Kinase mRNA Research Articles

Effects of dietary sodium butyrate on growth, diet conversion, body chemical compositions and distal intestinal health in yellow drum ( Nibea albiflora , Richardson)

The present study was conducted to investigate the effects of sodium butyrate (SB) on the physical barrier function, pro-inflammatory response and possible underlying mechanisms in the distal intestine (DI) of yellow drum when fed a high-SBM diet. Three iso-proteic and iso-lipidic diets were formulated with fish meal (FM, the control), 45% fish meal protein replaced by SBM (SBM) and 45% fish meal protein replaced by SBM but supplemented with 0.15% SB (SBM + SB). Fish were fed twice daily for 10 weeks. Results indicated that SB supplementation significantly increased the specific growth rate (SGR) and feed efficiency ratio (FER) and methionine content of muscle when compared with those of fish fed the SBM diet (p < .05). The morphologic histology results showed that SB dramatically improved the physical barrier structure, characterized as increases of fold height (FH) and muscular thickness (MT) (p < .05). RT-qPCR data were accordant with morphologic histology results, in which claudin 3, claudin 4 and occludin were increased while claudin 7 and myosin light chain kinase (MLCK) mRNA expression levels were decreased (p < .05). Sodium butyrate also exerted anti-inflammatory function, which may be attributed to the suppression of nucleus p65 protein expression. Results suggest SB can be incorporated in high-SBM diets to ameliorate the negative consequences of alternative dietary ingredients on yellow drum physiology.

Effects of thermal treatment on the physicochemical properties and osteogenic activity of lactoferrin

Lactoferrin as a key basic protein in bovine milk has attracted extensive attentions due to its multifunctional activities. Thermal treatment has been widely used during processing of dairy products, which might damage the bioactivity of heat-sensitive proteins. However, there has been little information on effect of heat treatment on the bioactivity of lactoferrin. In this study, the effects of four different thermal treatments (65°C/30 min, 72°C/10 s, 85°C/10 min, and 95°C/10 min) on lactoferrin were examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, circular dichroism spectrum, electron microscope, dynamic light scattering, and semiquantitative RT-PCR. Results revealed that these treatments altered its second structure and surface hydrophobicity. Moreover, the osteogenic activity of lactoferrin decreased by analyzing the effect of different samples on the mitogen-activated protein kinase mRNA expression. These findings provided theory data for the research and development of lactoferrin as a functional component in food industry. Practical applications As we know, thermal treatment has been widely used during processing of dairy products. Lactoferrin is a key basic protein component in bovine milk and has a variety of biological activities such as osteogenic activity. However, very few studies have focused on the effect of heat treatment on the osteogenic activity of lactoferrin, thereby limiting its applications. The objective of the present study was to investigate the effects of heating on the structure and osteogenic activity of lactoferrin. Results showed that the regular heat-processing parameters in dairy industry and structural change of lactoferrin led to the loss of its osteogenic activity. These results will provide some theoretical and practical data for the development of lactoferrin as ingredient in functional food industry.

Atezolizumab potentiates Tcell-mediated cytotoxicity and coordinates with FAK to suppress cell invasion and motility in PD-L1+ triple negative breast cancer cells

