BACKGROUNDSurfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.

METHODSTetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.

RESULTSHK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.

CONCLUSIONSThe present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.

Cell Line ; Cell Survival ; drug effects ; physiology ; Colorimetry ; Humans ; Kidney ; cytology ; metabolism ; Lipopolysaccharides ; toxicity ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Sulfonamides ; pharmacology ; Tetrazolium Salts ; chemistry ; Toll-Like Receptor 4 ; antagonists & inhibitors ; metabolism

BACKGROUNDVulvovaginal candidiasis is caused by Candida albicans. The vaginal epithelium, as the first site of the initial stage of infection by pathogens, plays an important role in resisting genital tract infections. Moreover, lactobacilli are predominant members of the vaginal microbiota that help to maintain a normal vaginal microenvironment. Therefore, Lactobacillus crispatus was explored for its capacity to intervene in the immune response of vaginal epithelial cells VK2/E6E7 to C. albicans.

METHODSWe examined the interleukin-2 (IL-2), 4, 6, 8, and 17 produced by VK2/E6E7 cells infected with C. albicans and treated with L. crispatus in vitro. The capacity of L. crispatus to adhere to VK2/E6E7 and inhibit C. albicans growth was also tested by scanning electron microscopy (SEM) and adhesion experiments.

RESULTSCompared with group VK2/E6E7 with C. albicans, when treated with L. crispatus, the adhesion of C. albicans to VK2/E6E7 cells decreased significantly by 52.87 ± 1.22%, 47.03 ± 1.35%, and 42.20 ± 1.55% under competition, exclusion, and displacement conditions, respectively. SEM revealed that the invasion of C. albicans into VK2/E6E7 cells was caused by induced endocytosis and active penetration. L. crispatus could effectively protect the cells from the virulence of hyphae and spores of C. albicans and enhance the local immune function of the VK2/E6E7 cells. The concentrations of IL-2, 6, and 17 were upregulated significantly (P < 0.01) and that of IL-8 were downregulated significantly (P < 0.01) in infected VK2/E6E7 cells treated with L. crispatus. The concentration of IL-4 was similar to that of the group VK2/E6E7 with C. albicans (24.10 ± 0.97 vs. 23.12 ± 0.76 pg/ml, P = 0.221).

CONCLUSIONSL. crispatus can attenuate the virulence of C. albicans, modulate the secretion of cytokines and chemokines, and enhance the immune response of VK2/E6E7 cells in vitro. The vaginal mucosa has a potential function in the local immune responses against pathogens that can be promoted by L. crispatus.

Candida albicans ; pathogenicity ; Cell Line, Tumor ; Epithelial Cells ; immunology ; metabolism ; microbiology ; ultrastructure ; Female ; Humans ; Interleukin-17 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Lactobacillus crispatus ; physiology ; Microscopy, Electron, Scanning ; Vagina ; cytology

To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates.AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC).On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all<0.01).Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.
Albumins ; biosynthesis ; metabolism ; Alginates ; Ammonia ; metabolism ; Animals ; Cell Aggregation ; physiology ; Cell Culture Techniques ; methods ; Cell Line, Transformed ; physiology ; Chitosan ; Diazepam ; metabolism ; Glucuronic Acid ; Hep G2 Cells ; cytology ; physiology ; Hepatocytes ; cytology ; physiology ; Hexuronic Acids ; Humans ; Liver, Artificial ; Rotation

