Icariin inhibits atherosclerosis progress in Apoe null mice by downregulating CX3CR1 in macrophage.

Horny Goat Weed is a commonly used in Chinese herbal medicine. And it is used in multiple kinds of diseases including cardiovascular diseases. Icariin is the major component isolated from Horny Goat Weed. It is reported to have lipid-lowering effect. In atherosclerosis, icariin attenuate the enhanced prothrombotic state independently of its lipid-lowering effects. However, its detail mechanism is remaining unclear. This study aimed to investigate the effect and mechanism of icariin on atherosclerosis. We performed gene expression profiling on icariin treated LPS-stimulated RAW264.7 and its control cells. Microarray analyses identified a list of genes significantly differentially expressed after icariin treated including downregulation of CX3CR1. Apoe null mice were assigned into 3 groups: control group, diet with 30 mg/kg/d icariin and diet with 60 mg/kg/d icariin. The results showed that icariin treatment significantly reduced lesion area and macrophage infiltration. Also icariin reduced CX3CR1 and CX3CL1 protein levels in the artery wall. In conclusion, icariin could be a potential anti-atherosclerosis agent by downregulating the expression of CX3CR1.

An extract from medical leech improve the function of endothelial cells in vitro and attenuates atherosclerosis in ApoE null mice by reducing macrophages in the lesions.

AIM: Medicinal leech has been widely used as a traditional Chinese medicine in cardiovascular diseases. However, its pharmaceutical effect is not fully revealed. The goal of this study was to determine whether a leech extract has the effect of anti-atherosclerosis in ApoE −/− mice and the mechanism of this effect. METHODS AND RESULTS: In vivo experiments: ApoE −/− mice fed on high-cholesterol diet were separated into 5 groups. Control group was administrated with normal water; leech extract of low dose treatment group was given a leech extract of 0.02 g/kg/d; leech extract of medium dose treatment group was given a leech extract of 0.1 g/kg/d; leech extract of high dose treatment group was given a leech extract of 0.5 g/kg/d; simvastatin group was given simvastatin of 10 mg/kg/d. Leech extract significantly reduced atherosclerotic lesions in aortic root compared with control group. And the number of macrophage in or around the atherosclerosis plaque is significantly reduced in the leech extract groups compared with control group. In vitro experiments: human endothelial cell line, EA.hy926, was induced with TNF-α to perform endothelial dysfunction. Control group: EA.hy926 cells with no special treatment; TNF-α group: EA.hy926 cells were induced by 10 ng/ml TNF-α for 6 h; leech extract only group: EA.hy926 cells were treated with 200 mg/ml leech extract only; leech extract and TNF-α group: 200 mg/ml leech extract was applied before TNF-α induction. Protein and mRNA level were detected in each group, leech extract can decrease the expression of intercellular adhesion factor (ICAM-1) and monocyte chemotactic protein (MCP-1) compared with TNF-α group. Furthermore, it showed less adhesion and migration of THP-1 cells to EA.hy926 cells in the adhesion assay and transwell assay. The NF-κB translation to nucleus was blocked by leech extract in the NF-κB translocation assay. CONCLUSIONS: Leech extract could obviously attenuate the area of atherosclerosis lesion in ApoE −/− mice. And this effect is dose dependent. The effect is mainly a result of reduced invasion of monocyte in artery walls by blocking NF-κB translocation.

[Optimization the formulation of Eisemia foetida protein burn spray by response surface methodology].

OBJECTIVE: To optimize the formulation of Eisemia foetida protein (EFP) burn spray. METHODS: A five-factor, three-level response surface method was employed; The response variable was the proliferation effect of EFP on NIH3T3 cells. RESULTS: The optimization formulation was as follows: the proportion of EFP, glycerol and mannitol was 0.91%, 1.42% and 5%, respectively; 0.02 mol/L Na2 HPO4 and 0.01 mol/L citric acid buffer system corresponding pH value was 7.0. CONCLUSION: The response surface method is reliable, efficient and suitable for optimizing the formulation of EFP burn spray.

A novel protein from Eupolyphaga sinensis inhibits adhesion, migration, and invasion of human lung cancer A549 cells.

Eupolyphaga sinensis Walker is an important insect used in Chinese traditional medicine. In this study, we purified a 72-kDa anticancer protein, designated as EPS72, from this species using ammonium sulfate precipitation, ultrafiltration, CM Sepharose Fast Flow cation exchange, Q Sepharose High Performance (HP) anion exchange, Butyl Sepharose HP hydrophobic chromatography, and Superdex 75 gel filtration chromatographic techniques. EPS72 exhibited a potent anticancer activity against the human lung cancer A549 cell line (IC50, 18.76 µg/mL). Further study showed that EPS72 could induce A549 cell detachment and apoptosis, inhibit cell adhesion to fibronectin and collagen IV, and restrain cell migration and invasion. Moreover, EPS72 significantly decreased the expression of ß1-integrin. This study suggests that EPS72 could potentially be developed as a novel anticancer therapeutic agent due to its possible antimetastatic activity.

[Protective effect of polysaccharides from sea cucumber on glu-induced neurotoxicity in PC12 cells].

