Modulation of binding to vascular endothelial growth factor and receptor by heparin derived oligosaccharide.

We investigated the mechanism of heparin-derived oligosaccharide on the proliferation of vascular smooth muscle cell (VSMC) induced by vascular endothelial growth factor (VEGF). Expression levels of VEGFR 1 and VEGFR 2 were examined by RT-PCR, and the corresponding protein expression levels were detected by Western blotting and immunocytochemistry. Western blotting was taken to identify the expression levels of mechanism proteins. The binding of VEGF and VEGFR 2 was measured by co-IP. Besides, HS competition assay was to detect the ability of HDO to compete with Heparin for VEGF165. HDO showed an inhibitory effect on the expression of VEGFR1/2 proteins and PKC, MAPK, PI3K/Akt pathways. In addition, HDO affected the binding of VEGF-VEGFR, which may be one of the most important mechanisms of HDO suppress the cell proliferation induced by growth factors. Thus HDO showed the ability as a VEGF antagonist.

Heparin-derived oligosaccharide inhibits vascular intimal hyperplasia in balloon-injured carotid artery.

The aims of the present study were to determine the effects of heparin-derived oligosaccharides (HDOs) on vascular intimal hyperplasia (IH) in balloon-injured carotid artery and to elucidate the underlying mechanisms of action. An animal model was established by rubbing the endothelia within the common carotid artery (CCA) in male rabbits. The rabbits were fed a high-cholesterol diet. Arterial IH was determined by histopathological changes to the CCA. Serum lipids were detected using an automated biochemical analysis. Expressions of mRNAs for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), scavenger receptor class B type I (SR-BI), and ATP-binding cassette transporter A1 (ABCA-1) were analyzed using reverse transcription polymerase chain reaction assays. Expressions of VEGF, VCAM-1, MCP-1, SR-BI and ABCA-1 proteins were analyzed by Western blotting. Enzyme-linked immunosorbent assays were used to quantify expression levels of VEGF and bFGF. Our results showed that administration of HDO significantly inhibited CCA histopathology and restenosis induced by balloon injury. The treatment with HDOs significantly decreased the mRNA and protein expression levels of VEGF, bFGF, VCAM-1, MCP-1, and SR-BI in the arterial wall; however, ABCA-1 expression level was elevated. HDO treatment led to a reduction in serum lipids (total cholesterol, triglycerides, high-density and low-density lipoproteins). Our results from the rabbit model indicated that HDOs could ameliorate IH and underlying mechanism might involve VEGF, bFGF, VCAM-1, MCP-1, SR-BI, and ABCA-1.

[The mechanisms of heparin-derived oligosaccharide on the inhibition of smooth muscle cells proliferation induced by platelet-derived growth factor].

In this study, the effect of heparin-derived oligosaccharide (HDO) on platelet-derived growth factor (PDGF) induced vascular smooth muscle cells (VSMCs) proliferation and the related signal transduction mechanisms were investigated. MTT assays were used to measure VSMCs proliferation. Cell cycle distribution was analyzed by flow cytometry. The level of key regulatory proteins in PKC, MAPK and Akt/PI3K pathways were determined by RT-PCR, Western blot and immunocytochemical methods. Meanwhile, mRNA expressions of some proto-oncogenes were assayed by RT-PCR method. Our data showed that HDO (0.01, 0.1 and 1 µmol · L(-1)) inhibited 30 ng · mL(-1) PDGF-induced VSMCs proliferation in a dose-dependent manner, blocked the G1/S transition and inhibited the level of key regulatory proteins and some proto-oncogenes (P < 0.05). The results showed that HDO may decrease the key regulatory proteins expression, hence suppress the transcription of proto-oncogene and G1/S transition, finally inhibiting VSMCs proliferation.

[Study on the degradation and transformation of nonylphenol in water containing algae].

