Knockdown of Bcl-3 alleviates psoriasis and dyslipidemia comorbidity by regulating Akt pathway

Background Psoriasis is considered as an inflammatory skin disease accompanied by dyslipidemia comorbidity. B-cell leukemia-3 (Bcl-3) belongs to IκB (inhibitor of nuclear factor kappa B [NF-κB]) family, and regulates inflammatory response through associating with NF-κB. The role of Bcl-3 in psoriasis was investigated in this study. Methods Apolipoprotein E (ApoE)-deficient mice were treated with imiquimod to induce psoriasis and dyslipidemia. Mice were injected intradermally in the back with lentiviral particles encoding Bcl-3 small hairpin RNA (shRNA). Hematoxylin and eosin were used to detect pathological characteristics. The blood lipid levels were determined by automatic biochemical analyzer, and inflammation was assessed by enzyme-linked-immunosorbent serologic assay and real-time quantitative reverse transcription polymerase chain reaction. Results Bcl-3 was elevated in imiquimod-induced ApoE-deficient mice. Injection with lentiviral particles encoding Bcl-3 shRNA reduced Psoriasis area and severity index (PASI) score in ApoE-deficient psoriatic mice. Knockdown of Bcl-3 also ameliorated imiquimod-induced psoriasiform skin lesions in ApoE-deficient mice. Moreover, loss of Bcl-3 enhanced expression of loricrin, an epidermal barrier protein, reduced expression of proliferating cell nuclear antigen (PCNA) and lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) in imiquimod-induced ApoE-deficient mice. The enhanced levels of blood lipid in ApoE-deficient mice were attenuated by silencing of Bcl-3 with increase of high-density lipoprotein, and reduction of total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Knockdown of Bcl-3 attenuated imiquimod-induced decrease of transforming growth factor beta (TGF-β), and increase of Interleukin (IL)-17A, IL-23, IL-6, and tumor necrosis factor-α (TNF-α) in ApoE-deficient mice. Protein expression of phospho-Akt (p-Akt) and p-GSK3β in ApoE-deficient psoriatic mice was decreased by silencing of Bcl-3 (AU)

CRYAB reduces cigarette smoke-induced inflammation, apoptosis, and oxidative stress by retarding PI3K/Akt and NF-kB signaling pathways in human bronchial epithelial cells

Background: Chronic obstructive pulmonary disease (COPD) is a familiar airway disease characterized by chronic immune response in the lungs. More and more evidences have assured that cigarette smoking is the primary reason for the progression of COPD, but its related regulatory mechanism requires further clarification. The α-B-crystallin (CRYAB) has been identified to exhibit vital functions in different diseases, and is down-regulated in the alveoli of mice mediated by cigarette smoke extract (CSE).Methods: The messenger RNA expression of CRYAB was assessed by reverse transcription--quantitative polymerase chain reaction. The proteins’ expressions were tested using Western blot method. The cytotoxicity was measured by lactate dehydrogenase assay. The levels of malondialdehyde, superoxide dismutase, catalase, myeloperoxidase, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were assessed through enzyme-linked-immunosorbent serologic assay (ELISA).Results: In this study, it was discovered that the expression of CRYAB was markedly decreased with the increased time of cigarette smoking. Moreover, CRYAB overexpression increased cell viability and decreased cell apoptosis induced by cigarette smoke. In addition, the strengthened oxidative stress and inflammation mediated by CSE treatment was relieved after overexpression of CRYAB. Eventually, results OF Western blot method confirmed that CRYAB retarded the activation of phosphatidylinositol 3-kinase–Ak strain transforming (PI3K–Akt) and nuclear factor kappa B (NF-κB) signaling pathways.Conclusion: Our results manifested that CRYAB reduced cigarette smoke-induced inflammation, apoptosis, and oxidative stress in normal and diseased bronchial epithelial (NHBE) and human bronchial epithelial (BEAS-2B) cells by suppressing PI3K/Akt and NF-κB signaling pathways, which highlighted the functioning of CRYAB in preventing or treating COPD (AU)

Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function.

