A pentapeptide monocyte locomotion inhibitory factor protects brain ischemia injury by targeting the eEF1A1/endothelial nitric oxide synthase pathway.

BACKGROUND AND PURPOSE: Ischemic stroke is a major cause of death worldwide but lacks viable treatment or treatment targets. Monocyte locomotion inhibitory factor (MLIF) is a small heat-stable pentapeptide produced by Entamoeba histolytica in axenic culture, which is supposed to protect the brain from ischemic injury; the mechanism, however, remains unknown. In this study, we further investigated the mechanism underlying the protective role of MLIF in brain ischemia. METHODS: A middle cerebral artery occlusion model in rats was used for detecting the effect of MLIF in the brain ischemia in vivo. To identify targets of MLIF in brain endothelial cells, we performed immunoprecipitation of biotin-conjugated MLIF and mass spectrometry. RESULTS: MLIF can protect the brain from ischemic injury in vivo, yielding decreased ischemic volume, prolonged survival, and improved neurological outcome. In vitro studies showed that MLIF displayed protective effects through inhibition of expression of pathological inflammatory adhesion molecules and enhancing endothelial nitric oxide synthase expression and nitric oxide release in the cerebrovascular endothelium. The target screening experiments demonstrated binding of MLIF to the ribosomal protein translation elongation factor eEF1A1. MLIF enhanced endothelial nitric oxide synthase expression through stabilization of endothelial nitric oxide synthase mRNA, and eEF1A1 was shown to be necessary for this enhanced expression. Knockdown of eEF1A1 or inhibition of endothelial nitric oxide synthase attenuated MLIF-mediated inhibition of adhesion molecule expression. CONCLUSIONS: In this study, we identified a new potential pharmacologically targetable mechanism underlying MLIF's protective effects in brain ischemia through the eEF1A1/endothelial nitric oxide synthase pathway.

CD133(+)CXCR4(+) colon cancer cells exhibit metastatic potential and predict poor prognosis of patients.

BACKGROUND: Colorectal cancer (CRC), which frequently metastasizes to the liver, is one of the three leading causes of cancer-related deaths worldwide. Growing evidence suggests that a subset of cells exists among cancer stem cells. This distinct subpopulation is thought to contribute to liver metastasis; however, it has not been fully explored in CRC yet. METHODS: Flow cytometry analysis was performed to detect distinct subsets with CD133 and CXCR4 markers in human primary and metastatic CRC tissues. The 'stemness' and metastatic capacities of different subpopulations derived from the colon cancer cell line HCT116 were compared in vitro and in vivo. The roles of epithelial-mesenchymal transition (EMT) and stromal-cell derived factor-1 (SDF-1) in the metastatic process were also investigated. A survival curve was used to explore the correlation between the content of CD133(+)CXCR4(+) cancer cells and patient survival. RESULTS: In human specimens, the content of CD133(+)CXCR4(+) cells was higher in liver metastases than in primary colorectal tumors. Clonogenic and tumorigenic cells were restricted to CD133(+) cells in the HCT116 cell line, with CXCR4 expression having no impact on the 'stemness' properties. We found that CD133(+)CXCR4(+)cancer cells had a high metastatic capacity in vitro and in vivo. Compared with CD133(+)CXCR4(-) cells, CD133(+)CXCR4(+)cancer cells experienced EMT, which contributed partly to their metastatic phenotype. We then determined that SDF-1/CXCL12 treatment could further induce EMT in CD133(+)CXCR4(+)cancer cells and enhance their invasive behavior, while this could not be observed in CD133(+)CXCR4- cancer cells. Blocking SDF-1/CXCR4 interaction with a CXCR4 antagonist, AMD3100 (1,10-[1,4-phenylenebis(methylene)]bis-1,4,8,11 -tetraazacyclotetradecane octahydrochloride), inhibited metastatic tumor growth in a mouse hepatic metastasis model. Finally, a high percentage of CD133(+)CXCR4(+)cells in human primary CRC was associated with a reduced two-year survival rate. CONCLUSIONS: Strategies targeting the SDF-1/CXCR4 interaction may have important clinical applications in the suppression of colon cancer metastasis. Further investigations on how high expression of CXCR4 and EMT occur in this identified cancer stem cell subset are warranted to provide insights into our understanding of tumor biology.

Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphate-activated kinase pathway.

OBJECTIVE: Stroke is a leading cause of mortality and disability. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in mammalian nicotinamide adenine dinucleotide (NAD)(+) biosynthesis and contributes to cell fate decisions. However, the role of Nampt in brain and stroke remains to be investigated. METHODS: We used lentivirus-mediated Nampt overexpression and knockdown to manipulate Nampt expression and explore the effects of Nampt in neuronal survival on ischemic stress both in vivo and in vitro. We also used adenosine monophosphate (AMP)-activated kinase-α2 (AMPKα2) and silent mating type information regulation 2 homolog 1 (SIRT1) knockout mice to investigate the underlying mechanisms of Nampt neuroprotection. RESULTS: Nampt inhibition by a highly-specific Nampt inhibitor, FK866, aggravated brain infarction in experimentally cerebral ischemia rats, whereas Nampt overexpression in local brain and Nampt enzymatic product nicotinamide mononucleotide (NMN) reduced ischemia-induced cerebral injuries. Nampt overexpression and knockdown regulated neuron survival via the AMPK pathway. Neuroprotection of Nampt was abolished in AMPKα2(-/-) neurons. In neurons, Nampt positively modulated NAD(+) levels and thereby controlled SIRT1 activity. SIRT1 coprecipitated with serine/threonine kinase 11 (LKB1), an upstream kinase of AMPK, and promoted LKB1 deacetylation in neurons. Nampt-induced LKB1 deacetylation and AMPK activation disappeared in SIRT1(-/-) neurons. In contrast, Ca(2+) /calmodulin-dependent protein kinase kinase-ß (CaMKK-ß), another upstream kinase of AMPK, was not involved in the neuroprotection of Nampt. More important, Nampt overexpression-induced neuroprotection was abolished in SIRT1(+/-) and AMPKα2(-/-) mice. INTERPRETATION: Our findings reveal that Nampt protects against ischemic stroke through rescuing neurons from death via the SIRT1-dependent AMPK pathway and indicate that Nampt is a new therapeutic target for stroke.

NDRG2 induced by oxidized LDL in macrophages antagonizes growth factor productions via selectively inhibiting ERK activation.

During atherogenesis, macrophage foam cells produce prodigious growth factors, cytokines, and chemokines, which play the central roles in inflammatory process in atherosclerotic plaque formation. In the present study, we identified a new protein marker, N-Myc downstream-regulated protein 2 (NDRG2), which is significantly up-regulated in oxidized low density lipoprotein (oxLDL) treated macrophages and in human atherosclerotic plaques. Over-expression and siRNA knockdown studies showed that NDRG2 is a negative regulator of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) productions in macrophages. Furthermore, we investigated the effects of NDRG2 on MAPK signal activation. Our results showed ERK1/2 activation, but not P38 or JNK1/2 activation, is responsible for regulation of NDRG2 on VEGF and PDGF productions. Consistent with the PDGF levels, the vascular smooth muscle cell (VSMC) proliferation was also regulated by the conditional medium of the oxLDL treated macrophages with NDRG2 knockdown or over-expression. Neutralizing anti-PDGF antibody can significantly inhibit the enhanced VSMC proliferation by macrophage medium with NDRG2 knockdown. Our present results demonstrate that NDRG2 participates in oxLDL-induced macrophage activation and modulates ERK1/2-dependent PDGF and VEGF production, which has potential application in atherogenesis.

