OBJECTIVETo investigate the host-defense role of short palate, lung, and nasal epithelium clone 1 (SPLUNC1) in Streptococcus pneumoniae (SP) infection and the effect of resveratrol (Res) on SPLUNC1 expression, and to provide new thoughts for the treatment of diseases caused by SP infection.

METHODSAccording to the multiplicity of infection (MOI), BEAS-2B cells with SP infection were divided into control group, MOI20 SP group, and MOI50 SP group. According to the different concentrations of Res, the BEAS-2B cells with MOI20 SP infection pretreated by Res were divided into 12.5Res+SP group, 25Res+SP group, and 50Res+SP group (the final concentrations of Res were 12.5, 25, and 50 μmol/L, respectively). Cell Counting Kit-8 was used to measure cell activity and determine the optimal concentration and action time of SP and Res. In the formal experiment, the cells were divided into control group, Res group, SP group, and Res+SP group. Real-time PCR and ELISA were used to measure the mRNA and protein expression of SPLUNC1.

RESULTSOver the time of SP infection, cell activity tended to decrease. Compared with the control group and the MOI20 SP group, the MOI50 SP group had a reduction in cell activity. Compared with the MOI20 SP group, the 25Res+SP group had increased cell activity and the 50Res+SP group had reduced cell activity (P<0.05). MOI20 SP bacterial suspension and 25 μmol/L Res were used for the formal experiment. Over the time of SP infection, the mRNA expression of SPLUNC1 in BEAS-2B cells firstly increased and then decreased in the SP group and the Res+SP group (P<0.05). Compared with the SP group, the Res+SP group had significant increases in the mRNA and protein expression of SPLUNC1 at all time points (P<0.05). Compared with the control group, the Res group had no significant changes in the mRNA and protein expression of SPLUNC1 (P>0.05).

CONCLUSIONSSP infection can induce SPLUNC1 expression and the host-defense role of SPLUNC1. Res can upregulate SPLUNC1 expression during the development of infection and enhance cell protection in a concentration- and time-dependent manner.

Bronchi ; metabolism ; Cells, Cultured ; Cytoprotection ; Epithelial Cells ; metabolism ; Glycoproteins ; analysis ; genetics ; physiology ; Humans ; Phosphoproteins ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Stilbenes ; pharmacology ; Streptococcus pneumoniae ; pathogenicity

OBJECTIVETo study the expression and significance of the mammalian target of rapamycin (mTOR)/eukaryote initiating factor 4E binding protein 1(4EBP1)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway in asthmatic mice.

METHODSForty SPF level 6-8 week-old female Balb/C mice were randomly divided into control, asthma, budesonide and mTOR inhibitor (rapamycin) intervention groups (n=10 each). The asthmatic mouse model was prepared via OVA induction and challenge test. The intervention groups were administered with rapamycin at the dosage of 3 mg/kg by an intraperitoneal injection or budesonide suspension at the dosage of l mg by aerosol inhalation respectively 30 minutes before the OVA challenge. The control and asthma groups were treated with normal saline instead. The concentrations of HIF-1α and VEGF in bronchoalveolar lavage fluid (BALF) were examined using ELISA 24 hours after the last challenge. The pathological changes of lung tissue were observed by hematoxylin-eosin (HE) staining. The p-mTOR and p-4EBP1 from the lung tissues were detected by immunohistochemistry and Western blot. Pearson analysis was used to study the correlation between p-mTOR, p-4EBP1, HIF-1α, and VEGF expression.

RESULTSCompared with the control group, inflammatory cell infiltration and secretions in the trachea increased in the asthma group. The levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues also increased (P<0.01). Compared with the asthma group, inflammatory cell infiltration and secretions in the trachea were reduced in the two intervention groups, and the levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues were also reduced (P<0.01). There were no significant differences in the above changes between the two intervention groups and control group (P>0.05). In the asthma group, there was a pairwise positive correlation between lung p-mTOR and p-4EBP1 expression and HIF-1α and VEGF levels in BALF (P<0.05). However, there were no correlations in the above indexes in the intervention groups and control group.

CONCLUSIONSp-mTOR, p-4EBP1, HIF-1α and VEGF together are involved in the pathogenesis of asthma. Rapamycin treatment can block this signaling pathway, suggesting that this pathway can be used as a novel target for asthma treatment.

Animals ; Asthma ; drug therapy ; metabolism ; Carrier Proteins ; analysis ; physiology ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; physiology ; Lung ; chemistry ; pathology ; Mice ; Mice, Inbred BALB C ; Phosphoproteins ; analysis ; physiology ; Signal Transduction ; physiology ; TOR Serine-Threonine Kinases ; analysis ; physiology ; Vascular Endothelial Growth Factor A ; analysis ; physiology

While several organs in mammals retain partial regenerative capability following tissue damage, the underlying mechanisms remain unclear. Recently, the Hippo signaling pathway, better known for its function in organ size control, has been shown to play a pivotal role in regulating tissue homeostasis and regeneration. Upon tissue injury, the activity of YAP, the major effector of the Hippo pathway, is transiently induced, which in turn promotes expansion of tissue-resident progenitors and facilitates tissue regeneration. In this review, with a general focus on the Hippo pathway, we will discuss its major components, functions in stem cell biology, involvement in tissue regeneration in different organs, and potential strategies for developing Hippo pathway-targeted regenerative medicines.
Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Homeostasis ; physiology ; Humans ; Phosphoproteins ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Regeneration ; physiology ; Signal Transduction ; physiology

Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.
Animals ; Chick Embryo ; Chickens ; Down-Regulation ; Fibroblasts ; virology ; Gene Targeting ; Lentivirus ; genetics ; metabolism ; Newcastle Disease ; virology ; Newcastle disease virus ; genetics ; physiology ; Phosphoproteins ; genetics ; metabolism ; Poultry Diseases ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology

PURPOSE: Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes. MATERIALS AND METHODS: Testes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed. RESULTS: Foxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter. CONCLUSION: FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function.
Animals ; Cell Aging/*physiology ; Cell Nucleus/metabolism ; Cytoplasm/metabolism ; Forkhead Transcription Factors/*metabolism ; HEK293 Cells ; Humans ; Leydig Cells/*drug effects/*enzymology/metabolism ; Luteinizing Hormone/blood ; Male ; Mice ; Phosphatidylinositol 3-Kinases ; Phosphoproteins/metabolism ; Phosphorylation ; Signal Transduction/drug effects ; Testosterone/blood/*metabolism

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 μmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 μmol·L(-1)), significantly blocked SCU (10-1 000 μmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 μmol·L(-1)), did not significantly change the effects of SCU (10-1 000 μmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 μmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 μmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.
Animals ; Apigenin ; pharmacology ; Cell Adhesion Molecules ; drug effects ; Cell Hypoxia ; Coronary Vessels ; drug effects ; Cyclic GMP ; analogs & derivatives ; metabolism ; pharmacology ; Cyclic GMP-Dependent Protein Kinases ; Glucuronates ; pharmacology ; Microfilament Proteins ; drug effects ; NG-Nitroarginine Methyl Ester ; metabolism ; pharmacology ; Phosphoproteins ; drug effects ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; physiopathology ; Signal Transduction ; drug effects ; Thionucleotides ; metabolism ; pharmacology ; Vasodilation ; drug effects ; physiology

Hippo signaling plays a crucial role in growth control and tumor suppression by regulating cell proliferation, apoptosis, and differentiation. How Hippo signaling is regulated has been under extensive investigation. Over the past three years, an increasing amount of data have supported a model of actin cytoskeleton blocking Hippo signaling activity to allow nuclear accumulation of a downstream effector, Yki/Yap/Taz. On the other hand, Hippo signaling negatively regulates actin cytoskeleton organization. This review provides insight on the mutual regulatory mechanisms between Hippo signaling and actin cytoskeleton for a tight control of cell behaviors during animal development, and points out outstanding questions for further investigations.
Actin Cytoskeleton ; physiology ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Cell Proliferation ; Drosophila Proteins ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.
3T3-L1 Cells ; AMP-Activated Protein Kinases/metabolism ; Adipocytes/*drug effects/physiology ; Adipogenesis/drug effects ; Animals ; Anti-Obesity Agents/*pharmacology/therapeutic use ; Antigens, Differentiation/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; Cell Size ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Enzyme Activation ; Gene Expression Regulation, Enzymologic/drug effects ; Glycogen Synthase Kinase 3/genetics/metabolism ; Lipase/genetics/metabolism ; Lipolysis/drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Obesity/etiology/metabolism/*prevention & control ; Phosphoproteins/genetics/metabolism ; Phosphorylation ; Propylene Glycols/*pharmacology/therapeutic use ; Protein Processing, Post-Translational ; Proto-Oncogene Proteins c-akt/metabolism ; Sphingosine/*analogs & derivatives/pharmacology/therapeutic use ; Sterol Esterase/metabolism

Early diabetic nephropathy is characterized by glomerular hyperpermeability as a result of impaired glomerular filtration structure caused by hyperglycemia, glycated proteins or irreversible advanced glycosylation endproducts (AGE). To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte ZO (zonula occludens)-1 protein and podocyte permeability induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, = control); 2) high glucose (HG, 30 mM); 3) AGE-added; or 4) HG plus AGE-added conditions and treated with GTS. HG and AGE increased the dextran filtration of monolayered podocytes at early stage (2-8 hr) in permeability assay. In confocal imaging, ZO-1 colocalized with actin filaments and beta-catenin at cell contact areas, forming intercellular filtration gaps. However, these diabetic conditions suppressed ZO-1 immunostainings and disrupted the linearity of ZO-1. In Western blotting, diabetic conditions also decreased cellular ZO-1 protein levels at 6 hr and 24 hr. GTS improved such quantitative and qualitative changes. These findings imply that HG and AGE have an influence on the redistribution and amount of ZO-1 protein of podocytes thereby causing hyperpermeability at early stage, which can be reversed by GTS.
Actin Cytoskeleton/metabolism ; Animals ; Cell Line ; Diabetic Nephropathies/physiopathology ; Glomerular Filtration Rate ; Glucose/*pharmacology ; Glycosylation End Products, Advanced/*pharmacology ; Hyperglycemia/physiopathology ; Membrane Proteins/*metabolism ; Mice ; *Panax ; Permeability/drug effects ; Phosphoproteins/*metabolism ; Plant Preparations/*pharmacology ; Podocytes/drug effects/pathology/physiology ; Saponins/*pharmacology ; beta Catenin/metabolism