To accommodate the changing needs of the developing brain, microglia must undergo substantial morphological, phenotypic, and functional reprogramming. Here, we examined whether cellular metabolism regulates microglial function during neurodevelopment. Microglial mitochondria bioenergetics correlated with and were functionally coupled to phagocytic activity in the developing brain. Transcriptional profiling of microglia with diverse metabolic profiles revealed an activation signature wherein the interleukin (IL)-33 signaling axis is associated with phagocytic activity. Genetic perturbation of IL-33 or its receptor ST2 led to microglial dystrophy, impaired synaptic function, and behavioral abnormalities. Conditional deletion of Il33 from astrocytes or Il1rl1, encoding ST2, in microglia increased susceptibility to seizures. Mechanistically, IL-33 promoted mitochondrial activity and phagocytosis in an AKT-dependent manner. Mitochondrial metabolism and AKT activity were temporally regulated in vivo. Thus, a microglia-astrocyte circuit mediated by the IL-33-ST2-AKT signaling axis supports microglial metabolic adaptation and phagocytic function during early development, with implications for neurodevelopmental and neuropsychiatric disorders.

Close association between the synergistic toxicity of zearalenone-deoxynivalenol combination and microRNA221-mediated PTEN/PI3K/AKT signaling in HepG2 cells.

Mycotoxins can impart different types of combined toxicity to humans and animals, therefore, it is critical to understand the underlying mechanisms to eliminate the harm. Herein a combination of zearalenone (ZEA) at 2 µM and deoxynivalenol (DON) at 0.1 µM decreased cell viability and increased ROS level in HepG2 cells, suggesting synergistic toxicity exerted by ZEA and DON even at their low toxic concentrations. Moreover, apoptosis and inflammatory response were promoted after the co-exposure of ZEA and DON, indicated by the increased expression of BAX, Caspase-3, IL-1ß and IL-6 genes. Such synergistic toxicity was closely associated with miR-221-mediated PTEN/PI3K/AKT signal pathway, with a negative regulatory relationship between PTEN and PI3K/AKT signaling. MiR-221 could influence cell viability and ROS level to counter the combined toxicity of ZEA and DON through targeting directly PTEN gene. This study demonstrated the toxicological impact of mycotoxin interactions on cells, and critical role of the interplay between miRNAs and PTEN in monitoring the synergistic toxicity of mycotoxin mixture.

Jolkinolide B inhibits proliferation or migration and promotes apoptosis of MCF-7 or BT-474 breast cancer cells by downregulating the PI3K-Akt pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The diterpenoids extracted from Euphorbia kansui S.L. Liou ex S.B.Ho, Euphorbia fischeriana Steud. have good antitumor effects. Jolkinolide B has anti-breast cancer effect, but it is unclear whether it has different therapeutic effects between luminal A subtype and luminal B subtype breast cancer. AIM OF THE STUDY: This study investigated the Jolkinolide B has different therapeutic, important targets and pathways effects between luminal A subtype and luminal B subtype breast cancer. MATERIALS AND METHODS: We used bioinformatics to predict the biological process and molecular mechanism of Jolkinolide B in treating two types of breast cancer. Then, in vitro, cultured MCF-7 cells and BT-474 cells were divided into control group, PI3K inhibitor + control group, Jolkinolide B group and PI3K inhibitor + Jolkinolide B group. The CCK-8 assay, Flow cytometric analysis and Transwell cell migration assay was used to detect the cell proliferation, apoptosis, and migration in each group, respectively. ELISA was used to measure the content of Akt and phosphorylated Akt (p-Akt) in cell lysis buffer. RESULTS: Compared to luminal A breast cancer, Jolkinolide B had more targets, proliferation, migration processes and KEGG pathways when treating luminal B subtype breast cancer. Jolkinolide B significantly prolonged the survival time of luminal B subtype breast cancer patients. Compared to the control group, the cell proliferation absorbance value (A value) and migration number of the two kinds of breast cancer cells in the Jolkinolide B group were decreased (P < 0.01, n = 6), and the number of apoptotic cells was increased (P < 0.01, n = 6). Compared to the Jolkinolide B group, the A value and migration number of the two types of breast cancer cells were significantly decreased in the PI3K inhibitor + Jolkinolide B group (P < 0.01, n = 6), and the number of apoptotic cells was significantly increased (P < 0.01, n = 6). In addition, compared to MCF-7 cells, the A value and migration number of BT-474 cells stimulated with Jolkinolide B were significantly decreased (P < 0.01, n = 6), and the number of apoptotic cells was significantly increased (P < 0.01, n = 6). Akt and p-Akt protein levels in the two breast cancer cell lines in the Jolkinolide B group were all decreased (P < 0.01, n = 6), especially in BT-474 cells stimulated by Jolkinolide B. CONCLUSION: Jolkinolide B regulates the luminal A and luminal B subtypes of breast cancer through PI3K-Akt, EGFR and other pathways. Jolkinolide B has more significant therapeutic effect on luminal B subtype breast cancer. In vitro, experiments verified that Jolkinolide B significantly inhibited the proliferation and migration activity of BT-474 breast cancer cells by downregulating the PI3K-Akt pathway.

