A stable hepatoma cell line(Hep G2 cell) insulin resistance model was established and used to analyze the effect of effective components of Mori Folium in alleviating insulin resistance,and preliminary explore the mechanism for alleviating insulin resistance. The Hep G2 insulin action concentration and the duration of action were investigated using the glucose oxidase method(GOD-POD method) to establish a stable Hep G2 insulin resistance model. Normal control group,model group,Mori Folium polysaccharide group,Mori Folium flavonoid group and rosiglitazone group were divided to determine the glucose consumption. The effect of Mori Folium effective components on Hep G2 insulin resistance was analyzed. The mRNA expressions of JNK,IRS-1 and PDX-1 in each group were detected by Real-time quantitative PCR(qRT-PCR). The protein expressions of p-JNK,IRS-1 and PDX-1 were detected by Western blot. And the mechanism of effective components of Mori Folium in alleviating insulin resistance was investigated. The results showed that the glucose consumption was significantly decreased in the insulin resistance cells after incubation with 25. 0 mg·L-1 insulin for 36 h(P<0. 01),and the model was relatively stable within 36 h. Mori Folium polysaccharides and flavonoids all alleviated insulin resistance,among which Mori Folium flavonoids had better effect in alleviating Hep G2 insulin resistance(P<0. 05). The qRT-PCR analysis showed that Mori Folium polysaccharides and flavonoids could inhibit JNK and IRS-1 mRNA expressions,while enhancing PDX-1 mRNA expression. Western blot analysis displayed that Mori Folium polysaccharides and flavonoids could inhibit p-JNK and IRS-1 protein expressions,while enhancing PDX-1 protein expression. Mori Folium polysaccharides and flavonoids can alleviate insulin resistance in Hep G2 cells,and its mechanism may be the alleviation of insulin resistance by inhibiting JNK signaling pathway.
Drugs, Chinese Herbal ; pharmacology ; Glucose ; Hep G2 Cells ; Homeodomain Proteins ; metabolism ; Humans ; Insulin ; Insulin Receptor Substrate Proteins ; metabolism ; Insulin Resistance ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; Morus ; chemistry ; Plant Leaves ; chemistry ; Trans-Activators ; metabolism

OBJECTIVE: To investigate the effect of a modified Wuzi Yanzong Pill (, WZYZP) on the male rats' testis after microwave radiation, as well as its potential mechanism. METHODS: Forty-five male rats were randomly assigned to three groups: the control group, the radiation group, and the WZYZP group. The rats in the radiation group and WZYZP group were exposed to microwave radiation for 15 min once, while the rats in the control group were not exposed to any radiation. The rats in the WZYZP group were given a modified of WZYZP by gavage daily for 7 days. Apoptosis in the testis was evaluated using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. Histopathological alterations of the testis were observed by haematoxylin-eosin (HE) staining. Tat-interactive protein, 60kD (Tip60) and p53 expressions were determined by Western blotting. RESULTS: The apoptosis index (AI) in the radiation group was higher than that of the WZYZP group and control group on day 1 (D1), day 7 (D7) day 14 (D14) after radiation (P<0.05). The seminiferous tubules were of normal morphology in the control group. In the radiation group, the partial seminiferous tubules were collapsed, basement membranes of the seminiferous epithelia became detached. WZYZP could restore the morphological changes. There was no expression of Tip60 among the three groups on D7 and D14. The expression of p53 was higher in the radiation group than in the control group (P<0.05). WZYZP could down-regulate the rising p53 induced by radiation on D7 and D14 (P<0.05). CONCLUSION A modified WZYZP may affect germ cells, and its protective effects may partly result from its ability to intervene in Tip60 mediated apoptosis.
Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Male ; Microwaves ; Rats, Wistar ; Testis ; drug effects ; metabolism ; pathology ; radiation effects ; Trans-Activators ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the effect of platelet-derived growth factor-BB (PDGFBBB) on rat corpus cavernosum smooth muscle (CCSM) cell proliferation, migration and phenotypic modulation and explore the underlying mechanisms.

