OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P < 0.01), while C/EBPβ and AP2 were significantly down-regulated (P < 0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.
3T3-L1 Cells ; Activating Transcription Factor 3/pharmacology* ; Adipocytes ; Adipogenesis/genetics* ; Animals ; CCAAT-Enhancer-Binding Protein-alpha/pharmacology* ; Cell Differentiation ; Goats ; Mice ; PPAR gamma/metabolism*

OBJECTIVETo study the expression of SUMO-modified CCAAT enhancer binding protein &alpha; (C/EBP&alpha;) in preterm rat model of bronchopulmonary dysplasisa (BPD) induced by hyperoxia exposure and its role.

METHODSEighteen preterm rats were randomly divided into an air group and a hyperoxia group (n=9 each). The model of BPD was prepared in preterm rats exposed to hyperoxia. The rats from the two groups were sacrificed on postnatal days 4, 7 and 14 respectively (3 rats at each time) and lung tissues were harvested. Periodic acid-Schiff (PAS) staining was used to observe the differentiation of rat lung tissues. Ki67 expression was detected by immunohistochemistry. Western blot was used to measure the protein expression of small ubiquitin-related modifier-1(SUMO1) and C/EBP&alpha;. A co-immunoprecipitation assay was performed to measure the protein expression of SUMO-modified C/EBP&alpha;.

RESULTSCompared with the air group, the hyperoxia group showed a decreased glycogen content in the lung tissue on postnatal day 4, and an increased content on postnatal days 7 and 14. Over the time of hyperoxia exposure, the hyperoxia group showed an increased expression of Ki67 in the lung tissue compared with the air group at all time points. Compared with the air group, the protein expression of C/EBP&alpha; increased on postnatal day 4 and decreased on postnatal days 7 and 14 in the hyperoxia group (P<0.05). The hyperoxia group had significantly upregulated expression of SUMO1 and SUMO-modified C/EBP&alpha; compared with the air group at all time points (P<0.05). In the hyperoxia group, the protein expression of SUMO-modified C/EBP&alpha; was positively correlated with the glycogen content (r=0.529, P<0.05) and the expression of Ki67 (r=0.671, P<0.05).

CONCLUSIONSHyperoxia may induce over-proliferation and differentiation disorders of alveolar epithelial cells in preterm rat model of BPD, possibly through an increased expression of SUMO-modified C/EBP&alpha.

Animals ; Animals, Newborn ; Bronchopulmonary Dysplasia ; etiology ; metabolism ; pathology ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Cell Proliferation ; Disease Models, Animal ; Hyperoxia ; complications ; pathology ; Ki-67 Antigen ; analysis ; Pulmonary Alveoli ; pathology ; Rats ; Rats, Sprague-Dawley ; Sumoylation

The CCAAT enhancer binding protein α (C/EBP α:p42 and p30),which encoded by CCAAT enhancer binding protein α () gene,plays a pretty crucial role in the regulation of myeloid hematopoiesis.The disorder of CEBPA gene expression is an pivotal mechanism of acute myeloid leukemia (AML). The result of uncontrolled expression of C/EBP α gene is the over-expression of p30 and the incomplete loss of p42, both of which contribute to the occurrence of AML. Restoring the expression ratio of C/EBP α such as over-expression of p42 or blocking the carcinogenic pathway of p30 seems to be important for the treatment of AML caused by such causes. In order to better guide medical decision-making, this article reviews research progress on C/EBPα in the pathogenesis of AML.
CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Gene Expression Regulation, Neoplastic ; Hematopoiesis ; genetics ; Humans ; Leukemia, Myeloid, Acute ; physiopathology

OBJECTIVETo investigate the expressions of inflammation- and fibrosis-related genes in perinephric and subcutaneous adipose tissues in patients with adrenocorticotropic hormone (ACTH)-independent Cushing's syndrome.

METHODSThe perinephric and subcutaneous adipose tissues adipose tissues were obtained from 8 patients with ACTH-independent Cushing's syndrome undergoing laparoscopic retroperitoneal adrenalectomy. Real-time PCR was used to detect the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor-&alpha; (TNF-&alpha;), matrix metallopeptidase 2 (MMP-2), TIMP metallopeptidase inhibitor 1 (TIMP-1), early growth response 1 (EGR1), CCAAT/enhancer binding protein β(CEBPβ), uncoupling protein 1(UCP-1), PPARγ coactivator 1 alpha (PGC1&alpha;) and cell death-inducing DFFA-like effector a (CIDEA).