Dihydroartemisinin inhibits Lewis Lung carcinoma progression by inducing macrophages M1 polarization via AKT/mTOR pathway.

Preclinical and clinical data show a close relationship between high infiltration of tumor-associated macrophages (TAMs) and a poor prognosis in most types of tumors, thus targeting TAMs stands out as promising anticancer immunotherapies. Recent studies have demonstrated the anti-tumor effects of artemisinin via enhancing anti-tumor immunity within tumor microenvironment, but the underlying mechanism is still not clear. In the present study we uncovered an important role of dihydroartemisinin (DHA) in regulating intratumoral TAM polarization and anti-tumor immune responses in mouse Lewis Lung carcinoma model. We found that DHA inhibited Lewis Lung carcinoma progress, moderately decreased the frequencies of TAMs within tumor stroma, and significantly increased CD86 expression while decreased CD206 expression on TAMs which indicates the role of DHA in polarizing TAMs into a M1-like phenotype. Then, our in vitro data confirmed that DHA dose-dependently promoted macrophage M1 phenotype transition by increasing M1 phenotype-related molecules, meanwhile decreasing the expression of M2 phenotype-related molecules. In addition, DHA increased proinflammatory cytokine production, enhanced the phagocytic capacity while decreased anti-inflammatory cytokine production. Finally, in order to prove that AKT/mTOR signaling potentially mediated DHA-induced macrophage differentiation, we used rapamycin to specifically block the activity of mTOR and stimulated macrophages under M1 stimuli. Our data clearly showed that rapamycin significantly decreased DHA-induced M1-related phenotypes and proinflammatory cytokine expression. In summary, our study highlighted DHA as one of future potential therapeutic options for the development of novel anticancer immunotherapies in lung cancer.

Tricin attenuates the progression of LPS-induced severe pneumonia in bronchial epithelial cells by regulating AKT and MAPK signaling pathways

Background Pneumonia is a continuous and widespread disease with higher incidence, the effects of it on human life can be fearful. Tricin has been demonstrated to take part in the progression and development of diseases. However, the function of Tricin and its related regulatory pathways remain unclear. This study was planned to investigate the effects of Tricin on severe pneumonia.Methods The cell viability was detected through CCK-8 assay. The TNF-α, IL-1β and IL-6 levels were assessed through ELISA and RT-qPCR. The levels of MDA, SOD and GSH were tested through corresponding commercial kits. The protein expressions were examined through western blot.Results In our study, the lipopolysaccharide (LPS) was firstly used to stimulate cell model for severe pneumonia. We discovered that Tricin had no toxic effects on BEAS-2B cells and the decreased cell viability induced by LPS was relieved by a dose-dependent Tricin treatment. Additionally, through ELISA and RT-qPCR, it was uncovered that Tricin reduced the LPS-induced inflammation through regulating TNF-α, IL-1β and IL-6. Furthermore, Tricin relieved LPS-induced oxidative stress through reducing MDA level and enhancing SOD and GSH levels. Finally, it was demonstrated that Tricin retarded LPS-activated AKT and MAPK pathways.Conclusion Our findings revealed that Tricin attenuated the progression of LPS induced severe pneumonia through modulating AKT and MAPK signaling pathways. This discovery might afford one novel sight for the treatment of severe pneumonia (AU)

Cross-regulation of notch/AKT and serum/glucocorticoid regulated kinase 1 (SGK1) in IL-4-stimulated human macrophages.

