OBJECTIVETo investigate the effects of Hedan Tablet () on serum lipid profile, proprotein convertase subtilisin/kexin type 9 (PSCK9) and high-density lipoprotein (HDL) subfractions in patients with hyperlipidemia.

METHODSThirty-seven patients with hyperlipidemia were randomized to treatment with Hedan Tablet 4.38 g/day as Hedan group (18 cases) or placebo (19 cases) as control group for 8 weeks. The lipid profile, PCSK9 and HDL subfractions were determined at day 0 and week 8 in both groups respectively.

RESULTSHedan treatment for 8 weeks mildly decreased serum low-density lipoprotein cholesterol (LDL-C) levels, while no changes were found in total cholesterol (TC), triglycerides (TG) and PCSK9 concentrations. Furthermore, Hedan treatment increased the concentration of large high-density lipoprotein cholesterol (HDL-C) and the percentage of large HDL subfraction, while decreased the concentration of small HDL-C and the percentage of small HDL subfraction without changing serum HDL-C levels in patients with hyperlipidemia.

CONCLUSIONHedan treatment of 4.38 g per day for 8 weeks could confer a favorable effects on serum LDL-C concentration as well as HDL subfractions.

Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Hyperlipidemias ; blood ; drug therapy ; enzymology ; Lipoproteins, HDL ; blood ; Male ; Middle Aged ; Proprotein Convertase 9 ; metabolism ; Subcellular Fractions ; metabolism

The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.
Antigens, Neoplasm ; metabolism ; Cell Line, Tumor ; Exosome Multienzyme Ribonuclease Complex ; metabolism ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; metabolism ; Neoplasm Proteins ; metabolism ; Protein Interaction Mapping ; RNA-Binding Proteins ; metabolism ; Subcellular Fractions ; metabolism ; WT1 Proteins ; metabolism

Arginine methylation has been implicated in the signal transduction pathway leading to cell growth. Here we show that a regenerating rat liver following partial hepatectomy exhibited elevated methyltransferase activity as shown by increased methylation of a subset of endogenous proteins in vitro. The 20-kDa protein was shown to be a major cytosolic protein undergoing methylation in regenerating hepatocytes. Methylation of the 20-kDa protein peaked at 1 d following partial hepatectomy, which gradually declined to a basal level within the next 14 d. Likewise, methylation of exogenously added bulk histones followed the similar time kinetics as the 20-kDa protein, reflecting time-dependent changes in methyltransferase activity in regenerating hepatocytes. Presence of exogenously added bulk histone in the in vitro methylation assay resulted in dose-dependent inhibition of methylation of the 20-kDa protein. All the histone subtypes tested, histone 1, 2A, 2B, 3 or 4, were able to inhibit methylation of the 20-kDa protein while addition of cytochrome C, a-lactalbumin, carbonic anhydrase, bovine serum albumin, and g globulin minimally affected methylation of the 20-kDa protein. Since methylation of the 20-kDa protein preceded proliferation of hepatocytes upon partial hepatectomy, it is tempting to speculate that the methylated 20-kDa protein by activated histone-specific methyltransferase may be involved in an early signal critical for liver regeneration.
Animals ; Cytoplasm/*chemistry ; *Hepatectomy ; Histones/metabolism ; Humans ; Liver Regeneration/*physiology ; Methylation ; Methyltransferases/metabolism ; Protein Isoforms/metabolism ; Proteins/*metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Signal Transduction/physiology ; Subcellular Fractions/chemistry/metabolism

OBJECTIVETo investigate the effects of drug-containing serum of Chinese herbal compound, Xiongshao Capsule (, XS, for activating-blood) and Huanglian Capsule (, HL, for dispellingtoxin) on the oxidized low-density lipoprotein (ox-LDL)-induced inflammatory factors in human umbilical vein endothelial cells (HUVECs).

METHODSThirty-two rats were randomly divided into four groups: the blank control group treated with distilled water, the positive control group treated with simvastatin (1.8 mg/kg), the test group I treated with Chinese herbal compound of XS (0.135 g/kg), and the test group II treated with Chinese herbal compound of XS (0.135 g/kg) and HL (0.135 g/kg). All the treatments were administered for 7 successive days by gastrogavage. Rats' blood serum was harvested 1 h after the last administration to prepare respective drugcontaining serum. HUVECs were exposed to ox-LDL (100 μg/mL) to induce cell injury model and incubated with corresponding drug-containing serum for 24 h. Untreated HUVECs were set for blank control. Levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and soluble intercellular adhesion molecule-1 (sICAM-1) in supernatant of cultured HUVECs were determined by enzyme-linked immunosorbent assay (ELISA). HUVEC surface expressions of ICAM-1 and E-selectin were determined by flow cytometry.

