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Background: Oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell dysfunction plays a crucial role in the progression of atherosclerosis (AS). Although miR-125b-5p is known to be involved in cardiovascular and cerebrovascular disorders, its function in ox-LDL-induced endothelial injury is still not well understood., Methods: An in vitro AS cell model was established by exposing human umbilical vein endothelial cells (HUVECs) to 100 µg/mL ox-LDL for 24 h. A series of functional assays, including CCK-8 assay, flow cytometry, MDA and SOD kits, capillary-like network formation assay and ELISA assay were performed in vitro. TNFSF4/TLR4/NF-κB pathway-related protein expressions were measured by Western blot. Molecular mechanisms were elucidated through quantitative real-time PCR, western blot analysis, and luciferase reporter assays., Results: Our investigation revealed that exposure to ox-LDL led to a downregulation in miR-125b-5p, while upregulating the expression of tumor necrosis factor (ligand) superfamily, member 4 (TNFSF4), TLR4, p-p65 and p-IkBa in HUVECs in a dose-dependent manner. We confirmed TNFSF4 as a direct target of miR-125b-5p. Ox-LDL exposure led to decreased cell viability and angiogenic capacity, along with increased apoptosis, inflammation, and oxidative stress in HUVECs. These effects were reversed by overexpressing miR-125b-5p or knocking down TNFSF4. Overexpression of TNFSF4 significantly reversed the effects brought about by miR-125b-5p in HUVECs exposed to ox-LDL. Moreover, miR-125b-5p inactivated the TLR4/NF-κB signaling pathway by negatively regulating TNFSF4., Conclusions: In summary, our findings demonstrate that miR-125b-5p possessed an anti-inflammatory and anti-apoptosis against ox-LDL-induced HUVEC injury by regulating the TNFSF4/TLR4/NF-κB signaling, indicating that miR-125b-5p may have an important therapeutic function for AS., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Diabetic retinopathy (DR), the most common ocular complication of diabetes mellitus (DM), has exhibited an increase in incidence over the past decade. S100 calcium-binding protein A9 (S100A9) plays a significant role in inflammation and cancer. Toll-like receptor 4 (TLR4), a transmembrane receptor, initiates signaling cascades upon ligand binding. S100A9 activates TLR4, and their involvement in various diseases is well-established. We found elevated S100A9/TLR4 pathway proteins in the vitreous of DR patients. Bioinformatics analysis revealed differential gene expression related to this pathway. These proteins were also detected in diabetic rat retinas and induced structural damage. Paquinimod, an S100A9 inhibitor, decreased pathway protein expression and reduced retinal damage. Our study validates the S100A9/TLR4 pathway in diabetic retinas and suggests its potential as a therapeutic target for DR. Targeting S100A9 could offer a novel approach to prevention and treatment., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plant diseases caused by fungal phytopathogens have led to significant economic losses in agriculture worldwide. The management of fungal diseases is mainly dependent on the application of fungicides, which are not suitable for sustainable agriculture, human health, and environmental safety. Thus, it is necessary to develop novel targets and green strategies to mitigate the losses caused by these pathogens. The target of rapamycin (TOR) complexes and key components of the TOR signalling pathway are evolutionally conserved in pathogens and closely related to the vegetative growth and pathogenicity. As indicated in recent systems, chemical, genetic, and genomic studies on the TOR signalling pathway, phytopathogens with TOR dysfunctions show severe growth defects and nonpathogenicity, which makes the TOR signalling pathway to be developed into an ideal candidate target for controlling plant disease. In this review, we comprehensively discuss the current knowledge on components of the TOR signalling pathway in microorganisms and the diverse roles of various plant TOR in response to plant pathogens. Furthermore, we analyse a range of disease management strategies that rely on the TOR signalling pathway, including genetic modification technologies and chemical controls. In the future, disease control strategies based on the TOR signalling network are expected to become a highly effective weapon for crop protection., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Dysregulation of cholesterol homeostasis occurs in multiple types of tumors and promotes cancer progression. Investigating the specific processes that induce abnormal cholesterol metabolism could identify therapeutic targets to improve cancer treatment. In this investigation, we observed upregulation of 7-dehydrocholesterol reductase (DHCR7), a vital enzyme involved in the synthesis of cholesterol, within bladder cancer tissues in comparison to normal tissues, which was correlated with increased bladder cancer metastasis. Increased expression of DHCR7 in bladder cancer was attributed to decreased mRNA degradation mediated by YTHDF2. Loss or inhibition of DHCR7 reduced bladder cancer cell invasion in vitro and metastasis in vivo. Mechanistically, DHCR7 promoted bladder cancer metastasis by activating the cAMP/protein kinase A/FAK pathway. Specifically, DHCR7 increased cAMP levels by elevating cholesterol content in lipid rafts, thereby facilitating the transduction of signaling pathways mediated by cAMP receptors. DHCR7 additionally enhanced the cAMP signaling pathway by reducing the concentration of 7-dehydrocholesterol and promoting the transcription of the G protein-coupled receptor, namely gastric inhibitory polypeptide receptor. Overall, these findings demonstrate that DHCR7 plays an important role in bladder cancer invasion and metastasis by modulating cholesterol synthesis and cAMP signaling. Furthermore, inhibition of DHCR7 shows promise as a viable therapeutic strategy for suppressing bladder cancer invasion and metastasis. Significance: Inhibiting DHCR7 induces cholesterol metabolism reprogramming and lipid raft remodeling to inactivate the cAMP/protein kinase A/FAK axis and suppress bladder cancer metastasis, indicating the therapeutic potential of targeting DHCR7., (©2024 American Association for Cancer Research.)

Backgrounds: Non-small cell lung carcinoma (NSCLC) is a common type of lung cancer. Prior investigations have elucidated the pivotal role of miR-29b-3p in restraining tumor growth and metastasis. Nonetheless, it remains to be determined whether miR-29b-3p can effectively suppress NSCLC progression and enhance the sensitivity of NSCLC cells to cisplatin. This investigation sought to determine the mechanism by which miR-29b-3p inhibited the advancement of NSCLC and mitigated resistance to cisplatin., Methods: We initially assessed miR-29b-3p and VEGF levels in NSCLC tissues and cell lines. Next, miR-29b-3p expression was elevated in NSCLC cell lines H1975 and A549 by overexpression plasmid transfection. Following this, a sequence of molecular biology experiments was conducted to evaluate the impact of miR-29b-3p on the biological behaviors of NSCLC cells and their resistance to cisplatin. Additionally, we predicted VEGF was a target gene of miR-29b-3p by bioinformatics analysis. We next employed western blot to evaluate the protein expression of Nrf2 and HO-1 in NSCLC cells. Finally, we elucidated the effects of VEGF and Nrf2/HO-1pathway on NSCLC progression and cisplatin resistance by in vitro assays., Results: In comparison to paracancerous tissues and human normal lung epithelial cells, the expression of miR-29b-3p was notably reduced and VEGF expression was clearly elevated in NSCLC tissues and cells. Moreover, miR-29b-3p upregulated obviously suppressed the biological activities of NSCLC cells and increased their sensitivity to cisplatin. Furthermore, in NSCLC cells, miR-29b-3p bound to VEGF and negatively regulate its transcription. Additionally, miR-29b-3p overexpression also inhibited the Nrf2/HO-1 signaling pathway. Finally, the overexpression of VEGF and the activation of the Nrf2/HO-1 pathway reversed miR-29b-3p-mediated inhibitory effect on biological behaviors of NSCLC cells and increased the cisplatin resistance., Conclusion: Our findings indicate that miR-29b-3p impedes NSCLC cells' biological behaviors and augments their sensitivity to cisplatin by targeting VEGF to modulate the Nfr2/HO-1 signaling pathway., (© 2024 Wiley Periodicals LLC.)