RESULTSThe mRNA level of CIDEA was significantly higher in the perinephric adipose tissue (peri-N) than in the subcutaneous adipose tissue (subQ) (P<0.05). The expressions of CEBPβ, UCP-1, and PGC1&alpha; mRNA in the peri-N were similar with those in the subQ. The expressions of IL-6, TIMP1 and EGR1 mRNA in the subQ were significantly higher than those in the peri-N (P<0.05). No significant difference in TNF-&alpha; and MMP-2 mRNA levels was found between peri-N and subQ.

CONCLUSIONThe expression levels of the inflammation- and fibrosis-related genes are higher in the subQ than in the peri-N of patients with ACTH-independent Cushing's syndrome, suggesting that chronic exposure to endogenous hypercortisolism may cause adipose tissue dysfunction.

Adrenalectomy ; Adrenocorticotropic Hormone ; CCAAT-Enhancer-Binding Protein-beta ; metabolism ; Cushing Syndrome ; metabolism ; surgery ; Early Growth Response Protein 1 ; metabolism ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; metabolism ; Real-Time Polymerase Chain Reaction ; Subcutaneous Fat ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Uncoupling Protein 1 ; metabolism

Obesity is associated with a number of metabolic abnormalities such as type 2 diabetes and has become a major health problem worldwide. In the present study, we investigated the effects of Epimedium koreanum Nakai (Herba Epimedii, HE) and its main constituent icariin on the adipocyte differentiation in 3T3-L1 preadipocytes. HE extract and icariin significantly reduced lipid accumulation and suppressed the expressions of PPARγ, C/EBPα, and SREBP-1c in 3T3-L1 adipocytes. They also inhibited fatty acid synthase (FAS), acyl-Co A synthase (ACS1), and perilipin. Moreover, HE extract and icariin markedly increased the phosphorylation of AMPK. These results indicated that HE extract and icariin can inhibit the adipocyte differentiation through downregulation of the adipogenic transcription factors, suggesting that HE containing icariin may be used as a potential therapeutic agent in the treatment and prevention of obesity.
3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Adipogenesis ; drug effects ; Animals ; CCAAT-Enhancer-Binding Protein-alpha ; genetics ; metabolism ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Lipid Metabolism ; drug effects ; Mice ; PPAR gamma ; genetics ; metabolism ; Plant Extracts ; pharmacology ; Sterol Regulatory Element Binding Protein 1 ; genetics ; metabolism

OBJECTIVETo investigate the expression and the possible mechanism of the transcription factor C/EBP&alpha; in chronic myeloid leukemia（CML).

METHODSBone marrow samples from 50 CML patients（including 33 patients in chronic phase, 7 in accelerated phase and 10 in blast crisis）and peripheral blood specimens of 20 healthy donors were collected. The expression of C/EBP&alpha; gene and the effect of Imatinib on its expression was detected by RT- PCR. C/EBP&alpha; gene was inserted into lentivirus expression vector pLVX- EGFP- 3FLAG- Puro by recombinant DNA technology to construct C/EBP&alpha; stable expression in K562 cells. Cell proliferation was assayed by CCK-8. The expressions of Foxo3a and Bim genes were detected by RT-PCR.

RESULTSThe level of C/EBP&alpha; expression was significantly declined in CML patients compared with that of normal control group（P<0.01）and had negative correlation with bcr- abl expression（Spearman r=－ 0.505, P<0.01). The stable K562- C/EBP&alpha; cell line was successfully established and confirmed by RT-PCR and Western blot. Cell proliferation ability was lower in the K562- C/EBP&alpha; group than that in the non- transfection and mock-vehicle groups. The expressions of Foxo3a and Bim genes were 1.06 ± 0.06 and 0.53 ± 0.07, respectively, which was higher than that of nontransfection and mock-vehicle groups（P<0.01, P<0.05).

CONCLUSIONC/EBP&alpha; expression was decreased in CML patients, overexpression of C/EBP&alpha; could inhibit K562 cell growth.