The photodegradation of nonylphenol induced by two common freshwater algae was investigated. The mechanism of nonylphenol photodegradation induced by algae was analyzed. The synergistic induction of nonylphenol degradation by algae and substances in water such as humic acid and ferric ions was also investigated. Results showed that the algae could induce the photodegradation of nonylphenol. The degradation of nonylphenol in water in the presence of algae, humic acid and ferric ions was obvious and the efficiency of degradation could reach 58% after 4 h illumination. Based on the results, it was speculated that the algae, humic acid and ferric ions system could produce more active oxygen after illumination, which could promote the photodegradation of the organic contaminants in water.

Effect of heparin-derived oligosaccharide on vascular smooth muscle cell proliferation through inhibition of PKC-alpha expression.

In this study, the effect of heparin-derived oligosaccharide (HDO) on bovine vascular smooth muscle cell (VSMC) proliferation and signal transduction mechanism involved were investigated. The levels of PKC-alpha protein and mRNA were determined by cell-based ELISA, RT-PCR, Western blotting and immunocytochemical methods. Meanwhile, mRNA levels of c-jun, c-myc and c-fos were assayed by RT-PCR method. The results showed that HDO inhibited newborn calf serum (NCS)-induced expression of PKC-alpha and proto-oncogenes, which may be one of the mechanisms for the inhibition of VSMC proliferation by HDO. Flow cytometry analysis indicated that HDO blocked NCS-induced cell cycle progression by arresting cells at G0/G1 phase. The results imply that HDO inhibits VSMC proliferation by moderating the gene level of PKC-alpha, eventually inhibiting proto-oncogene mRNA expression and blocking G1/S transition.

Pathway redesign for deoxyviolacein biosynthesis in Citrobacter freundii and characterization of this pigment.

Violacein (Vio) is an important purple pigment with many potential bioactivities. Deoxyviolacein, a structural analog of Vio, is always synthesized in low concentrations with Vio in wild-type bacteria. Due to deoxyviolacein's low production and difficulties in isolation and purification, little has been learned regarding its function and potential applications. This study was the first effort in developing a stable and efficient biosynthetic system for producing pure deoxyviolacein. A recombinant plasmid with vioabce genes was constructed by splicing using an overlapping extension-polymerase chain reaction, based on the Vio-synthesizing gene cluster of vioabcde, originating from Duganella sp. B2, and was introduced into Citrobacter freundii. With the viod gene disrupted in the Vio synthetic pathway, Vio production was completely abolished and the recombinant C. freundii synthesized only deoxyviolacein. Interestingly, vioe gene expression was strongly stimulated in the viod-deleted recombinant strain, indicating that viod disruptions could potentially induce polar effects upon the downstream vioe gene within this small operon. Deoxyviolacein production by this strain reached 1.9 g/L in shaker flasks. The product exhibited significant acid/alkali and UV resistance as well as significant inhibition of hepatocellular carcinoma cell proliferation at low concentrations of 0.1-1 µM. These physical characteristics and antitumor activities of deoxyviolacein contribute to illuminating its potential applications.

Protein kinase C pathway is involved in the inhibition by crocetin of vascular smooth muscle cells proliferation.

Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. Our previous study showed that crocetin inhibits angiotensin II (Ang II)-induced proliferation of vascular smooth muscle cells (VSMCs). The present study investigated the involvement of the protein kinase C (PKC) pathway in the growth-inhibitory action of crocetin in VSMCs. The findings showed that PKC activity in the membrane fraction of VSMCs increased following stimulation with Ang II, which was suppressed significantly by pretreating the cells with crocetin. Inhibition of PKC activity by crocetin appeared to be associated with growth inhibition in VSMCs, because chelerythrine chloride, a specific PKC inhibitor, likewise decreased cell proliferation. PKC-a, a conventional PKC isoform, was detected in bovine aorta VSMCs by RT-PCR and western blotting analysis. Crocetin inhibited Ang II-induced membrane translocation of PKC-a, and the inhibition of crocetin on PKC activity in membrane fraction coincided with its suppression on membrane translocation of PKC-a. In addition, Ang II-induced mRNA expressions of c-fos, c-jun and c-myc were also decreased by crocetin. Taken together, the data suggest that the inhibition by crocetin of PKC activity, at least in part due to inactivation of PKC-a, and the subsequent suppression of proto-oncogene expressions might mediate its inhibitory effect on VSMCs proliferation.