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

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation

Cementum is critical for anchoring the insertion of periodontal ligament fibers to the tooth root. Several aspects of cementogenesis remain unclear, including differences between acellular cementum and cellular cementum, and between cementum and bone. Biomineralization is regulated by the ratio of inorganic phosphate (Pi) to mineral inhibitor pyrophosphate (PPi), where local Pi and PPi concentrations are controlled by phosphatases including tissue-nonspecific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). The focus of this study was to define the roles of these phosphatases in cementogenesis. TNAP was associated with earliest cementoblasts near forming acellular and cellular cementum. With loss of TNAP in the Alpl null mouse, acellular cementum was inhibited, while cellular cementum production increased, albeit as hypomineralized cementoid. In contrast, NPP1 was detected in cementoblasts after acellular cementum formation, and at low levels around cellular cementum. Loss of NPP1 in the Enpp1 null mouse increased acellular cementum, with little effect on cellular cementum. Developmental patterns were recapitulated in a mouse model for acellular cementum regeneration, with early TNAP expression and later NPP1 expression. In vitro, cementoblasts expressed Alpl gene/protein early, whereas Enpp1 gene/protein expression was significantly induced only under mineralization conditions. These patterns were confirmed in human teeth, including widespread TNAP, and NPP1 restricted to cementoblasts lining acellular cementum. These studies suggest that early TNAP expression creates a low PPi environment promoting acellular cementum initiation, while later NPP1 expression increases PPi, restricting acellular cementum apposition. Alterations in PPi have little effect on cellular cementum formation, though matrix mineralization is affected.
Alkaline Phosphatase ; metabolism ; Animals ; Cell Line, Transformed ; Dental Cementum ; cytology ; metabolism ; physiology ; Gene Expression Profiling ; Humans ; Mice ; Models, Animal ; Phosphoric Diester Hydrolases ; metabolism ; Pyrophosphatases ; metabolism ; Tooth Root ; metabolism ; physiology ; X-Ray Microtomography

OBJECTIVETo explore the effect of colon cancer cell-derived interleukin-1α on the migration and proliferation of human umbilical vein endothelial cells as well as the role of IL-1α and IL-1ra in the angiogenesis process.

METHODSWestern blot was used to detect the expression of IL-1α and IL-1R1 protein in the colon cancer cell lines with different liver metastatic potential. We also examined how IL-1α and IL-1ra influence the proliferation and migration of umbilical vascular endothelial cells assessed by PreMix WST-1 assay and migration assay, respectively. Double layer culture technique was used to detect the effect of IL-1α on the proliferation and migration of vascular endothelial cells and the effect of IL-1ra on the vascular endothelial cells.

RESULTSWestern blot analysis showed that IL-1α protein was only detected in highly metastatic colon cancer HT-29 and WiDr cells, but not in the lowly metastatic CaCo-2 and CoLo320 cells.Migration assay showed that there were significant differences in the number of penetrated cells between the control (17.9±3.6) and 1 ng/ml rIL-1α group (23.2±4.2), 10 ng/ml rIL-1α group (31.7±4.5), and 100 ng/ml rIL-1α group (38.6±4.9), showing that it was positively correlated with the increasing concentration of rIL-1α (P<0.01 for all). The proliferation assay showed that the absorbance values were 1.37±0.18 in the control group, and 1.79±0.14 in the 1 ng/ml rIL-1α group, 2.14±0.17 in the 10 ng/ml rIL-1α group, and 2.21±0.23 in the 100 ng/ml rIL-1α group, showing a positive correlation with the increasing concentration of rIL-1α(P<0.01 for all). IL-1ra significantly inhibited the proliferation and migration of vascular endothelial cells (P<0.01). The levels of VEGF protein were (1.697±0.072) ng/ml, (3.507±0.064)ng/ml and (4.139±0.039)ng/ml in the control, HUVECs+ IL-1α and HUVECs+ HT-29 co-culture system groups, respectively, showing a significant difference between the control and HUVECs+ 10 pg/ml rIL-1α groups and between the control and HUVECs+ HT-29 groups (P<0.01 for both).

CONCLUSIONSOur findings indicate that colon cancer cell-derived IL-1α plays an important role in the liver metastasis of colon cancer through increased VEGF level of the colon cancer cells and enhanced vascular endothelial cells proliferation, migration and angiogenesis, while IL-1ra can suppress the effect of IL-1α and inhibit the angiogenesis in colon cancer.