ER-residential Nogo-B accelerates NAFLD-associated HCC mediated by metabolic reprogramming of oxLDL lipophagy.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome that elevates the risk of hepatocellular carcinoma (HCC). Although alteration of lipid metabolism has been increasingly recognized as a hallmark of cancer cells, the deregulated metabolic modulation of HCC cells in the NAFLD progression remains obscure. Here, we discovers an endoplasmic reticulum-residential protein, Nogo-B, as a highly expressed metabolic modulator in both murine and human NAFLD-associated HCCs, which accelerates high-fat, high-carbohydrate diet-induced metabolic dysfunction and tumorigenicity. Mechanistically, CD36-mediated oxLDL uptake triggers CEBPß expression to directly upregulate Nogo-B, which interacts with ATG5 to promote lipophagy leading to lysophosphatidic acid-enhanced YAP oncogenic activity. This CD36-Nogo-B-YAP pathway consequently reprograms oxLDL metabolism and induces carcinogenetic signaling for NAFLD-associated HCCs. Targeting the Nogo-B pathway may represent a therapeutic strategy for HCC arising from the metabolic syndrome.

Adenoviral gene transfer of viral interleukin-10 protects cerebrovascular impairment induced by lysophosphatidylcholine.

Cerebrovascular disease is a significant cause of morbidity and mortality in the world. Inflammatory processes induce several pathological responses such as atherosclerosis, which have fundamental roles in stroke in the etiology of ischemic cerebrovascular disease and the pathophysiology of cerebral ischemia. Viral interleukin-10 (vIL-10), a potential anti-inflammatory cytokine, has been studied extensively. However, the efficacy of vIL-10 on cerebrovascular dysfunction is not well known. Our goal in this study was to explore the effect of gene transfer of vIL-10 mediated by adenovirus (Ad/vIL-10) on cerebrovascular function using a model of vasocontraction of isolated basilar artery from mongrel dogs induced by lysophosphatidylcholine (lysoPC), a proinflammatory and atherogenic serum lysophospholipid. To clarify the relation between contraction of basilar aorta and cell adhesion and adhesion molecules, our further study explored effects of Ad/vIL-10 on monocyte-cerebrovascular endothelial cells adhesion and expression of cell adhesion molecule by cultured cerebromicrovascular endothelial cells, bEnd.3, after incubation by lysoPC. Our results showed that Ad/vIL-10 significantly decreased contractive response of basilar aorta produced by lysoPC and augmented vasorelaxation to acetylcholine. Further studies showed the Ad/vIL-10 significantly depressed adherence of monocytes to cerebrovascular endothelial cells and inhibited up-regulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) which are bio-markers in inflammatory progress. These data demonstrated the protective effects of Ad/vIL-10 on cerebrovascular dysfunction induced by inflammation, and proved that inhibition of expression of cell adhesion molecules should be one of ways of vIL-10 to protect vascular function during inflammation.

Lentiviral vector-mediated siRNA knockdown of SR-PSOX inhibits foam cell formation in vitro.

AIM: To investigate the expression of scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXC chemokine ligand 16 (CXCL16) in the human monocyte-derived cell line THP-1, and the effect of lentiviral vectors for the stable delivery of SR-PSOX/CXCL16 short hairpin RNA on foam cell formation. METHODS: A lentiviral expression vector containing enhanced green fluorescence protein (GFP) and SR-PSOX small interfering RNA (siRNA) (Lenti-SR-PSOXsi), or the control siRNA (Lenti-NC) gene was constructed. A human monocyte-derived cell line THP-1 was transfected with a different multiplicity of infection (MOI) of Lenti-SR-PSOXsi or Lenti-NC, and cultured to obtain stably-transfected THP- 1KD and THP-1NC cells. After incubation with oxidatively-modified, low-density lipoprotein (Ox-LDL), the expression of SR-PSOX/CXCL16 mRNA was determined by real-time PCR. The expression of the SR-PSOX/CXCL16 protein was detected by flow cytometry analysis. The effect of Lenti-SR-PSOXsi on foam cell formation was assessed by Oil red O-stain analysis. RESULTS: Ox-LDL increased the expression of SR-PSOX/CXCL16 mRNA in a time- and dose-dependent manner in THP-1 cells. Four days after transfection with Lenti-SR-PSOXsi (MOI: 100), the percentage of GFP expression cells was over 89.3%. The expression of the SR-PSOX/ CXCL16 mRNA and protein in THP-1KD cells significantly decreased compared with the parent cells, even the THP-1KD cells stimulated with 40 mg/L Ox-LDL. Ox-LDL uptake experiments in THP-1- and THP-1KD-derived macrophages indicated that SR-PSOX/CXCL16 deficiency decreased the development of macrophage- derived foam cell formation. CONCLUSION: The above data showed that SRPSOX siRNA delivered by using lentiviral vectors in THP-1 cells was a powerful tool for studying the effect of SR-PSOX, and decreased the expression of the SRPSOX gene by inhibiting macrophage-derived foam cell formation.

