChR expression and the effect of sinomenine. FLS was isolated from synovial tissues of adjuvant-induced-arthritis (AIA) rat. Tumor necrosis factor(TNF)-α was used to induce the aggressive proliferation of FLS. MTT assay was applied to evaluate the proliferation of FLS. The messenger RNA (mRNA) and protein levels of α7nAChR and early growth response gene-1 (Egr-1) were measured. The results showed that TNF-α induced FLS proliferation in vitro (P < .01) and increased the phosphorylation of ERK1/2 and the expression of Egr-1 and α7nAChR (P < .05 or P < .01). U0126, the inhibitor of ERK1/2 inhibited α7nAChR expression and FLS proliferation significantly (P < .05 or P < .01). Specific short interference RNA(siRNA) of α7nAChR decreased α7nAChR expression and inhibited FLS proliferation as well. SIN inhibited the proliferation of FLS and decreased the phosphorylation of ERK1/2, and the expression of Egr-1 and α7nAChR induced by TNF-α (P < .05). In conclusion, the expression of α7nAChR involved in the aggressive proliferation of FLS induced by TNF-α and was regulated by ERK/Egr-1 signal pathway. SIN inhibited FLS proliferation and α7nAChR expression through inhibiting ERK/Egr-1 signal pathway, this may contribute to the anti-inflammatory and anti-arthritic effect of SIN.

Arecoline activates latent transforming growth factor ß1 via mitochondrial reactive oxygen species in buccal fibroblasts: Suppression by epigallocatechin-3-gallate.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) is a premalignant condition caused by the chewing of areca nut (AN). Transforming growth factor ß (TGFß) plays a central role in the pathogenesis of OSF. Connective tissue growth factor (CTGF or CCN2) and early growth response-1 (Egr-1) are important mediators in the fibrotic response to TGFß in several fibrotic disorders including OSF. Arecoline, a major AN alkaloid, induced the synthesis of CCN2 and Egr-1 in human buccal mucosal fibroblast (BMFs). The aims of this study were to investigate whether arecoline-induced CCN2 and Egr-1 syntheses are mediated through TGFß1 signaling and to inspect the detailed mechanisms involved. METHODS: Western blot and TGFß1 Emax® ImmunoAssay were used to measure the effect of arecoline on the TGFß signaling pathways. 2',7'-dichlorodihydrofluorescein diacetate and MitoSOX™ Red were used to measure the effect of arecoline on the cellular and mitochondrial reactive oxygen species (ROS). RESULTS: Arecoline induced latent TGFß1 activation, Smad2 phosphorylation, and mitochondrial and total cellular ROS in BMFs. TGFß-neutralizing antibody completely inhibited the arecoline-induced synthesis of CCN2 and Egr-1. Mito-TEMPO, a mitochondria-targeted antioxidant, completely suppressed arecoline-induced latent TGFß1 activation and mitochondrial and total cellular ROS. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited arecoline-induced TGFß1 activation and mitochondrial ROS in BMFs. CONCLUSION: Our results indicated that arecoline-induced mitochondrial ROS plays pivotal roles in the activation of latent TGFß1 leading to the initiation of TGFß1 signaling and subsequent increase in the synthesis of CCN2 and Egr-1. EGCG can be a useful agent in the chemoprevention and treatment of OSF.

Attenuation of pCREB and Egr1 expression in the insular and anterior cingulate cortices associated with enhancement of CFA-evoked mechanical hypersensitivity after repeated forced swim stress.

The perception and response to pain are severely impacted by exposure to stressors. In some animal models, stress increases pain sensitivity, which is termed stress-induced hyperalgesia (SIH). The insular cortex (IC) and anterior cingulate cortex (ACC), which are typically activated by noxious stimuli, affect pain perception through the descending pain modulatory system. In the present study, we examined the expression of phospho-cAMP response element-binding protein (pCREB) and early growth response 1 (Egr1) in the IC and ACC at 3h (the acute phase of peripheral tissue inflammation) after complete Freund's adjuvant (CFA) injection in naïve rats and rats preconditioned with forced swim stress (FS) to clarify the effect of FS, a stressor, on cortical cell activities in the rats showing SIH induced by FS. The CFA injection into the hindpaw induced mechanical hypersensitivity and increased the expression of the pCREB and Egr1 in the IC and ACC at 3h after the injection. FS (day 1, 10min; days 2-3, 20min) prior to the CFA injection enhanced the CFA-induced mechanical hypersensitivity and attenuated the increase in the expression of pCREB and Egr1 in the IC and ACC. These findings suggested that FS modulates the CFA injection-induced neuroplasticity in the IC and ACC to enhance the mechanical hypersensitivity. These findings are thought to signify stressor-induced dysfunction of the descending pain modulatory system.