Genotyping of 30 kinds of cutaneous human papillomaviruses by a multiplex microfluidic loop-mediated isothermal amplification and visual detection method.

BACKGROUND: Human papillomaviruses (HPVs), a group of non-enveloped small viruses with double-stranded circular DNA which lead to multiple skin diseases such as benign warts, are commonly seen in clinics. The current HPV detection systems aim mainly at mucosal HPVs, however, an efficient clinical approach for cutaneous HPVs detection is lacking. OBJECTIVES: To establish a rapid detection system for cutaneous HPVs using a colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue (HNB) dye in combination with microfluidic technology. METHODS: L1 DNA sequences of the 30 cutaneous HPVs were chemically synthesized, and LAMP primers against L1 DNA were designed with use of an online LAMP designing tool. Isothermal amplification was performed with use of a water bath and the amplification results were inspected with the naked eye. Using PCR sequencing as a control method, the specificity and sensitivity of the new detection system were obtained by detecting clinical samples. RESULTS: The lower detection limit of the LAMP assay was 107 viral DNA copies/µl when tested on synthesized L1 DNA sequences, which was better than the conventional PCR. Compared to PCR sequencing, the sensitivity of HPV27, HPV2, HPV1, HPV57, HPV3, HPV4, HPV7 and HPV75 genotypes detections were 100%, whereas the specificity was 34.55, 45.12, 95.83, 98.59 and 97.62% respectively, when tested on clinical samples. CONCLUSIONS: The new cutaneous type HPV detection system is characterized by both a good sensitivity and specificity compared to conventional methods.

[Research progress and secondary development ideas of Astragali Radix polysaccharides for injection].

Astragali Radix( AR) polysaccharide for injection( Guoyao Zhunzi Z20040086) is a traditional Chinese medicine for intravenous powder injection developed by Shanxi Academy of Traditional Chinese Medicine in early 1990 s by taking advantage of AR resources in Shanxi province. The effective parts of AR polysaccharides were obtained by advanced technology. The hemogram of patients with radiotherapy and chemotherapy showed alleviations in clinic. However,due to the technical bottleneck in separation of the complex polysaccharides mixture and the difficulties in accurate measurement of the polysaccharide structures,the pharmacodynamic mechanism of the drug remained unclear,and the side effect was hard to control. In recent years,the theoretical studies for polysaccharide receptors have indicated that when polysaccharides bound to protein receptors,only the oligosaccharide fragments of the polysaccharide molecule bound to the receptors,and one or more active sites of oligosaccharide fragments may existed in the polysaccharide molecule.Therefore,the active center of polysaccharides can be studied based on the level of oligosaccharides through degradation of the polysaccharides,which provided a new strategy for breaking through the bottleneck in polysaccharide structure determination. Therefore,this paper reviews the current status of studies for AR polysaccharides for injection,the polysaccharide receptors theory and successful cases,in order to propose the secondary development ideas of AR polysaccharides for injection. The study results will lay a material foundation for the development of new drugs of polysaccharides from traditional Chinese medicine,and provide a basis for the resolution of international difficulties in quality control of polysaccharide drugs and molecular models,so as to further study of glycobiology,and enrich the polysaccharide receptors theory.

Characterization of a new endo-type polyM-specific alginate lyase from Pseudomonas sp.

An alginate lyase gene, algA, encoding a new poly ß-D-mannuronate (polyM)-specific alginate lyase AlgA, was cloned from Pseudomonas sp. E03. The recombinant AlgA with (His)6-tag, consisting of 364 amino acids (40.4 kDa),was purified using Ni-NTA Sepharose. The purified lyase had maximal activity (222 EU/mg) at pH 8 and 30 °C and also maintained activity between pH 7-9 and below 45 °C. It exclusively and endolytically depolymerized polyM by ß-elimination into oligosaccharides with degrees of polymerization (DP) of 2-5. Due to its high substrate specificity, AlgA could be a valuable tool for production of polyM oligosaccharides with low DP and for determining the fine structure of alginate.