Melanoma is a prevalent malignant skin tumor known for its high invasive ability and a high rate of metastasis, making clinical treatment exceptionally challenging. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment and play a crucial role in tumor survival and development. Cold atmospheric plasma (CAP) is an emerging tool for tumor treatment that has garnered attention from scholars due to its interaction with non-tumor cells in the tumor microenvironment. Here, we used the macrophage lines THP-1 and RAW264.7, as well as the melanoma cell lines A375 and MV3, as research subjects to investigate the effect of plasma-activated liquid (PAL) on macrophage differentiation and its inhibitory effect on melanoma cell proliferation. We confirmed that the killing effect of PAL on melanoma cells was selective. Using flow cytometry and PCR, we discovered that PAL can influence macrophage differentiation. Through in vitro cell coculture, we demonstrated that PAL-treated macrophages can significantly impede tumor cell development and progression, and the effect is more potent than that of PAL directly targeting tumor cells. Furthermore, we have proposed the hypothesis that PAL promotes the differentiation of macrophages into the M1 type through the ROS/JAK2/STAT1 pathway. To test the hypothesis, we employed catalase and fludarabine to block different sites of the pathway. The results were then validated through Western Blot, qPCR and ELISA. This study illustrates that PAL therapy is an effective tumor immunotherapy and expands the scope of tumor immunotherapy. Furthermore, these findings establish a theoretical foundation for potential clinical applications of PAL., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Environmental arsenic exposure is one of the major global public health problems. Studies have shown that arsenic exposure can cause renal fibrosis, but the underlying mechanism is still unclear. Integrating the in vivo and in vitro models, this study investigated the potential molecular pathways for arsenic-induced renal fibrosis. In this study, SD rats were treated with 0, 5, 25, 50, and 100 mg/L NaAsO 2 for 8 weeks via drinking water, and HK2 cells were treated with different doses of NaAsO 2 for 48 h. The in vivo results showed that arsenic content in the rats' kidneys increased as the dose increased. Body weight decreased and kidney coefficient increased at 100 mg/L. As a response to the elevated NaAsO 2 dose, inflammatory cell infiltration, renal tubular injury, glomerular atrophy, tubulointerstitial hemorrhage, and fibrosis became more obvious indicated by HE and Masson staining. The kidney transcriptome profiles further supported the protein-protein interactions involved in NaAsO 2 -induced renal fibrosis. The in vivo results, in together with the in vitro experiments, have revealed that exposure to NaAsO 2 disturbed mitochondrial dynamics, promoted mitophagy, activated inflammation and the TGF-β1/SMAD signaling pathway, and finally resulted in fibrosis. In summary, arsenic exposure contributed to renal fibrosis via regulating the mitochondrial dynamics and the NLRP3-TGF-β1/SMAD signaling axis. This study presented an adverse outcome pathway for the development of renal fibrosis due to arsenic exposure through drinking water., (© 2024 Wiley Periodicals LLC.)

Pentraxin 3 (PTX3) has been documented to be involved in the development of chemoresistance, however, the mechanisms by which it regulates cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC) have never been elucidated. Quantitative reverse transcriptase polymerase chain reaction and Western blot were carried to determine the expression of PTX3, ATP-binding cassette sub-family B member 1 (ABCB1)/P-glycoprotein 1 (p-gp), protein kinase B (Akt), phosphorylated Akt and nuclear factor-kappa B (NF-кB) p65. The biological roles of PTX3 in NSCLC progression and NSCLC cell resistance to DDP were evaluated using enzyme-linked immunosorbent assay, cell count kit-8, colony formation assay, flow cytometry, as well as xenograft tumor assay. The expression of PTX3 was increased in the serum of NSCLC patients as well as in NSCLC cell lines. Lower PTX3 level was associated with longer overall survival in lung adenocarcinoma and lung squamous cell carcinoma patients. Furthermore, PTX3 expression was greatly higher in DDP-resistant NSCLC cells than that in NSCLC cells. Silencing of PTX3 restrained the proliferation and promoted the apoptosis of NSCLC cells, as well as sensitized DDP-resistant NSCLC cells to DDP. Additionally, knockdown of PTX3 inhibited the growth of NSCLC tumors in vivo. Upregulation of PTX3 expression was dependent on the activation of Akt/NF-κB signaling. The induction of apoptosis by PTX3 knockdown was enhanced by MK-2206 or JSH-23. In conclusion, knockdown of PTX3 restrained the progression of NSCLC and sensitized NSCLC cells towards DDP, which provides a potential target to restore DDP chemoresponse., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ovarian cancer (OC) is one of the leading causes of female deaths. However, the molecular pathogenesis of OC has still remained elusive. This study aimed to explore the potential genes associated with the progression of OC. In the current study, 3 data sets of OC were downloaded from the GEO database to identify hub gene. Somatic mutation data obtained from TCGA were used to analyse the mutation. Immune cells were used to estimate effect of the hub gene to the tumour microenvironment. RNA-seq and clinical data of OC patients retrieved from TCGA were used to investigate the diagnostic and prognostic values of hub gene. A series of in vitro assays were performed to indicate the function of hub gene and its possible mechanisms in OC. As a result, RAD51AP1 was found as a hub gene, which expression higher was mainly associated with poor survival in OC patients. Up-regulation of RAD51AP1 was closely associated with mutations. RAD51AP1 up-regulation accompanied by accumulated Th2 cells, but reduced CD4 + T cells and CD8 + T cells. Nomogram demonstrated RAD51AP1 increased the accuracy of the model. Down-regulation of RAD51AP1 suppressed proliferation, migration and invasion capabilities of OC cells in vitro. Additionally, scatter plots showed that RAD51AP1 was positively correlated with genes in TGF-β/Smad pathway. The above-mentioned results were validated by RT-qPCR and Western blotting. In conclusion, up-regulation of RAD51AP1 was closely associated with mutations in OC. RAD51AP1 might represent an indicator for predicting OS of OC patients. Besides, RAD51AP1 might accelerate progression of OC by TGF-β/Smad signalling pathway., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Preclinical mouse models that recapitulate some characteristics of coronavirus disease (COVID-19) will facilitate focused study of pathogenesis and virus-host responses. Human agniotensin-converting enzyme 2 (hACE2) serves as an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect people via binding to envelope spike proteins. Herein we report development and characterization of a rapidly deployable COVID-19 mouse model. C57BL/6J (B6) mice expressing hACE2 in the lung were transduced by oropharyngeal delivery of the recombinant human adenovirus type 5 that expresses hACE2 (Ad5-hACE2). Mice were infected with SARS-CoV-2 at Day 4 after transduction and developed interstitial pneumonia associated with perivascular inflammation, accompanied by significantly higher viral load in lungs at Days 3, 6, and 12 after infection compared with Ad5-empty control group. SARS-CoV-2 was detected in pneumocytes in alveolar septa. Transcriptomic analysis of lungs demonstrated that the infected Ad5-hACE mice had a significant increase in IFN-dependent chemokines Cxcl9 and Cxcl10 , and genes associated with effector T-cell populations including C d3 g, Cd8a, and Gzmb . Pathway analysis showed that several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the data set, including cytokine-cytokine receptor interaction, the chemokine signaling pathway, the NOD-like receptor signaling pathway, the measles pathway, and the IL-17 signaling pathway. This response is correlative to clinical response in lungs of patients with COVID-19. These results demonstrate that expression of hACE2 via adenovirus delivery system sensitized the mouse to SARS-CoV-2 infection and resulted in the development of a mild COVID-19 phenotype, highlighting the immune and inflammatory host responses to SARS-CoV-2 infection. This rapidly deployable COVID-19 mouse model is useful for preclinical and pathogenesis studies of COVID-19.

