Revealing the mechanism of ethyl acetate extracts of Semen Impatientis against prostate cancer based on network pharmacology and transcriptomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer (PCa) is the most common malignancy of the male genitourinary system and currently lacks effective treatment. Semen Impatientis, the dried ripe seed of Impatiens balsamina L., is described by the Chinese Pharmacopoeia as a traditional Chinese medicine (TCM) and is used in clinical practice to treat tumors, abdominal masses, etc. In our previous study, the ethyl acetate extracts of Semen Impatientis (EAESI) was demonstrated to be the most effective extract against PCa among various extracts. However, the biological effects of EAESI against PCa in vivo and the specific antitumor mechanisms involved remain unknown. AIM OF THE STUDY: In this study, we aimed to investigate the antitumor effect of EAESI on PCa in vitro and in vivo by performing network pharmacology analysis, transcriptomic analysis, and experiments to explore and verify the underlying mechanisms involved. MATERIALS AND METHODS: The antitumor effect of EAESI on PCa in vitro and in vivo was investigated via CCK-8, EdU, flow cytometry, and wound healing assays and xenograft tumor models. Network pharmacology analysis and transcriptomic analysis were employed to explore the underlying mechanism of EAESI against PCa. Activating transcription factor 3 (ATF3) and androgen receptor (AR) were confirmed to be the targets of EAESI against PCa by RT‒qPCR, western blotting, and rescue assays. In addition, the interaction between ATF3 and AR was assessed by coimmunoprecipitation, immunofluorescence, and nuclear-cytoplasmic separation assays. RESULTS: EAESI decreased cell viability, inhibited cell proliferation and migration, and induced apoptosis in AR+ and AR- PCa cells. Moreover, EAESI suppressed the growth of xenograft tumors in vivo. Network pharmacology analysis revealed that the hub targets of EAESI against PCa included AR, AKT1, TP53, and CCND1. Transcriptomic analysis indicated that activating transcription factor 3 (ATF3) was the most likely critical target of EAESI. EAESI downregulated AR expression and decreased the transcriptional activity of AR through ATF3 in AR+ PCa cells; and EAESI promoted the expression of ATF3 and exerted its antitumor effect via ATF3 in AR+ and AR- PCa cells. CONCLUSIONS: EAESI exerts good antitumor effects on PCa both in vitro and in vivo, and ATF3 and AR are the critical targets through which EAESI exerts antitumor effects on AR+ and AR- PCa cells.

Co-catalpol alleviates fluoxetine-induced main toxicity: Involvement of ATF3/FSP1 signaling-mediated inhibition of ferroptosis.

BACKGROUND: Fluoxetine is often used as a well-known first-line antidepressant. However, it is accompanied with hepatogenic injury as its main organ toxicity, thereby limiting its application despite its superior efficacy. Fluoxetine is commonly traditionally used combined with some Chinese antidepressant prescriptions containing Rehmannia glutinosa (Dihuang) for depression therapy and hepatoprotection. Our previous experiments showed that co-Dihuang can alleviate fluoxetine-induced liver injury while efficiencies, and catalpol may be the key ingredient to characterize the toxicity-reducing and synergistic effects. However, whether co-catalpol can alleviate fluoxetine-induced liver injury and its toxicity-reducing mechanism remain unclear. PURPOSE: On the basis of the first recognition of the dose and duration at which pre-fluoxetine caused hepatic injury, co-catalpol's alleviation of fluoxetine-induced hepatic injury and its pathway was comprehensively elucidated. METHOD AND RESULTS: The hepatoprotection of co-catalpol was evaluated by serum biochemical indexes sensitive to hepatic injury and multiple staining techniques for hepatic pathologic analysis. Subsequently, the pathway by which catalpol alleviated fluoxetine-induced hepatic injury was predicted by network pharmacology to be predominantly the inhibition of ferroptosis. These were validated and confirmed in subsequent experiments with key technologies and diagnostic reagents related to ferroptosis. Further molecular docking showed that activating transcription factor 3 (ATF3) and ferroptosis suppressor protein 1 (FSP1) were the the most prospective molecules for catalpol and fluoxetine among many ferroptosis-related molecules. The critical role of ATF3/FSP1 signaling was further observed by surface plasmon resonance, diagnostic reagents, transmission electron microscopy, Western blot, real-time PCR, immunofluorescence, and immunohistochemistry. Results showed that fluoxetine directly bound to ATF3 and FSP1; agonisting ATF3 or blocking FSP1 abolished the alleviation of catalpol on fluoxetine-induced liver injury, and both exacerbated ferroptosis. Moreover, co-catalpol significantly enhanced the antidepressant efficacy of fluoxetine against depressive behaviours in mice. CONCLUSION: The hepatic impairment properties of fluoxetine were largely dependent on ATF3/FSP1 target-mediated ferroptosis. Co-catalpol alleviated fluoxetine-induced hepatic injury while enhancing its antidepressant efficacy, and that ATF3/FSP1 signaling-mediated inhibition of ferroptosis was involved in its co-administration detoxification mechanism. This study was the first to reveal the hepatotoxicity characteristics, targets, and mechanisms of fluoxetine; provide a detoxification and efficiency regimen by co-catalpol; and elucidate the detoxification mechanism.