ABSTRACTImmune check point inhibitors targeting programmed cell death protein-1 (PD-1) and its ligand (PD-L1) have shown clinical success in treatment of human malignancies. Triple negative breast cancer (TNBC), which is primarily characterized by high heterogeneity and presence of tumor infiltrating lymphocytes, remains therapeutic challenge due to unavailability of approved targeted therapy. Therapeutic potential of immune check point inhibitors for TNBC patients is under active clinical investigation. In this study, we show that FDA-approved anti-PD-L1 antibody, atezolizumab (ATE), potentiates T cell-mediated cytotoxicity and apoptosis of TNBC cells that express higher levels of PD-L1, but does not have significant effect on TNBC cells expressing low levels of PD-L1. PD-L1 knockdown further confirmed that ability of ATE to promote T cell-induced cytotoxicity is PD-L1 expression dependent. Combination of ATE with PD-L1 upregulating agents, such as HDAC, proteasomal, and lysosomal inhibitors, further augmented cytotoxic activity of T cells toward TNBC cells. Based on analysis of breast cancer tissue samples deposited in The Cancer Genome Atlas (TCGA), we found a positive correlation between PD-L1 and focal adhesion kinase (FAK) mRNA expression in PD-L1-positive (PD-L1+) TNBC, suggesting a functional association of FAK and immune checkpoints. We further demonstrate that ATE dramatically downregulates phosphorylation status of FAK, an important regulator of cell invasion and migration, and significantly enhances FAK inhibitor mediated inhibition of cell motility and invasion of PD-L1+ TNBC cells independent of T cells. Taken together, our data suggest that ATE shows promising anti-tumor activity in PD-L1+ TNBC via both T cell-dependent and -independent mechanisms.

Effect of electroacupuncture on neurological deficit and activity of TLR2/NF-κB signaling in ce-rebral ischemia /reperfusion injury rats

To observe the effect of electroacupuncture (EA) on neurological behavior and activity of Toll-like receptor 2 / nuclear factor kappa B (TLR2/NF-κB) signaling of the ischemic cerebral area in cerebral ischemia-reperfusion injury (CIRI) rats, so as to explore its mechanisms underlying improvement of CIRI. A total of 120 male SD rats were randomly divided into blank control, sham operation, model, EA and EA＋NF-κB inhibitor (Pyrrolidine Dithiocarbamate Hydrochloride, PDTC, EA＋PDTC) groups which were further divided into 3, 7, 14 and 28 d subgroups (n＝6 in each subgroup). The CIRI model was established by occlusion of the middle cerebral artery for 90 min, followed by reperfusion. EA (1－20 Hz, 6 V) was applied to "Shuigou" (GV26), "Neiguan" (PC6), "Sanyinjiao" (SP6) and "Weizhong" (BL40) for 30 min, once a day for 28 days. For rats of the EA＋PDTC group, PDTC solution (120 mg/kg) was intraperitoneally injected on the 3rd day after successful modeling and before EA intervention. The neurological deficit severity (Zea Longa score) was assessed 3, 7, 14 and 28 days after modeling. The expression levels of TLR2, Interleukin-1 receptor-associated kinase (IRAK) and NF-κB mRNAs in the ischemic penumbra region of brain tissue were detected by real-time fluorescence quantitative PCR. Following modeling, the neurological deficit scores were significantly increased from the 3rd day on after CIRI (P<0.05), the expression levels of TLR2 mRNA on day 3, 7, 14 and 28, and IRAK mRNA on day 3 and 7, as well as NF-κB mRNA on day 3, 7 and 14 were significantly up-regulated in the model group relevant to the blank control group (P<0.05). After EA intervention, the neurological deficit scores were significantly decreased in the EA group on day 3, 7 and 28 and in the EA＋PDTC group on day 3, 7, 14 and 28 in comparison with those of the model group (P<0.05). In addition, the expression levels of TLR2 mRNA and NF-κB mRNA on day 3, 7 and 14 in the EA group, and on day 3, 7, 14 and 28 in the EA＋PDTC group, IRAK mRNA on day 3 in the EA and EA＋PDTC group were significantly down-regulated (P<0.05), but those of IRAK mRNA on day 14 and 28 in the EA group were significantly up-regulated in comparison with those of the model group (P<0.05). The effect of the EA＋PDTC was obviously superior to that of simple EA in down-regulating the expression of TLR2 (on day 28), and IRAK (on day 3, 14, 28), and NF-κB (on day 3, 7 and 14) (all P<0.05). EA stimulation can improve the symptoms of neurological deficits in CIRI rats, which may be related to its effect in suppressing the expression of TLR2, NF-κB and IRAK mRNAs of the ischemic cerebral tissue, i．e., down-regulating the activity of TLR2/NF-κB signaling.