Notch signaling regulates the responses of macrophages to different stimuli in a context-dependent manner. The roles of Notch signaling in proinflammatory macrophages are well characterized, whereas its involvement, if any, in IL-4-stimulated macrophages (M(IL-4)) is still unclear. We observed that Notch signaling is functional in human M(IL-4). We performed transcriptome analysis of the Notch1 intracellular domain (NIC1)-overexpressing human monocytic cell line THP-1 with or without IL-4 stimulation to understand the global impact of Notch signaling in M(IL-4). The results revealed that NIC1-overexpressing THP-1 upregulated proinflammatory-associated genes and target genes of IL-4 signaling. We identified serum/glucocorticoid regulated kinase 1 (SGK1) as one of the genes increased by NIC1 overexpression in M(IL-4). To dissect the signaling pathway leading to SGK1 upregulation, we pretreated THP-1-derived macrophages with specific inhibitors of Notch (DAPT), AKT (LY294002) or ERK (U0126). Among these inhibitors, only LY294002 decreased the SGK1 mRNA levels in M(IL-4), indicating that the AKT pathway plays a key role in SGK1 transcription in M(IL-4). Furthermore, treatment of THP-1-derived macrophages with the SGK1 inhibitor (GSK650394) suppressed AKT phosphorylation, but not STAT6, in response to IL-4, indicating that SGK1 positively regulates AKT pathway in M(IL-4). Finally, GSK650394 treatment of human M(IL-4) increased the levels of PPARG mRNA and its protein, indicating a negative role of SGK1 in M(IL-4) function. Overall, we report that the Notch signaling and AKT pathways cooperatively regulate SGK1 expression in M(IL-4) where SGK1, in turn, plays an important role in suppressing IL-4-induced PPARγ expression.

MiRNA-494-3p Regulates Bupivacaine-Induced Neurotoxicity by the CDK6-PI3K/AKT Signaling.

Bupivacaine (BUP) is a long-acting amide local anesthetic that may induce strong neurotoxicity and neurological complications. In this study, we elucidate the influence of microRNA-494-3p (miR-494-3p) in BUP-induced neurotoxicity in primary mouse hippocampal neuronal cells. In this study, primary hippocampal neurons were isolated from neonatal C57BL/6 mice. The isolated neurons were treated with various doses of BUP. MTT assay was conducted to analyze neuronal viability. Gene expression measurement was done by RT-qPCR. The impact of miR-494-3p in BUP-mediated neural injury was examined using TUNEL, flow cytometry, western blotting, and ROS activity detection. The regulatory relationship between miR-494-3p and cyclin-dependent kinases 4 and 6 (CDK6) was identified using a luciferase reporter assay. BUP treatment led to neurotoxicity and miR-494-3p upregulation in primary cultured hippocampal neurons. Functionally, miR-494-3p depletion alleviated neuronal apoptosis and oxidative damage induced by BUP. We verified that miR-494-3p targeted and negatively modulated CDK6. MiR-494-3p depletion also activated PI3K/AKT signaling by elevating CDK6 expression in BUP-treated neurons. Furthermore, CDK6 knockdown or PI3K/AKT inactivation attenuated the neuroprotective role of miR-494-3p depletion. Silencing miR-494-3p exerts neuroprotective function in hippocampal neuronal cells against BUP-induced injury by the CDK6-PI3K/AKT pathway.

Bixin protects mice against bronchial asthma though modulating PI3K/Akt pathway.

Accumulating evidence has implicated the potential of natural compounds in treatment of asthma. Bixin is a natural food coloring isolated from the seeds of Bixa Orellana, which possesses anti-tumor, anti-inflammatory and antioxidative properties. Nevertheless, its therapeutic effect in asthma has not been elucidated. Our present study demonstrated that administration of Bixin suppressed allergic airway inflammation and reversed glucocorticoids resistance, as well as alleviated airway remodeling and airway hyperresponsiveness (AHR) in asthmatic mice. In vitro studies showed that Bixin treatment could inhibit the development of epithelial-mesenchymal transition (EMT) mediated by transforming growth factor beta (TGF-ß) signaling. Importantly, Bixin antagonized activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway both in vitro and in vivo. Above all, our findings reveal that Bixin functions as a potent antagonist of PI3K/Akt signaling to protect against allergic asthma, highlighting a novel strategy for asthma treatment based on natural products.