The phytohormone abscisic acid (ABA) plays pivotal roles in triggering stomatal closure and facilitating adaptation of plants to drought stress. Hydrogen sulfide (H 2 S), a small signaling gas molecule, is involved in ABA-dependent stomatal closure. However, how H 2 S regulates ABA signaling remains largely unclear. Here, we show that ABA induces the production of H 2 S catalyzed by L-CYSTEINE DESULFHYDRASE1 (DES1) in guard cells, and H 2 S in turn positively regulates ABA signaling through persulfidation of Open Stomata 1 (OST1)/SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6). Two cysteine (Cys) sites, Cys131 and Cys137, which are exposed on the surface of SnRK2.6 and close to the activation loop, were identified to be persulfidated, which promotes the activity of SnRK2.6 and its interaction with ABA response element-binding factor 2 (ABF2), a transcription factor acting downstream of ABA signaling. When Cys131, Cys137, or both residues in SnRK2.6 were substituted with serine (S), H 2 S-induced SnRK2.6 activity and SnRK2.6-ABF2 interaction were partially (SnRK2.6 C131S and SnRK2.6 C137S ) or completely (SnRK2.6 C131SC137S ) compromised. Introduction of SnRK2.6 C131S , SnRK2.6 C137S , or SnRK2.6 C131SC137S into the ost1-3 mutant could not rescue the mutant phenotype: less sensitivity to ABA- and H 2 S-induced stomatal closure and Ca 2+ influx as well as increased water loss and decreased drought tolerance. Taken together, our study reveals a novel post-translational regulatory mechanism of ABA signaling whereby H 2 S persulfidates SnRK2.6 to promote ABA signaling and ABA-induced stomatal closure., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

Calycosin-7-O-β-D-glucoside (CG) is the component of Astragali Radix, and the aim of the present study is to investigate whether CG protects myocardium from I/R-induced damage by the regulation of IL-10/JAK2/STAT3 signaling pathway. H9C2 cells were subjected to I/R treatment and pretreated with 1 μm CG in vitro. In addition, a mouse model of myocardial I/R injury was induced by left anterior descending (LAD) coronary artery ligation and administrated with 30 mg/kg CG by intravenous injection before I/R surgery. In vitro and in vivo results showed that CG up-regulated IL-10 level, activated the JAK2/STAT3 pathway, and protected myocardial cells from I/R-induced apoptosis. The hemodynamic measurement, TTC staining, TUNEL staining, and western blot results in vivo showed that the protective effects of CG on myocardial function and cell apoptosis were all reversed by the IL-10R α neutralizing antibody. CG-induced phosphorylation activation of JAK2/STAT3 signaling pathway was also suppressed by the blocking of IL-10. In summary, these findings suggest that CG might alleviate myocardial I/R injury by activating the JAK2/STAT3 signaling pathway via up-regulation of IL-10 secretion, which provides us insights into the mechanism underlying the protective effect of CG on myocardial I/R injury.

Constitutive activation of FGFR1 as a result of chromosome translocations is responsible for the development of a hematopoietic stem cell disorder that progresses to AML. We have developed a syngeneic mouse model of BCR-FGFR1 driven AML and used RNASeq to define gene expression signatures associated with disease progression. The development of the leukemic stem cells (LSC) is associated with a profound downregulation of specific transcription factors that normally maintain stem cell quiescence as well as cell adhesion and motility gene sets related to confinement to the stem cell niche. A prominent feature of the LSCs is the upregulation of genes involved in T-cell function, activation, migration and development. Despite this apparent T-cell priming in the LSCs, however, the majority of these genes are subsequently inactivated in the leukemic blast cells that derive from them. These studies provide insights into the molecular etiology of development and progression of FGFR1 driven AML., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors., Methods: Human lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src 418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-Src Y530F to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA, and Vimentin were examined at 48 h by RT-PCR and western blot. At 48 h and 72 h of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assays., Results: Activity of c-Src kinase, which causes the expression of p-Src 418 , was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-Src Y530F , the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src 418 increased. The expressions of both E-cadherin and ZO-1 were suppressed, while the expressions of vimentin and α-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src 418 expressions were knocked down. The expressions of E-cadherin and ZO-1 increased, but the expressions of Vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced., Conclusions: The c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.

From scientific advances in medical research to the plethora of anti-aging products available, our obsession with slowing the aging process and increasing life span is indisputable. A large research effort has been levied towards this perpetual search for the fountain of youth, yet the molecular mechanisms governing an organism's life span and the causes of aging are only beginning to emerge in animals and remain largely unanswered in plants. As one central pathway in eukaryotes controlling cell growth, development, and metabolism, the target of rapamycin (TOR) plays an evolutionarily conserved role in aging and the determination of life span. The modulation of TOR pathway components in a wide range of species, including the model plant Arabidopsis thaliana, has effects on life span. However, the mechanisms enabling some of the longest living species to endure, including trees that can live for millennia, have not been defined. Here, we introduce key TOR research from plant systems and discuss its implications in the plant life cycle and the broader field of life span research. TOR pathway functions in plant life cycle progression and life span determination are discussed, noting key differences from yeast and animal systems and the importance of 'omics' research for the continued progression of TOR signaling research., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The traditional view holds that apoptosis is non-immunogenic and does not induce an inflammatory response. However, recent studies have suggested that certain chemotherapeutic drugs that induce tumor cell apoptosis can induce immunogenic cell death (ICD) in cancer cells. This process is characterized by not only up-regulation of a series of signaling molecules in cancer cells, including expose of calreticulin (CRT), secretion of adenosine triphosphate (ATP) and release of high mobility group box 1 (HMGB1). In this review, we summarize recent progress in identifying and classifying ICD inducers; concepts and molecular mechanisms of ICD; and the impact and potential applications of ICD in clinical studies. We also discuss the contributions of ICD inducers in combination with other anticancer drugs in clinical applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The present study assessed the effects of dietary arginine on intestinal antioxidant status and immunity involved in Nrf2 and NF-κB signaling pathway in juvenile blunt snout bream. Fish were fed three practical diets with graded arginine levels (0.87%, 1.62% and 2.70%) for 8 weeks. Compared with the control group (0.87%), the counts of white blood cell (WBC), red blood cell (RBC) and hemoglobin (HGB) content were significantly improved at dietary arginine levels of 1.62% (P<0.05). Plasma albumin (ALB) levels and alkaline phosphatase (ALP) activities were significantly improved at dietary arginine levels of 1.62% and 2.70% (P < 0.05). Alanine transaminase (ALT) activity was decreased in fish fed with 1.62% dietary arginine level (P<0.05). Plasma glutathione peroxidase (GPx) activities, copper-zinc superoxide dismutase (Cu/Zn-SOD) activities, total antioxidant capacity (T-AOC) activities and glutathione (GSH) levels were significantly increased at dietary arginine levels of 1.62% and 2.70% (P<0.05). Plasma total superoxide dismutase (T-SOD) activities and catalase (CAT) activities were significantly improved in fish fed with 1.62% dietary arginine level. Significantly higher manganese superoxide dismutase (Mn-SOD) activity was observed in fish fed with 1.62% dietary arginine level compared with 2.70% dietary arginine level (P<0.05). 1.62% and 2.70% dietary arginine levels significantly lowered malondialdehyde (MDA) levels. The relative expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was significantly increased in fish fed with 1.62% dietary arginine level, inversely, the relative expression of Kelch-like ECH-associated protein 1 (Keap1) showed a converse trend. 1.62% and 2.70% dietary arginine levels significantly improved the relative expressions of Cu/Zn-SOD, GPx and CAT. Furthermore, 2.70% dietary arginine level significantly lowered the relative expression of Mn-SOD compared with the control group and 1.62% dietary arginine levels. The relative expressions of Interleukin 1β (IL-1β), tumour necrosis factor-α (TNF-α) and nuclear factor-kappa B (NF-κB) were lowered in fish fed with 1.62% dietary arginine level. 1.62% and 2.70% dietary arginine levels significantly improved the relative expressions of transforming growth factor-β (TGF-β). Hematocrit (HCT), aspartate aminotransferase (AST) activities, interleukin 8 (IL-8) and interleukin 10 (IL-10) expressions were not significantly affected by the graded dietary arginine levels. These results suggest that the optimal dietary arginine level plays an important role in enhancing antioxidant and immune status to maintain the intestinal health of juvenile blunt snout bream., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Patients with Hutchinson-Gilford progeria syndrome (HGPS) have low bone mass and an atypical skeletal geometry that manifests in a high risk of fractures. Using both in vitro and in vivo models of HGPS, we demonstrate that defects in the canonical WNT/β-catenin pathway, seemingly at the level of the efficiency of nuclear import of β-catenin, impair osteoblast differentiation and that restoring β-catenin activity rescues osteoblast differentiation and significantly improves bone mass. Specifically, we show that HGPS patient-derived iPSCs display defects in osteoblast differentiation, characterized by a decreased alkaline phosphatase activity and mineralizing capacity. We demonstrate that the canonical WNT/β-catenin pathway, a major signaling cascade involved in skeletal homeostasis, is impaired by progerin, causing a reduction in the active β-catenin in the nucleus and thus decreased transcriptional activity, and its reciprocal cytoplasmic accumulation. Blocking farnesylation of progerin restores active β-catenin accumulation in the nucleus, increasing signaling, and ameliorates the defective osteogenesis. Moreover, in vivo analysis of the Zmpste24-/- HGPS mouse model demonstrates that treatment with a sclerostin-neutralizing antibody (SclAb), which targets an antagonist of canonical WNT/β-catenin signaling pathway, fully rescues the low bone mass phenotype to wild-type levels. Together, this study reveals that the β-catenin signaling cascade is a therapeutic target for restoring defective skeletal microarchitecture in HGPS. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