METHODSWistar rat CCSM cells were obtained through a modified tissue culture method and identified by immunofluorescence assay. The effect of PDGFBB on the proliferation of CCSM cells was investigated using a CCK-8 kit and the optimum PDGFBB concentration for cell treatment was determined. CCSM cells were treated with vehicle or PDGF-BB at the optimum concentration, and the cell migration was examined using scratch assay; the mRNA expression of the transcription factor myocardin and the contractile phenotype markers αSMA and SMMHC in CCSM cells were determined by qRT-PCR at 24 h and 48 h. The protein expression of myocardin in CCSM cells incubated with PDGFBB for 0, 24 and 48 h was examined by Western blotting.

RESULTIn CCSM cell culture, 96.5%and 96% of the cells were positive for αSMA and smoothelin, respectively. PDGFBB at different concentrations markedly promoted the proliferation of CCSM cells; the optimum PDGFBB concentration for enhancing cell proliferation was 12.5 ng/mL, which induced the migration of CCSM cells and significantly reduced the mRNA expressions of myocardin, αSMA and SMMHC (P<0.01). Exposure to PDGFBB decreased the protein expression of myocardin as the exposure time extended (within 48 h).

CONCLUSIONCCSM cells of a high purity can be obtained by the modified tissue culture method. PDGFBB can promote the proliferation and migration of CCSM cells and cause a phenotypic conversion from the contractile to the synthetic type possibly by down-regulating myocardin.

Actins ; metabolism ; Animals ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Down-Regulation ; Male ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Myosin Heavy Chains ; metabolism ; Nuclear Proteins ; metabolism ; Penis ; cytology ; Phenotype ; Proto-Oncogene Proteins c-sis ; pharmacology ; RNA, Messenger ; Rats ; Rats, Wistar ; Trans-Activators ; metabolism

OBJECTIVETo explore the inhibition effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on myofibroblast differentiation of MRC-5 human fetal lung fibroblasts induced by angiotensin (Ang) II.

METHODSThe study was divided into 2 step: (1) MRC-5 human fetal lung fibroblasts was induced for 48 h at different dose of Ang II and at different time point by 100 nmol/L Ang II. Then the expression of collagen type I and α-smooth muscle actin (α-SMA) were mesaured by western blot. (2) MRC-5 human fetal lung fibroblasts were divided into 4 group: (1) control, (2) Ang II, (3) Ang II+Ac-SDKP, (4) Ang II+8-Me-cAMP (a specific activator of Epac). The α-SMA expression was observed by immnocytochemical stain. The protein expression of collagen type I, α-SMA, serum response factor (SRF), myocardin-related transcription factor (MRTF)-A, exchange protein directly activated by cAMP (Epac) 1, 2 were measured by Westen blot.

RESULTSMyofibroblast differentiation could be induced by Ang II from MRC-5 cells with a dose- and time-dependent manner. The up-regulation of SRF and MRTF-A were observed in MRC-5 cells induced by Ang II and accompanied with collagen I and α-SMA increased. Pre-treatment with 8-Me-cAMP or Ac-SDKP could attenuated all this changes induced by Ang II, and promoted the expression of Epac1.

CONCLUSIONAc-SDKP can inhibit the myofibroblast differentiation of MRC-5 cells induced by Ang II via Epac1 activating.

Actins ; Angiotensin II ; Cell Differentiation ; drug effects ; Collagen ; Collagen Type I ; Cyclic AMP ; analogs & derivatives ; Fetus ; cytology ; Fibroblasts ; cytology ; Guanine Nucleotide Exchange Factors ; Humans ; Lung ; cytology ; Myofibroblasts ; drug effects ; Oligopeptides ; pharmacology ; Serum Response Factor ; Trans-Activators

OBJECTIVETo explore the involvement of hepatitis B X protein (HBx) in promoter 3 (P3)-driven mRNA overexpression of the insulin-like growth factor II gene (IGF-II) and investigate the underlying epigenetic mechanism.