Timosaponin B-II improves memory and learning dysfunction induced by cerebral ischemia in rats.

Sapogenins from Anemarrhenae asphodeloides was reported to improve the learning and memory abilities. In this study, we investigated the effect of Timosaponin B-II(TB-II), a purified extract from A. asphodeloidesb on rat vascular dementia (VD) produced by transient (2h) middle cerebral artery occlusion. The learning and memory abilities of rats were measured by water maze task and passive avoidance task. Daily oral administration of TB-II at two different dose levels of 100 and 200 mg/kg resulted in a significant improvement of the deficit in the learning of the water maze task, beginning 14 days after ischemia. Shortened mean escape latency was detected in TB-II group compared with model group during the same trial days. TB-II treatment also significantly reversed the ischemia-induced retention deficit determined by a one trial step-down type of passive avoidance task. Meanwhile, the expression of interleukin-10, an anti-inflammatory cytokine, and its receptor were significantly increased in TB-II treated VD rats. The results presented the first evidence of a neuroprotective effect of TB-II in the model of vascular dementia. We suggest that the anti-dementia effect by TB-II is derived at least in part from its anti-inflammatory properties.

Proteomic analysis of foam cells.

Foam cells are characteristic pathological cells in the lesions of atherosclerosis. Previous works have established macrophage-derived foam cell model to study the central role of the foam cells, and analyzed the protein expression profiles in foam cells. The reported in vitro foam cell model was established by incubating the human U937 cells with oxidized low-density lipoprotein. The global changes in protein expressions between U937 foam cell and normal U937 cells were measured with two-dimensional gel electrophoresis, and some interested proteins were tryptic-digested and then identified via mass spectrometry after capillary liquid chromatography separation. Some of the identified proteins were validated via the Internet links to the U937 proteomic map provided from the Expasy Proteomics server (http://us.expasy.org). The experimental data can provide potential markers during the inflammatory reactions for atherosclerotic studies.

Effects of Ginkgo biloba extract on expressions of IL-1beta, TNF-alpha, and IL-10 in U937 foam cells.

This study is to investigate the protein and mRNA expressions of pro-inflammatory and anti-inflammatory cytokines in U937 foam cells and effects of Ginkgo biloba extract (GbE) on the cytokines. U937 cells were cultured with different concentrations of GbE (0.1, 1, and 10 microg x L(-1)), and stimulated by 100 mg x L(-1) oxidized low density lipoprotein (ox-LDL) for 24 h. The expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in culture solution were detected by enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that incubated with 100 mg x L(-1) ox-LDL for 24 h, the U937 cells became foam cells, the protein or mRNA expressions of IL-1beta, TNF-alpha, IL-10, and its receptor IL-10R in U937 foam cells were higher markedly than those in normal U937 cells. When the cells were pretreated with GbE (0.1, 1, and 10 microg x L(-1)), the increases of IL-1beta and TNF-alpha in U937 foam cells were remarkably inhibited, but IL-10 expression increased greatly. Especially when cells were pretreated with 10 microg x L(-1) GbE, the protein and mRNA expressions of IL-1beta and TNF-alpha were markedly lower than those in U937 foam cells. The protein expression of IL-10 and mRNA expressions of IL-10 and its receptor IL-10R were markedly higher than those in U937 foam cells. GbE inhibited production of pro-inflammatory cytokines IL-1beta and TNF-alpha, but up-regulated the production of anti-inflammatory cytokine IL-10 and its receptor IL-10R in U937 foam cells, which might be related with its anti-atherosclerotic actions.