Expression and activity of pyruvate kinase in bladder interstitial cells of cajal in rats with diabetic cystopathy

Objective To investigate the expression and activity of pyruvate kinase in interstitial cells of Cajal (ICCs) in the bladder of rats with diabetic cystopathy (DCP). Methods Forty 8-week-old female rats were divided into two groups randomly, 31 as the experiment group and the rest 9 as the control group. The experiment group was intraperitoneally injected with streptozotocin (STZ) to establish diabetic models and 18 rats were succeeded to be induced. Nine diabetic rats were further raised for 12 weeks getting DCP rats. ICCs from bladder muscle were cultured. The expression of pyruvate kinase protein and mRNA was detected by Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR) respectively, and pyruvate kinase activity was measured. Results The expression levels of pyruvate kinase protein and mRNA and activity in DCP rats were reduced significantly as compared with those of the diabetic rats and control rats [(0.537±0.015), (0.624±0.026), (0.098±0.010) U/g·prot respectively, vs. (0.722±0.026), (0.739±0.045), (0.178±0.017) U/g·prot, vs. (0.923±0.031), (1.000±0.022), (0.277±0.030) U/g·prot; P<0.01]. Conclusion The reduction of pyruvate kinase activity in ICCs may play an important role in the development of DCP. Key words: Diabetic cystopathy; Pyruvate kinase; Interstitial cells of cajal

Effects of Electroacupuncture on Expression of PI3K/Akt/Foxo3a in Granulosa Cells from Women with Shen (Kidney) Deficiency Syndrome Undergoing in vitro Fertilization-Embryo Transfer.

To observe the effects of electroacupuncture (EA) on reproductive outcomes in women with Shen (Kidndy) deficiency syndrome after in vitro fertilization-embryo transfer (IVF-ET), and explore the underlying molecular mechanism. Sixty-six infertile patients with Shen deficiency syndrome undergoing IVF-ET were divided into EA or control groups according to a random table, 33 cases in each group. Before undergoing IVF, patients in the EA and control groups received EA therapy and placebo needle puncture, respectively, for 3 menstrual cycles. Shen deficiency syndrome scores were assessed. Other outcome measures included the number of retrieved oocytes and fertilization, high-quality embryo and clinical pregnancy rates. Follicular fluid was collected on the day of oocyte retrieval, and granulosa cell expression of phosphatidylinositide 3-kinases (PI3K), serine-threonine kinase (Akt) and forkhead box O3 (Foxo3a) mRNA were measured by reverse transcribed and quantitative real-time polymerase chain reaction. Syndrome scores for pre- versus post-treatments decreased significantly (16.53±1.75 to 8.67±1.61) in the EA group (P<0.05), but showed no significant change in the control group (17.18±1.58 to 14.74±1.58). A significant difference in score change was found between the EA and control groups (P<0.05). High-quality embryo and clinical pregnancy rates were both increased in the EA group compared with the control group [69.15% (195/282) vs. 60.27% (176/292) and 66.67% (22/33) vs. 42.42% (14/33), respectively, P<0.05]. The fertilization rate was equivalent in EA and control groups. No difference was found in the number of retrieved oocytes between the two groups. Granulosa cell expression levels of PI3K and Akt mRNA were significantly increased in the EA group compared with the control group, while the expression of Foxo3a was reduced (all P<0.05). For infertile patients with Shen deficiency syndrome undergoing IVF, EA for tonifying Shen as an adjunct treatment may alleviate clinical symptoms and improve the high-quality embryo rate. The EA-induced mechanism may involve regulation of PI3K/Akt/Foxo3a expression in granulosa cells to improve the developmental microenvironment of oocytes and inhibit granulosa cell apoptosis, possibly contributing to the improved clinical pregnancy rate (Registration No. ChiCTR 1800016217).