Blast Crisis ; Bone Marrow ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Case-Control Studies ; Cell Cycle ; Cell Proliferation ; Humans ; Imatinib Mesylate ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Transfection

This study was aimed to explore the possible mechanisms of hepcidin increase in multiple myeloma patients. The clinical information and peripheral venous blood of eligible patients with previously untreated multiple myeloma were collected. Serum concentration of IL-6 was detected by ELISA. Peripheral blood monocytes were isolated by CD14⁺ magnetic beads. The expression of hepcidin, IL-6 and C/EBP&alpha; mRNA of monocytes were detected by real time quantitative PCR. The results indicated that the hemoglobin level was reduced in 17 multiple myeloma patients enrolled in study (97.8 ± 27.5 g/L), showing the characteristics of anemia of chronic disease. The hepcidin and C/EBP&alpha; expression of peripheral blood monocytes significantly increased (P < 0.01), serum IL-6 was also higher than that in normal controls (P < 0.01). Serum IL-6 positively correlated with monocyte hepcidin and C/EBP&alpha; expression (P < 0.05); monocyte C/EBP&alpha; expression positively correlated with monocyte hepcidin expression (P < 0.05). It is concluded that the elevated IL-6 may induce hepcidin expression through up-regulating C/EBP&alpha; in untreated myeloma patients.
Anemia ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Chronic Disease ; Hepcidins ; metabolism ; Humans ; Interleukin-6 ; Monocytes ; Multiple Myeloma ; metabolism ; RNA, Messenger ; Up-Regulation

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 microg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 microg/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARgamma and C/EBPalpha expression as shown in in vitro and in vivo, and the suppression of PPARgamma activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.
3T3-L1 Cells ; Adipogenesis/*drug effects ; Adipose Tissue/drug effects/metabolism ; Adipose Tissue, White ; Animals ; Anti-Obesity Agents/administration & dosage/pharmacology/*therapeutic use ; Body Weight/drug effects ; CCAAT-Enhancer-Binding Protein-alpha/genetics ; Cell Differentiation/drug effects ; Eating/drug effects ; Fatty Acids/metabolism ; Gene Expression/drug effects ; Lipid Metabolism/*drug effects ; Lipids ; Lipogenesis/drug effects ; Mice ; Mice, Inbred C57BL ; Obesity/prevention & control ; PPAR gamma/antagonists & inhibitors/genetics ; Plant Extracts/*pharmacology ; Plants, Medicinal ; Primulaceae/*chemistry

OBJECTIVETo study the effects and mechanisms of Huanglian Jiedu Decoction (HJD) and its disassembled recipes containing serums on the proliferation and differentiation of preadipocytes.

METHODSHJD and its disassembled recipes containing serums were prepared. The 3T3-L1 preadipocytes were cultured. The proliferation of 3T3-L1 preadipocytes was detected by methyl thiazolyl tetrazolium (MTT) method. The accumulation of lipid droplets in the cytoplasm of differentiated preadipocytes was observed by oil red O staining and quantitatively analyzed by colorimetry. The mRNA expressions of peroxisome proliferation activated receptor y (PPAR gamma) and CAAT/enhancer binding protein (C/EBP alpha) were detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSIntervention with serum containing HJD, Phellodendron amurense Rupr plus Gardenia jasminoides Ellis, or Gardenia jasminoides Ellis showed significantly stimulative effects on the proliferation of preadipocytes, as compared with that in the blank control group (P<0.05, P<0.01). The preadipocytes treated with serum containing HJD, Phellodendron amurense Rupr plus Gardenia jasminoides Ellis, Coptis chinensis Franch or Gardenia jasminoides Ellis showed that the lipid droplets in the cytoplasm were significantly lessened, so did the mRNA expressions of PPAR gamma and C/EBP alpha when compared with the blank control group (P<0.05, P<0.01).

CONCLUSIONSHJD promoted the proliferation of preadipocytes, decreased the accumulation of lipid droplets during the differentiation of adipocytes, and inhibited the differentiation of adipocytes, which might be associated with its effects on decreasing the mRNA expressions of PPAR gamma and C/EBP alpha. Phellodendron amurense Rupr and Gardenia jasminoides Ellis were the main components of HJD playing these roles.

3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Mice ; PPAR gamma ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum