Sp1-mediated miR-193b suppresses atopic dermatitis by regulating HMGB1.

Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease. Keratinocyte dysfunction plays a central role in AD development. MicroRNA is a novel player in many inflammatory and immune skin diseases. In this study, we investigated the potential function and regulatory mechanism of miR-193b in AD. Inflamed human keratinocytes (HaCaT) were established by tumor necrosis factor (TNF)-α/interferon (IFN)-γ stimulation. Cell viability was measured using MTT assay, while the cell cycle was analyzed using flow cytometry. The cytokine levels were examined by enzyme-linked immunosorbent assay. The interaction between Sp1, miR-193b, and HMGB1 was analyzed using dual luciferase reporter and/or chromatin immunoprecipitation (ChIP) assays. Our results revealed that miR-193b upregulation enhanced the proliferation of TNF-α/IFN-γ-treated keratinocytes and repressed inflammatory injury. miR-193b negatively regulated high mobility group box 1 (HMGB1) expression by directly targeting HMGB1. Furthermore, HMGB1 knockdown promoted keratinocyte proliferation and inhibited inflammatory injury by repressing nuclear factor kappa-B (NF-κB) activation. During AD progression, HMGB1 overexpression abrogated increase of keratinocyte proliferation and repression of inflammatory injury caused by miR-193b overexpression. Moreover, transcription factor Sp1 was identified as the biological partner of the miR-193b promoter in promoting miR-193b expression. Therefore, Sp1 upregulation promotes keratinocyte proliferation and represses inflammatory injury during AD development via promoting miR-193b expression and repressing HMGB1/NF-κB activation.

Metformin improved oxidized low-density lipoprotein-impaired mitochondrial function and increased glucose uptake involving Akt-AS160 pathway in raw264.7 macrophages.

BACKGROUND: Macrophage accumulation in the vascular wall is a hallmark of atherosclerosis. Studies showed that shifting of oxidized lipids-induced inflammatory macrophages towards an anti-inflammatory phenotype by promoting oxidative metabolism attenuated atherosclerosis progression. Therefore, this study aimed to investigate whether metformin, which has ameliorated atherosclerosis in animal models and clinical trials, modulated oxidized low-density lipoprotein (Ox-LDL) induced inflammatory status in macrophages by regulating cellular oxidative metabolism. METHODS: Murine raw264.7 macrophages were incubated with Ox-LDL (50 µg/mL) in the presence or absence of metformin (15 µmol/L) for 24 h. Real-time polymerase chain reaction was used to quantify the transcription of classically activated (M1) pro-inflammatory and alternatively activated (M2) anti-inflammatory markers and mitochondrial DNA copy numbers. Cellular reactive oxygen species (ROS) production and mitochondrial membrane potential were detected by immunofluorescence. Cellular adenosine triphosphate (ATP) synthesis, glucose uptake, and lactic acid production were measured by commercial kit and normalized to cellular lysates. Western blotting analysis was performed to detect the expression of mitochondrial fusion/fission related proteins, enzymes mediating lipid metabolism and signaling pathway of glucose transport. Differences between groups were analyzed using one-way analysis of variance. RESULTS: Metformin improved Ox-LDL-impaired anti-inflammatory phenotype in raw264.7 macrophages as shown by up-regulated transcription of anti-inflammatory markers including interleukin 10 (0.76 ±â€Š0.04 vs. 0.94 ±â€Š0.01, P = 0.003) and Resistin-like molecule alpha (0.67 ±â€Š0.08 vs. 1.78 ±â€Š0.34, P = 0.030). Conversely, Ox-LDL-diminished phosphorylation of Akt was up-regulated by metformin treatment (0.47 ±â€Š0.05 vs. 1.02 ±â€Š0.08, P = 0.040), associated with an improvement of mitochondrial function, characterized by decreased ROS generation (2.50 ±â€Š0.07 vs. 2.15 ±â€Š0.04, P = 0.040), increased lipid oxidation, and elevated cellular ATP production (0.026 ±â€Š0.001 vs. 0.035 ±â€Š0.003, P = 0.020). Moreover, metformin-mediated Akt activation increased Akt substrate of 160 kDa (AS160) phosphorylation (0.51 ±â€Š0.04 vs. 1.03 ±â€Š0.03, P = 0.0041), promoted membrane translocation of glucose transporter 1, and increased glucose influx into the cells (4.78 ±â€Š0.04 vs. 5.47 ±â€Š0.01, P < 0.001). CONCLUSION: This study suggested that targeting macrophage metabolism with new or existing drugs had therapeutic potential for the prevention and treatment of diabetes-accelerated atherosclerosis.