The relationship between food exposure frequency and expression of AMP‐activated protein kinase (AMPK) mRNA in the brain, liver, and muscle of channel catfish

Obesity poses serious challenges to our healthcare system with serious consequences of associated diseases. Our laboratory has been examining how the expression of genes involved in food intake and tissue nutrient metabolism is associated with the development of obese‐like phenotypes in channel catfish. The objective of the study was to examine changes in expression of AMPK mRNA in response to changes in feeding frequency. Fish were fed every 12 (overfed), 24 (control), or 48 (underfed) hours (n= 4 tanks per treatment, 8 fish per tank) for 28 days. Brain, liver, and muscle tissue were collected at the end of the study. The expression of the AMPK mRNA was examined using quantitative RT‐PCR. The muscle expression of all AMPK subunit mRNA was unaffected by feeding frequency. In contrast, brain expression of alpha 1 and beta 1 subunit mRNA had a linear tendency for increase in response to increased feeding frequency. Hepatic expression of alpha 2 and beta 1 mRNA was lower in the overfeed group compared to those assigned as control or underfeed. Hepatic expression of alpha 1 mRNA decreased linearly in response to increased feeding frequency. Our results demonstrated that the hepatic and the brain expression of AMPK mRNA was influenced by increase in exposure to food in a transcript‐specific manner. Given that the AMPK serves as a regulator of tissue nutrient sensing and metabolism in mammals, it is possible that AMPK may share a similar function in the brain and liver but not in the muscle of channel catfish.Support or Funding InformationKansas IDeA Network of Biomedical Research Excellence Network (NIH/NIGMS grant number P20GM103418)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

THE RELATIONSHIP OF THE BRAF-V600E MUTATION WITH THE EXPRESSION OF TRANSCRIPTIONAL, GROWTH FACTORS, COMPONENTS OF THE AKT / M-TOR SIGNALING PATHWAY IN THE TISSUE OF PAPILLARY THYROID CANCER

One of the initiating mutations in the development of thyroid cancer is BRAF-V600E, which leads to the various signaling cascades activation and changes in the production of transcription and growth factors. It is known that papillary thyroid cancer is characterized by activation of the expression of transcription factors NF-kB and HIF-2a and AKT/m-TOR signaling cascade. However, the relationship of the studied molecular markers in patients with the wild and mutant BRAF gene has not yet been studied. The aim of the work was to study the expression of transcription factors NF-65B p65 and NF-kB p50, HIF-1a, HIF-2a, growth factors VEGF, CAIX and VEGFR2, components of the AKT/m-TOR signaling pathway in patients with thyroid papillary cancer depending on the presence of mutations BRAF-V600E. Material and methods. It was included 40 patients with papillary thyroid cancer with the stage of the tumor process T1-4N0-2M0. The expression of the indicators was determined by real-time PCR. BRAF-V600E mutation was revealed by allele-specific PCR in real-time. Results and discussion. It was found that patients with the absence and presence of the BRAF-V600E mutation had similar clinical and morphological parameters of the disease, which was accompanied by a change in the molecular-biological characteristics of the tumor. The presence of the mutant form of the gene in the tumor led to a decrease in the AKT, cRAF, GSK-3P kinases mRNA levels and the overexpression of NF-kB p65, HIF-1 and VEGF. Conclusion. Patients with papillary thyroid cancer have no differences in the clinical and morphological characteristics of the disease, depending on the status of the BRAF-V600E gene, is characteristic. It was identified biological parameters associated with this somatic mutation. An increase in the mRNA level of growth and transcription factors was observed with a decrease in the activity of the AKT / m-TOR signaling pathway.

Sirtuin-mediated deacetylation of hnRNP A1 suppresses glycolysis and growth in hepatocellular carcinoma.

