[Effects of a new houttuyfonate derivative on proliferation of NIH3T3 cell and expression of syndecan-4 induced by tumor necrosis factor alpha].

OBJECTIVE: To investigate the effect of a new houttuyfonate derivative (NHD) on proliferation of NIH3T3 cell and expression of Syndecan-4 induced by TNF-alpha in vitro. METHODS: NIH3T3 cells were cultured and exposed to TNF-alpha or NHD respectively, and then cotreated with TNF-alpha and NHD. All the groups were cultured for 24 hour in vitro, in addition to the untreated control group established for comparison. The ratio of proliferation of NIH3T3 cell was determined by non-radioactive MTS/PMS assay and the expression of Syndecan-4 was evaluated by western blot using anti-Syndecan-4 antibody. RESULTS: Statistical analysis showed that, compared with the control group, NHD had no effect on VSMCs growth, but significantly inhibited NIH3T3 cell proliferation while induced by TNF-alpha. It also showed that compared with control group, NHD had no effect on the expression of Syndecan-4, but significantly inhibited its expression while induced by TNF-alpha (P < 0.05). CONCLUSIONS: NHD can inhibit the proliferation of NIH3T3 cell and the expression of syndecan-4 protein induced by TNF-alpha in vitro.

[Effects of flavone from leaves of Diospyros kaki on expression of apoptosis signal-regulating kinase 1 and rat vascular smooth muscle cells proliferation by tumor necrosis factor alpha in vitro].

OBJECTIVE: To observe the effects of flavone from leaves of Diospyros kaki on expression of apoptosis signal-regulating kinase 1 (ASK1) and rat vascular smooth muscle cells (VSMCs) proliferation by tumor necrosis factor alpha in vitro. METHODS: Rat aortic VSMCs were cultured in vitro and treated with tumor necrosis factor alpha (TNF-alpha) and flavone from leaves of Diospyros kaki, respectively, and were observed in comparison with the control group. The ratio of cell proliferation was determined by non-radioactive MTS/PES as-say. The expression of ASK1 protein was evaluated by the immunoblotting technique using anti-ASKL antibody. RESULTS: The ratio of cell proliferation was 0.817 +/- 0.074 in the control group, and was 1.865 +/- 0.093 in TNF-alpha20 ng/ml group. It was shown that TNF-alpha significantly induced rat VSMCs proliferation (P < 0.05). The ratio of cell proliferatioh was 0.905 +/- 0.044 in flavone from leaves of Diospyros kaki group corresponding to concentration of 50 microg/ml. It was shown that flavone from leaves of Diospyros kaki alone had no effect on rat VSMCs proliferation (p > 0.05). With TNF-alpha stimulation, flavone from leaves of Diospyros kaki significantly inhibited rat expression of ASK1 protein enhanced by TNF-alpha was significantly inhibited rat-VSMNCs proliferation (1.247 +/- 0.061 vs. 1.865 +/- 0.093, p < 0.05). The expression of ASK1 protein enhanced by TNF-alpha was significantly inhibited by flavone from leaves of Diospyros kaki in VSMCs. CONCLUSION: Flavone from leaves of Diospyros kaki can significantly inhibit expression of ASK1 protein stimulated by TNF-alpha of rat VSMcs in vitro.

[Green tea polyphenols inhibit low-density lipoprotein-induced proliferation of rat vascular smooth muscle cells].