The present study assessed the effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream. Fish were fed six practical diets with graded arginine levels ranging from 0.87% to 2.70% for 8 weeks. The results showed that compared with the control group (0.87% dietary arginine level), significantly higher mRNA levels of adenosine monophosphate activated protein kinase (AMPK) and nitric oxide synthetase (NOS), activities of total nitric oxide synthetase (T-NOS) and nitric oxide synthetase (iNOS), and plasma nitric oxide (NO) contents were observed in fish fed with 1.62%-2.70% dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.22%-2.70%, the mRNA levels of iNOS were significantly improved. Dietary arginine also significantly influenced plasma interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) contents. Furthermore, dietary arginine significantly affected the activity and mRNA level of glutathione peroxidase (GPx), the mRNA levels of pro-inflammatory factor including IL-8 and TNF-α and plasma malondialdehyde (MDA) content. However, total superoxide dismutase (T-SOD) activity, plasma complement component 3 (C3) content, plasma immunoglobulin M (IgM) content, plasma interleukin 1β (IL-1β) content and the mRNA levels of copperzinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD) and IL-1β were not significantly affected by dietary arginine. After Aeromonas hydrophila challenge, the death rate was significantly lowered in fish fed with 1.62%-1.96% dietary arginine levels. Furthermore, the mRNA levels of AMPK, NOS and iNOS, plasma NO content and the activities of T-NOS and iNOS showed an upward trend with increasing dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.96%-2.31%, T-SOD activities were significantly improved. Significantly higher GPx activities were observed in fish fed with 1.22%-2.70% dietary arginine levels. At dietary arginine levels of 1.22%-2.31%, the plasma TNF-α and IL-8 contents were significantly decreased. Significantly lower plasma IL-1β contents were observed in fish fed 1.62%-1.96% dietary arginine levels. Dietary arginine significantly influenced the mRNA levels of antioxidant and pro-inflammatory genes including Cu/Zn-SOD, Mn-SOD, GPx, IL-8, TNF-α and IL-1β. Significantly higher plasma C3 contents and significantly lower plasma MDA contents were observed in fish fed with 1.62%-1.96% arginine levels. Furthermore, plasma IgM contents were significantly improved at dietary arginine levels of 1.62%-2.31%. However, high dietary arginine group (2.70%) significantly improved the mRNA levels of pro-inflammatory genes including IL-8, TNF-α and IL-1β and plasma MDA, IL-8, TNF-α and IL-1β contents as compared with optimal dietary arginine levels (1.62% and 1.96%). The present results indicate that optimal arginine level (1.62% and 1.96%) could improve antioxidant capacity, immune response and weaken tissues inflammatory involved in arginine-AMPK-NO signaling pathway, while high arginine level resulted in excessive NO production, leading to increase oxidative stress damage and inflammatory response in juvenile blunt snout bream., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The present study assessed the effects of dietary leucine on growth performance, antioxidant status and immunity in juvenile blunt snout bream. Fish were fed six practical diets of graded leucine levels ranging from 0.90% to 2.94% of dry basis for 8 weeks. Trail results showed that compared to control group (0.90%), 1.72% dietary leucine level significantly improved final weight (FW), weight gain rate (WG) and specific growth rate (SGR), and significantly lowered feed conversion ratio (FCR). Based on WG and SGR, the optimal dietary leucine level was obtained at 1.40% and 1.56%, respectively. Whole body crude lipid and protein contents were improved with increasing dietary leucine up to 2.14% and thereafter showed a downward trend, while whole body moisture content showed a converse trend. No significant change was found in whole body ash content. 1.72% dietary leucine level significantly improved the antioxidant capacity of fish by regulating the plasma superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, aspartate aminotransferase (AST) activities and malondialdehyde (MDA) content, furthermore, 1.72% dietary leucine level also significantly improved the antioxidant genes expressions of associated with Nrf2 signaling pathway by regulating heme oxygenase-1 (HO-1), GPx, copperezinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), 2.14% dietary leucine levels also significantly improved glutathione transferase (GST) mRNA level. Dietary leucine levels significantly affected plasma immunity parameters such as the contents of plasma complement component 3 (C3), immunoglobulin M (IgM) and lowered the hepatopancreas genes expressions of pro-inflammatory factor by regulating interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels. The present study indicated that optimal dietary leucine level plays an important role in improving growth, enhancing antioxidant and immune status to maintain the health in juvenile blunt snout bream., (Copyright © 2017. Published by Elsevier Ltd.)

This study evaluated the mechanisms governing insulin resistance, glucose metabolism and lipogenesis in juvenile fish fed with graded levels of dietary arginine. The results showed that, compared with the control group (0.87%), 2.31% dietary arginine level resulted in the upregulation of the relative gene expression of IRS-1, PI3K and Akt in the insulin signaling pathway, while 2.70% dietary arginine level led to inhibition of these genes. 1.62% dietary arginine level upregulated glycolysis by increasing GK mRNA level; 2.70% dietary arginine level upregulated gluconeogenesis and resulted in high plasma glucose content by increasing PEPCK and G6P mRNA level. Furthermore, 2.70% dietary arginine level significantly lowered GLUT2 and increased PK mRNA levels. 1.62% dietary arginine level significantly upregulated ACC, FAS and G6PDH mRNA levels in the fat synthesis pathway and resulted in high plasma TG content. These results indicate that 1.62% dietary arginine level improves glycolysis and fatty acid synthesis in juvenile blunt snout bream. However, 2.70% dietary arginine level results in high plasma glucose, which could lead to negative feedback of insulin resistance, including inhibition of IRS-1 mRNA levels and activation of gluconeogenesis-related gene expression. This mechanism seems to be different from mammals at the molecular level.

Upon exposure to environmental engineered nanomaterials (ENMs), animals will activate certain response signals to protect themselves from the toxic effects. However, the underlying molecular mechanisms for this response are still largely unclear. Using in vivo assay system of Caenorhabditis elegans, we here found that antimicrobial proteins of LYS-1, LYS-8, SPP-1, DOD-6, and F55G11.4 were activated by graphene oxide (GO) exposure. These antimicrobial proteins functioned as molecular targets of transcriptional factor DAF-16 in insulin signaling pathway, and acted in intestine to regulate the response to GO. Among these antimicrobial proteins, DOD-6, F55G11.4, and SPP-1 participated in the formation of signaling cascade of DAF-16-DOD-6-SOD-3-F55G11.4/SPP-1 in response to GO exposure by activating the antioxidation system. Different from this, LYS-1 and LYS-8, two lysozymes, mediated TUB-2 signaling and DAF-8-DAF-5 signaling cascade, respectively, to regulate the response to GO exposure. During the regulation of response to GO exposure, LYS-1 and LYS-8 acted synergistically, which could be largely explained by the observed synergistic interaction between TUB-2 and DAF-8. Therefore, our results demonstrate the crucial protection role of antimicrobial proteins for animals in response to environmental ENMs' exposure. The elucidated different signaling cascades mediated by antimicrobial proteins provide important molecular targets for future toxicity assessment and chemical modification of GO.

Target of rapamycin (TOR) acts as an important regulator of cell growth, development and stress responses in most examined diploid eukaryotes. However, little is known about TOR in tetraploid species such as cotton. Here, we show that TORC1-S6K-RPS6, the major signaling components, are conserved and further expanded in cotton genome. Though the cotton seedlings are insensitive to rapamycin, AZD8055, the second-generation inhibitor of TOR, can significantly suppress the growth in cotton. Global transcriptome analysis revealed that genes associated with jasmonic acid (JA) biosynthesis and transduction were significantly altered in AZD8055 treated cotton seedlings, suggesting the potential crosstalk between TOR and JA signaling. Pharmacological and genetic approaches have been employed to get further insights into the molecular mechanism of the crosstalk between TOR and JA. Combination of AZD8055 with methyl jasmonate can synergistically inhibit cotton growth, and additionally JA levels were significantly increased when cotton seedlings were subjected to AZD8055. JA biosynthetic and signaling mutants including jar1, coi1-2 and myc2-2 displayed TOR inhibitor-resistant phenotypes, whereas COI1 overexpression transgenic lines and jaz10 exhibited sensitivity to AZD8055. Consistently, cotton JAZ can partially rescue TOR-suppressed phenotypes in Arabidopsis. These evidences revealed that the crosstalk between TOR and JA pathway operates in cotton and Arabidopsis.

The components of the target of rapamycin (TOR) signaling pathway have been well characterized in heterotrophic organisms from yeast to humans. However, because of rapamycin insensitivity, embryonic lethality in tor null mutants and a lack of reliable ways of detecting TOR protein kinase in higher plants, the key players upstream and downstream of TOR remain largely unknown in plants. Using engineered rapamycin-sensitive Binding Protein 12-2 (BP12-2) plants, the present study showed that combined treatment with rapamycin and active-site TOR inhibitors (asTORis) results in synergistic inhibition of TOR activity and plant growth in Arabidopsis. Based on this system, we revealed that TOR signaling plays a crucial role in modulating the transition from heterotrophic to photoautotrophic growth in Arabidopsis. Ribosomal protein S6 kinase 2 (S6K2) was identified as a direct downstream target of TOR, and the growth of TOR-suppressed plants could be rescued by up-regulating S6K2. Systems, genetic, and biochemical analyses revealed that Brassinosteriod Insensitive 2 (BIN2) acts as a novel downstream effector of S6K2, and the phosphorylation of BIN2 depends on TOR-S6K2 signaling in Arabidopsis. By combining pharmacological with genetic and biochemical approaches, we determined that the TOR-S6K2-BIN2 signaling pathway plays important roles in regulating the photoautotrophic growth of Arabidopsis., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Connexin43 (Cx43) containing gap junctions play an important role in bone homeostasis, yet little is known about the second messengers communicated by Cx43 among bone cells. Here, we used MC3T3-E1 pre-osteoblasts and UMR106 rat osteosarcoma cells to test the hypothesis that cAMP is a second messenger communicated by bone cells through Cx43 containing gap junctions in a manner that is sufficient to impact osteoblast function. Overexpression of Cx43 markedly enhanced the activity of a cAMP-response element driven transcriptional luciferase reporter (CRE-luc) and increased phospho-CREB and phospho-ERK1/2 levels following expression of a constitutively active Gsα or by treatment with prostaglandin E2 (PGE2), 3-Isobutyl-1-methyl xanthine (IBMX) or forskolin. The Cx43-dependent potentiation of signaling in PGE2 treated cells was not accompanied by a further increase in cAMP levels, suggesting that the cAMP was shared between cells rather than Cx43 enhancing cAMP production. To support this, we developed a novel assay in which one set of cells expressing constitutively active Gsα (donor cells) were co-cultured with a second set of cells expressing a CRE-luc reporter (acceptor cells). Using this assay, activation of a CRE-luc reporter in the acceptor cells was both Cx43- and cell contact-dependent, indicating communication of cAMP among cells. Finally, we showed that Cx43 increased the cAMP-dependent mRNA expression of receptor activator of nuclear factor kappa B ligand (RANKL) and enhanced the repression of the sclerostin mRNA, implying a potential mechanism for the modulation of tissue remodeling. In total, these data demonstrate that Cx43 can communicate cAMP between cells and, more importantly, that the communicated cAMP is sufficient to impact signal transduction cascades and the expression of key bone effector molecules between interconnected cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate the effects and mechanisms of calycosin‑7‑O‑β‑D‑glucoside (CG) on ischemia‑reperfusion (I/R) injury in vivo. Hemodynamic parameters, including ejection fraction (EF), fractional shortening (FS), left ventricular end‑systolic pressure (LVESP) and left ventricular end‑diastolic pressure (LVEDP) were monitored using an ultrasound system, and infarct size was measured using Evans blue/tetrazolium chloride double staining. The activities of serum creatine kinase (CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA) were determined to assess the degree of myocardial injury and oxidative stress‑induced damage. The protein expression levels of cleaved‑caspase‑3, cleaved‑caspase‑9, phosphorylated (p)‑phosphatidylinositol 3‑kinase (PI3K) p85, PI3K p85, p‑Akt and Akt were determined using western blotting. The results demonstrated that pretreatment with high dose (H)‑CG markedly improved cardiac function, as evidenced by upregulated EF, FS and LVESP, and downregulated LVEDP. In addition, administration of CG resulted in significant decreases in infarct size in the I/R+low dose‑CG and I/R+H‑CG groups, compared with the I/R group. The activities of CK and LDH, and the levels of MDA in the I/R+H‑CG group were reduced, compared with those in the I/R group, whereas SOD activity was elevated. Treatment with CG inhibited the cleavage and activity of caspase‑3 and caspase‑9, and enhanced the phosphorylation of PI3K p85 and Akt. Notably, administration of the PI3K inhibitor, LY294002, markedly lowered the levels of p‑PI3K p85/p‑Akt, and eradicated the inhibitory effects of H‑CG on infarct size, myocardial injury and oxidative stress‑induced damage. Taken together, the results suggested that CG may alleviate I/R injury by activating the PI3K/Akt signaling pathway.

Background: The flowers of Carthamus tinctorius L. are widely used in traditional Chinese medicine to treat cerebrovascular and cardiovascular diseases. Hydroxysafflor yellow A (HSYA), the main constituent of C. tinctorius L. flowers, is known for its multiple biological activities. The present study investigated the effects of HSYA on angiogenesis in vitro and in a mouse hindlimb ischemia model., Methods: Using human umbilical vein endothelial cells (HUVEC) in vitro and a mouse hindlimb ischemia model in vivo, the angiogenic role of HSYA was evaluated., Results: HSYA significantly increased the capillary-like tube formation and migration of HUVEC. HSYA not only induced a rise in the expression of angiopoietin 1 and Tie-2 but it also increased phosphorylation of Tie-2, Akt, and extracellular signal-regulated kinase 1/2. Furthermore, an anti-Tie-2 neutralizing antibody significantly inhibited HSYA-induced HUVEC tube formation and migration. In vivo, the recovery of perfusion of ischemic hindlimb tissue after femoral artery interruption was significantly increased in HSYA-treated mice compared to vehicle controls. Consistent with these results, the arteriole and capillary densities in ischemic gastrocnemius muscles were significantly increased in HSYA-treated mice., Conclusions: These results indicate the potential utility of HSYA for the treatment of ischemic diseases., (© 2016 S. Karger AG, Basel.)