OBJECTIVE: To investigate the protective effect of polysaccharides from sea cucumber (HS) on Glu-induced neurotoxicity in PC12 cells. METHODS: The toxicity model was established by treating PC12 cells with Glu in vitro. Cell viability was assayed with MTT method. The vitality of antioxidant enzymes (SOD) and the level of malondialdehyde (MDA) and lactic dehydrogenase (LDH) were evaluated by chromatometry. RESULTS: The vitality of SOD increased and the levels of MDA and LDH significantly decreased in PC12 cells of HS group compared with the model group. HS in different concentration showed protective effects as it increased the vitality of SOD and decreased the content of MDA and LDH in a dose-dependent manner. CONCLUSION: Polysaccharide from sea cucumber can protect PC12 cells from the injury induced by Glu.

Expression and secretion of recombinant ZZ-EGFP fusion protein by the methylotrophic yeast Pichia pastoris.

We constructed a fusion protein ZZ-EGFP by fusing the ZZ domains of staphylococcal protein A (SpA) and enhanced green fluorescent protein (EGFP). ZZ-EGFP was secreted in the yeast, Pichia pastoris, with a hexahistidine tag. Its expression level was determined by measuring the fluorescence of EGFP. When the recombinant yeast cells in shake-flasks were induced with 0.5% methanol for 96 h, a maximum yield of 115 mg ZZ-EGFP/l was obtained. The resulting ZZ-EGFP fusion protein retained immunoglobulin G (IgG)-binding capacity and EGFP fluorescence. ZZ-EGFP was then used in immunofluorescence assays for detecting antinuclear antibodies (ANA); it produced a good signal that was comparable in its brightness and fluorescence pattern to that generated with fluorescein isothiocyanate (FITC)-labelled anti-human IgG. Thus, ZZ-EGFP showed great potential in immunological applications due to its ability to bind to various IgG from different animal sources.

Effect of Glycine and Triton X-100 on secretion and expression of ZZ-EGFP fusion protein.

Two-factor and three-level fractional factorial design was employed for evaluation of the effect of Glycine and Triton X-100 on the secretion and expression of ZZ-EGFP fusion proteins. Varying contents of glycine (0%, 1%, 2%) and Triton X-100 (0%, 1%, 2%) were added into shaking flasks, respectively, and supplied with appropriate volume of ampicillin (total 9 combinations; group at concentration zero serving as control) to promote more ZZ-EGFP diffuse into liquid culture medium. Fluorescent intensity in the culture supernatant was detected. A standard curve could be generated on the basis of fluorescent intensity and protein concentration. The expression level of ZZ-EGFP fusion proteins was estimated by checking the protein standard curve concentration fluorescene intensity. Results show that when the culture medium contains 2% Glycine and 1% Triton X-100, the expression level of ZZ-EGFP was able to be greatly increased. Further experiments revealed that absorbance value (A600) in the experiment group, whose culture medium contains 2% Glycine and 2% Triton X-100, is significantly lower than other groups in the present experiment. These results indicate that the culture medium containing appropriate quantity of Glycine and Triton X-100 is favourable to the secretion and expression level of ZZ-EGFP in gene-engineering bacteria Escherichia coli HB101.

Structure-efficacy relationships of immunostimulatory activity of CpG-containing oligodeoxynucleotides on mouse spleen cells.

AIM: To study the relationship between primary structures of oligodeoxynucleotides (ODN) containing unmethylated deoxycytidyldeoxyguanosine (CpG) dinucleotide motifs and their immunostimulatory activities in mouse spleen cells. METHODS: A series of CpG ODN with different primary structures were synthesized. Their capabilities to stimulate mouse spleen cell proliferation were determined by [3H]thymidine incorporation assay. Cytokine (interleukin [IL]-6, IL-12, and IFN-alpha) secretion spectra induced by CpG ODN were assessed by ELISA. The ability of CpG ODN to activate natural killer cells was evaluated by standard 4 h (51)Cr-release assay. Flow cytometry was utilized to examine the expressions of various lymphocyte surface molecules on diverse immunocytes. An effective CpG ODN for murine, ODN1826, was set as the template of modification and the positive control. RESULTS: The immunostimulatory activities of CpG ODN with different sequences and compositions varied markedly, both in character and in extent. It was useless for improving the immunostimulatory activity of ODN1826 by simply increasing the functional hexameric CpG motif number, modifying the site of CpG motifs, or changing the distance between multi-CpG motifs. However, an addition of a self-complementary palindrome structure at the 3'-end, but not the 5'-end of CpG ODN, aroused marked improvement in its activity. Several designed ODN had superior comprehensive immunostimulatory properties compared to ODN1826. CONCLUSION: The immunostimulatory activity of a CpG ODN was relevant to its primary structure. It was useless for promoting immunostimulatory activity to simply change CpG motif number, space, or distance. The 3'-end palindrome structure of CpG ODN is associated with enhanced immunostimulatory activity.

[The extraction, purification and assaying of Gynostemma pentaphyllum polysaccharides].

By orthogonal design, and considering extracting efficiency and cost, optimizing the extract method of Gynostemma pentaphyllum polysaccharides. We purified the crude Gynostemma pentaphyllum polysaccharides initially, and assayed the polysaccharides content of Gynostemma pentaphyllum polysccharides. The content of Gynostemma pentaphyllum polysaccharides was sigificantly higher than the predecessor. It would provide conditions for the deep exploitation of Gynostemma pentaphyllum.