Effect of puerarin in promoting fatty acid oxidation by increasing mitochondrial oxidative capacity and biogenesis in skeletal muscle in diabetic rats.

BACKGROUND: Type 2 diabetes is characterized by dyslipidemia and the accumulation of lipids in non-adipose tissue, including skeletal muscle. Puerarin, which is a natural isoflavonoid isolated from the root of the plant Pueraria lobata, has been shown to have antidiabetic activity. However, the lipid-reducing effect of puerarin, in particular in skeletal muscle, has not yet been addressed. METHODS: We examined the effect of puerarin on mitochondrial function and the oxidation of fatty acids in the skeletal muscle of high-fat diet/streptozotocin-induced diabetic rats. RESULTS: Puerarin effectively alleviated dyslipidemia and decreased the accumulation of intramyocellular lipids by upregulating the expression of a range of genes involved in mitochondrial biogenesis, oxidative phosphorylation, the detoxification of reactive oxygen species, and the oxidation of fatty acids in the muscle of diabetic rats. Also, the effect of puerarin on mitochondrial biogenesis might partially involve the function of the µ-opioid receptor. In addition, puerarin decreased the trafficking of fatty acid translocase/CD36 to the plasma membrane to reduce the uptake of fatty acids by myocytes. In vitro studies confirmed that puerarin acted directly on muscle cells to promote the oxidation of fatty acids in insulin-resistant myotubes treated with palmitate. CONCLUSIONS: Puerarin improved the performance of mitochondria in muscle and promoted the oxidation of fatty acids, which thus prevented the accumulation of intramyocellular lipids in diabetic rats. Our findings will be beneficial both for elucidating the mechanism of the antidiabetic activity of puerarin and for promoting the therapeutic potential of puerarin in the treatment of diabetes.

Laser Ablation of 6H-SiC Single Crystals and Spectral Characterization.

Laser micromachining has proven to be a useful tool for precision processing of semiconductors. For Silicon Carbide (SiC) single crystals, ablation with ultraviolet wavelength laser could lead to the maximum absorption efficiency of incident energy. In this paper, laser ablations were performed on 6H-SiC single crystals through a 355 nm solid state laser. Different confining media were also employed to find the optimal processing condition. The surface of SiC after laser ablation was characterized by Raman spectroscopy. Amorphous silicon and nanocrystalline graphite were found to be the main compositions left. For SiC wafers ablation in air, the amorphous silicon exhibited mainly around rather than inside the ablated crater. However, the amorphous silicon showed opposite spatial distribution features for samples processing under liquid. Through analysis of the compositions left on the ablated surface, the ablation mechanism was investigated from another point of view. For liquid confined laser processing,previous studies mainly concentrate on the thickness and viscosity of the liquids, little information has been done on the reducibility of liquids. To investigate the influence of liquid reducibility, the surface morphology and oxygen content of ablation under different confining media were checked by confocal laser scanning microscopy and energy dispersive spectroscopy. Results showed that the reducibility of confining liquid also played a vital role in the ablation process under liquid. Utilizing liquids with deoxidizing ability as confining media will result in a remarkable reduction of surface oxygen content and a more regular morphology.

Connective tissue growth factor, a regulator related with 10-hydroxy-2-decenoic acid down-regulate MMPs in rheumatoid arthritis.

10-hydroxy-2-decenoic acid (10H2DA) is suggested to be a potential medication for rheumatoid arthritis (RA) by activation of matrix metalloproteinases (MMPs) via mitogen-activated protein kinase signaling pathways. The aim of the present work was to seek differentially expressed proteins in rheumatoid arthritis synovial fibroblasts (RASFs) treated with 10H2DA by comparative proteomics analysis. Two-dimensional electrophoresis (2-DE) and LC-MS/MS were performed to identify changes in protein expression after 24-h 10H2DA treatment. Differentially expressed proteins were identified by real-time PCR and Western blot analysis. Influence of down-regulation of connective tissue growth factor (CTGF) expression on MMPs was studied by RNAi. Ten proteins were up-regulated and 9 proteins were down-regulated after 24-h 10H2DA treatment. A total of 19 differentially expressed proteins were identified and found to be associated with glycolysis, lipid metabolism, cell adhesion, ATP synthesis, oxidation reduction, and anti-apoptosis. CTGF, a member of the C-terminal cystein-rich proteins (CCN) family, was down-regulated after 24-h 10H2DA treatment. MMPs were down-regulated after RNAi (CTGFi). These results suggest that CTGF is a regulator factor in the expression of MMPs, and 10H2DA down-regulate the concentration of MMPs probably by down-regulating the expression of CTGF.