The aim of this paper is to study the participation of transforming growth factor-β (TGF-β) signaling pathway in mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs), via the interaction between TGF-β and ER signaling pathways. The UL cells were prepared by primary culture and subculture methods. To validate the role of TGF-β3 (5 ng/ml) for the viability of human uterine leiomyoma cells, CCK-8 assay was performed in each of five treatment groups including E2 group (E2 10(9) mol/l), BPA group (bisphenol A 10 μmol/l), NP group (nonylphenol 32 μmol/l), OP group (octylphenol 8 μmol/l), or control group (DMSO only). Subsequently, qRT-PCR was applied to detect mRNA expressions of ERα and c-fos, while western blot assay was used to test the expressions of p-Smad3, SnoN, and c-fos proteins in all settings mentioned above; the expressions were compared among different groups, and also in settings with and without synchronous treatment of ICI 182,780. Primarily cultured UL cells were successfully established. Compared with the control group, there were statistically significant increases in the proliferation rate of the UL cells in all EE groups or treated with TGF-β3 only (p < 0.05). Nevertheless, a slight decrease in proliferation rate of UL was detected in coexistence with TGF-β3 in all EE groups (p > 0.05). Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs compared to isolated EE treatment (p < 0.05). Compared with the control group, the expression of p-Smad3 and c-fos proteins significantly decreased (p < 0.05) in each of E2, BPA, NP, and OP group, and the expression of SnoN protein also significantly reduced only in BPA and NP groups (p < 0.05), followed by TGF-β3 treatment. When adding ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group (p < 0.05), but slightly increased in E2, BPA, NP, and OP groups (p > 0.05). However, compared with the control group, the expressions of SnoN and c-fos proteins significantly decreased (p < 0.05) after adding ICI182,780. Moreover, there was a significant statistical difference in the expression of p-Smad3, SnoN, and c-fos proteins between pre- and post-treatment of ICI 182,780 in all groups (p < 0.05). The ERα signaling pathway and TGF-β signaling pathway have different roles in the control of UL cell proliferation. The phenolic EEs can be a promoter of UL cell proliferation, which is mediated by ERα signaling pathway and its cross-talking with TGF-β signaling pathway. Both less exposure to EEs and blockade of TGF signaling pathway are necessary strategies to prevent UL.

Thymoquinone (TQ) has been reported to possess anti-tumor activity in various types of cancer. However, its effects and molecular mechanism of action in hepatocellular carcinoma (HCC) are still not completely understood. We observed that TQ inhibited tumor cell growth in vitro, where treatment with TQ arrested the cell cycle in G1 by upregulating p21 and downregulating cyclinD1 and CDK2 expression; moreover, TQ induced apoptosis by decreasing expression of Bcl-2 and increasing expression of Bax. Simultaneously, TQ demonstrated a suppressive impact on the Notch pathway, where overexpression of NICD1 reversed the inhibitory effect of TQ on cell proliferation, thereby attenuating the repressive effects of TQ on the Notch pathway, cyclinD1, CDK2 and Bcl-2, and also diminishing upregulation of p21 and Bax. In a xenograft model, TQ inhibited HCC growth in nude mice; this inhibitory effect in vivo, as well as of HCC cell growth in vitro, was associated with a discernible decline in NICD1 and Bcl-2 levels and a dramatic rise in p21 expression. In conclusion, TQ inhibits HCC cell growth by inducing cell cycle arrest and apoptosis, achieving these effects by repression of the Notch signaling pathway, suggesting that TQ represents a potential preventive or therapeutic agent in HCC patients.

Astrocyte elevated gene-1 (AEG-1) has been reported to regulate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and is also regulated by it. This study investigated how AEG-1 participates in the survival pathway of motor neurons in amyotrophic lateral sclerosis (ALS). We found reduced levels of AEG-1 in ALS motor neurons, both in vivo and in vitro, compared to wild type controls. Moreover, AEG-1 silencing demonstrated inhibition of the PI3K/Akt pathway and increased cell apoptosis. Additionally, the PI3K/Akt pathway in mSOD1 cells was unresponsive under serum deprivation conditions compared to wtSOD1 cells. These results suggest that AEG-1 deficiency, together with the inhibited PI3K/Akt pathway was associated with decreased viability of ALS motor neurons. However, the mRNA levels of AEG-1 were still lower in mSOD1 cells compared to the control groups, though the signaling pathway was activated by application of a PI3-K activator. This suggests that in ALS motor neurons, some unknown interruption exists in the PI3K/Akt/CREB/AEG-1 feedback loop, thus attenuating the protection by this signaling pathway. Together, these findings support that AEG-1 is a critical factor for cell survival, and the disrupted PI3K/Akt/CREB/AEG-1cycle is involved in the death of injured motor neurons and pathogenesis of ALS., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is an idiopathic and lethal neurodegenerative disease that currently has no effective treatment. A recent study found that the Notch signaling pathway was up-regulated in a TAR DNA-binding protein-43 (TDP-43) Drosophila model of ALS. Notch signaling acts as a master regulator in the central nervous system. However, the mechanisms by which Notch participates in the pathogenesis of ALS have not been completely elucidated. Recent studies have shown that the mood stabilizers lithium and valproic acid (VPA) are able to regulate Notch signaling. Our study sought to confirm the relationship between the Notch pathway and ALS and whether the Notch pathway contributes to the neuroprotective effects of lithium and VPA in ALS. We found that the Notch pathway was activated in in vitro and in vivo models of ALS, and suppression of Notch activation with a Notch signaling inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and Notch1 siRNA significantly reduced neuronal apoptotic signaling, as evidenced by the up-regulation of Bcl-2 as well as the down-regulation of Bax and cytochrome c. We also found that lithium and VPA suppressed the Notch activation associated with the superoxide dismutase-1 (SOD1) mutation, and the combination of lithium and VPA produced a more robust effect than either agent alone. Our findings indicate that the Notch pathway plays a critical role in ALS, and the neuroprotective effects of lithium and VPA against mutant SOD1-mediated neuronal damage are at least partially dependent on their suppression of Notch activation., (Copyright © 2015. Published by Elsevier Ltd.)

Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1β and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

Objective: Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 downregulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin-dependent cooperative binding interaction between VEGFR-2 and αVβ3., Approach and Results: We studied effects of PAI-1 on VEGF signaling in human umbilical vein endothelial cells. PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in human umbilical vein endothelial cells grown on vitronectin, but not on fibronectin or collagen. PAI-1 inhibited the binding of VEGFR-2 to β3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to vitronectin and another requiring binding to very low-density lipoprotein receptor. PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption., Conclusions: PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a vitronectin-dependent proangiogenic binding interaction involving αVβ3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, vitronectin, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling., (© 2014 American Heart Association, Inc.)

Tubeimoside-1 (TBMS1) is one of the important components in Bolbostemma paniculatum (Maxim.) Franque. In the study, its immunosuppressive effects on murine T lymphocyte responses were evaluated in vitro and in vivo. The data showed that TBMS1 inhibited ConA-induced T lymphocyte proliferation, decreased the ratio of CD4(+)/CD8(+), suppressed IL-2, IFN-γ, IL-4 and IL-6 production and mRNA expression, down-regulate activation of NF-κB, NFAT2 and AP-1 signal transduction pathways in vitro. In addition, administration of TBMS1 significantly inhibited T cell-mediated DTH response in vivo. These findings indicated that TBMS1 inhibits the proliferation and activation of T lymphocytes in mice.

Leucine has been shown to influence intestinal protein metabolism, cell proliferation and migration. Furthermore, our previous study demonstrated that branched-chain amino acids could modulate the intestinal amino acid and peptide transporters in vivo. As the possible mechanisms are still largely unknown, in the present work, we studied the transcriptional and translational regulation of leucine on amino acid transporter production in IPEC-J2 cells and the signaling pathways involved. Treatment of IPEC-J2 cells with 7.5 mM leucine enhanced the mRNA expression of the Na(+)-neutral AA exchanger 2 (ASCT2) and 4F2 heavy chain (4F2hc) and caused an increase in ASCT2 protein expression. Leucine also activated phosphorylation of 4E-BP1 and eIF4E through the phosphorylation of mTOR, Akt and ERK signaling pathways in IPEC-J2 cells. Pre-treatment of IPEC-J2 cells with inhibitors of mTOR and Akt (rapamycin and wortmannin) or an inhibitor of ERK (PD098059) for 30 min before leucine treatment attenuated the positive effect of leucine in enhancing the protein abundance of ASCT2. These results demonstrate that leucine could up-regulate the expression of the amino acid transporters (ASCT2) through transcriptional and translational regulation by ERK and PI3K/Akt/mTOR activation.