Pedobacter hainanensis sp. nov., isolated from seaside soil.

A Gram-negative, aerobic, rod-shaped, motile, non-spore-forming bacterial strain, designated 13-Q(T), was isolated from seaside soil under the stacks of the red algae in Hainan province in China. Identification was carried out on the basis of polyphasic taxonomy. Phylogenetic analysis of 16S rRNA gene sequences showed that strain 13-Q(T) belonged to the genus Pedobacter, and the highest similarity was 94.4 % with Pedobacter terricola KCTC 12876(T). Strain 13-Q(T) was able to grow at 10-40 °C, in pH 5.0-10.0, in the presence of 0-2.0 % NaCl. The major fatty acids were iso-C(15:0) (40.4 %), summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16:1) ω7c) (18.9 %) and iso-C(17:0) 3-OH (18.4 %). The predominant menaquinone was MK-7. The G+C content of the genomic DNA was 42.7 mol%. Strain 13-Q(T) could be distinguished from the nearest phylogenetic neighbors by various chemotaxonomic and phenotypic properties. The results of the polyphasic analyses suggested that strain 13-Q(T) should be considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter hainanensis sp. nov. is proposed. The type strain is 13-Q(T) (=CCTCC AB 2012076(T) = NRRL B-59850(T)).

Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro.

Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50~200 µg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.

Relationship Between the Structure and Immune Activity of Components From the Active Polysaccharides APS-II of Astragali Radix by Enzymolysis of Endo α-1,4-Glucanase.

Astragali Radix polysaccharides (APSs) have a wide range of biological activities. Our preliminary experiment showed that APS-Ⅱ (10 kDa) was the main immunologically active component of APSs. However, the characteristic structure related to activity of APS-Ⅱ needs further verification and clarification. In this study, APS-II was degraded by endo α-1,4-glucosidase. The degraded products with different degrees of polymerization [1-3 (P1), 3-6 (P2), 7-14 (P3), and 10-18 (P4)] were obtained using a polyacrylamide gel chromatography column. The structural features of the different products were characterized by HPGPC, monosaccharide composition, Fourier transform infrared spectrum, GC-MS, nuclear magnetic resonance, and UPLC-ESI-QTOF-MS analysis. Specific immune and non-specific immune cell tests were used to identify the most immunogenic fractions of the products. The backbone of P4 was speculated to be α-D-1,4-linked glucans and rich in C2 (25.34%) and C6 (34.54%) branches. Immune screening experiments indicated that the activity of P4 was better than that of APS-II and the other three components. In this research, the relationship between the structure of APS-Ⅱ and the immune activity from the degradation level of polysaccharides was studied, laying a foundation for the quality control and product development of APSs.

Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases.

AIM: To investigate whether and how COS inhibited IL-8 production in LPS-induced human umbilical vein endothelial cells (HUVECs). METHODS: RT-PCR, enzyme-linked immunosorbent assays (ELISA) and Western blotting were used to study IL-8 expression and related signaling pathway. Wound healing migration assays and monocytic cell adhesion analysis were used to explore the chemotactic and adhesive activities of HUVECs. RESULTS: COS 50-200 µg/mL exerted a significant inhibitory effect on LPS 100 ng/mL-induced IL-8 expression in HUVECs at both the transcriptional and translational levels. In addition, COS 50-200 µg/mL inhibited LPS-induced HUVEC migration and U937 monocyte adhesion to HUVECs in a concentration-dependent manner. Signal transduction studies suggest that COS blocked LPS-induced activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) as well as phosphorylation of p38 mitogen-activated protein kinase (MAPK) and phosphokinase Akt. Further, the over-expression of LPS-induced IL-8 mRNA in HUVECs was suppressed by a p38 MAPK inhibitor (SB203580, 25 µmol/L) or a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002, 50 µmol/L). CONCLUSION: COS inhibited LPS-induced IL-8 expression in HUVECs through the blockade of the p38 MAPK and PI3K/Akt signaling pathways.