Tumor cells undergo a metabolic shift in order to adapt to the altered microenvironment, although the underlying mechanisms have not been fully explored. HnRNP A1 is involved in the alternative splicing of the pyruvate kinase (PK) mRNA, allowing tumor cells to specifically produce the PKM2 isoform. We found that the acetylation status of hnRNP A1 in hepatocellular carcinoma (HCC) cells was dependent on glucose availability, which affected the PKM2-dependent glycolytic pathway. In the glucose-starved HCC cells, SIRT1 and SIRT6, members of deacetylase sirtuin family, were highly expressed and deacetylated hnRNP A1 after direct binding. We identified four lysine residues in hnRNP A1 that were deacetylated by SIRT1 and SIRT6, resulting in significant inhibition of glycolysis in HCC cells. Deacetylated hnRNP A1 reduced PKM2 and increased PKM1 alternative splicing in HCC cells under normal glucose conditions, thereby reducing the metabolic activity of PK and the non-metabolic PKM2-β-catenin signaling pathway. However, under glucose starvation, the low levels of acetylated hnRNP A1 reduced HCC cell metabolism to adapt to the nutrient deficiency. Taken together, sirtuin-mediated hnRNP A1 deacetylation inhibits HCC cell proliferation and tumorigenesis in a PKM2-dependent manner. These findings point to the metabolic reprogramming induced by hnRNP A1 acetylation in order to adapt to the nutritional status of the tumor microenvironment.

Prognostic values of inhibitory κB kinases mRNA expression in human gastric cancer.

Background: Inhibitory κB kinases (IKKs) play a key role in modulating proinflammatory and growth stimulating signals through their regulation of the nuclear factor κB (NF-κB) cascade. Therefore, the level of expression of IKKs represents a viable prognostic predictor with regard to various pathological processes. The prognostic value of IKKs expression in gastric cancer remains unclear. Methods: We used the ‘Kaplan–Meier plotter’ (KM plotter) online database, to explore the predictive prognostic value of individual IKKs members’ mRNA expression to overall survival (OS) in different clinical data including pathological staging, histology, and therapies employed. Results: Our results revealed that a higher mRNA expression of inhibitor of NF-κB kinase subunit α (IKKα) was correlated to better OS, whereas higher mRNA expression of IKKβ, inhibitor of NF-κB kinase subunit γ (IKKγ), inhibitor of NF-κB kinase subunit ε (IKKε), and suppressor of IKKε (SIKE) were generally correlated to unfavorable OS in gastric cancer. Increased mRNA expression of IKKε also showed better outcomes in stage IV gastric cancer. Further a correlation between elevated levels of mRNA expression of both IKKε and SIKE was found to have favorable OS in diffuse type gastric cancer. It was also revealed that high expression of SIKE had favorable OS when treated with other adjuvant therapies, while worse OS when treated only with 5FU therapy. Conclusion: Our results suggest that mRNA expression of individual IKKs and SIKE are associated with unique prognostic significance and may act as valuable prognostic biomarkers and potential targets for future therapeutic interventions in gastric cancer.

Effects of spermine on liver barrier function, amino acid transporters, immune status, and apoptosis in piglets.

This study investigated the effects of spermine supplementation and its extended duration on amino acid transporters, immune status, barrier function, and apoptosis in the liver. Eighty piglets were randomly assigned to a group receiving either a diet supplemented with spermine (0.4 mmol kg−1 of body weight) or a restricted nutrient intake supplemented with saline in pairs for 7 h, 3 days, 6 days, and 9 days. Regardless of treatment time, spermine increased the levels of amino acid transporters, immunoglobulin M, antimicrobial peptides, cellular immune components, anti-inflammatory cytokines, mammalian target of rapamycin, ribosomal protein S6 kinase 1, signal transducer and activator of transcription 2 and 3, Janus kinase 2, zonula occludens 1 and 2, occluding, claudin-1, claudin-2, claudin-16 and Bcl-2 mRNA levels, whereas it decreased the levels of pro-inflammatory cytokines, inducible nitric oxide synthase, nuclear factor-kappa B P65, eukaryotic IF4E-binding protein 1, myosin light chain kinase, Bax, and caspase-3 mRNA in the liver (P < 0.05). These effects were also found in cases of prolonged spermine intake (P < 0.05). Spermine can decrease pro-inflammatory cytokines and caspase-3 levels. In conclusion, spermine may promote barrier function and improve amino acid transport, and can increase immune status and inhibit apoptosis in the liver.

Ammonia-N exposure alters neurohormone levels in the hemolymph and mRNA abundance of neurohormone receptors and associated downstream factors in the gills of Litopenaeus vannamei.