SIRT1 activated by AROS sensitizes glioma cells to ferroptosis via induction of NAD+ depletion-dependent activation of ATF3.

Ferroptosis is a type of programmed cell death resulting from iron overload-dependent lipid peroxidation, and could be promoted by activating transcription factor 3 (ATF3). SIRT1 is an enzyme accounting for removing acetylated lysine residues from target proteins by consuming NAD+, but its role remains elusive in ferroptosis and activating ATF3. In this study, we found SIRT1 was activated during the process of RSL3-induced glioma cell ferroptosis. Moreover, the glioma cell death was aggravated by SIRT1 activator SRT2183, but suppressed by SIRT inhibitor EX527 or when SIRT1 was silenced with siRNA. These indicated SIRT1 sensitized glioma cells to ferroptosis. Furthermore, we found SIRT1 promoted RSL3-induced expressional upregulation and nuclear translocation of ATF3. Silence of ATF3 with siRNA attenuated RSL3-induced increases of ferrous iron and lipid peroxidation, downregulation of SLC7A11 and GPX4 and depletion of cysteine and GSH. Thus, SIRT1 promoted glioma cell ferroptosis by inducting ATF3 activation. Mechanistically, ATF3 activation was reinforced when RSL3-induced decline of NAD+ was aggravated by FK866 that could inhibit NAD + synthesis via salvage pathway, but suppressed when intracellular NAD+ was maintained at higher level by supplement of exogenous NAD+. Notably, the NAD + decline caused by RSL3 was enhanced when SIRT1 was further activated by SRT2183, but attenuated when SIRT1 activation was inhibited by EX527. These indicated SIRT1 promoted ATF3 activation via consumption of NAD+. Finally, we found RSL3 activated SIRT1 by inducing reactive oxygen species-dependent upregulation of AROS. Together, our study revealed SIRT1 activated by AROS sensitizes glioma cells to ferroptosis via activation of ATF3-dependent inhibition of SLC7A11 and GPX4.

Salvia miltiorrhiza Extract and Individual Synthesized Component Derivatives Induce Activating-Transcription-Factor-3-Mediated Anti-Obesity Effects and Attenuate Obesity-Induced Metabolic Disorder by Suppressing C/EBPα in High-Fat-Induced Obese Mice.

Pharmacological studies indicate that Salvia miltiorrhiza extract (SME) can improve cardiac and blood vessel function. However, there is limited knowledge regarding the effects (exerted through epigenetic regulation) of SME and newly derived single compounds, with the exception of tanshinone IIA and IB, on obesity-induced metabolic disorders. In this study, we administered SME or dimethyl sulfoxide (DMSO) as controls to male C57BL/J6 mice after they were fed a high-fat diet (HFD) for 4 weeks. SME treatment significantly reduced body weight, fasting plasma glucose, triglyceride levels, insulin resistance, and adipogenesis/lipogenesis gene expression in treated mice compared with controls. Transcriptome array analysis revealed that the expression of numerous transcriptional factors, including activating transcription factor 3 (ATF3) and C/EBPα homologous protein (CHOP), was significantly higher in the SME group. ST32db, a novel synthetic derivative similar in structure to compounds from S. miltiorrhiza extract, ameliorates obesity and obesity-induced metabolic syndrome in HFD-fed wild-type mice but not ATF3-/- mice. ST32db treatment of 3T3-L1 adipocytes suppresses lipogenesis/adipogenesis through the ATF3 pathway to directly inhibit C/EBPα expression and indirectly inhibit the CHOP pathway. Overall, ST32db, a single compound modified from S. miltiorrhiza extract, has anti-obesity effects through ATF3-mediated C/EBPα downregulation and the CHOP pathway. Thus, SME and ST32db may reduce obesity and diabetes in mice, indicating the potential of both SME and ST32db as therapeutic drugs for the treatment of obesity-induced metabolic syndrome.

Transcriptome analysis upon potassium usnate exposure reveals ATF3-induced apoptosis in human gastric and colon cancer cells.