Senescence marker protein 30 (SMP30) protects against high glucose-induced apoptosis, oxidative stress and inflammatory response in retinal ganglion cells by enhancing Nrf2 activation via regulation of Akt/GSK-3ß pathway.

Senescence marker protein 30 (SMP30) is an aging-related protein that participates in the regulation of tissue damage under various pathological conditions. However, the role of SMP30 in mediating high glucose (HG)-induced injury of retinal ganglion cells (RGCs) has not been fully determined. We found that SMP30 expression declined during HG stimulation in RGCs. Cellular functional studies showed that the up-regulation of SMP30 dramatically prohibited HG-evoked apoptosis, oxidative stress and inflammatory response in RGCs. Mechanism research reported that SMP30 overexpression led to the enhancement of nuclear factor erythroid 2-related factor (Nrf2) activation in HG-stimulated RGCs. Moreover, SMP30 overexpression enhanced the phosphorylation of Akt and glucogen synthase kinase-3ß (GSK-3ß), and the suppression of Akt markedly abolished SMP30-mediated Nrf2 activation in HG-stimulated RGCs. Additionally, the suppression of Nrf2 substantially reversed SMP30-overexpression-induced anti-HG injury effects in RGCs. Overall, these findings suggest that SMP30 protects against HG injury of RGCs by potentiating Nrf2 through regulation of the Akt/GSK-3ß pathway. Our work underscores that SMP30/Akt/GSK-3ß/Nrf2 may exert a vital role in mediating the injury of RGCs during diabetic retinopathy.

Wild-Type KRAS Allele Effects on Druggable Targets in KRAS Mutant Lung Adenocarcinomas.

KRAS mutations are one of the most common oncogenic drivers in non-small cell lung cancer (NSCLC) and in lung adenocarcinomas in particular. Development of therapeutics targeting KRAS has been incredibly challenging, prompting indirect inhibition of downstream targets such as MEK and ERK. Such inhibitors, unfortunately, come with limited clinical efficacy, and therefore the demand for developing novel therapeutic strategies remains an urgent need for these patients. Exploring the influence of wild-type (WT) KRAS on druggable targets can uncover new vulnerabilities for the treatment of KRAS mutant lung adenocarcinomas. Using commercially available KRAS mutant lung adenocarcinoma cell lines, we explored the influence of WT KRAS on signaling networks and druggable targets. Expression and/or activation of 183 signaling proteins, most of which are targets of FDA-approved drugs, were captured by reverse-phase protein microarray (RPPA). Selected findings were validated on a cohort of 23 surgical biospecimens using the RPPA. Kinase-driven signatures associated with the presence of the KRAS WT allele were detected along the MAPK and AKT/mTOR signaling pathway and alterations of cell cycle regulators. FoxM1 emerged as a potential vulnerability of tumors retaining the KRAS WT allele both in cell lines and in the clinical samples. Our findings suggest that loss of WT KRAS impacts on signaling events and druggable targets in KRAS mutant lung adenocarcinomas.

Lactobacillus rhamnosus GG Promotes Early B Lineage Development and IgA Production in the Lamina Propria in Piglets.

Gut microbes play an important role in the development of host B cells. It has been controversial whether GALT is the development site of B cells in pigs. By investigating the relationship between gut microbes and the development of B cells in the GALT of piglets, we found, to our knowledge for the first time, that early B cells exist in the gut lamina propria (LP) in pigs at different ages. We further used Lactobacillus rhamnosus GG (LGG) to treat piglets. The results showed that LGG promotes the development of the early B lineage, affects the composition of the Ig CDR3 repertoires of B cells, and promotes the production of IgA in the intestinal LP. Additionally, we found that the p40 protein derived from LGG can activate the EGFR/AKT and NF-κB signaling pathways, inducing porcine intestinal epithelial cells (IPEC-J2) to secrete a proliferation-inducing ligand (APRIL), which promotes IgA production in B cells. Finally, we identified ARF4 and DIF3 as candidates for p40 receptors on IPEC-J2 by GST pull-down, liquid chromatography-mass spectrometry/mass spectrometry analysis, and coimmunoprecipitation. In conclusion, LGG could promote early B cell differentiation and development in the intestinal LP in piglets and might contribute to promoting IgA production via secretion of p40, which interacts with the membrane receptors on IPEC-J2 and induces them to secrete APRIL. Our study will provide insight to aid in better utilization of probiotics to increase human health.