Effects of ammonia-N (0.05, 2, 10 and 20 mg l-1) on the neuroendocrine regulation of ammonia transport were investigated in Litopenaeus vannamei The results showed that corticotrophin-releasing hormone, adrenocorticotropic hormone, dopamine, noradrenaline and 5-hydroxytryptamine concentrations in all ammonia-N groups increased significantly between 3 and 12 h. Cortisol increased significantly between 3 and 24 h. All hormones except crustacean hyperglycemic hormone were reduced to control levels. mRNA abundance of guanylyl cyclase increased significantly during the experiment. Dopamine receptor D4 and α2 adrenergic receptor mRNA abundance in treatments decreased significantly at the beginning, and eventually returned to the control level, whereas mRNA abundance of the 5-HT7 receptor increased significantly only within the first 12 h. Changes in protein kinase (PKA, PKG) mRNA abundance were similar to the patterns of biogenic amines and crustacean hyperglycemic hormone, peaking at 6 and 12 h, respectively, whereas PKC mRNA abundance decreased within 24 h. 14-3-3 protein, FXYD2 and cAMP-response element binding protein mRNA abundance increased significantly and peaked at 6 h. β-catenin and T-cell factor mRNA abundance increased significantly throughout the experiment and peaked at 12 h. The upregulation of Rh protein, K+ channel, Na+/K+-ATPase, V-type H+-ATPase and vesicle associated membrane protein (VAMP) mRNA, together with downregulation of Na+/K+/2Cl- cotransporter mRNA, indicated an adjustment of general branchial ion-/ammonia-regulatory mechanisms. Meanwhile, hemolymph ammonia concentration was significantly increased in most ammonia-N exposure groups. Histological investigation revealed the hepatopancreatic damage caused by ammonia-N. Results suggest that hormones, biogenic amines and Wnt/β-catenin play a principal role in adapting to ammonia-N exposure and facilitating ammonia transport.

IL-1β enhances cell adhesion through laminin 5 and β4 integrin in gingival epithelial cells.

The junctional epithelium and dental enamel adhere because of hemidesmosomes containing laminin 5 and α6β4 integrin, which are important adhesion molecules in the internal basal lamina. Interleukin (IL)-1 is important in the pathogenesis of periodontal disease. IL-1β induces bone resorption by activating osteoclasts; however, its effects on adhesion of epithelial cells remain to be clarified. Laminin β3, β4 integrin, and focal adhesion kinase mRNA levels were higher after 1 h and 3 h of stimulation with IL-1β (1 ng/mL), and IL-1β, type I α1, and type IV α1 collagen mRNA levels were higher after 1 h and lower after 3 h of stimulation with IL-1β. After IL-1β stimulation, colocalization of laminin 5 and β4 integrin was increased after 1 h, colocalization of β4 integrin and plectin was increased after 1 h and decreased after 3 h, and colocalization of β4 integrin and type IV collagen was decreased after 3 h. Wound healing assays showed that IL-1β treatment (3 h) delayed wound healing. These results suggest that IL-1β enhances cell adhesion by altering localization of epithelial adhesion molecules.

Inula Japonica Thunb. Flower Ethanol Extract Improves Obesity and Exercise Endurance in Mice Fed A High-Fat Diet.

Inula japonica Thunb. (Asteraceae) is a flowering plant that grows mainly in Korea, Japan, and China and its flower extract has diverse biological effects such as anti-inflammatory and antioxidative activities. However, the effects on obesity and enhancement of endurance capacity have not been explored yet. This study aims to reveal the effects of I. japonica flower ethanol extract (IJE) on obesity and endurance capacity in high-fat diet (HFD) fed C57BL/6J mice and the mechanism. IJE inhibited lipid accumulation in 3T3-L1 adipocytes in vitro. Also, IJE-fed mice showed reduced body weight gain, hepatic lipid, and body fat mass, and increased muscle weight. IJE reduced lipid accumulation in the liver and adipose tissue by decreasing lipogenic and adipogenic gene expression. Additionally, consumption of low-dose IJE significantly enhanced endurance capacity via increasing AMP-activated protein kinase activity and mRNA levels of Myh7 and Myh2. Luteolin and 1β-hydroxyalantolactone (1β-HA), compounds of IJE, are involved in anti-adipogenesis in the 3T3-L cells and only luteolin increased the protein levels of MHC during C2C12 myoblast differentiation. Collectively, our results suggest that consumption of IJE not only helps to prevent obesity but also enhances endurance capacity reduced by HFD.