Blotting, Western ; Caco-2 Cells ; Cell Line, Tumor ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Coculture Techniques ; Colonic Neoplasms ; blood supply ; metabolism ; pathology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Interleukin 1 Receptor Antagonist Protein ; metabolism ; physiology ; Interleukin-1alpha ; metabolism ; physiology ; Liver Neoplasms ; secondary ; Neovascularization, Pathologic ; etiology

BACKGROUNDAbnormal neuronal differentiation plays an important role in central nervous system (CNS) development abnormalities such as Down syndrome (DS), a disorder that results directly from overexpression of genes in trisomic cells. Receptor-interacting protein 140 (RIP140) is significantly upregulated in DS brains, suggesting its involvement in DS CNS development abnormalities. However, the role of RIP140 in neuronal differentiation is still not clear. The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a) neuroblastoma cells, in vitro.

METHODSStably RIP140-overexpressing N2a (N2a-RIP140) cells were used as a neurodevelopmental model, and were constructed by lipofection and overexpression validated by real-time polymerase chain reaction and Western blot. Retinoic acid (RA) was used to stimulate N2a differentiation. Combining the expression of Tuj1 at the mRNA and protein levels, the percentage of cells baring neurites, and the number of neurites per cell body was semi-quantified to determine the effect of RIP140 on differentiation of N2a cells. Furthermore, western blot and the ERK1/2 inhibitor U0126 were used to identify the specific signaling pathway by which RIP140 induces differentiation of N2a cells. Statistical significance of the differences between groups was determined by one-way analysis of variance followed by the Dunnett test.

RESULTSCompared to untransfected N2a cells RIPl40 expression in N2a-RIP140 cells was remarkably upregulated at both the mRNA and protein levels. N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05). In addition, Tuj1, a neuronal biomarker, was strongly upregulated in N2a-RIP140 cells (P < 0.05) and phosphorylated ERK1/2 (p-ERK1/2) levels in N2a-RIP140 cells were dramatically increased, while differentiation was inhibited by the ERK1/2-specific inhibitor U0126.

CONCLUSIONSRIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.

Blotting, Western ; Cell Differentiation ; physiology ; Cell Line ; Humans ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neurons ; cytology ; metabolism ; Nuclear Receptor Co-Repressor 1 ; metabolism ; Signal Transduction ; physiology

OBJECTIVEInterferon-γ (IFN-γ) plays an important role in apoptosis and was shown to increase the risk of diabetes. Visfatin, an adipokine, has anti-diabetic, anti-tumor, and regulating inflammatory properties. In this study we investigated the effect of visfatin on IFN-γ-induced apoptosis in rat pancreatic β-cells.

METHODSThe RINm5F (rat insulinoma cell line) cells exposed to IFN-γ were treated with or without visfatin. The viability and apoptosis of the cells were assessed by using MTT and flow cytometry. The expressions of mRNA and protein were detected by using real-time PCR and western blot analysis.

RESULTSThe exposure of RINm5F cells to IFN-γ for 48 h led to increased apoptosis percentage of the cells. Visfatin pretreatment significantly increased the cell viability and reduced the cell apoptosis induced by IFN-γ. IFN-γ-induced increase in expression of p53 mRNA and cytochrome c protein, decrease in mRNA and protein levels of anti-apoptotic protein Bcl-2 were attenuated by visfatin pretreatment. Visfatin also increased AMPK and ERK1/2 phosphorylation and the anti-apoptotic action of visfatin was attenuated by the AMPK and ERK1/2 inhibitor.

CONCLUSIONThese results suggested that visfatin protected pancreatic islet cells against IFN-γ-induced apoptosis via mitochondria-dependent apoptotic pathway. The anti-apoptotic action of visfatin is mediated by activation of AMPK and ERK1/2 signaling molecules.