[A novel severe aplastic anemia mouse model induced by IFN-γ plus busulphan].

OBJECTIVE: To establish a novel severe aplastic anemia (SAA) mouse model by interferon-γ (IFN-γ) plus busulphan. METHODS: Thirty clean-class BALB/c female mice were intraperitoneally injected with IFN-γ and intragastrically administrated with busulphan (group I), meanwhile busulphan alone group (n = 30, group II) and normal control group (n = 30, group III). Multi-parameters were compared among the three groups. RESULTS: In group I at day 10 after treatment, the incidence of SAA was 100% and mortality 20% respectively; the WBC, HGB, PLT, absolute reticulocyte count (Ret) and tibial nucleated cell count (TNCC) were (0.8 ± 0.3) × 10(9)/L, (45 ± 20) g/L, (10 ± 8) × 10(9)/L, (15.2 ± 10.2) × 10(9)/L, (12 ± 7) × 10(6)/tibia, respectively, which were significantly different from the other two groups (all P < 0.05). The bone marrow smears and patho-histological examinations showed marked reductions of marrow cell proliferation, and increases of the percentages of non-hematopoietic cells and cellular adipose. The depression was severe and irreversible. In group II, the blood cells count, TNCC and marrow proliferation recovered gradually with erythroid hyperplasia and hematopoietic dysplasia. CONCLUSIONS: IFN-γ plus busulphan can establish a SAA mouse model in a relatively short period, which is more resemble with human SAA.

[Application of proteomic models for the diagnosis of inflammatory and non-inflammatory preterm delivery].

OBJECTIVE: To establish the serum proteomic models for the identification of premature delivery with inflammation or non-inflammation. METHODS: The laboring patients from 2008 to 2010 at our hospital were divided into 5 groups according to placental pathology, including inflammatory preterm group, non-inflammatory preterm group and blind test group (n = 50 each). The control group was normal full-term. The preterm models with or without inflammation were established by the methods of SELDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight-mass spectrometry) and bioinformatics. And statistical analysis was performed after a blind test. Then differential protein fingerprints were compared and analyzed. RESULTS: A total of 36 different proteins were harvested after a comparison of inflammatory preterm and control groups. The model was established by eight markers. The rates of specificity, sensitivity, positive predictive value and negative predictive value were 80.85%, 84.44%, 93.33% and 46.67% respectively. Fifteen different proteins existed between non-inflammatory preterm and control groups. There were 4 marker proteins. The rates of specificity, sensitivity, positive predictive value and negative predictive value were 75.68%, 64.44%, 80.00% and 66.67% respectively. Different marker proteins existed between the identification models of inflammatory preterm labor and inflammatory full-term. CONCLUSION: Different serum proteomic models may be used for the diagnosis of preterm labor and the differentiation of preterm labor with or without inflammation. And different proteins are expressed during different stages of pregnancy with chorioamnionitis.

Identification of a stem-like cell population by exposing metastatic breast cancer cell lines to repetitive cycles of hypoxia and reoxygenation.

INTRODUCTION: The irregular vasculature of solid tumors creates hypoxic regions, which are characterized by cyclic periods of hypoxia and reoxygenation. Accumulated evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seem to provide an advantage to tumor growth. Although the development of hypoxia tolerance in tumors predicts poor prognosis, mechanisms contributing to hypoxia tolerance remain to be elucidated. Recent studies have described a subpopulation of cancer stem cells (CSC) within tumors, which have stem-like properties such as self-renewal and the ability to differentiate into multiple cell types. The cancer stem cell theory suggests CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Since hypoxia is considered to be one of the critical niche factors to promote invasive growth of tumors, we hypothesize that repetitive cycles of hypoxia/reoxygenation also play a role in the enrichment of breast CSCs. METHODS: Two metastatic human breast cancer cell lines (MDA-MB 231 and BCM2) were used to optimize the conditions of hypoxia and reoxygenation cycles. The percentage of CSCs in the cycling hypoxia selected subpopulation was analyzed based on the CD44, CD24, ESA, and E-cadherin expression by three-color flow cytometry. Colony formation assays were used to assess the ability of this subpopulation to self-renew. Limiting dilution assays were performed to evaluate the tumor-initiating and metastatic ability of this subpopulation. Induction of EMT was examined by the expression of EMT-associated markers and EMT-associated microRNAs. RESULTS: Using an optimized hypoxia and reoxygenation regimen, we identified a novel cycling hypoxia-selected subpopulation from human breast cancer cell lines and demonstrated that a stem-like breast cancer cell subpopulation could be expanded through repetitive hypoxia/reoxygenation cycles without genetic manipulation. We also found that cells derived from this novel subpopulation form colonies readily, are highly tumorigenic in immune-deficient mice, and exhibit both stem-like and EMT phenotypes. CONCLUSIONS: These results provide the validity to the newly developed hypoxia/reoxygenation culture system for examining the regulation of CSCs in breast cancer cell lines by niche factors in the tumor microenvironment and developing differential targeting strategies to eradicate breast CSCs.