BACKGROUND: Potassium usnate (KU), a water-soluble form of usnic acid, shows anticancer activity. However, the underlying mechanisms have not been fully elucidated. PURPOSE: We aimed to identify the pathways involved in anticancer effects of KU in human gastric cancer (GC) and colorectal cancer (CRC) cells using RNA-sequencing (RNA-seq) based transcriptome analysis. STUDY DESIGN: We analyzed the cytotoxic effects of KU to identify the common molecular events in GC and CRC cells upon KU exposure using unbiased approaches. METHODS: Cell viability assays and western blot experiments were used to examine apoptotic changes, cell cycle arrest, and endoplasmic reticulum (ER) stress-induced cellular responses in KU-treated cells. Total RNA from KU-treated human GC and CRC cells was prepared for RNA-seq analysis. Gene ontology term and gene set enrichment analyses were used to identify the key mediators of the cytotoxic effects of KU. The expression of ER stress-induced apoptotic markers was evaluated using quantitative reverse-transcription PCR and western blot analysis. Chromatin immunoprecipitation assays for ATF3 and H3K27ac, and ATF3 knockdown were employed to verify the underlying molecular mechanisms. The inhibitory effect of KU on tumor growth in vivo was validated with metastatic tumor nodule formations in a mouse liver model. RESULTS: KU exerted cytotoxicity in human GC and CRC cells through the activation of the ER stress-induced apoptotic pathway. KU stimulated ATF3 expression, an important mediator of molecular events of apoptosis. ATF3 binds to the promoter region of ATF3, CHOP, GADD34, GADD45A, DR5, and PUMA genes and subsequently promoted apoptotic events. Knockdown of ATF3 significantly reduced the expression of ATF3 target genes and the cytotoxic effects of KU. The intraperitoneal injection of KU induced ATF3 and the apoptosis of implanted colon cancer cells, resulting in reduced metastatic tumor growth in the mouse livers. CONCLUSION: KU exerts cytotoxic effects in human GC and CRC cells by triggering ER stress-induced apoptosis via an ATF3 dependent pathway.

Citrus alkaline extracts prevent endoplasmic reticulum stress in type II alveolar epithelial cells to ameliorate pulmonary fibrosis via the ATF3/PINK1 pathway.

BACKGROUND: Idiopathic pulmonary fibrosis is a chronic, progressive, fibrotic disease. Although the pathogenesis remains unclear, the effect of endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AEC IIs) is increasingly thought to be a critical mechanism. PURPOSE: We investigated the effects of citrus alkaline extracts (CAE) on AEC IIs and elucidated the underlying mechanism for their possible use in ameliorating pulmonary fibrosis (PF). METHODS: A bleomycin-induced mouse model of PF, and an in vitro tunicamycin (TM) -induced ER stress model in A549 cells were successfully established. Accumulation of collagen in lung tissues in vivo was assessed using histological analysis and western blotting. The expression levels of the ER-stress marker BiP and other related proteins were assessed by western blotting and immunofluorescence staining. Mitochondrial membrane potential was assessed to evaluate mitochondrial homeostasis. RESULTS: CAE mitigated collagen deposition to ameliorate PF in vivo. CAE suppressed the bleomycin or TM-induced increases in ER-stress biomarker, BiP, and PERK pathway proteins, resulting in a decrease in ER stress in mouse lung tissues and A549 cells, respectively. Additionally, CAE treatment suppressed the bleomycin or TM-induced increase in the ER-stress downstream proteins, activating ATF3 and increased the levels of PINK1 in AEC IIs, both in vivo and in vitro. The reduced mitochondrial homeostasis induced by TM was restored by CAE-treatment in A549 cells. Furthermore, conditioned media from TM-treated A549 cells increased collagen deposition in MRC5 cells mainly via TGF-ß1. The increased collagen deposition was not seen using conditioned media from CAE-treated A549 cells. CONCLUSION: These results provide novel insights into the potential mechanism of CAE in inhibiting ER stress in AEC IIs, and suggests that it has great potential to ameliorate PF via the ATF3/PINK1 pathway.

Aqueous extract of Taxus chinensis var. mairei regulates the Hippo-YAP pathway and promotes apoptosis of non-small cell lung cancer via ATF3 in vivo and in vitro.

Taxus chinensis var. mairei (TC) is a traditional Chinese ornamental and medicinal plant, the leaves and twigs of which are used in anti-tumor therapy in southern China. However, the mechanism and role of aqueous extract of TC (AETC) in promoting apoptosis in non-small cell lung cancer (NSCLC) cell lines has remained unclear. In this research, we observed that AETC inhibited the suppression of the proliferation of NSCLC cells and highly inhibited the proliferation of NCI-1975 cells. Furthermore, AETC exerted minimal inhibitory effects on normal human lung epithelial cells and induced apoptosis in NCI-1975 and A549 cells. The findings of RNA sequencing, qRT-PCR, western blotting, and immunofluorescence showed that upregulated ATF3 expression and ATF3 gene knockdown, respectively, increased and decreased the anti-tumor effects of AETC associated with Hippo pathway inhibition and decreased YAP degradation. Furthermore, AETC reduced the tumor volume and weight in nude mice; upregulated ATF3, p-MOB1, and p-YAP (Ser397); and actively regulated cleaved PARP and cleaved caspase-9/8/3. These findings suggest that AETC induced NSCLC cell apoptosis via the ATF3-Hippo-YAP pathway in vivo and in vitro. We also found that AETC is non-toxic to normal cells and nude mice. Thus, AETC might represent a promising adjuvant for anti-tumor therapy against NSCLC.