Identification and activity evaluation of Astragalus Radix from different germplasm resources based on specific oligosaccharide fragments.

Objective: Based on trifluoroacetic acid (TFA) hydrolysis, polyacrylamide gel electrophoresis (PAGE) and high performance thin layer chromatography (HPTLC) analysis, the carbohydrate responsible for immunomodulatory activity are used as quality indicators for Astragalus Radix (AR). Methods: In this study, 24 batches of AR from different germplasm resources were selected as the research object, and AR polysaccharides were extracted. PAGE and HPTLC methods were used to analyze the partial acid hydrolyzate of AR polysaccharides and obtain a series of saccharide fingerprints. The data were analyzed by principal component analysis to obtain the difference between AR from different germplasm resources. Results: The results showed that trisaccharide and tetrasaccharide could be used as differential fragments to distinguish AR of different cultivation methods; Disaccharides and trisaccharides can be used as differential fragments to distinguish different species of AR. The immunological activity analysis of the specific oligosaccharide fragment of AR showed that the specific oligosaccharide fragment of AR could promote the secretion of TNF-α, IL-1ß, IL-6, and NO in THP-1 cells in a concentration-dependent manner. Conclusion: Both PAGE and HPTLC methods can be used to evaluate AR from different germplasm resources. This study laid the foundation for the quality evaluation of AR medicinal herbs.

Geniposide inhibits interleukin-6 and interleukin-8 production in lipopolysaccharide-induced human umbilical vein endothelial cells by blocking p38 and ERK1/2 signaling pathways.

OBJECTIVE: The aim of this study was to investigate the inhibitory effect of geniposide on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and interleukin-8 (IL-8) production in human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: Primary HUVECs were used. The mRNA/protein levels of IL-6 and IL-8 was determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). LPS-induced HUVEC migration and adhesion of monocytes to HUVECs were studied by monolayer wound healing experiments and monocytic cell adhesion assay, respectively. Expression of nuclear factor kappaB (NF-kappaB), inhibitory factor kappaB-alpha (IkappaB-alpha), p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were determined by Western blot analysis. RESULTS: Geniposide effectively inhibited LPS-induced expression of IL-6 and IL-8 in HUVECs at the transcription and translation levels. Additionally, geniposide suppressed LPS-induced HUVEC migration and U937 monocyte adhesion to HUVECs. Signal transduction studies indicate that geniposide blocked the activation of NF-kappaB, degradation of IkappaB-alpha, and phosphorylation of p38 MAPK and ERK1/2 in HUVECs challenged by LPS. CONCLUSION: The results show that geniposide can inhibit LPS-induced IL-6 and IL-8 production in HUVECs by blocking p38 MAPK and ERK1/2 signaling pathways.

UHPLC Q-Exactive MS-Based Serum Metabolomics to Explore the Effect Mechanisms of Immunological Activity of Astragalus Polysaccharides With Different Molecular Weights.

Astragalus polysaccharides (APS) have a wide range of biological activities. Most researchers discuss total APS as the main research object. However, because the relative molecular weight of APS has a wide distribution, in-depth studies on the mechanisms of the biological activity of notable molecules are limited. For example, the relationship between the immunomodulatory effect of APS and its relative molecular weight has not been clearly defined. Therefore, in this paper, we separated and obtained APS of different molecular weights by ultrafiltration technology and then constructed a mouse cyclophosphamide-induced immunosuppression model to investigate the immune activity of APS of different molecular weights. The immune enhancement mechanism of APS was explored by examining changes in routine blood indicators, body weight, immune organs, and differential metabolites in mouse serum. Results showed that APS-I (molecular weight, >2,000 kDa), APS-II (molecular weight, 1.02 × 104 Da) and APS-III (molecular weight, 286 Da) could increase the number of immune cells in mouse serum and improve immune organ damage to varying degrees. Among the samples obtained, APS-II showed the best effects. Compared with those in the blank group, 29 metabolites determined by UHPLC Q-Exactive MS in the serum of the model group changed remarkably, and APS-I, APS-II, and APS-III respectively restored 13, 25, and 19 of these metabolites to normal levels. Metabolomics analysis revealed that APS-II is mainly responsible for the immunomodulatory activity of APS. Metabolomics analysis revealed that the mechanisms of this specific molecule may involve the regulation of phenylalanine metabolism, cysteine and methionine metabolism, tricarboxylic acid cycle (TCA cycle) and arginine and proline metabolism.

Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus.

Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 µM min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase.

[Expression of tobacco SKP1 gene induced by oligochitosan].

Oligochitosan is an effective inductor for plant resistance. To understand the induced resistance mechanism, mRNA differential display was used to isolate genes from Nicotiana tabacum var. Samsun NN and four enhanced-expression gene fragments were obtained and were reamplified. The reamplified products of appropriate size were isolated and purified before they were subcloned into PMD18-T vector. The results of plasmids digestion by EcoRI and HindIII and analysis of reverse Northern blot indicated that the expression of the four genes was enhanced by oligochitosan induction. Sequence and homology analysis show that they share 82% identity in nucleotide sequence with Nicotiana benthamiana SKP1 gene. Because the SKP1 protein as the core component of SCF (ubiquitin ligase enzyme E3) is relevant to plant resistance to virus, so these results suggested that oligochitosan can induce plant resistance and its mechanism may be relevant to ubiquitination.

[Chemical constituents of Cyanotis arachnoidea].

AIM: To investigate the chemical constituents of Cyanotis arachnoidea. METHODS: By using chromatographic methods for separation and combination with spectral analysis, their chemical structures were determined. RESULTS: Six compounds were identified as ajugasterone C-20, 22-acetonide (1), 20-hydroxyecdysone-20, 22-acetonide (2), 22-oxo-ajugasterone C (3), 22-oxo-20-hydroxyecdysone (4), beta-sitosterol (5), daucosterol (6). CONCLUSION: Compound 3 is a new compound, 4 was a new natural compound.

[Study on the chemical constituents from the herb of Gentianopsis paludosa].

OBJECTIVE: To study the chemical constituents from the herb of Gentianopsis paludosa. METHOD: Column chromatogrophy and spectral analysis were used to isolate and identify the constituents. RESULT: Six compounds were isolated and identified as 1,7-dihydroxy-3,8-dimethoxyxanthone (I), 1-hydroxy-3, 7, 8-trimethoxyxanthone (II), 1, 8-dihydroxy-3, 7-dimethoxyxanthone (III), 1-hydroxy-3, 7-dimethoxyxanthone (IV), beta-sitosterol (V), daucosterol (VI). CONCLUSION: Compounds III-VI were isolated from the plant for the first time.

Chitosan oligosaccharides block LPS-induced O-GlcNAcylation of NF-κB and endothelial inflammatory response.

It is known that chitosan oligosaccharides (COS) suppress LPS-induced vascular endothelial inflammatory response by mechanism involving NF-κB blockade. It remains unknown how COS inhibit NF-κB. We provided evidence both in cultured endothelial cells and mouse model supporting a new mechanism. Regardless of the endothelial cell types, the LPS-induced NF-κB-dependent inflammatory gene expression was suppressed by COS, which was associated with reduced NF-κB nucleus translocation. LPS enhanced O-GlcNAc modification of NF-κB/p65 and activated NF-κB pathway, which could be prevented either by siRNA knockdown of O-GlcNAc transferase (OGT) or pretreatment with COS. Inhibition of either mitogen-activated protein kinase or superoxide generation abolishes LPS-induced NF-κB O-GlcNAcylation. Consistently, aortic tissues from LPS-treated mice presented enhanced NF-κB/p65 O-GlcNAcylation in association with upregulated gene expression of inflammatory cytokines in vascular tissues; however, pre-administration of COS prevented these responses. In conclusion, COS decreased OGT-dependent O-GlcNAcylation of NF-κB and thereby attenuated LPS-induced vascular endothelial inflammatory response.