10-Hydroxy-2-decenoic acid from Royal jelly: a potential medicine for RA.

AIM OF THE STUDY: Rheumatoid arthritis synovial fibroblasts (RASFs) are known to produce matrix metalloproteinases (MMPs) and cause joint destruction. The purpose of this study is to develop a potential medicine for rheumatoid arthritis (RA). MATERIALS AND METHODS: To this end, first, the MMPs inhibition factor was purified from an alkali-solubilized fraction of RJ (Apis mellifera) by C18 reverse-phase column chromatography and identified as 10-hydroxy-2-decenoic acid (10H2DA) by LTQ XL analysis. Next, Experimental test 10H2DA how to inhibited the activities of MMPs: with RASFs isolated from rheumatoid tissues by enzymatic digestion, cultures in monolayers were treated with 10H2DA (0.5mM, 1mM, and 2mM) or PBS for 2h followed by stimulation with TNF-alpha (10 ng/ml) for 2h, mRNA. Protein levels of MMP-1 and MMP-3 were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), the DNA-binding activity of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) by electrophoretic mobility shift assay (EMSA), and the protein kinase activity of p38, ERK and JNK by kinase assay. RESULTS: The molecular investigation revealed that the 10H2DA-mediated suppression was likely to occur through blocking p38 kinase and c-Jun N-terminal kinase-AP-1 signaling pathways. In contrast, 10H2DA had no effect on extracellular signal-regulated kinase activity, NF-kappaB DNA-binding activity and IkappaBalpha degradation. CONCLUSION: These results suggest that 10H2DA may be of potential therapeutic value in inhibiting joint destruction in RA.

[The Effects of IFN-γ on AKT activated 32D Cells and its Mechanisms].

OBJECTIVE: To investigate the effects of activated AKT on murine myeloid precursor cells (32D cells), and the effects of IFN-γ on 32D cells and its mechanisms. METHODS: Plasmid transduction was used to enhance the expression of AKT on 32D cells. After the transfected cells treated with IFN-γ for 24 hours, proliferation rate was tested by WST-1, apoptosis by flow cytometry, expression of phosphorylated Erk1/2, Stat3 and phosphorylated Stat3 was determined by Western blot. RESULTS: (1) IFN-γ at low concentration (100 U/ml) enhanced the growth and proliferation of 32D cells, while at high concentration (1000 U/ml) suppressed them. (2) Compared with control groups, low concentration IFN-γ increased (1124 ± 13) Stat3 phosphorylation in 32D-cell, while it high concentration IFN-γ decreased (601 ± 13). 32D cells transfected with activated Akt grew rapidly (0.287 ± 0.010) and had a low apoptotic rate [(9.57 ± 0.17)% (P < 0.05)]. (3) The expression of p-Erk1/2 in transfected 32D-cell was significantly reduced (P < 0.05). (4) Apoptosis rate of IFN-γ treated group was significantly decreased in transfected 32D cells (P < 0.05). CONCLUSIONS: IFN-γ has dual effects on 32D cells, namely, at low concentration enhanced the growth and proliferation of 32D cells, while at high concentration suppressed them. Its mechanisims is possibly through Stat3 pathway. Activated Akt can significantly promote the growth and proliferation of 32D cell and significantly inhibit apoptosis and IFN-γ can regulate cell proliferation and apoptosis through AKT. AKT activation can inhibit the Erk signal pathway, which may be affected by inhibition the modificaton of Raf1.