METHODSLevels of P3 and HBx mRNA and status of P3 methylation were analyzed in human hepatocellular carcinoma (HCC) samples, with and without hepatitis B virus (HBV) infection, using quantitative reverse transcription-PCR and bisulfite sequencing. In addition, the levels of P3 mRNA and P3 methylation were examined in HepG2 cells stably overexpressing HBx (HepG2-HBx). Finally, P3 promoter-luciferase constructs were cotransfected into HepG2 cells along with an HBx-expressing plasmid, and the effects of HBx on transcriptional activity and methylation of P3 were analyzed. Statistical analyses of the data were conducted by chi square test, Fisher's exact test, Student's t-test, Marn-Whitney U test, and Pearson's correlation coefficient test.

RESULTSThe HBV-positive HCC specimens had significantly higher levels of P3 mRNA than the HBV-negative HCC specimens (-9.59 ± 3.22 vs. -12.97 ± 3.08 delta CT; P=0.006) but significantly lower levels of P3 methylation (mean values for the 17 CpG sites (36.9% ± 15.5% vs. 52.1% ± 19.1%; P=0.025). The P3 transcript abundance was positively correlated with the level of HBx expression and negatively correlated with the level of P3 methylation. The epigenetic results from experiments with the HepG2-HBx cells were similar. Transfection of HBx significantly decreased P3 methylation level and increased its activity.

CONCLUSIONHBx expression may promote IGF-II expression by inducing hypomethylation of its P3 promoter in hepatocellular carcinoma.

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; Epigenesis, Genetic ; Female ; Gene Expression ; Hep G2 Cells ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; Male ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Trans-Activators ; pharmacology

OBJECTIVETo investigate the effect of Pseudomonas quinolone signal (PQS) on the virulence of Pseudomonas aeruginosa.

METHODSPseudomonas aeruginosa strain PAO1 was treated with PQS alone, PQS plus farnesol, or farnesol alone. The transcriptional levels of the regulator gene ExsA and virulence protein gene ExoS of type III secretion system were examined using quantitative real-time PCR, and spectrophotometry was employed to detect pyocyanin production in the bacteria. The adhesion and invasiveness of the treated PAO1 in cultured alveolar epithelial cells A549 were assessed on plate count agar, and their effects on the survival of a mouse model of peritonitis was compared.

RESULTSThe increase or decrease of PQS did not affect the growth of PAO1. Compared with the untreated bacteria, PQS-treated PAO1 showed obviously increased transcription levels of ExsA and ExoS (P<0.01) and pyocyanin production, which was significantly lowered by farnesol (P<0.01). In A549 cell cultures, farnesol-treated PAO1 exhibited significantly lowered adhesion and invasiveness, while PQS-treated PAO1 caused a significantly decreased survival time of mice with peritonitis (P<0.01). Farnesol treatment did not obviously affected ExsA transcription (P>0.05) but caused a significant reduction in the transcriptional level of Exos (P<0.05) in PAO1. PQS showed no significant effect on the adhesion and invasiveness of PAO1 (P<0.05).

CONCLUSIONPQS can maintain the adhesion and invasiveness of Pseudomonas aeruginosa, and in the hosts of the bacteria, PQS concentration is positively correlated with pyocyanin production and hence negatively with the survival time of the hosts.

ADP Ribose Transferases ; genetics ; metabolism ; Animals ; Bacterial Adhesion ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Cell Line ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Peritonitis ; microbiology ; Pseudomonas aeruginosa ; genetics ; metabolism ; pathogenicity ; Quinolones ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Transcription, Genetic ; Virulence

OBJECTIVETo investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma.

METHODSA human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting.

RESULTSThe HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR.

CONCLUSIONHBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Genes, p16 ; Hep G2 Cells ; Hepatitis B virus ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic ; Trans-Activators ; pharmacology