Oxidized low-density lipoprotein (ox-LDL), one of the most important risk factors of atherosclerosis, is a highly antigenic, potent chemoattractant that facilitates the development of atherosclerosis. Gap junctions also play an important in the development of atherosclerosis. In this study, we investigated the effects of ox-LDL on connexin43 and the mechanisms of connexin43 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cell (HUVEC), to clarify the role of connexin43 in atherosclerosis. Our results showed that ox-LDL significantly inhibited the growth and promoted apoptosis of HUVEC in a dose-dependent manner. Also, ox-LDL upregulated the expression of connexin43. Furthermore, knockdown connexin43 by siRNA promoted proliferation and inhibited apoptosis in ox-LDL-stimulated HUVEC. Moreover, the level of phosphor-ERK1/2 and connexin43 was remarkably attenuated by a ERK pathway inhibitor (PD98059). These results suggest that connexin43 siRNA promotes HUVEC proliferation and inhibits apoptosis induced by ox-LDL, and ERK signaling pathway appears to be involved in these processes.

The demonstration of bystander effect, which means injured cells propagate damage to neighboring cells, in whole organisms has clear implication of the potential relevance of the non-targeted response to human health. Here we show that 10 μM lead acetate, the optimum concentration for inducing apoptosis confirmed by the expression levels of Bax and Bcl-2, can also induce rat pheochromocytoma (PC12) cells to exert bystander effects to neighboring cells. In a novel co-culture system, GFP-PC12 (Pb(2+)) cells, which were stable transfected with EF1A-eGFP and pre-exposed with lead acetate, were co-cultured with unexposed PC12 cells at a 1:5 ratio. Parachute assays demonstrated the functional gap-junctional intercellular communication (GJIC) formed between Pb(2+)-exposed and unexposed cells. The Pb(2+)-exposed cells induced very similar effects on neighboring unexposed cells to apoptosis coincide with intracellular ROS generation and the collapse of mitochondrial membrane potential (Δψm). Furthermore, carbenoxolone (CBX), a blocker of GJIC, inhibited the bystander effects. The results indicate that the Pb(2+)-induced insults propagate through GJIC between PC12 cells, while inducing the bystander cells to apoptosis via ROS-mitochondria-dependent apoptotic signaling., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

Myeloid and lymphoid neoplasm associated with FGFR1 is an aggressive disease, and resistant to all the current chemotherapies. To define the molecular etiology of this disease, we have developed murine models of this disease, in syngeneic hosts as well as in nonobese diabetic/severe combined immunodeficiency/interleukin 2Rγ(null) mice engrafted with transformed human CD34+ hematopoietic stem/progenitor cells. Both murine models mimic the human disease with splenohepatomegaly, hypercellular bone marrow, and myeloproliferative neoplasms that progresses to acute myeloid leukemia. Molecular genetic analyses of these model mice, as well as primary human disease, demonstrated that CNTRL-FGFR1, through abnormal activation of several signaling pathways related to development and differentiation of both myeloid and T-lymphoid cells, contribute to overt leukemogenesis. Clonal evolution analysis indicates that myeloid related neoplasms arise from common myeloid precursor cells that retain potential for T-lymphoid differentiation. These data indicate that simultaneously targeting these pathways is essential to successfully treating this almost invariably lethal disease.

Background: Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines., Methods: We conducted a detailed histopathological characterization of lymphomas developed in p80HT transgenic mice and microarray gene expression profiling of p80HT B cells with the goal of identifying genes that drive plasma cell tumor development. We further verified the significance of our findings in human multiple myeloma cell lines., Results: Approximately 40% of p80HT mice showed elevated levels of monoclonal immunoglobulin (M-protein) in the serum and developed plasma cell tumors. Some of these mice displayed key features of human multiple myeloma with accumulation of plasma cells in the bone marrow, osteolytic bone lesions and/or diffuse osteoporosis. Gene expression profiling of B cells from M-protein-positive p80HT mice revealed aberrant expression of genes known to be important in the pathogenesis of multiple myeloma, including cyclin D1, cyclin D2, Blimp1, survivin, IL-10 and IL-15. In vitro assays demonstrated a critical role of Stat3, a key downstream component of IL-10 signaling, in the survival of human multiple myeloma cells., Conclusions: These findings provide a mouse model for human multiple myeloma with aberrant NF-κB2 activation and suggest a molecular mechanism for NF-κB2 signaling in the pathogenesis of plasma cell tumors by coordinated regulation of plasma cell generation, proliferation and survival.

Background and Purpose: Pyruvate is known to be cytoprotective through antioxidant and anti-inflammatory mechanisms. We tested the hypothesis that pyruvate protects the brain against ischemia-reperfusion injury by inducing endogenous erythropoietin (EPO) expression., Methods: Pyruvate's protective effect was evaluated in C6 glioma cells and HT22 neuronal cells subjected to transient oxygen glucose deprivation. Cell viability (calcein AM assay) and expression of hypoxia-inducible factor-1α, EPO, Akt and Erk (immunoblot), and EPO receptor (reverse transcription-polymerase chain reaction) were analyzed. Transient focal cerebral ischemia in rats was induced by 2 hours middle cerebral artery occlusion followed by 24 hours reperfusion. Pyruvate or saline was infused from 60 minutes occlusion until 30 minutes reperfusion. Lesion volume and DNA fragmentation were assessed by 2,3,5-triphenyltetrazolium staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, respectively. Immunoblots were conducted to determine cerebral EPO contents., Results: Pyruvate increased cell viability, hypoxia-inducible factor-1α, EPO, and Akt phosphorylation. Small interfering RNA suppression of hypoxia-inducible factor-1α and EPO abolished pyruvate-induced cytoprotection. In the rat stroke model, pyruvate reduced lesion volume by 84% and DNA fragmentation by 77% versus controls; increased EPO content paralleled these cerebroprotective actions of pyruvate., Conclusions: Pyruvate activation of the hypoxia-inducible factor-1α-EPO signaling cascade in neurons and glia could protect the brain from ischemia-reperfusion injury.

Natriuretic peptides (NPs) are cyclic vasoactive peptide hormones with high therapeutic potential. Three distinct NPs (ANP, BNP, and CNP) can selectively activate natriuretic peptide receptors, NPR-A and NPR-B, raising the cyclic GMP (cGMP) levels. Insulin-degrading enzyme (IDE) was found to rapidly cleave ANP, but the functional consequences of such cleavages in the cellular environment and the molecular mechanism of recognition and cleavage remain unknown. Here, we show that reducing expression levels of IDE profoundly alters the response of NPR-A and NPR-B to the stimulation of ANP, BNP, and CNP in cultured cells. IDE rapidly cleaves ANP and CNP, thus inactivating their ability to raise intracellular cGMP. Conversely, reduced IDE expression enhances the stimulation of NPR-A and NPR-B by ANP and CNP, respectively. Instead of proteolytic inactivation, IDE cleavage can lead to hyperactivation of BNP toward NPR-A. Conversely, decreasing IDE expression reduces BNP-mediated signaling. Additionally, the cleavages of ANP and BNP by IDE render them active with NPR-B and a reduction of IDE expression diminishes the ability of ANP and BNP to stimulate NPR-B. Our kinetic and crystallographic analyses offer the molecular basis for the selective degradation of NPs and their variants by IDE. Furthermore, our studies reveal how IDE utilizes its catalytic chamber and exosite to engulf and bind up to two NPs leading to biased stochastic, non-sequential cleavages and the ability of IDE to switch its substrate selectivity. Thus, the evolutionarily conserved IDE may play a key role in modulating and reshaping the strength and duration of NP-mediated signaling.