Alginate oligosaccharides enhanced Triticum aestivum L. tolerance to drought stress.

Alginate oligosaccharides (AOS) prepared from degradation of alginate is a potent plant elicitor. Hydroponic experiments were carried out to investigate the mechanism of AOS on improving Triticum aestivum L. resistant ability to drought stress. Drought model was simulated by exposing the roots of wheat to polyethylene glycol-6000 (PEG-6000) solution (150 g L(-1)) for 4 days and the growth of wheat treated with PEG was significantly decreased. However, after AOS application, seedling and root length, fresh weight and relative water content of wheat were increased by 18%, 26%, 43% and 33% under dehydration status compared with that of PEG group, respectively. Moreover, the antioxidative enzymes activities were obviously enhanced and malondialdehyde (MDA) content was reduced by 37.9% in samples treated by AOS. Additionally, the drought resistant related genes involved in ABA signal pathway, such as late embryogenesis abundant protein 1 gene (LEA1), psbA gene, Sucrose non-fermenting 1-related protein kinase 2 gene (SnRK2) and Pyrroline-5-Carboxylate Synthetase gene (P5CS) were up-regulated by AOS. Our results suggested that AOS might regulate ABA-dependent signal pathway to enhance drought stress resistance of wheat during growth period.

Chitosan oligosaccharides protect mice from LPS challenge by attenuation of inflammation and oxidative stress.

Sepsis and its derivative syndromes are major causes of morbidity and mortality in the intensive care unit. Recently, lots of studies have shown that the progression of sepsis is attributed to redox imbalance and overproduction of proinflammatory cytokines. In previous studies, we have reported the anti-oxidative and anti-inflammatory effects of chitosan oligosaccharides in vitro. In the light of these findings, we applied the model of sepsis to mice by LPS injection to investigate whether chitosan oligosaccharides have a protective effect on LPS-induced sepsis. We found that treatment by chitosan oligosaccharides not only attenuated organ dysfunction but also improved survival rate after LPS injection. To further understand how it works, we examined several proinflammatory markers including neutrophil infiltration in organs and TNF-α and IL-1ß in serum, and found that these cytokines were significantly reduced by chitosan oligosaccharide treatment. In addition to this, anti-oxidants including glutathione (GSH) and catalase (CAT) levels were depleted and malondialdehyde (MDA) levels were increased in LPS-induced sepsis, while chitosan oligosaccharides smoothed out the redox imbalance. Furthermore, we also assessed c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase signal activation by LPS-stimulation, and found both of them were attenuated by chitosan oligosaccharide treatment. Collectively, our data demonstrated that chitosan oligosaccharides can protect mice from the LPS challenge by virtue of anti-inflammatory effects as well as anti-oxidation properties, which might offer beneficial effects for patients with sepsis.

Chitosan oligosaccharides inhibit the expression of interleukin-6 in lipopolysaccharide-induced human umbilical vein endothelial cells through p38 and ERK1/2 protein kinases.