Medical ozone induces proliferation and migration inhibition through ROS accumulation and PI3K/AKT/NF-kB suppression in human liver cancer cells in vitro

Background Hepatocellular carcinoma is one of the most common malignancies and leading cancer-associated deaths worldwide. Ozone has been proposed as a promising therapeutic agent in the treatment of various disorders. Purpose The purpose of this paper is to assess the potential anticancer effects of the ozone on liver cancer cells. Method The liver cancer cell line of bel7402 and SMMC7721 was used in this study. Proliferation was evaluated using the CCK-8 and the colony formation assay. Wond healing assay and transwell assay without Matrigel were used to evaluate their migration ability. Flow cytometry was used for cell cycle analysis and reactive oxygen species (ROS) determination. Glutathione detection kit was used for measurement of glutathione level. Protein expression was estimated by western blot analysis. Results Ozone treatment inhibited liver cancer cell proliferation, colony formation. Ozone induced G2/M phase cell cycle arrest, which could be elucidated by the change of protein levels of p53, p21, Cyclin D1, cyclin B1, cdc2, and CDK4. We also found that ozone treatment inhibited migration ability by inhibiting EMT-relating protein. Ozone also induced ROS accumulation and decreased glutathione level decreased, which contributed to the inactivation of the PI3K/AKT/NF-κB pathway. Finally, we found that pre-treatment of liver cancer cells with N-acetylcysteine resisted ozone-induced effects. Conclusions Ozone restrains the proliferation and migration potential and EMT process of liver cancer cells via ROS accumulation and PI3K/AKT/NF-κB suppression (AU)

(-)-Syringaresinol suppressed LPS-induced microglia activation via downregulation of NF-κB p65 signaling and interaction with ERß.

Albiziae Cortex (AC) is a well-known traditional Chinese medicine with sedative-hypnotic effects and neuroprotective ability. However, the bioactive components of AC responsible for the neuro-protective actitivity remain unknown. Here, we investigated the anti-neuroinflammatory effects of (-)-syringaresinol (SYR) extracted from AC in microglia cells and wild-type mice. As a result, (-)-SYR significantly reduced lipopolysaccharide (LPS)-induced production of interleukin - 6 (IL-6), tumor necrosis factor α (TNF-α), interleukin -1 beta (IL-1ß), cycloxygenase-2 (COX-2), and nitric oxide (NO) in BV2 microglia cells. (-)-SYR also significantly reduced M1 marker CD40 expression and increased M2 marker CD206 expression. Moreover, we found that (-)-SYR inhibited LPS-induced NF-κB activation by suppressing the translocation of NF-κB p65 into the nucleus in a concentration-dependent manner. Meanwhile, estrogen receptor ß (ERß) was found to be implied in the anti-inflammatory activity of (-)-SYR in BV2 microglia. In vivo experiments revealed that administration of (-)-SYR in mice significantly reduced microglia/astrocytes activation and mRNA levels of proinflammatory mediators. Taken together, our data indicated that (-)-SYR exerted the anti-neuroinflammatory effects by inhibiting NF-κB activation and modulation of microglia polarization, and via interaction with ERß. The anti-neuroinflammatory activity of (-)-SYR may provide a new therapeutic avenue for the treatment of brain diseases associated with inflammation.

Giardia duodenalis extracellular vesicles regulate the proinflammatory immune response in mouse macrophages in vitro via the MAPK, AKT and NF-κB pathways.

BACKGROUND: Giardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host's innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored. METHODS: In this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs. RESULTS: The results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1ß, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects. CONCLUSIONS: The results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis-host interactions.

Decorin-An Antagonist of TGF-ß in Astrocytes of the Optic Nerve.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-ß) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-ßs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-ß2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-ß1, TGF-ß2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-ß1, and TGF-ß2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-ß and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-ß and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

Novel RAF/MEK inhibitor CH5126766/VS-6766 has efficacy in combination with eribulin for the treatment of triple-negative breast cancer.