Crocetin inhibits cell cycle G1/S transition through suppressing cyclin D1 and elevating p27kip1 in vascular smooth muscle cells.

Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. Our previous study shows that crocetin inhibits angiotensin II (Ang II)-induced vascular smooth muscle cells (VSMCs) proliferation. To further explore the mechanism by which crocetin inhibits VSMCs proliferation, in the present study we examined the effect of crocetin on cell cycle progression and cell cycle regulatory proteins. Flow cytometry analysis showed that Ang II elicited significant increase in the percentage of VSMCs in the S phase, with a concomitant decline in the percentage of VSMCs in the G(0)/G(1) phase. However, on pretreatment of VSMCs with crocetin, the percentage of VSMCs in the S phase decreased, while that in the G(0)/G(1) phase increased significantly. In addition, Ang II-induced increase of cell proliferation index was also decreased by crocetin. Western blotting analysis indicated that crocetin markedly inhibited the protein expression of cyclin D1 but not cyclin E. Crocetin also increased the level of cyclin-dependent kinase inhibitor (CDKI) p27(kip1) but not CDKI p21(waf1/cip1). In conclusion, our present results suggest that the inhibition of cell cycle G(1)/S transition in VSMCs by crocetin can be attributed, at least in part, to its suppression of cyclin D1 and elevation of CDKI p27(kip1).

Anti-inflammatory effects and gastrointestinal safety of NNU-hdpa, a novel dual COX/5-LOX inhibitor.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with a risk of serious adverse events. Now, the development of dual inhibitors of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) has become a hot area in searching for safer NSAIDs. NNU-hdpa, 2-(4-hydroxylphenyl)-3-(3,5-dihydroxylphenyl) propenoic acid, a newly synthesized compound, is expected to have COX/5-LOX dual inhibition with an improved gastrointestinal profile. In this study, NNU-hdpa was subjected to in vitro and in vivo experiment protocols. In vitro COX/5-LOX inhibition assays showed that NNU-hdpa exhibits a dual inhibitory activity against the COX and 5-LOX enzymes. Anti-inflammatory activity in vivo was evaluated using two animal edema model tests. Pretreatment with NNU-hdpa (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice and carrageenan-induced paw edema in rats respectively. In gastric lesion test, NNU-hdpa was gastric-sparing in that it elicited markedly fewer stomach lesions as compared to the stomach lesions caused by aspirin in rats. In further studies, NNU-hdpa was found to significantly inhibit the productions of PGE(2) and LTB(4) in LPS-challenged RAW 264.7, which is parallel to its prevention of the nuclear translocation of the NF-kappaB p50 and p65 subunits. These data indicate that NNU-hdpa comprises a novel class of dual inhibitors of COX and 5-LOX having therapeutic potential with an enhanced gastric safety profile.

[Expression of Cx43 and Pax3 in the small intestinal muscular layers of early human embryos].

OBJECTIVE: To explore the patterns of Cx43 and Pax3 protein expressions in the small intestinal muscular layers of human embryo during early development. METHODS: Immunohistochemistry with SABC method was employed to examine the expression of Cx43 and Pax3 proteins in the muscular layers of the small intestine in early human embryos in the second to fourth months of gestation. RESULTS: In the second month of gestation, the muscle layer of the small intestine was negative for Cx43 and Pax3 protein expressions. In the third month, Cx43 and Pax3 expressions were negative in the inner circular muscle layer, but some positive cells were found in the longitudinal muscle layer and the myenteric plexus. In the fourth month, positive expression of Cx43 and Pax3 proteins were seen in the entire muscle layer. CONCLUSION: Cx43 and Pax3 proteins are closely related to the growth and development of the cells and tissues in the small intestinal muscle layer in human embryos.