Chitosan oligosaccharides (COS) have been reported to exert anti-fungal activities, antitumour activities and immuno-enhancing effects. However, the potential roles of COS in the treatment of vascular inflammations remain unknown. In the present study, we examined the effects of COS on interleukin-6 (IL-6) production in human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). Induction of HUVECs with LPS (100 ng/ml) increased the mRNA expression and protein secretion of IL-6 (versus the vehicle-treated group, p < 0.01), which were significantly reverted by the pre-treatment with COS (50-200 microg/ml) for 24 hr before LPS exposure (versus the LPS-treated group, p < 0.05 or 0.01). Signal transduction studies showed that the pre-treatment of HUVECs with COS (50-200 microg/ml) for 24 hr markedly inhibited the LPS-induced over-expression of phosphorylated p38 mitogen-activated protein kinase (MAPK), phosphorylated ERK1/2 and nuclear factor kappaB (NF-kappaB). Moreover, the LPS-induced NF-kappaB activation was suppressed by the specific ERK1/2 inhibitor PD98059 (30 microM) (versus the LPS-treated group, p < 0.01), but not by the specific p38 MAPK inhibitor SB203580 (25 microM). Additionally, both MAPK inhibitors markedly suppressed LPS-induced IL-6 mRNA expression in HUVECs (versus the LPS-treated group, p < 0.01). In conclusion, our results suggest that COS inhibit LPS-induced up-regulation of IL-6 in HUVECs, and this can be regulated by at least two parallel signalling pathways: one via p38 MAPK pathway independent of NF-kappaB activation and one via ERK1/2 pathway dependent on NF-kappaB activation.

Tetramethylpyrazine inhibits production of nitric oxide and inducible nitric oxide synthase in lipopolysaccharide-induced N9 microglial cells through blockade of MAPK and PI3K/Akt signaling pathways, and suppression of intracellular reactive oxygen species.

AIM OF THE STUDY: To determine the inhibitory effect of tetramethylpyrazine (TMP) on lipopolysaccharide (LPS)-induced over-production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in N9 microglial cells. MATERIALS AND METHODS: N9 cells were pretreated with vehicle or TMP and then exposed to LPS for the time indicated. Cell viability was determined by methylthiazoyltetrazolium (MTT) assay. Nitrite assay was performed by Griess reaction. Expression of iNOS mRNA was examined by RT-PCR. Protein levels of iNOS, p38 mitogen-activated protein kinase (MAPK), ERK1/2, JNK, phosphatidylinositol 3-kinase (PI3K) and Akt were determined by western blot analysis. Formation of reactive oxygen species (ROS) was evaluated by fluorescence image system. RESULTS: TMP inhibited LPS-induced over-production of NO and iNOS in N9 cells. TMP also inhibited the NF-kappaB translocation from cytoplasm into nucleus of N9 cells. In addition, TMP showed blocking effect on the phosphorylation of p38 MAPK, ERK1/2, JNK and Akt, but not PI3K. Further, TMP suppressed the formation of intracellular ROS in LPS-induced N9 cells. CONCLUSIONS: TMP inhibited production of NO and iNOS in LPS-induced N9 cells through blocking MAPK and PI3K/Akt activation and suppressing ROS production.

Chitosan oligosaccharides attenuate hydrogen peroxide-induced stress injury in human umbilical vein endothelial cells.

Chitosan oligosaccharides (COS) have been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we investigated the protective effects of COS against hydrogen peroxide (H(2)O(2))-induced oxidative damage on human umbilical vein endothelial cells (HUVEC, ECV304 cells). After 24h pre-incubation with COS (25-200 microg/ml), the viability loss in ECV304 cells induced by H(2)O(2) (300 microM) for 12h was markedly restored in a concentration-dependent manner as measured by MTT assay. This effect was accompanied by a marked decrease in intracellular reactive oxygen species (ROS) by measuring intensity of DCFH fluorescence. COS also exerted preventive effects on suppressing the production of lipid peroxidation such as malondialdehyde (MDA), restoring activities of endogenous antioxidants including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), along with the capacity of increasing levels of nitric oxide (NO) and nitric oxide synthase (NOS), as were determined by commercial regent kits. In addition, pre-incubation of COS with ECV304 cells for 24h resulted in the reduction of apoptosis and the induction of cell cycle arrest in G(1)/S+M phase as assayed quantitatively by Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit using flow cytometry. Taken together, our findings suggest that COS can effectively protect HUVECs against oxidative stress by H(2)O(2), which might be of importance in the treatment of cardiovascular diseases.