Bisphenol A Induces Agrp Gene Expression in Hypothalamic Neurons through a Mechanism Involving ATF3.

BACKGROUND: Bisphenol A (BPA) is a ubiquitous endocrine disrupting chemical and obesogen. Although limited evidence exists of the effects of BPA on hypothalamic agouti-related peptide (AgRP) levels, the mechanisms underlying these effects remain unknown. Given that AgRP is a potent orexigenic neuropeptide, determining the mechanism by which BPA increases AgRP is critical to preventing the progression to metabolic disease. METHODS: Using quantitative reverse transcriptase polymerase chain reaction, we investigated the response of Agrp-expressing mouse hypothalamic cell lines to BPA treatment. The percentage of total BPA entering hypothalamic cells in culture was quantified using an enzyme-linked immunosorbent assay. In order to identify the mechanism underlying BPA-mediated changes in Agrp, siRNA knockdown of transcription factors, FOXO1, CHOP, ATF3, ATF4, ATF6, and small-molecule inhibitors of endoplasmic reticulum stress, JNK or MEK/ERK were used. RESULTS: BPA increased mRNA levels of Agrp in six hypothalamic cell lines (mHypoA-59, mHypoE-41, mHypoA-2/12, mHypoE-46, mHypoE-44, and mHypoE-42). Interestingly, only 18% of the total BPA in the culture medium entered the cells after 24 h, suggesting that the exposure concentration is much lower than the treatment concentration. BPA increased pre-Agrp mRNA levels, indicating increased Agrp transcription. Knockdown of the transcription factor ATF3 prevented BPA-mediated increase in Agrp, pre-Agrp, and in part Npy mRNA levels. However, chemical chaperone, sodium phenylbutyrate, JNK inhibitor, SP600125, or the MEK/ERK inhibitor PD0352901 did not block BPA-induced Agrp upregulation. CONCLUSION: Overall, these results indicate that hypothalamic Agrp is susceptible to dysregulation by BPA and implicate ATF3 as a common mediator of the orexigenic effects of BPA in hypothalamic neurons.

Panax Notoginseng Saponins Inhibits Ventricular Remodeling after Myocardial Infarction in Rats Through Regulating ATF3/MAP2K3/p38 MAPK and NF κ B Pathway.

OBJECTIVE: To explore whether Panax notoginseng saponins (PNS) exhibits heart protective effect in myocardial infarction (MI) rats and to identify the potential signaling pathways involved. METHODS: MI rats induced by ligating the left anterior descending (LAD) coronary artery were assigned to sham coronary artery ligation or coronary artery ligation. Totally 36 Sprague-Dawley rats were randomly divided into sham group (distilled water, n=9), MI group (distilled water, n=9), PNS group (PNS, 40 mg/kg daily, n=9) and fosinopril group (FIP, 1.2 mg/kg daily, n=9) according to a random number table. The left ventricular morphology and function were conducted by echocardiography. Histological alterations were evaluated by the stainings of HE and Masson. The serum levels of C reactive protein (CRP), tumor necrosis factor α (TNF-α), growth differentiation factor-15 (GDF-15) and the ratio of metalloproteinase-9 (MMP-9) and tissue inhibitor of MMP-9 (TIMP-1) were determined by ELISA. The levels of activating transcription factor 3 (ATF3), mitogen-activated protein kinase kinase 3 (MAP2K3), p38 mitogen-activated protein kinase (p38 MAPK), phosphorylation of p38 MAPK (p-p38 MAPK), transforming growth factor-ß (TGF-ß1), collagen I, nuclear factor kappa B p65 (NFκB p65), phosphorylation of NFκB p65 (p-NFκB p65), and phosphorylation of inhibitory kappa Bα (p-Iκ Bα) in hearts were measured by Western blot and immunohistochemical staining, respectively. RESULTS: PNS improved cardiac function and fibrosis in MI rats (P<0.05). The serum levels of CRP, TNF-α, GDF-15 and the ratio of MMP9/TIMP1 were reversed by PNS in MI rats. The expressions of TGF-ß1, collagen I, MAP2K3, p38 MAPK, p-p38 MAPK, NFκB p65, p-NFκB p65, and p-IκBα were down-regulated, while ATF3 increased with the treatment of PNS (P<0.05). CONCLUSIONS: PNS may improve cardiac function and fibrosis in MI rats via regulating ATF3/MAP2K3/p38 MAPK and NFκB signaling pathways. These results suggest the potential of PNS in preventing the development of ventricular remodeling in MI rats.

Anti­inflammatory effects of leaf and branch extracts of honeyberry (Lonicera caerulea) on lipopolysaccharide­stimulated RAW264.7 cells through ATF3 and Nrf2/HO­1 activation.