C-reactive protein induced expression of adhesion molecules in cultured cerebral microvascular endothelial cells.

Ischemic stroke can trigger an acute phase response resulting in a rise of plasma concentration of C-reactive protein (CRP). Clinical data about the relationship between CRP and prognosis suggest that CRP might be involved in the pathogenesis of cerebral ischemia. In the present work, a significant increase of circulating level of CRP was observed in an vivo rat brain ischemia model of middle cerebral artery occlusion. To determine the possible effects of CRP on brain microvessel endothelium, we performed a dose-dependent experiment in mouse brain microvascular endothelial cells (bEnd.3 cells) with emphasis on its relation to cell adhesions molecules. Incubation with CRP (1-75 mg/L) for 24 h significantly increased Lactate dehydrogenase (LDH) leakage from bEnd.3 cells (P<0.01) in a dose-dependent manner, and induced significant up-regulations of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions analyzed by Western blotting (P<0.01). In contrast to earlier report, CRP also induced significant increase in ICAM-1 expression in the absence of serum (P<0.01). In conclusion, the present results suggest that CRP may be involved directly in the development of inflammation in response to cerebral ischemia.

EF1A1/HSC70 Cooperatively Suppress Brain Endothelial Cell Apoptosis via Regulating JNK Activity.

AIMS: In our previous study, eEF1A1 was identified to be a new target for protecting brain ischemia injury, but the mechanism remains largely unknown. In this study, we screened the downstream cellular protein molecules interacted with eEF1A1 and found mechanism of eEF1A1 in brain ischemia protection. METHODS AND RESULTS: Through co-immunoprecipitation and mass spectrometry for searching the interaction of proteins with eEF1A1 in bEnd3 cells, HSC70 was identified to be a binding protein of eEF1A1, which was further validated by Western blot and immunofluorescence. eEF1A1 or HSC70 knockdown, respectively, increased OGD-induced apoptosis of brain vascular endothelial cells, which was detected by Annexin V-FITC/PI staining. HSC70 or eEF1A1 knockdown enhances phosphorylated JNK, phosphorylation of c-JUN (Ser63, Ser73), cleaved caspase-9, and cleaved caspase-3 expression, which could be rescued by JNK inhibitor. CONCLUSION: In summary, our data suggest that the presence of chaperone forms of interaction between eEF1A1 and HSC70 in brain vascular endothelial cells, eEF1A1 and HSC70 can play a protective role in the process of ischemic stroke by inhibiting the JNK signaling pathway activation.

Time courses of vascular endothelial growth factor and intercellular adhesion molecule-1 expressions in aortas of atherosclerotic rats.

An increasing number of studies have focused on the expressions of growth factors and adhesion molecules in atherosclerotic lesions, which are confirmed to play central roles in angiogenesis and endothelial dysfunction, including vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). However, the difference of growth factor and adhesion molecule expression time courses has not been determined in vivo. This study aimed to determine the expression patterns and expression curves of ICAM-1 and VEGF in atherosclerotic rats during the time course. An experiment atherosclerotic model in rats was established by combining the high fat/cholesterol diets with injection of vitamin D3. In situ hybridization was used to determine the expression patterns of VEGF and ICAM-1 in aortas of normal or atherosclerotic rats in 8 weeks. There was a massive increase in reactivity for both ICAM-1 and VEGF in atherosclerotic plaques. Northern blot, Western blot and ELISA analysis were used to quantify VEGF and ICAM-1 expressions in time course. In rat aorta, the expression curves in time course showed that ICAM-1, not VEGF, was up-regulated in mRNA levels significantly in 2 weeks; while VEGF expression was hysteresis than ICAM-1, which showed maximum expression level in 8 weeks. Our results provide the evidence of VEGF and ICAM-1 expression curves in time course in atherosclerotic rats, which indicated different regulatory mechanisms of VEGF and ICAM-1 expression in atherogenesis.

Intercellular adhesion molecule-1 and vascular endothelial growth factor expression kinetics in macrophage-derived foam cells.

Macrophage-derived foam cells seem to play an important role during inflammatory response of atherosclerosis, in which the overexpression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF) are associated with the early and later pathological changes in foam cell formation. In this study, we investigated the expression kinetics of ICAM-1 and VEGF in macrophage-derived foam cells. The foam cell model was established through incubating the human monocyte line (U937 cells) with oxidized-low density lipoprotein (ox-LDL). Up-regulated expressions of ICAM-1 and VEGF were analyzed in protein and mRNA levels in U937 foam cells by flow cytometry, ELISA, and Northern blot. Kinetic studies showed the deferent kinds of expression curves in dose response and time course. The expression dose-kinetics demonstrated that the ICAM-1 showed the peak expression induced by ox-LDL 50 mg/L, while VEGF levels increased in a dose-dependent manner with the maximum level induced by ox-LDL 200 mg/L. Time-kinetic studies revealed that the ICAM-1 levels showed the peak expression in 12 h while VEGF expression increased in a time-dependent manner with the maximum level in 48 h. These results proved that both ICAM-1 and VEGF expressions were enhanced in the macrophage-derived foam cells, but ICAM-1 expression increased earlier than the up-regulation of VEGF; low dose of ox-LDL mainly up regulated ICAM-1 expression, while high dose mainly increased the VEGF expression.

Comparative analysis of the proteome of left ventricular heart of arteriosclerosis in rat.

Despite the worldwide occurrence of coronary atherosclerotic heart disease (CAHD), the pathogenic mechanisms underlying this disease remain largely unknown. In this study, the experimental model of atherosclerosis in rat (CAHD rat) was established by the injection of vitamin D3 associated with high fat diet for 6 weeks. By using the proteomic approach, we comparatively analyzed the proteome of the control and CAHD rat left ventricular myocardial tissues. We reproducibly separated over 2500 polypeptides by using two-dimensional electrophoresis (2-DE) at pH range of 3-11. Among these proteins, 26 proteins with large amount were identified using micro high performance liquid chromatography mass spectrometer/mass spectrometer (micro-HPLC-MS/MS). Using PDQUEST software to process the 2-DE gel images, 38 protein spots that significantly altered in CAHD were detected. Of these, 12 proteins were identified with high confidence by using 2-DE and matrix-associated laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The identification of protein alterations specify to CAHD would clarify the pathogenetic mechanisms involved in the disease and might be of prognostic and therapeutic benefit.

[Expression of VEGF in endothelial cells and the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside].

AIM: To determine the effect of lysophosphatidylcholine (LPC) on the expression of vascular endothelial growth factor (VEGF) in human umbilical veins endothelial cell line (ECV304) and the inhibitory effect of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside (ST I) in vitro. METHODS: Exposure to 2.5 mg x L(-1) LPC or LPC + ST I for 24 hours, VEGF protein was determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, VEGF mRNA expression in ECV304 was examined by in situ hybridization. VEGF165 mRNA was examined by RT-PCR and Realtime RT-PCR. RESULTS: LPC upregulated VEGF protein and VEGF mRNA expression in the ECV304 cells. ST I was shown to markedly inhibit the LPC-induced increase of VEGF protein and VEGF165 mRNA (P < 0.001). CONCLUSION: LPC can induce a strong expression of VEGF in ECV304 cells and ST I can inhibit it.

[Expression of vascular endothelial growth factor in U937 foam cells and the inhibitory effect of drugs].

AIM: To study the expression of vascular endothelial growth factor (VEGF) in U937 foam cells and the inhibitory effect of salvianolic acid B and Ginkgo biloba extract in vitro. METHODS: U937 cells were incubated with 80 mg.L-1 oxidized low density lipoprotein (OX-LDL) for 48 h and a macrophage-derived foam cell model was established. The VEGF concentration in the media was determined by ELISA; the VEGF protein expression in cells was measured with immunohistochemistry; the VEGF mRNA level in cells was measured by in situ hybridization; the positive ratio detected by a morphometrical analysis system was used as the amount of the VEGF protein expression and the mRNA level. RESULTS: After U937 cells were incubated with OX-LDL, VEGF expression level increased greatly both in the cells and in the media. Salvianolic acid B and Ginkgo biloba extract were shown to remarkably inhibit the increase of VEGF. After treated with 10 micrograms/L-1 salvianolic acid B and Ginkgo biloba extract, the VEGF protein concentration in the media and positive ratio in the cells decreased compared with foam cells. After treated with 10 micrograms.L-1 salvianolic acid B and 100 micrograms.L-1 Ginkgo biloba extract, the VEGF mRNA level decreased measured by in situ hybridization. CONCLUSION: A high VEGF expression level was determined in U937 foam cells. Salvianolic acid B and Ginkgo biloba extract were found to inhibit VEGF expression significantly in U937 foam cells in vitro.