The pathogenesis of Acanthamoeba keratitis (AK) is complicated. In our previous studies, TLR4 was found involved in the process of infection by Acanthamoeba in human corneal cells. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signalling pathway in Wistar rats challenged with Acanthamoeba. The rat model of AK was established. Corneas were collected and analysed by real-time PCR to assess the mRNA levels of TLR 2, 4, myeloid differentiation protein (MyD)88, nuclear factor (NF)-κB, extracellular signal-regulated kinase (ERK), interleukin (IL)-8, tumour necrosis factor (TNF)-α and interferon (IFN) -β. Immunocytochemistry and Western blot were conducted to examine the proteins of TLR2, TLR4, p-Erk1/2 and p-IκB. Specific inhibitors PDTC and U0126 were used to pretreat the animals to determine the exact receptor and signalling pathway involved in pathogenesis. Expressions of TLR4, MyD88, all three cytokines, NF-κB, p-IκB and p-Erk1/2 were increased in Acanthamoeba-treated rat corneas. PDTC inhibited the production of IL-8 and TNF-α, while U0126 inhibited the synthesis of IFN-β. TLR4 was involved in sensing the challenge of Acanthamoeba and inducing production of cytokines through TLR4-NF-κB and TLR4-Erk1/2 pathways in corneas of Wistar rats., (© 2010 Blackwell Publishing Ltd.)

Aim: Insulin-like growth factor-1 (IGF-1) is an important hypertrophic and cell cycle progression factor for a number of cell types. It has been proven that IGF-1 is involved in the regulation of thyroid proliferation and cell cycle progression; however, the exact mechanism of this regulation has not been fully elucidated. In the present study, we investigated the effect of IGF-1 on the expression of cyclin D1, an important cell cycle regulatory protein, and a signaling pathway involved in IGF-1's effect on cyclinD1 expression in FRTL thyroid cells., Methods: FRTL thyroid cells were treated with IGF-1 or vector control for 24 h. As appropriate to individual experiments, a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and/or a nuclear factor-kappaB (NF-kappaB) inhibitor, BAY11-7082, were added 1 h prior to IGF-1 treatment. Western blotting was used to detect cyclin D1 protein expression. Immunofluorescence was performed to analyze the expression of IkappaBalpha, an NF-kappaB inhibitory protein. Cell cycle analysis was performed by fluorescence activated cell sorting (FACS)., Results: IGF-1 increased the cyclin D1 expression in thyroid cells. This increase was blocked by pretreatment with LY294002 or BAY11-7082. Further studies showed that IGF-1 specifically induced NF-kappaB activity. Treatment with IGF-1 could accelerate cell cycle progression from G(0)/G(1) to S phase, whereas this progression was inhibited by the presence of LY294002 or BAY11-7082., Conclusion: In summary, the results of the present study show that in FRTL cells, IGF-1 promotes cell cycle progression via an upregulation of cyclin D1 expression, at least partially through the PI3K/NF-kappaB signaling pathway.

It is known that decreased apoptosis of thyrocytes may be involved in the formation of goiters in patients with Graves' disease, and growth factors are involved in regulating the size of the thyroid gland. The purpose of our study was to investigate mRNA and protein levels of an antiapoptotic protein, namely, Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). The results showed that in FRTL thyroid cells, treatment with IGF-I upregulated mRNA and protein levels of FLIP in a dose-dependant manner. While a specific nuclear factor-kappaB (NF-kappaB) inhibitor, BAY11-7082, blocked this effect. Further study demonstrated that IGF-I induced the DNA-binding activity of NF-kappaB in association with decreased expression of the NF-kappaB inhibitory protein IkappaBalpha . These findings implied that IGF-I increased FLIP expression by enhancing the activation of NF-kappaB in FRTL thyroid cells. Using a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, we also found that PI3K was involved in the pathway by which IGF-I activated NF-kappaB and increased FLIP expression. When treated with IGF-I and LY294002, decreased NF-kappaB DNA binding activity and increased expression of IkappaBalpha protein were detected in cultured thyroid cells, which further confirmed that NF-kappaB was under the control of the PI3K pathway. Taken together, our results suggest that IGF-I regulates the expression of FLIP in FRTL cells by activating the PI3K/NF-kappaB cascade.

Resistance to the growth-inhibitory action of retinoic acid (RA), the bioactive derivative of vitamin A, is common in human tumors. One form of RA resistance has been associated with silencing and hypermethylation of the retinoic acid receptor beta2 gene (RARbeta2), an RA-regulated tumor suppressor gene. The presence of an epigenetically silent RARbeta2 correlates with lack of the RA receptor alpha (RARalpha). Normally, RARalpha regulates RARbeta2 transcription by mediating dynamic changes of RARbeta2 chromatin in the presence and absence of RA. Here we show that interfering with RA signal through RARalpha (which was achieved by use of a dominant-negative RARalpha, by downregulation of RARalpha by RNA interference, and by use of RARalpha antagonists) induces an exacerbation of the repressed chromatin status of RARbeta2 and leads to RARbeta2 transcriptional silencing. Further, we demonstrate that RARbeta2 silencing causes resistance to the growth-inhibitory effect of RA. Apparently, RARbeta2 silencing can also occur in the absence of DNA methylation. Conversely, we demonstrate that restoration of RA signal at a silent RARbeta2 through RARalpha leads to RARbeta2 reactivation. This report provides proof of principle that RARbeta2 silencing and RA resistance are consequent to an impaired integration of RA signal at RARbeta2 chromatin.

Cinnamon has been shown to potentiate the insulin effect through upregulation of the glucose uptake in cultured adipocytes. In the present study, we evaluated the effect of the cinnamon extract on the insulin action in awaked rats by the euglycemic clamp and further analyzed possible changes in insulin signaling occurred in skeletal muscle. The rats were divided into saline and cinnamon extract (30 and 300 mg/kg BW-doses: C30 and C300) oral administration groups. After 3-weeks, cinnamon extract treated rats showed a significantly higher glucose infusion rate (GIR) at 3 mU/kg per min insulin infusions compared with controls (118 and 146% of controls for C30 and C300, respectively). At 30 mU/kg per min insulin infusions, the GIR in C300 rats was increased 17% over controls. There were no significant differences in insulin receptor (IR)-beta, IR substrate (IRS)-1, and phosphatidylinositol (PI) 3-kinase protein content between C300 rats and controls. However, the skeletal muscle insulin-stimulated IR-beta and the IRS-1 tyrosine phosphorylation levels in C300 rats were 18 and 33% higher, respectively, added to 41% higher IRS-1/PI 3-kinase association. These results suggest that the cinnamon extract would improve insulin action via increasing glucose uptake in vivo, at least in part through enhancing the insulin-signaling pathway in skeletal muscle.

This study investigated the effects of the traditional herbal medicine, Keishi-ka-jutsubu-to (KJT) on insulin action in vivo and insulin signaling in skeletal muscle in STZ-induced diabetes. Rats were divided into single and 7-days oral administration groups. Euglycemic clamp (insulin infusion rates: 3 and 30 mU/kg/min) was used in awaked rats and the insulin signaling in skeletal muscle was evaluated. At low-dose insulin infusion, the decreased metabolic clearance rates of glucose (MCR) in diabetic rats were improved by a single and 7-days administration of KJT (800 mg/kg BW, p.o.; acute effect: 6.7 +/- 0.6 vs. 12.3 +/- 1.2, and 7-days effect: 6.3 +/- 0.5 vs. 13.9 +/- 1.0 ml/kg/min, P<0.001, respectively). During high-dose insulin infusion, the MCR was increased in 7-days KJT treated diabetes compared with saline diabetes, but, these changes were not observed after a single KJT treatment. About 90% of the increasing effect in MCR induced by the 7-days KJT treatment was blocked by L-NMMA. However, no further additive effects were seen in KJT + SNP treatment. IRbeta protein increase and decreased IRS-1 protein expression in diabetes were significantly improved by KJT treatment. KJT had no effect on the GLUT4 protein content. The increased tyrosine phosphorylation level of IRbeta, IRS-1, and IRS-1 associated with PI 3-kinase were significantly inhibited in KJT treated diabetes. The present study suggests that the improvement of impaired insulin action in STZ-diabetes by administration of KJT may be due, at least in part, to enhanced insulin signaling, which may be involved with production of nitric oxide (NO).