Various molecular-targeting drugs have markedly improved the treatment of patients with breast cancer. As yet, therapies for triple-negative breast cancer are mainly cytotoxic agents. To investigate the novel therapy for triple-negative breast cancer, we herein examined the effects of a new combination therapy comprising a RAF/MEK inhibitor CH5126766, also known as VS-6766, which we originally discovered, and eribulin. The combination of CH5126766 and eribulin potently inhibited cell growth in the triple-negative breast cancer cell lines tested. The underlying mechanism in the efficacy of this combination treatment in vitro and in vivo was due to enhanced apoptosis through the suppression of survivin and Bcl-2 family proteins. We also showed the suppressed expression of programmed cell death ligand 1 (PD-L1) in combination therapy in vivo. We found that combination therapy with eribulin and CH5126766 for triple-negative breast cancer inhibited cell growth by apoptosis and raised a possibility that immune responses through suppression of PD-L1 might partially contribute to inhibition of tumor growth, indicating the potential of this combination as a novel strategy for triple-negative breast cancer.

"Olfactory three-needle" acupuncture enhances synaptic function in Aß1-42-induced Alzheimer's disease via activating PI3K/AKT/GSK-3ß signaling pathway.

Synaptic dysfunction and neuronal loss are related to cognitive impairment of Alzheimer's disease. Recent evidence indicates that regulating the phosphatidylinositol 3-Kinase (PI3K)/AKT/GSK-3ß pathway is a therapeutic strategy for improving synaptic plasticity in Alzheimer's disease. Here, we investigated "olfactory three-needle" effects on synaptic function and the PI3K/AKT/GSK-3ß signaling pathway in ß-amyloid1-42 (Aß1-42)-induced Alzheimer's disease rats. A three-needle olfactory bulb insertion for 28 days alleviated Aß1-42-induced Alzheimer's disease rats' cognitive impairment as assessed by performance in the Morris water maze test. Furthermore, the three-needle electrode inhibited neuro-apoptosis and neuro-inflammation. It significantly upregulated the protein expression of postsynaptic density protein 95, synaptophysin, and GAP43, indicating a protective effect on hippocampal synaptic plasticity. Additionally, the activation level of PI3K/AKT signaling and the phosphorylation inactivation of GSK-3ß were significantly enhanced by the "olfactory three-needle". Our findings suggested that the three-needle acupuncture is a potential alternative to improve synaptic plasticity and neuronal survival of Alzheimer's disease brain in rodents.

Blockage of PAK1 alleviates the proliferation and invasion of NSCLC cells via inhibiting ERK and AKT signaling activity

Purpose P21-activated kinase 1 (PAK1), a serine/threonine protein kinase which functions downstream of RAC and CDC42 GTPase, is activated by a variety of stimuli, including RAS and other growth signaling factors. The extracellular signal kinase (ERK) and protein kinase B (AKT) signal pathways have been implicated in the pathogenesis of cancers. Whether PAK1 is sensitive to KRAS mutation signals and plays a role through ERK and AKT signaling pathways in NSCLC needs to be studied. Methods The expression of PAK1, ERK and AKT was detected in both lung cancer cell lines and clinical samples. PAK1 RNA interference and specific inhibitor of PAK1(IPA-3) were applied to lung cancer cell lines and mouse xenograft tumors. Cell growth was measured by MTT and colony formation assays. Cell migration and invasion were detected by wound healing and transwell assays. RAS mutation was detected by Taqman probe method. Correlation between KRAS, PAK1, ERK and AKT activities was analyzed in lung cancer patients. Results PAK1 was highly expressed not only in RAS mutant but also in RAS wild-type lung cancer cells. Using specific inhibitor of PAK1, IPA-3 and PAK1 RNA interference, cell proliferation, migration and invasion of lung cancer cells were reduced significantly, accompanied by decreased activities of ERK and AKT. Dual inhibition of ERK and AKT suppressed these cellular processes to levels comparable to those achieved by reduction in PAK1 expression. In NSCLC patients, PAK1 was not correlated with KRAS mutation but was significantly positively correlated with pERK and pAKT. Conclusion PAK1 played roles in NSCLC proliferation and invasion via ERK and AKT signaling and suggested a therapeutic target for NSCLC (AU)