Adenylate Kinase ; metabolism ; Animals ; Apoptosis ; physiology ; Cell Line ; Cytokines ; physiology ; Flow Cytometry ; Interferon-gamma ; physiology ; Islets of Langerhans ; cytology ; MAP Kinase Signaling System ; Nicotinamide Phosphoribosyltransferase ; physiology ; Rats ; Real-Time Polymerase Chain Reaction ; Signal Transduction

OBJECTIVETo explore the effects of different concentrations of putrescine on proliferation, migration, and apoptosis of human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were routinely cultured in vitro. The 3rd to the 5th passage of HUVECs were used in the following experiments. (1) Cells were divided into 500, 1 000, and 5 000 µg/mL putrescine groups according to the random number table (the same grouping method was used for following grouping), with 3 wells in each group, which were respectively cultured with complete culture solution containing putrescine in the corresponding concentration for 24 h. Morphology of cells was observed by inverted optical microscope. (2) Cells were divided into 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, 1 000.0 µg/mL putrescine groups, and control group, with 4 wells in each group. Cells in the putrescine groups were respectively cultured with complete culture solution containing putrescine in the corresponding concentration for 24 h, and cells in control group were cultured with complete culture solution with no additional putrescine for 24 h. Cell proliferation activity (denoted as absorption value) was measured by colorimetry. (3) Cells were divided (with one well in each group) and cultured as in experiment (2), and the migration ability was detected by transwell migration assay. (4) Cells were divided (with one flask in each group) and cultured as in experiment (2), and the cell apoptosis rate was determined by flow cytometer. Data were processed with one-way analysis of variance, Kruskal-Wallis test, and Dunnett test.

RESULTS(1) After 24-h culture, cell attachment was good in 500 µg/mL putrescine group, and no obvious change in the shape was observed; cell attachment was less in 1 000 µg/mL putrescine group and the cells were small and rounded; cells in 5 000 µg/mL putrescine group were in fragmentation without attachment. (2) The absorption values of cells in 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, 1 000.0 µg/mL putrescine groups, and control group were respectively 0.588 ± 0.055, 0.857 ± 0.031, 0.707 ± 0.031, 0.662 ± 0.023, 0.450 ± 0.019, 0.415 ± 0.014, 0.359 ± 0.020, 0.204 ± 0.030, and 0.447 ± 0.021, with statistically significant differences among them (χ(2) = 6.86, P = 0.009). The cell proliferation activity in 0.5, 1.0, 5.0, and 10.0 µg/mL putrescine groups was higher than that in control group (P < 0.05 or P < 0.01). The cell proliferation activity in 500.0 and 1 000.0 µg/mL putrescine groups was lower than that in control group (with P values below 0.01). The cell proliferation activity in 50.0 and 100.0 µg/mL putrescine groups was close to that in control group (with P values above 0.05). (3) There were statistically significant differences in the numbers of migrated cells between the putrescine groups and control group (F = 138.662, P < 0.001). The number of migrated cells was more in 1.0, 5.0, and 10.0 µg/mL putrescine groups than in control group (with P value below 0.01). The number of migrated cells was less in 500.0 and 1 000.0 µg/mL putrescine groups than in control group (with P value below 0.01). The number of migrated cells in 0.5, 50.0, and 100.0 µg/mL putrescine groups was close to that in control group (with P values above 0.05). (4) There were statistically significant differences in the apoptosis rate between the putrescine groups and control group (χ(2)=3.971, P=0.046). The cell apoptosis rate was lower in 0.5, 1.0, 5.0, and 10.0 µg/mL putrescine groups than in control group (with P values below 0.05). The cell apoptosis rate was higher in 500.0 and 1 000.0 µg/mL putrescine groups than in control group (with P values below 0.01). The cell apoptosis rates in 50.0 and 100.0 µg/mL putrescine groups were close to the cell apoptosis rate in control group (with P values above 0.05).

CONCLUSIONSLow concentration of putrescine can remarkably enhance the ability of proliferation and migration of HUVECs, while a high concentration of putrescine can obviously inhibit HUVECs proliferation and migration, and it induces apoptosis.

Apoptosis ; drug effects ; Biological Products ; Cell Line ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Putrescine ; administration & dosage ; adverse effects ; pharmacology ; physiology ; Skin ; cytology ; Wound Healing