RESULTSLevels of IL-6, TNF-α, and sICAM-1 in the supernatant of HUVECs as well as the cell surface expressions of ICAM-1 and E-selectin significantly increased after 24-h ox-LDL stimulation (P<0.01), while the abnormal elevations, except sICAM-1 in the test group I, were all reduced in the treated groups (the positive control and the two test groups) significantly (P<0.01 or P<0.05). Besides, the effect in the test group II seemed somewhat higher than that in the test group I but with no statistical significance (P>0.05).

CONCLUSIONDrug-containing serum of XS plus HL has a certain inhibitory effect on the vascular endothelial inflammation response induced by ox-LDL.

Animals ; Capsules ; Cell Membrane ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; E-Selectin ; metabolism ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Inflammation Mediators ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-6 ; metabolism ; Lipoproteins, LDL ; metabolism ; Rats ; Rats, Wistar ; Solubility ; drug effects ; Subcellular Fractions ; drug effects ; metabolism ; Toxins, Biological ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Genetic transformation was adopted to analyze the subcellular localization and the resistance to fungal pathogens of Arabidopsis lipid transfer protein AtDHyPRP1. The coding sequence of AtDHyPRP1 amplified by PCR from Ws ecotype was used to construct the plant binary expression vector pRI101-AN-AtDHyPRP1 and the fusion expression vector pCAMBIA1302-AtDHyPRP1-GFP. Transgenic tobacco and Arabidopsis plants were produced by leaf disc and floral dip protocols, respectively. AtDHyPRP1 could improve the resistance of tobacco to Botrytis cinerea remarkably and the infection sites on transgenic tobacco leaves accumulated large amounts of H2O2. Observation under laser scanning confocal microscope showed that AtDHyPRP1 was localized to cell surface. It suggested that AtDHyPRP1 might play special function after secretion to outside of the cell and was involved in plant defense system against pathogens.
Amino Acid Sequence ; Antigens, Plant ; genetics ; metabolism ; Arabidopsis ; genetics ; metabolism ; microbiology ; Arabidopsis Proteins ; genetics ; metabolism ; Botrytis ; Carrier Proteins ; genetics ; metabolism ; Disease Resistance ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Plant Diseases ; immunology ; microbiology ; Plant Proteins ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; microbiology ; Recombinant Proteins ; genetics ; metabolism ; Subcellular Fractions ; metabolism ; Tobacco ; genetics ; metabolism ; microbiology

OBJECTIVETo investigate the role of the TLR4-NFκB-TNFα inflammation pathway on: lipopolysaccharide (LPS)-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B (Sal B).

METHODSWistar rat (1-2 days old) cardiomyocytes were isolated and cultured. Sal B 10(-5)mol/L, 10(-6)mol/L and 10(-7)mol/L were pre-treated for 6 h in the culture medium. LPS (1 μg/mL) was added to mol/the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase (LDH) in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were detected by immunohistochemistry. The mRNA expressions of TLR4 and NFκB were detected by real-realtime reverse transcription polymerase chain reaction (RT-PCR).

RESULTS(1) The concentrations of LDH and: TNFα in the LPS control group were significantly higher than those in the control group (561.41±67.39 U/L and 77.94±15.08 pg/mL, versus 292.13±26.02 U/L and 25.39±16.53 pg/mL, respectively, P<0.01, P<0.05). Compared with the LPS control group, the concentrations of LDH and TNFα were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (451.76±83.96 U/L and 34.00±10.38 pg/mL, respectively, P<0.05). (2) The TLR4 and NFκB protein expression area in the LPS control group were significantly higher than those in the control group (1712.41±410.12 μm(2) and 2378.15±175.29 μm(2), versus 418.62±24.42 μm(2) and 1721.74±202.87 μm(2), respectively, P<0.01). The TLR4 and NFκB protein expression internal optical density (IOD) values in the LPS control group were also significantly higher than those in the control group (3.06±0.33 and 7.20±1.04, versus 0.91±0.21 and 4.24±0.48, respectively, P<0.05 and P<0.01). Compared with the LPS control group, the TLR4 and NFκB protein expression areas were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (1251.54±133.82 μm(2) and 1996.37±256.67 μm(2), respectively, P<0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.92±0.28 and 5.17±0.77, respectively, treated P<0.05). (3) The TLR4 and NFκB mRNA expressions (2(-ΔΔ)CT value) in the LPS control group were significantly higher than those in the control group (3.16±0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P<0.01). Compared with the LPS control group, the TLR4 and NFκB mRNA expressions (2(-ΔΔ) -CT value) were significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.34±0.22 and 1.74±0.26, respectively, treated P<0.05). The concentration of HSP70 did not show any 0.05).

CONCLUSIONSThe TLR4-NFκB-TNFα pathway was quickly activated: and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NFκB-TNFα pathway and are dose-dependent.