HepaCAM Regulates Warburg Effect of Renal Cell Carcinoma via HIF-1α/NF-κB Signaling Pathway

ObjectiveTo investigate how hepatocyte cell adhesion molecule (hepaCAM) regulates cancer energy metabolism through hypoxia-inducible factor (HIF-1α) in renal cell carcinoma (RCC). Materials and MethodsThe expression of hepaCAM and HIF-1α in RCC tissue samples was examined by immunohistochemistry. Glucose consumption and lactate production assays were used to detect metabolic activity in RCC cell lines. P65 and IκB kinase (IKKβ) mRNA and protein expression were detected using quantitative real-time polymerase chain reaction and western blotting, respectively. Nuclear translocation of P65 was observed by immunofluorescence staining after re-expressing hepaCAM. The luciferase reporter assay was applied to validate the transcriptional activity of HIF-1α. ResultsHIF-1α expression was elevated and hepaCAM suppressed in RCC compared with adjacent normal tissues. Furthermore, hepaCAM re-expression significantly decreased glycolytic metabolism in RCC cell lines, and reduced HIF-1α, IKKβ, and P65 expression. The expression of HIF-1α, GLUT1, LDHA, and PKM2 were further reduced with combined hepaCAM overexpression and treatment with the NF-κB inhibitor BAY11-7082, compared to hepaCAM overexpression alone. Additionally, hepaCAM decreased the transcriptional activity of HIF-1α and blocked P65 nuclear translocation by the NF-κB pathway. ConclusionOur data suggest that hepaCAM suppresses the Warburg effect via the HIF-1α/NF-κB pathway in RCC, which is a facilitating factor in hepaCAM-reduced tumorigenesis.

Atorvastatin improves left ventricular remodeling and cardiac function in rats with congestive heart failure by inhibiting RhoA/Rho kinase-mediated endothelial nitric oxide synthase.

The aim of the present study was to investigate the effects and possible mechanisms of atorvastatin (Ato) against chronic heart failure (CHF). A rat model of CHF was established and cardiac functions were assessed using Echocardiography. The expression of RhoA/Rho kinase and endothelial nitric oxide synthase (eNOS) was assessed using western blotting and reverse transcription polymerase chain reaction following 4 weeks of treatment. The three groups assessed in the present study were as follows: The control group (no treatment), the Ato + isopropylnoradrenaline (ISO) group (subcutaneous injections of 340 mg/kg ISO + orally administered 50 mg/kg Ato dissolved in saline; administered once daily) and the ISO group (subcutaneous injections of 340 mg/kg ISO + orally administered with an equal volume of saline; administered once daily). Heart volume and weight in the ISO group were significantly increased compared with the control (C) group (P<0.01), whereas contractility was decreased. The results were reverse for the Ato group when compared with the ISO group (P<0.05). Levels of RhoA/Rho kinase protein and mRNA were significantly increased in the ISO group (P<0.01); however. The mRNA and protein expression of eNOS was significantly decreased (P<0.05) when compared with the C group. The mRNA and protein expression of RhoA/Rho kinase was significantly reduced in the Ato+ISO group compared with the ISO group (P<0.01), whereas the mRNA and protein expression of eNOS was significantly increased (P<0.05). RhoA protein expression was increased in the cytoplasm of the C group and on the cell membrane of the ISO group; however, in the Ato+ISO group, RhoA protein expression on the cell membrane was significantly downregulated when compared with the ISO group (P<0.05). The results of the present study suggest that Ato upregulates eNOS by inhibiting RhoA/Rho kinase overexpression in the myocardial tissue of rats with CHF, thus improving left ventricular remodeling and cardiac function.