[Microbial community structure of beer wastewater treatment plants analyzed by PCR-DGGE technique].

The microbial community structure of beer wastewater treatment plants with hydrolyze-acidification and SBR process was studied by PCR amplification and denaturing gradient gel electrophoresis (DGGE) based 16S rDNA. Activated sludge samples were collected from both hydrolyze-acidification tank and SBR tank at different depth and disposal period. The total DNA was extracted and the 16S rDNA was amplified by universal primer. The microbial community structure was analyzed by denaturing gradient gel electrophoresis, compared with the DGGE band pattern of the screened high efficient bacteria. The PCR products were cloned and sequenced to analyze the microbial community in phylogenic evolution. The DGGE pattern showed that the microbial community in hydrolyze-acidification tank changed with the variation of depth both in microbial structure and population, and at the depth of two-meter the microbial community was relatively rich, the dominant bands were notable. The microbial community in SBR tank at different depths and different disposal periods (precipitation period, inflow period, aeration period) were consistent respectively, but the dominant bands were different. Sequences of y2, 23, 25, 31, h5, 15 yielded high homogeneity with the strains uncultured Thermotogales sp., Comamonas sp. WT OTU1, Agrobacterium tumefaciens, Bacillus subtilis, Bdellovibrio bacteriovorus, Comamonas testosteroni from GenBank. The 16S rDNA sequences of the dominant bands were not completely same with that of the high efficient bacteria, which indicated that the strains screened were not the dominant strains in actual process.

Influence of Crocetin on experimental atherosclerosis in hyperlipidamic-diet quails.

Antioxidants have been expected to have potential as antiatherogenic agents. Crocetin is a natural carotenoid antioxidant isolated from Gardenia jasminoids Ellis. Therefore, in the present study, we investigated the inhibitory effect of Crocetin on experimental atherosclerosis in quails. The atherosclerosis model was established by feeding hyperlipidamic diet to quail and Crocetin (25, 50, 100 mg/kg/day) was administered by oral gavage. At the 9th week, serum lipids, malondialdehyde and nitric oxide were measured, and Hematoxylin-Eosin (H&E) stains was used to investigate the histopathological changes of aorta. Results showed that Crocetin could reduce the levels of serum total cholesterol, triglyceride, low density lipoprotein cholesterol and inhibit the formation of aortic plaque. Crocetin could also reduce malondialdehyde and inhibit the descending of nitric oxide in serum. The results suggested that Crocetin could inhibit the formation of atherosclerosis in quails, which might be related to the hypolipidemic effects along with the antioxidative properties of Crocetin.

Involvement of Ca2+ in the inhibition by crocetin of angiotensin II-induced ERK1/2 activation in vascular smooth muscle cells.

Crocetin, a carotenoid compound, was isolated from Gardenia jasminoids Ellis. Our recent study shows that crocetin inhibits angiotensin II-induced extracellular signal-regulated kinases 1/2 (ERK1/2) activation and subsequent proliferation in vascular smooth muscle cells (VSMCs). To further explore the mechanism involved, in the present study, we investigated the effect of Ca(2+) in the activation of ERK1/2 and whether Ca(2+) is involved in the suppression by crocetin of angiotensin II-induced ERK1/2 activation. Our findings showed that crocetin pretreatment partially attenuated both the intracellular Ca(2+) mobilization and the extracellular Ca(2+) influx induced by angiotensin II. Moreover, angiotensin II-induced ERK1/2 activation was completely abolished by acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N ',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator, and partially inhibited by EGTA, an extracellular Ca(2+) chelator, or verapamil, an L-type Ca(2+) channel blocker. These findings suggest that Ca(2+) may play an important role in angiotensin II-induced ERK1/2 activation in VSMCs, and Ca(2+)-dependent pathway may be involved in the inhibitory effect by crocetin of angiotensin II-induced ERK1/2 activation.