Honeyberry (Lonicera caerulea) has long been used as a traditional medicine in China, Japan and northern Russia. Functional studies of honeyberry have mainly focused on the fruits, which have been reported to exert various pharmacological activities, including anti­inflammatory activity, with limited or no studies on the other parts of the plant, such as the leaves and branches. In the present study, the anti­inflammatory effects of extracts of the leaves (HBL), branches (HBB) and fruit (HBF) of honeyberry plant were evaluated in lipopolysaccharide (LPS)­stimulated RAW264.7 cells. HBL and HBB significantly inhibited the production of pro-inflammatory mediators in LPS­stimulated RAW264.7 cells, and the inhibitory effects of HBL and HBB were stronger than those of HBF. HBL and HBB blocked the nuclear accumulation of p65 independently of IκB­α. HBL did not inhibit the phosphorylation of ERK1/2 or p38; however, HBB effectively inhibited the phosphorylation of p38 but not ERK1/2. HBL and HBB increased the expression of heme oxygenase­1 (HO­1) protein by inducing the nuclear accumulation of nuclear factor erythroid 2­related factor 2 (Nrf2) through the activation of the reactive oxygen species (ROS)/p38 pathway; the reduction in inducible nitric oxide synthase (iNOS) and interleukin­1ß (IL­1ß) expression by HBL and HBB was inhibited by HO­1 knockdown. In addition, HBL and HBB increased the expression of activating transcription factor­3 (ATF3), and the reduction in iNOS and IL­1ß expression by HBL and HBB was inhibited by ATF3 knockdown. Collectively, HBL and HBB inhibited LPS­induced nuclear factor­κB activation by blocking the nuclear accumulation of p65, increasing HO­1 expression through activation of the ROS/p38/Nrf2 pathway, and increasing ATF3 expression. Furthermore, HBB inhibited LPS­induced p38 phosphorylation. These findings suggest that HBL and HBB may have great potential as natural products for the development of anti­inflammatory drugs.

Anticancer Activity of a Novel High Phenolic Sorghum Bran in Human Colon Cancer Cells.

Human colon cancer is the third leading cause of mortality in the United States and worldwide. Chemoprevention using diet is widely accepted as a promising approach for cancer management. Numerous population studies indicate a negative correlation between the incidence of colon cancer and consumption of whole grains with a high content of bioactive phenolic compounds. In the current study, we evaluated the anticancer properties of a high phenolic sorghum bran extract prepared using 70% ethanol with 5% citric acid solvent at room temperature. A significant dose-dependent suppression of cell proliferation was observed in human colon cancer cells treated with the high phenolic sorghum bran extract. Apoptosis and S phase growth arrest were induced, while cell migration and invasion were inhibited by this treatment; these effects were accompanied by altered expression of apoptosis, cell cycle, and metastasis-regulating genes. We also found that the high phenolic sorghum bran extract stimulated DNA damage in association with induction of extracellular signal-regulated kinase (ERK) and c-Jun-NH2-terminal kinase (JNK) and subsequent expression of activating transcription factor 3 (ATF3). The present study expands our understanding of the potential use of high phenolic sorghum bran to prevent human colon cancer.

Xiao-Ai-Ping Injection Enhances Effect of Paclitaxel to Suppress Breast Cancer Proliferation and Metastasis via Activating Transcription Factor 3.

Chemotherapy is an effective treatment for invasive breast cancer. Paradoxically, many recently published findings showed that the first-line chemotherapeutic agent paclitaxel (PTX) showed pro-metastatic effects in the progress of treating breast cancer. Xiao-Ai-Ping (XAP) injection, composed of a traditional herbal medicine, Marsdenia tenacissimae extract, is known to exert antitumor effects on various cancers. However, there are few experimental studies on breast cancer. The underlying mechanism of the antitumor effect of XAP combined with chemotherapy agents has not been fully understood. In the present study, we sought to find the antitumor effects of XAP combined with PTX in vitro and in vivo. The data demonstrated that the combination of XAP with PTX resulted in remarkable enhancement of the pro-apoptotic, migration-inhibiting, and anti-invasive effects of PTX in vitro. Significantly, further study showed the overexpression of ATF3 in PTX-treated cell, while XAP counteracted the change of ATF3 induced by PTX. Moreover, it showed that combination treatment could promote the inhibition of tumor growth in MDA-MB-231 cell xenograft mouse model. Compared with PTX treatment, the downregulation of ATF3 indicated that ATF3 played a pivotal role in the combination of XAP with PTX to exert a synergistic effect. Overall, it is expected that PTX combined with XAP may serve as an effective agent for antitumor treatment, and dampening ATF3 maybe a potential strategy to improve the efficacy of PTX.

Adipocyte browning and resistance to obesity in mice is induced by expression of ATF3.