Animals ; Animals, Newborn ; Benzofurans ; chemistry ; pharmacology ; Gene Expression Regulation ; drug effects ; HSP70 Heat-Shock Proteins ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; NF-kappa B ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; enzymology ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription, Genetic ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.
Acetylglucosamine/*analogs & derivatives/metabolism/pharmacology ; Adipocytes/*metabolism ; Animals ; Deoxyglucose/pharmacokinetics ; Glycosylation ; Immunoprecipitation ; *Insulin Resistance ; Male ; Monosaccharide Transport Proteins/metabolism ; Oximes/*pharmacology ; Phenylcarbamates/*pharmacology ; Phosphoproteins/*metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/*metabolism ; Proto-Oncogene Proteins/*metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Subcellular Fractions/metabolism ; beta-N-Acetylhexosaminidase/antagonists & inhibitors

OBJECTIVETo observe the effects of Yinian Jiangya Decoction (YNJYD) on cytokine secretion in spontaneoulsy hypertensive rats (SHRs) vascular endothelium.

METHODSAortic endothelial cells (ECs) were primarily cultured from SHRs; male SD rats were treated with different doses (high, medium, and low doses) of YNJYD, the blood was collected on the 21st day, and then, the serum was separated. ECs were cocultured with the serum for different time courses, and the culture supernatant concentrations of endothelin (ET)-1, nitric oxide (NO), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1) were determined by ABC-ELISA methods.

RESULTSET-1, NO, t-PA, and PAI-1 levels in endothelial cell culture supernatant were increased in a time-dependent manner; YNJYD could significantly elevate NO and t-PA expressions in ECs, while ET-1 and PAI-1 expressions were dramatically decreased; these effects of YNJYD were in a concentration-dependent manner.

CONCLUSIONThe therapeutic effect of YNJYD on hypertension is attributed to its effect on regulating vessel dilation and blood coagulation, in which ET-1/NO and PAI-1/t-PA are two pairs of pivotal mediators.

Animals ; Cytokines ; secretion ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Endothelin-1 ; metabolism ; Endothelium, Vascular ; drug effects ; secretion ; Male ; Nitric Oxide ; metabolism ; Plasminogen Activator Inhibitor 1 ; metabolism ; Rats ; Rats, Inbred SHR ; Subcellular Fractions ; drug effects ; metabolism ; Time Factors ; Tissue Plasminogen Activator ; metabolism

OBJECTIVETo study the experimental efficacy of Qushi Huayu Decoction (祛湿化瘀方,QHD) on, protein and gene expression of cathepsin B (ctsb) in HepG2 cells induced by free fatty acids (FFAs).

METHODSThe model of HepG2 steatosis and tumor necrosis factor-α (TNF-α) secretion was induced by long-chain FFAs. HepG2 cells were divided into 4 groups: control group (group C), model group (group M), low-dose QHD group (group L) and high-dose QHD group (group H). Long-chain FFAs were added to groups M, L and H. The 10% blank-control serum was added to group C and M, while 5% and 10% QHD-containing sera were added to group L and H, respectively. The levels of serum TNF-α and cellular triglyceride (TG) were detected. Cellular p-IκB and ctsb expression were detected using Western blot and PCR. The expression and distribution of ctsb were observed by immunofluorescence.

RESULTSAfter incubating with FFA for 24 h, TG deposition in HepG2, TNF-α content in cell supernatant, the protein expression of cellular ctsb and P-IκB, as well as mRNA expression of ctsb increased markedly in group M compared with group C (P<0.05, P<0.01). Compared with group M, TG deposition, the expression of cellular ctsb, P-IκB and ctsb mRNA in groups L and H, as well as TNF-α content in group H, decreased significantly (P<0.05). Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA. In this study, these above-mentioned changes were inhibited markedly in groups L and H.

CONCLUSIONQHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNFα pathway of hepatic lipotoxicity.

Cathepsin B ; genetics ; metabolism ; Cell Death ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fatty Acids ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Hep G2 Cells ; Humans ; I-kappa B Proteins ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; NF-KappaB Inhibitor alpha ; RNA, Messenger ; genetics ; metabolism ; Subcellular Fractions ; drug effects ; enzymology ; Triglycerides ; metabolism ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs.

METHODSDCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis.

RESULTSThe proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P <0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P <0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P <0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P <0.05). However, there was no significant difference between the two CM groups (P >0.05).

CONCLUSIONSTwo kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.

Carcinoma, Hepatocellular ; pathology ; ultrastructure ; Cell Line, Tumor ; Cell Shape ; Dendritic Cells ; drug effects ; immunology ; Glucans ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Liver Neoplasms ; pathology ; ultrastructure ; Lymphocyte Culture Test, Mixed ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; Transcription Factor RelA ; metabolism