Effect of crocin on experimental atherosclerosis in quails and its mechanisms.

In the present study, we examined the prophylaxis effect of crocin on experimental atherosclerosis and its possible mechanisms. The atherosclerosis formation was induced by hyperlipidamic diet in quails. At the 9th week, serum lipid, MDA and NO were measured, and HE staining was used to investigate the histopathological changes of aorta. Bovine aortic endothelial cells (EC) were obtained from the thoracic aorta of newborn calves. After incubation of the cells with Ox-LDL (50 mg x L(-1)) for 24 h, the activities of LDH, NO in culture media and activity of NOS in endothelial cells were measured, flow cytometer was used to determine the rate of endothelial cells apoptosis. Peritoneal macrophages were obtained from thioglycolate-injected mice. Cholesterol and free cholesterol in cells were assayed after incubation of the cells with Ox-LDL. Bovine aortic smooth muscle cells (SMC) were obtained from the thoracic aorta of newborn calf. Proliferation was induced by 100 microg x L(-1) Ox-LDL and antiproliferative effect of crocin on SMCs were observed. SMCs cycle phases were measured by flow cytometry. SMCs were loaded with Fluo-3/AM and [Ca2+]i was measured by Laser Scanning Confocal Microscope (LSCM). Crocin could reduce the level of serum TC, TG, LDL-C and inhibit the formation of aortic plaque. Crocin could reduce MDA and inhibit the descending of NO in serum. Compared with control, Ox-LDL group could increase the activity of LDH and decrease activity of NO in culture media and activity of NOS in endothelial cells, preincubated with crocin, the effects of Ox-LDL were inhibited. Crocin could decrease the EC apoptosis induced by Ox-LDL. Crocin concentration-dependently inhibited the TC and CE elevation induced by Ox-LDL in macrophages. Crocin could inhibit the proliferation of SMCs induced by Ox-LDL. In the presence or absence of extracellular Ca2+, crocin concentration-dependently inhibited the [Ca2+]i elevation induced by 120 mg x L(-1)Ox-LDL, In the absence of extracellular Ca2+, crocin could inhibit the [Ca2+]i elevation induced by CHCl3 in a concentration-dependent manner. The results indicated that crocin could inhibit the formation of atherosclerosis in quails. Crocin had protective effects on endothelial cells. Crocin could decrease CE in macrophages and uptake of Ox-LDL, inhibiting the formation of foam cell, which would promote the initiation and progression of atherosclerosis. Crocin could inhibit the [Ca2+]i elevation in smooth muscle cell, Ca2+ is an important second messenger that regulates a variety of cellular processes, including smooth muscle cell proliferation and gene expression . Crocin exerted antiatherosclerotic effects through decreasing the level of Ox-LDL that plays an important role in the initiation and progression of atherosclerosis.

Effect of crocin on intracellular calcium concentration in cultured bovine aortic smooth muscle cells.

AIM: To study the effect of crocin on intracellular calcium concentration ([Ca2+]i) in cultured bovine aortic smooth muscle cells (BASMCs). METHODS: Cells were loaded with fluorescence probe Fluo-3/AM and [Ca2+]i was measured by laser scanning confocal microscope (LSCM). RESULTS: In the presence or absence of extracellular Ca2+, crocin (1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol x L(-1)) concentration-dependently inhibited the [Ca2+]i elevation induced by 1 x 10(-2) mol x L(-1) H2O2 (for the former, the inhibition rates were 34.1%, 57.1% and 74.3%, while for the latter were 26.2%, 32.1%, 50.0%). In the absence of extracellular Ca2+, crocin (1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol x L(-1)) could inhibit the [Ca2+]i elevation induced by 70 mmol x L(-1) CHCl3, the inhibition rates were 27.8%, 27.8% and 50.0% respectively. CONCLUSION: Crocin could inhibit the extracellular Ca2+ influx and release of intracellular Ca2+ stores in endoplasmic reticulum.