Effect of neurotrophic factor-3 on the proliferation and apoptosis of bone marrow mesenchymal stem cells and mechanisms / 中华行为医学与脑科学杂志

Objective To investigate the effect of neurotrophin-3(NT-3)on the proliferation and apoptosis of bone marrow mesenchymal stem cells(BMSCs)in rats and possible mechanisms. Methods The NT-3 overexpression and lentiviral transfection of BMSCs were co-cultured with neuronal cells respectively and then they were divided into overexpression control group,NT-3 transfection group and shRNA-NT-3 transfection group(NT-3 silencing group).MTT assay was used to detect the cell culture for 24 h,48 h and 72 h. Cell cycle and apoptosis were detected by flow cytometry for 48 h. Real-time quantitative PCR was used to detect the expression of C/EBPβmRNA.The expression of C/EBPβprotein was detected by Western blot. Results MTT results showed that the proliferation ability of BMSCs in the NT-3 overexpres-sion group was significantly higher than that in the control group(0.650±0.042,0.826±0.074)at 48 h and 72 h(P<0.05).Compared with the control group(P<0.05),the cell cycle and apoptosis of BMSCs in NT-3 silencing group were significantly decreased at 48 h and 72 h(P<0.05). The results of 48 h cell cycle and apoptosis showed that the percentage of G1 phase in BMSCs was decreased,G2 and S were increased and the apoptosis was decreased. The percentage of G1 phase in G2-S phase and the increase of apoptosis were in-creased in NT-3 silencing group. The results of Western Blot showed that C/EBPβ mRNA and protein levels were significantly up-regulated in BMSCs of NT-3 overexpression group and significantly decreased in NT-3 silencing group(P<0.05).Conclusion NT-3 may promote the expression of C/EBP beta and affect the ex-pression of its downstream target genes,which can inhibit the apoptosis of BMSCs cells.

Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Behçet Disease Depending on Disease Activity

BACKGROUND: Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known. OBJECTIVE: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs). METHODS: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) β was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPβ and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs. CONCLUSION: We found differential expression of C/EBPβ, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

A study of the lipoprotein lipase inhibitory mechanism of Poncirus trifoliata water extracts / 한국영양학회지

PURPOSE: Poncirus trifoliata has been reported to have anti-inflammatory, antioxidant, and immune activities. However, its anti-obesity activity and the mechanism by which the water extract of dried, immature fruit of Poncirus trifoliata (PF-W) acts are not clear. This study suggests a potential mechanism associated with the anti-obesity activity of PF-W. METHODS: We measured the effect of PF-W on lipoprotein lipase (LPL) regulation using enzyme-linked immunosorbent assay (ELISA) and an activity assay. The LPL regulation mechanism was examined by reverse transcription polymerase chain reaction (RT-PCR) to measure the mRNA expression of biomarkers related to protein transport and by western blot for analysis of the protein expression of the transcription factor CCAAT-enhancer-binding protein (C/EBPbeta) RESULTS: The total polyphenol and flavonoid content of PF-W was 52.15 +/- 4.02 and 6.56 +/- 0.47 mg/g, respectively. PF-W treatment decreased LPL content in media to 58 +/- 5% of that in control adipocyte media, and increased LPL content to 117 +/- 3.5% of that in control adipocytes, but did not affect the mRNA expression of LPL. PF-W also increased the mRNA expression of sortilin-related receptor (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL, in a concentration- and time-dependent manner. Finally, cell fractionation revealed that PF-W treatment induced the expression of C/EBPbeta, a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. CONCLUSION: The LPL secretion and activity assay showed PF-W to be an LPL secretion inhibitor, and these results suggest the potential mechanism of PF-W involving inhibition of LPL secretion through C/EBPbeta-mediated induction of SorLA expression.

Therapeutic effects of CCAAT/enhancer-binding protein α gene on liver fibrosis in mice / 中华消化杂志

Objective To investigate the difference of CCAAT/enhancer-binding protein α (C/EBP-α) gene induced apoptosis between hepatocytes and hepatic stellate cells (HSC) in mice with liver fibrosis.Methods Sixty BALB/c mice were evenly divided into normal group,model group,treatment group,blank control group and negative control group,12 mice in each group.Except the mice of normal control group,the mice of other groups were treated with intraperitoneal injection of CCl4 to establish liver fibrosis mice model.Mice of treatment group,blank control group and negative control group were administrated with C/EBP-α carried adenovirus (Ad-C/EBP-α),phosphate buffered solution and empty vector of adenovirus (Ad-EGFP) respectively through tail vein for the first week.The expression of C/EBP-α and α-smooth muscle actin (α-SMA) was detected by immunohistochemistry method.Sinusoidal endothelial structure of peri-portal regions and far from portal regions was observed by transmission electron microscope (TEM).Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was applied to detect apoptosis of cells in liver tissue.The degree of liver fibrosis in mice was determined with sirius red staining and hydroxyproline content measurement.Single factor variance analysis was performed for comparison among multiple groups,and t test was used for comparison between two groups.Results C/EBP-α was expressed in nucleus of hepatocyte in normal control group mice.The expression decreased in model group,blank control group and negative control group.However,the expression of C/EBP-α of treatment group increased,and mainly expressed in cells located in perisinusoidal and perivascular.Hepatic sinusoids was distorted,blood vessel wall thickened.Hepatocyte degeneration and lots of lipid droplets was found in model group,blank control group and negative control group.The thicken degree of endothelial layer of blood vessel of treatment group was lower than that of model group.The percentage of sirius red positive cells of normal group,model group,treatment group,blank control group and negative control group was (0.10±0.03)％,(5.81±0.32)％,(2.32±0.45)％,(6.34± 0.81)％ and (6.10± 0.92)％,respectively; content of hydroxyproline was (0.07±0.00) μg/mg,(0.69 ± 0.10) μg/mg,(0.19±0.06) μg/mg,(0.56±0.03) μg/mg and (0.64±0.08) μg/mg,respectively; the percentage of α-SMA positive cells was (0.50 ±0.03)％,(5.30 ± 0.52)％,(2.15 ± 0.29)％,(5.53 ± 0.43) ％ and (5.42 ± 0.25) ％,respectively; the number of TUNEL positive cells was (0.25 ± 0.08),(0.15±0.02),(7.10±1.53),(0.13±0.03) and (0.18±0.07),respectively.The differences between the groups were statistically significant (F=113.74,148.29,292.43 and 140.25,all P＜0.05).The difference between normal group and model group,between model group and treatment group,between treatment group and blank control group,between treatment group and negative control group were statistically significant (tarirus positive cell =-52.54,-16.20,-10.60 and-7.99,thydroxyproline content =-168.00,11.53,11.07 and 12.54,ta SMA pusitive cells-24.77,-13.82,15.94 and 18.37,tTUNEL positive cells =3.26,-11.91,-11.95 and-11.88,all P＜ 0.05),there was no statistically significant difference between model group and blank control group,between model group and negative control group (both P＞0.05).TUNEL positive cells mainly located in perisinusoidal and perivascular of liver in mice,which was consistent with the distribution of α-SMA-positive cells.Conclusion C/EBP-α could effectively relieve CCl4 induced liver fibrosis in mice mainly through inducing HSC apoptosis,however no apoptosis effect on hepatocytes.