The proliferation and migration of vascular smooth muscle cells (VSMCs) is one of the major mechanisms of intimal thickening in atherosclerosis and post-angioplasty restenosis. Elevated plasma levels of low-density lipoprotein (LDL) have been implicated in the pathogenesis of atherosclerotic vascular diseases. The purpose of this study was to determine the effects of green tea polyphenols on the proliferation and p44/42 mitogen-activated protein kinase (MAPK) activity in rat VSMCs simulated by native LDL. Rat aortic VSMCs were cultured and treated with LDL (100 microg/ml) in the absence or presence of green tea polyphenols, and the cell proliferation was subsequently quantified by non-radioactive MTS/PES assay and the cell cycle analyzed by flow cytometry. The p44/42 MAPK activity was evaluated by immunoblotting using anti-p44/42 phospho-MAPK antibody. Compared with the cells without polyphenol treatment, the proliferation of the VSMCs induced by LDL was dose-dependently inhibited by green tea polyphenols (P<0.05), with more numerous cells in G(0)G(1) phase (P<0.05) as shown by flow cytometry analysis. LDL significantly enhanced the p44/42 MAPK activity, an effect obviously inhibited by green tea polyphenols (at 100 microg/ml). These results suggest that green tea polyphenols can inhibit high levels of LDL-induced proliferation of phosphorylated p44/42 MAPK expression in rat VSMCs. Green tea polyphenols may, therefore, offer vascular protection by inhibiting VSMC growth in response to hypercholesterolemia.

A comparative molecular field analysis of cytotoxic beta-carboline analogs.

AIM: To derive a model that could be used in drug design. METHODS: Beta-carbolines are reported to have antitumor activities on cultured cancer cell lines. A comparative molecular field analysis (CoMFA) was undertaken to elucidate the correlation of cytotoxities and structural parameters of 16 beta-carboline analogs (1-16). The compound 12 was finally used as a template for the other compounds in the dataset because of its highest biological activity. RESULTS: The CoMFA applied to the final alignment resulted in a q2(cv) of 0.656 and it showed that the steric fields contributed 43.3% of the model information while the electrostatic fields represented the other 56.7%. CONCLUSION: Three designed compounds, which were predicted to have high, moderate and low activities respectively, were synthesized. The IC50 values of these compounds indicated the significance of the analysis in this study. The model derived from the current study could be further used in design for more active compounds.

Green tea polyphenols inhibit advanced glycation end product-induced rat vascular smooth muscle cell proliferation.

OBJECTIVE: To determine the effects of green tea polyphenols(GTP) on advanced glycation end products (AGEs)-induced proliferation and expression of p44/42 mitogen-activated protein kinase (MAPK) of rat vascular smooth muscle cells (VSMCs) METHODS: Rat aortic VSMCs isolated and cultured in vitro were stimulated with AGEs in the presence or absence of GTP at different concentrations, followed by quantitative analysis of the cell proliferation with colorimetric assay. The p44/42 MAPK activity was evaluated by immunoblotting technique using anti-p44/42 phospho-MAPK antibody. RESULTS: Compared with the control cells(without GTP treatment), GTP dose-dependently inhibited AGE-stimulated VSMC proliferation (P<0.05), and the p44/42 MAPK activity was significantly enhanced. The effects of AGEs were antagonized by GTP (372+/-41 vs 761+/-56, P<0.05). CONCLUSION: GTP can inhibit the AGE-induced proliferation and p44/42 MAPK expression of rat VSMCs.

[Effects of flavone from leaves of Diospyros kaki on adventitial fibroblast proliferation induced by advanced glycation end-products in vitro].

OBJECTIVE: To observe the effects of flavone from the leaves of Diospyros kaki on proliferation of adventitial fibroblasts induced by advanced glycation end-products (AGEs) in vitro. METHODS: NIH/3T3 cells cultured in vitro were treated with both AGEs and flavone from the leaves of Diospyros Kaki for observation in comparison with the cells that received treatments with either AGEs or flavone, or neither. The ratio of cell proliferation was determined by non-radioactive MTS/PES assay. RESULTS: The ratio of cell proliferation was 0.840+/-0.061 in the untreated control group, and was 1.330+/-0.055, 1.210+/-0.119, 1.029+/-0.076 and 0.792+/-0.060 in AGEs groups corresponding to AGE concentrations of 100, 50, 10 and 1 microg/ml respectively. AGEs significantly induced fibroblast proliferation in a dose-dependent manner when the concentration was above 10 microg/ml (P<0.05), as compared with the untreated control group. (P<0.05). The ratio of cell proliferation was 0.829+/-0.056 in cells treated with flavone at the concentration of 50 microg/ml, which alone failed to affect fibroblast proliferation (P>0.05). With AGEs stimulation, however, flavone from the leaves of Diospyros kaki significantly inhibited the proliferation of the fibroblasts (P<0.05). CONCLUSION: Flavone from the leaves of Diospyros kaki can significantly inhibit the proliferation of adventitial fibroblasts stimulated by AGEs in vitro.