Billions of people have obesity-related metabolic syndromes such as diabetes and hyperlipidemia. Promoting the browning of white adipose tissue has been suggested as a potential strategy, but a drug still needs to be identified. Here, genetic deletion of activating transcription factor 3 (ATF3-/- ) in mice under a high-fat diet (HFD) resulted in obesity and insulin resistance, which was abrogated by virus-mediated ATF3 restoration. ST32da, a synthetic ATF3 inducer isolated from Salvia miltiorrhiza, promoted ATF3 expression to downregulate adipokine genes and induce adipocyte browning by suppressing the carbohydrate-responsive element-binding protein-stearoyl-CoA desaturase-1 axis. Furthermore, ST32da increased white adipose tissue browning and reduced lipogenesis in HFD-induced obese mice. The anti-obesity efficacy of oral ST32da administration was similar to that of the clinical drug orlistat. Our study identified the ATF3 inducer ST32da as a promising therapeutic drug for treating diet-induced obesity and related metabolic disorders.

The MRVI1-AS1/ATF3 signaling loop sensitizes nasopharyngeal cancer cells to paclitaxel by regulating the Hippo-TAZ pathway.

Long non-coding RNA (lncRNA) plays an important role in malignant tumor occurrence, development, and chemoresistance, but the mechanism of how they affect nasopharyngeal cancer (NPC) paclitaxel chemosensitivity is unclear. In this study, lncRNA array of CNE-1 and HNE-2 paclitaxel-resistant cells and their parental strains revealed that the paclitaxel-resistant strains had significantly lower MRVI1-AS1 (murine retrovirus integration site 1 homolog antisense RNA 1) expression than the parental strains, and that MRVI1-AS1 overexpression in vitro and in vivo increased paclitaxel chemosensitivity. Further, MRVI1-AS1 upregulated ATF3 (activating transcription factor 3) by simultaneously inhibiting miR-513a-5p (microRNA-513a-5p) and miR-27b-3p expression levels to increase NPC paclitaxel chemosensitivity. Chromatin immunoprecipitation and quantitative real-time PCR showed that ATF3 could feed-back MRVI1-AS1 regulation positively. Furthermore, MRVI1-AS1 and ATF3 could form a positive feedback loop, which promoted the expression of RASSF1 (Ras association domain family member 1), a Hippo-TAZ (tafazzin) signaling pathway regulatory factor, thereby inhibiting TAZ expression. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay and flow cytometry showed that the decreased TAZ increased NPC cell paclitaxel chemosensitivity. Overall, the results indicate that the MRVI1-AS1/ATF3 signaling pathway can increase NPC paclitaxel chemosensitivity by modulating the Hippo-TAZ signaling pathway. Therefore, targeting the loop may be a new NPC treatment strategy.

Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat.

Electroacupuncture has been shown to effectively reduce chronic pain in patients with nerve injury. The underlying mechanisms are not well understood. Accumulated evidence suggests that purinergic P2X3 receptors (P2X3Rs) in dorsal root ganglion neurons play a major role in mediating chronic pain associated with nerve injury. The aim of this study is to determine if electroacupuncture stimulation alters P2X3R activity in dorsal root ganglia to produce analgesia under neuropathic pain condition. Peripheral neuropathy was produced by ligation of the left lumbar 5 (L5) spinal nerve in rats. Low-frequency (2 Hz) electrical stimulation was applied to ipsilateral ST36 and BL60 acupoints in rats. The P2X3R agonist (α,ß-meATP)-induced flinch responses were reduced after electroacupuncture treatment. Western analyses showed that P2X3R expression was upregulated in nerve-uninjured lumbar 4 (L4) dorsal root ganglion neurons ipsilateral to the spinal nerve ligation. Electroacupuncture-stimulation reversed the upregulation. In nerve-injured L5 dorsal root ganglia, P2X3R expression was substantially reduced. Electroacupuncture had no effect on the reduction. We also determined the injury state of P2X3R expressing dorsal root ganglion neurons using the neuronal injury marker, activating transcription factor 3 (ATF3). Immunohistochemical assay showed that in L4 dorsal root ganglia, almost all P2X3Rs were expressed in uninjured (ATF3-) neurons. Spinal nerve ligation increased the expression of P2X3Rs. Electroacupuncture reduced the increase in P2X3R expression without affecting the percentage of ATF + neurons. In ipsilateral L5 dorsal root ganglion neurons, spinal nerve ligation reduced the percentage of P2X3R + neurons and markedly increased the percentage of ATF3 + cells. Almost all of P2X3Rs were expressed in damaged (ATF3+) neurons. Electroacupuncture had no effect on spinal nerve ligation-induced changes in the percentage of P2X3R or percentage of ATF3 + cells in L5 dorsal root ganglia. These observations led us to conclude that electroacupuncture effectively reduces injury-induced chronic pain by selectively reducing the expression of P2X3Rs in nerve-uninjured L4 dorsal root ganglion neurons.