Antitumor effect of enhancer binding protein C/EBPα on the leukemic BALB/c nude mice / 白血病·淋巴瘤

Objective To explore the role of tumor inhibition of enhancer binding protein C/EBPα in the leukemic mice. Methods BALB/c nude mice were randomly divided into three groups. Three kinds of cells including pEGFP-C/EBPα-K562 cells, pEGFP-K562 cells and K562 cells as the control were injected into mice separately through the subcutaneous and tail vein, and subcutaneous tumors and leukemic models were formed. The changes of tumors were observed and the apoptosis of cells was detected by TUNEL; The capacity of proliferation of leukemia cells was observed in the bone marrow and the peripheral blood by Wright-Giemsa staining. The expression of genes of related to proliferation was detected by RT-PCR. Results The quality and the max diameter of tumors in the pEGFP-C/EBPα-K562 group were smaller than that of pEGFP-K562 group and K562 control group [(2.4±0.1) g vs (5.1±0.3) g and (5.7±0.4) g, both P ＜0.05; (11+2)mm vs (19+3) mm and (23+3) mm, both P ＜0.05]. More apoptosis cells were found in the pEGFP-C/EBPα-K562 group leukemic cells were found in the peripheral blood of leukemic models, and the proliferation of leukemic cells in the pEGFP-C/EBPo-K562 group were lower than that of other groups, accompany by the conspicuous cell differentiation. p53 was significantly elevated by RT-PCR, while down-regulated of c-myc.Conclusion Enhancer binding protein C/EBPα promote the apoptosis of cells and inhibit the proliferation of leukemia cells in leukemia mice, and further induce the cell differentiation. The inhibition of enhancer binding protein C/EBPα in the leukemia may have effect through the regulation of related genes.

Ginsenoside Rb_1 facilitates adipocyte differentiation and inhibits lipolysis in 3T3-L1 adipocytes / 中华内分泌代谢杂志

Objective To observe the effect of ginsenoside Rb1,the most abundant ginsenoside in ginseng root,on differentiation and lipolysis of 3T3-L1 cells and to explore its anti-diabetic mechanism.Methods 3T3-L1 preadipoeytes were induced under standard differentiation process in the presence of 0.1,1,10,100?mol/L ginsenoside Rb_1 for 6 days.Oil red O staining,measurement of triglyceride contents and glucose uptake assay were performed.The expressions of mRNA and protein of PPAR?2,C/EBP?,ap2,glucose transporter (Glut) 1,and Glut4 were analysed with quantitative real time-PCR and Western blot.The binding affinity of Rb_1 to PPAR?-LBD was evaluated by Surface Plasmon Resonance (SPR).Lipolysis of adipocytes was examined by the measurement of glycerol released from adipoeytes treated with Rb_1 for 1 h.Results Ginsenoside Rb_1 facilitated differentiation of 3T3-L1 preadipoeytes in a dose-depondent manner.10?mol/L ginsenoside Rb_1 increased lipid accumulation by about 56%.Treatment of differentiating adipocytes with 10?mol/L ginsenoside Rb_1 increased the expressions of PPAR?2 and C/EBP?mRNA and protein,as well as mRNA expression of ap2,one of their target genes.After treatment of differentiating adipoeytes with Rb_1,basal and insulin-mediated glucose transport augmented significantly accompanied by up-regulations of mRNA and protein level of Glut4,but not of Glutl.SPR showed Rb_1 could bind to PPAR?which suggested Rb_1 was a ligand of PPAR?.Ginsenoside Rb_1 inhibited basal lipolysis in adipoeytos in a dose-dependent manner.However,it did not affect isoproterenol-stimulated lipolysis.Conclusion As a PPAR?ligand,ginsenoside Rb_1 promotes adipogenesis,inhibitas basal lipolysis and inereasos basal and insulin-mediated glucose transport in cultured adipoeytes.Therefore,anti-diabetic and insulin-sensitizing activity of ginsenosides is,at least in part,involved in the enhancing effect on PPAR?2 and C/EBP?expressions,hence promoting adipogenesis and glucose uptake,and inhibiting lipolysis in adipocytes.