Glucagon-like peptide-1 (GLP-1) is a potent glucoincretin hormone and an important agent for the treatment of type 2 diabetes. Here we demonstrate that B-cell translocation gene 2 (BTG2) is a crucial regulator in GLP-1-induced insulin gene expression and insulin secretion via upregulation of pancreatic duodenal homeobox-1 (PDX-1) in pancreatic beta-cells. GLP-1 treatment significantly increased BTG2, PDX-1 and insulin gene expression in pancreatic beta-cells. Notably, adenovirus-mediated overexpression of BTG2 significantly elevated insulin secretion, as well as insulin and PDX-1 gene expression. Physical interaction studies showed that BTG2 is associated with increased PDX-1 occupancy on the insulin gene promoter via a direct interaction with PDX-1. Exendin-4 (Ex-4), a GLP-1 agonist, and GLP-1 in pancreatic beta-cells increased insulin secretion through the BTG2-PDX-1-insulin pathway, which was blocked by endogenous BTG2 knockdown using a BTG2 small interfering RNA knockdown system. Finally, we revealed that Ex-4 and GLP-1 significantly elevated insulin secretion via upregulation of the BTG2-PDX-1 axis in pancreatic islets, and this phenomenon was abolished by endogenous BTG2 knockdown. Collectively, our current study provides a novel molecular mechanism by which GLP-1 positively regulates insulin gene expression via BTG2, suggesting that BTG2 has a key function in insulin secretion in pancreatic beta-cells.
Animals ; Gene Expression Regulation/drug effects ; Glucagon-Like Peptide 1/*pharmacology ; Homeodomain Proteins/*genetics/metabolism ; Humans ; Immediate-Early Proteins/genetics/*metabolism ; Insulin/genetics/*secretion ; Insulin-Secreting Cells/drug effects/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Peptides/pharmacology ; Promoter Regions, Genetic/genetics ; Protein Binding/drug effects/genetics ; Rats ; Trans-Activators/*genetics/metabolism ; Tumor Suppressor Proteins/genetics/*metabolism ; Venoms/pharmacology

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.
Animals ; Biological Markers/metabolism ; *Cell Culture Techniques ; *Cell Differentiation ; Cell Proliferation/drug effects ; Cell Separation ; Cells, Cultured ; Dermis/*cytology/drug effects ; Diabetes Mellitus, Experimental/*surgery ; Female ; Fibroblasts/*cytology/drug effects ; Genitalia, Female/*cytology ; Glucose/metabolism ; Hepatocyte Nuclear Factor 3-beta/metabolism ; Homeodomain Proteins/metabolism ; Humans ; Insulin/pharmacology/secretion ; Insulin-Secreting Cells/*cytology/metabolism ; *Islets of Langerhans Transplantation ; Mesenchymal Stem Cells/*cytology/drug effects/metabolism ; Mice ; Mice, Nude ; Niacinamide/pharmacology ; Recovery of Function ; SOXF Transcription Factors/metabolism ; Sodium Selenite/pharmacology ; Trans-Activators/metabolism ; Transferrin/pharmacology

Six transmembrane protein of prostate 2 (STAMP2) plays a key role in linking inflammatory and diet-derived signals to systemic metabolism. STAMP2 is induced by nutrients/feeding as well as by cytokines such as TNFalpha, IL-1beta, and IL-6. Here, we demonstrated that STAMP2 protein physically interacts with and decreases the stability of hepatitis B virus X protein (HBx), thereby counteracting HBx-induced hepatic lipid accumulation and insulin resistance. STAMP2 suppressed the HBx-mediated transcription of lipogenic and adipogenic genes. Furthermore, STAMP2 prevented HBx-induced degradation of IRS1 protein, which mediates hepatic insulin signaling, as well as restored insulin-mediated inhibition of gluconeogenic enzyme expression, which are gluconeogenic genes. We also demonstrated reciprocal expression of HBx and STAMP2 in HBx transgenic mice. These results suggest that hepatic STAMP2 antagonizes HBx-mediated hepatocyte dysfunction, thereby protecting hepatocytes from HBV gene expression.
Animals ; Female ; Gene Expression ; Gluconeogenesis/genetics ; Hep G2 Cells ; Humans ; Insulin/pharmacology/physiology ; Insulin Receptor Substrate Proteins/genetics/metabolism ; Insulin Resistance ; *Lipid Metabolism ; Liver/*metabolism/physiopathology ; Male ; Membrane Proteins/metabolism/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Mice, Transgenic ; Oxidoreductases/metabolism/*physiology ; Phosphorylation ; Protein Binding ; Protein Processing, Post-Translational ; Proteolysis ; Receptor, Insulin/metabolism ; Trans-Activators/*physiology ; Transcriptional Activation