Anti-inflammatory effects of naturally occurring retinoid X receptor agonists isolated from Sophora tonkinensis Gagnep. via retinoid X receptor/liver X receptor heterodimers.

Retinoid X receptor (RXR) ligands have a wide range of beneficial effects in mouse models of Alzheimer's disease (AD). Recently accumulated evidence suggests that early neuroinflammation may be a therapeutic target for AD treatment. We therefore investigated the anti-inflammatory effects of the prenylated flavanoids SPF1 and SPF2, which were previously isolated from root of Sophora tonkinensis and identified as potent ligands for RXR, and potential mechanisms involved. SPF1 and SPF2 efficiently reduced interleukin (IL)-1ß messenger RNA (mRNA) and IL-6 mRNA levels in lipopolysaccharide-stimulated and tumor necrosis factor-α-stimulated RAW264.7 cells, whereas SPF3-which has a structure similar to SPF1 and SPF2 but no RXR ligand activity-did not exhibit such effects. Intriguingly, the liver X receptor (LXR) ligand T0901317 reduced proinflammatory cytokine mRNA levels, and these effects were potentiated by SPF1. With regard to the mechanism underlying the anti-inflammatory effects, SPF1 induced significant amounts of activating transcription factor 3 (ATF3) mRNA and protein, and this effect was potentiated by T0901317. SPF1 also reduced translocation of nuclear factor κB (NF-κB) into nuclei. The production of proinflammatory cytokines was significantly inhibited by SPF1, and this effect was primarily exerted via RXR/LXR heterodimers. The effects of SPF1 may partly depend on the induction of ATF3, which may bind to the p65 subunit of NF-κB, resulting in reduced translocation of NF-κB into nuclei and reduced NF-κB transcription. Although inflammatory effects mediated by RXR/LXR heterodimers have not been thoroughly investigated, the above-described results shed light on the mechanism of the anti-inflammatory effect via RXR/LXR heterodimer.

Tabebuia aurea decreases hyperalgesia and neuronal injury induced by snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore is used as anti-inflammatory, analgesic and antiophidic in traditional medicine, though its pharmacological proprieties are still underexplored. In the bothropic envenoming, pain is a key symptom drove by an intense local inflammatory and neurotoxic event. The antivenom serum therapy is still the main treatment despite its poor local effects against pain and tissue injury. Furthermore, it is limited to ambulatorial niches, giving space for the search of new and more inclusive pharmacological approaches. AIM OF THE STUDY: evaluation of Tabebuia aurea hydroethanolic extract (HEETa) in hyperalgesia and neuronal injury induced by Bothrops mattogrossensis venom (VBm). MATERIALS AND METHODS: Stem barks from Tabebuia aurea were extracted with ethanol and water (7:3, v/v) to yield the extract HEETa. Then, HEETa was analyzed by LC-DAD-MS and its constituents were identified. Snake venoms were extracted from adult specimens of Bothrops mattogrossensis, lyophilized and kept at -20 °C until use. Male Swiss mice, weighting 20-25 g, were used to hyperalgesia (electronic von Frey), motor impairment (Rotarod test) and tissue injury evaluation (histopatology and ATF-3 immunohistochemistry). Therefore, three experimental groups were formed: VBm (1 pg, 1 ng, 0.3 µg, 1 µg, 3 and 6 µg/paw), HEETa orally (180, 540, 720, 810 or 1080 mg/kg; 10 mL/kg, 30 min prior VBm inoculation) and VBm neutralized (VBm: HEETa, 1:100 parts, respectively). In all set of experiments a control (saline group) was used. First, we made a dose-time-response course curve of VBm's induced hyperalgesia. Next, VBm maximum hyperalgesic dose was employed to perform HEETa orally dose-time-response course curve and analyses of VBm neutralized. Paw tissues for histopathology and DRGs were collected from animals inoculated with VBm maximum dose and treated with HEETa antihyperalgesic effective dose or neutralized VBm. Paws were extract two or 72 h after VBm inoculation and DRGs, in the maximum expected time expression of ATF-3 (72 h). RESULTS: From HEETa extract, glycosylated iridoids were identified, such as catalpol, minecoside, verminoside and specioside. VBm induced a time and dose dependent hyperalgesia with its highest effect seen with 3 µg/paw, 2 h after venom inoculation. HEETa effective dose (720 mg/kg) decreased significantly VBm induced hyperalgesia (3 µg/paw) with no motor impairment and signs of acute toxicity. HEETa antihyperalgesic action starts 1.5 h after VBm inoculation and lasted up until 2 h after VBm. Hyperalgesia wasn't reduced by VBm: HEETa neutralization. Histopathology revealed a large hemorragic field 2 h after VBm inoculation and an intense inflammatory infiltrate of polymorphonuclear cells at 72 h. Both HEETa orally and VBm: HEETa groups had a reduced inflammation at 72 h after VBm. Also, the venom significantly induced ATF-3 expression (35.37 ±â€¯3.25%) compared with saline group (4.18 ±â€¯0.68%) which was reduced in HEETa orally (25.87 ±â€¯2.57%) and VBm: HEETa (19.84 ±â€¯2.15%) groups. CONCLUSION: HEETa reduced the hyperalgesia and neuronal injury induced by VBm. These effects could be related to iridoid glycosides detected in HEETa and their intrinsic reported mechanism.