Polarity Acquisition in Cortical Neurons Is Driven by Synergistic Action of Sox9-Regulated Wwp1 and Wwp2 E3 Ubiquitin Ligases and Intronic miR-140

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.

Ghrelin Receptor Is Required for the Effect of Nesfatin-1 on Glucose Metabolism.

Studies of nesfatin-1 in glucose metabolism have become a topic of interest recently, however, the specific receptor for nesfatin-1 has not yet been identified. Some studies hinted at a connection between nesfatin-1 and the ghrelin receptor, growth hormone secretagogue receptor. Therefore, we aimed to study the role of GHSR in the glycemic effects of nesfatin-1 as well as its downstream pathways. We employed C57/BL6 mice (wild type and GHSR knockout mice) eating a normal chow diet and a high fat diet in this study, and the experimental technique included western blot, real-time PCR, immunofluorescence and ELISA. We found that in mice fed a normal chow diet (NCD), nesfatin-1 improved glucose tolerance, up-regulated AKT kinase (AKT) mRNA levels and phosphorylation and GLUT4 membrane translocation in skeletal muscle. These effects were blocked by co-injection of GHSR antagonist [D-Lys3]-GHRP-6 and were attenuated in GHSR knockout mice. In mice fed high-fat diet (HFD), nesfatin-1 not only exerted the effects observed in NCD mice, but also suppressed appetite and raised AKT levels in liver tissues that also required GHSR. Peripheral nesfatin-1 suppressed c-fos expression of GHSR immunoreactive neurons induced by fasting in hypothalamic nuclei, indicating that nesfatin-1 inhibited the activation of central GHSR. We concluded that the effects of nesfatin-1 on food intake and glucose metabolism were GHSR-dependent, and that the glycemic effect was associated with AKT and GLUT4. This study should stimulate further exploration of the nesfatin-1 receptor.

Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice

Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. Mice with disruption of Tmem173 (STINGgt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STINGgt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STINGgt mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STINGgt mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STINGgt, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STINGgt macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages. Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

The Effect of Prim-O-Glucosylcimifugin on Tryptase-Induced Intestinal Barrier Dysfunction in Caco-2 Cells.

The intestinal barrier dysfunction is a critical pathological change in irritable bowel syndrome (IBS). The objective of this study was to evaluate the effect of Prim-O-glucosylcimifugin (POG) on intestinal barrier dysfunction and reveal possible molecular mechanisms. Human colon adenocarcinoma cell line (Caco-2) cell monolayers induced by tryptase (TRYP) were used to establish an intestinal barrier dysfunction model. Caco-2 cell monolayers from both functional and dysfunctional samples were treated with POG (30, 60 and 120 µg/mL) for 2, 8, 24, 36, 48 and 72 h. The Caco-2 cell monolayers were assessed by measurement of trans-epithelial electrical resistance (TEER) and percentage of fluorescein permeation (PFP). The expression of Protease Activated Receptor 2 (PAR-2) and myosin light chain kinase (MLCK) mRNA was analyzed by RT-PCR and the level of Zonula Occludens-1 (ZO-1) protein expression was determined by Western blot. In addition, the impact of POG on the distribution of the tight juction protein of Occludin was performed by immunofluorescence. Our results showed that POG elevated the TEER and decreased the PFP of the functional Caco-2 cell monolayers, as well as the dysfunctional Caco-2 cell monolayers. Furthermore, POG inhibited the expression of PAR-2 mRNA and MLCK mRNA and increased the level of ZO-1 protein expression in dysfunctional Caco-2 cells. The distribution of the Occludin proteins was ameliorated simultaneously. This study demonstrates that POG can enhance the intestinal barrier function of Caco-2 cell monolayers by inhibiting the expression of PAR-2 and MLCK and up-regulating the expression of ZO-1 protein, and ameliorated the distribution of Occludin protein.