In vitro effects of flavone from leaves of Diospyros kaki on rat cardiac myocyte apoptosis induced by hypoxia-reoxygenation and advanced glycation end products.

OBJECTIVE: To investigate the effects of flavone extracted from the leaves of Diospyros kaki on the apoptosis of rat cardiac myocytes induced by hypoxia-reoxygenation and advanced glycation end products in vitro. METHODS: The cardiac myocytes were isolated from neonatal SD rats and cultured in vitro for 72 h. Apoptosis of the cultured cells was induced by hypoxia-reoxygenation and advanced glycation end products (AGEs) respectively, and the effects of flavone extracted from the leaves of Diospyros kaki on the cell apoptosis was observed by measuring the apoptotic rates using flow cytometry. RESULTS: Apoptosis of neonatal rat cardiac myocytes was induced by hypoxia-reoxygenation in a time-dependent manner (6.05% +/-0.46% vs 12.45% +/-1.66%, P< 0.05), and flavone treatment of the cells significantly lowered the apoptotic rates. AGEs alone also induced apoptosis of neonatal rat cardiac myocytes (11.03+/-0.39 vs 6.25+/-0.48, P< 0.05), which was inhibited by flavone (8.40+/-0.47 vs 11.03+/-0.39, P < 0.05). CONCLUSION: Flavone extracted from the leaves of Diospyros kaki can inhibit the apoptosis of in vitro cultured neonatal rat cardiac myocytes induced by hypoxia-reoxygenation and advanced glycation end products.

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3',5'-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (), having 61.7% beta-sheet and no alpha-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD(50) of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5+/-160.5%. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.

Recombinant human interleukin-10 inhibits proliferation of vascular smooth muscle cells stimulated by advanced glycation end products and neointima hyperplasia after carotid injury in the rat.

The purposes of this study was to determine the effects of recombinant human interleukin-10 (rhIL-10) on proliferation of vascular smooth muscle cells (VSMCs) stimulated by advanced glycation end products (AGE) and neointima hyperplasia after rat carotid arterial injury. Rat aortic VSMCs were cultured and treated with rhIL-10 or AGE respectively, and then co-treated with rhIL-10 and AGE. Proliferation of VSMCs was quantified by colormetric assay. Cell cycle analysis was performed by flow cytomertry. Sprague-Dawley rats were treated with recombinant human IL-10 (rhIL-10) for 3 d after carotid arteries injury. The ratio of neointima to media area at the site of arterial injury was measured 28 d after balloon injury. The p44/42 MAPK activity was evaluated by the immunoblotting technique using anti-p44/42 phospho-MAPK antibody. Compared to control, AGE stimulated VSMCs proliferation. rhIL-10 alone had no effect on VSMCs growth. With AGE stimulation, rhIL-10, at dose as low as 10 ng/ml, inhibited VSMCs growth (P<0.05). The cell number in G(0)/G(1) phase of AGE and rhIL-10 co-treatment group was higher than that of AGE treatment alone (P<0.01) by flow cytometry analysis. Compared with the control group of neointima hyperplasia in rats, the ratio of neointima to media area of recombinant human IL-10 group was reduced by 45% (P<0.01). The p44/42 MAPK activity was significantly enhanced by AGE. The AGE effects were opposed by rhIL-10. The anti-inflammatory cytokine rhIL-10 inhibits AGE-induced VSMCs proliferation. Recombinant human IL-10 also inhibited neointima hyperplasia after carotid artery injury in rats. The results suggest the possibility that recombinant human IL-10, as a potential therapeutic approach, prevents neointimal hyperplasia.

[The construction of cDNA expression library from the tentacles of Sagartia rosea].

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.