Botanical Formulation HX109 Ameliorates TP-Induced Benign Prostate Hyperplasia in Rat Model and Inhibits Androgen Receptor Signaling by Upregulating Ca2+/CaMKKß and ATF3 in LNCaP Cells.

Benign prostatic hyperplasia (BPH) is a common disease in the elderly male population throughout the world. Among other factors, androgen dysregulation has been known to play major roles in its pathogenesis. HX109 is a botanical formulation prepared from a mixture of Taraxacum officinale, Cuscuta australis, and Nelumbo nucifera, which have traditionally been used-usually along with other plants-to treat urinary diseases. An ethanol extract was prepared from a mixture of these three plants, and its quality was controlled through cell-based bioassays and by quantification of several marker compounds by high-performance liquid chromatography (HPLC). In the testosterone propionate (TP)-induced prostate hyperplasia rat model, oral administration of HX109 ameliorated prostate enlargement and histological changes induced by TP. In LNCaP cells, a human prostate epithelial cell line, HX109 repressed AR-mediated cell proliferation and the induction of androgen receptor (AR) target genes at the transcriptional level without affecting the translocation or expression of AR. Such effects of HX109 on AR signaling were mediated through the control of activating transcriptional factor 3 (ATF3) expression, phosphorylation of calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß), and increases in intracellular calcium, as evidenced by data from experiments involving ATF3-specific siRNA, CaMKKß inhibitor, and calcium chelator, respectively. Taken together, our data suggest that HX109 might be used as a starting point for developing therapeutic agents for the treatment of BPH.

Interesterified soybean oil promotes weight gain, impaired glucose tolerance and increased liver cellular stress markers.

Interesterified fats have largely replaced hydrogenated vegetable fat, which is rich in trans fatty acids, in the food industry as an economically viable alternative, generating interest to study their health effects. The aim of this study was to evaluate the effect that interesterification of oils and fat has on lipid-induced metabolic dysfunction, hepatic inflammation and ER stress. Five week-old male Wistar rats were randomly divided into three experimental groups, submitted to either normocaloric and normolipidic diet containing 10% of lipids from unmodified soybean oil (SO) or from interesterified soybean oil (ISO), and one more group submitted to a high fat diet (HFD) containing 60% of fat from lard as a positive control, for 8 or 16 weeks. Metabolic parameters and hepatic gene expression were evaluated. The HFD consumption led to increased body mass, adiposity and impaired glucose tolerance compared to SO and ISO at both time points of diet. However, the ISO group showed an increased body mass gain, retroperitoneal WAT mass, fasting glucose, and impaired glucose tolerance during ipGTT at 16 weeks compared to SO. Moreover, at 8 weeks, hepatic gene expression of Atf3 and Tnf were increased in the ISO group compared to the SO group. Thus, replacement of natural fat with interesterified fat on a normocaloric and normolipidic diet negatively modulated metabolic parameters and resulted in impaired glucose tolerance in rats.

Anti-inflammatory effect of the extracts from the branch of Taxillus yadoriki being parasitic in Neolitsea sericea in LPS-stimulated RAW264.7 cells.

Mistletoe has been used as the herbal medicine to treat hypertension, diabetes mellitus, inflammation, arthritis and viral infection. In this study, we evaluated the anti-inflammatory effect of extracts of branch from Taxillus yadoriki being parasitic in Neolitsea sericea (TY-NS-B) using in vitro model. TY-NS-B significantly inhibited LPS-induced secretion of NO and PGE2 in RAW264.7 cells. TY-NS-B was also observed to inhibit LPS-mediated iNOS COX-2 expression. In addition, TY-NS-B attenuated production of inflammatory cytokines such as TNF-α and IL-1ß induced by LPS. TY-NS-B blocked LPS-mediated inhibitor of IκB-α, and inhibited p65 translocation to the nucleus and NF-κB activation. Furthermore, TY-NS-B reduced the phosphorylation of MAPKs such as p38 and JNK, but not ERK1/2. In addition, TY-NS-B increased ATF3 expression and ATF3 knockdown by ATF3 siRNA attenuated TY-NS-B-mediated inhibition of pro-inflammatory mediator expression. Collectively, our results suggest that TY-NS-B exerts potential anti-inflammatory effects by suppressing NF-κB and MAPK signaling activation, and increasing ATF3 expression. These findings indicate that TY-NS-B could be further developed as an anti-inflammatory drug.