[Studies on the simvastatin effect on the artery of atherosclerotic rabbit using proteomics approaches].

AIM: To evaluate the inhibitory effect of simvastatin via investigating the overall expression level of proteins in the artery of atherosclerotic rabbit. METHODS: Experimental model was established by feeding the rabbits a high fat diet (cholesterol 0.5 g.kg-1.d-1, lard 0.5 mL.kg-1.d-1) for 8 weeks. Then simvastatin (5 mg.kg-1) were fed for 4 weeks to the rabbits. The overall protein levels were measured using two-dimensional gel electrophoresis and a PDQUEST data processing. RESULTS: Twenty nine protein spots showed significant quantitative changes in comparison with the normal and the diseased rabbits. Furthermore, after the diseased rabbit having taken simvastatin, an obvious decay of symptom of fatty liver was observed, and the intensity of most spots has not been back-regulated. CONCLUSION: Simvastatin facilitates the metabolism of fat in the blood, but the lesion of the internal wall of the atherosclerotic artery cannot be restored.

[DNA microarray analysis of protective mechanism of buyang huanwu decoction on focal cerebral ischemia/reperfusion in rats].

OBJECTIVE: To analyse the effects of buyang huanwu decoction (BYHWT) on differentially expressed genes during cerebral ischemia/reperfusion in rats with DNA microarray. METHOD: cDNA microarray chips containing 512 cDNAs were made by Biostar Genechip Inc. Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) with an filament. Saline or BYHWT was given p.o. after onset of cerebral ischemia and brains were removed after 24 h of recirculation for mRNAs isolation. A differential measurment of mRNAs from post-ischemic and BYHWT treated animals was performed with microarray. RESULT: Up-and down-regulated genes were 69 and 80 in ischemic group. Up-and down-regulated genes were 25 and 6 in BYHWT treated group. CONCLUSION: BYHWT regulates the differential expression genes after focal brain ischemia/reperfusion in rats, due to its mechanism of protecting cerebral ischemia/reperfusion injury.

[Effect of madecassoside on depression behavior of mice and activities of MAO in different brain regions of rats].

OBJECTIVE: To evaluate the effects of madecassoside (MC) on the depression behavior of mice and the activities of monoamine oxidase (MAO) in different rat brain regions. METHODS: Imipramine as the positive contrast medicine, effects of MC on the depression behavior of mice were observed by forced swimming test and reserpine antagonist test. Moclobemide and pargyline as the positive controlled medicines, the activities of monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B) in different rat brain regions were determined after intragastric administration of MC in 3 different dosages for 3 days or 21 days. RESULTS: (1) The low, middle and high dosages of MC (i.g.) significantly reduced the immobility time of mice in forced swimming test (P<0.05). (2) MC in dosages of 10 mg/kg and 20 mg/kg prevented the lowering of temperature induced by reserpine (P<0.05), while 40 mg/kg had no significant effects on it (P>0.05). (3) With acute administration (3 days), the low, middle and high dosagey of MC (i.g.) significantly inhibited the activity of MAO-A in hippocampus (P<0.01), and the high dosage significantly inhibited the activity of MAO-A in hypothalamus (P<0.01), while the 3 dosages had no significant effects on the activity of MAO-A in cortex (P>0.05). With chronic administration (21 days), MC in 3 dosages had no significant effects on the activities of MAO-A in cortex and hypothalamus (P>0.05), and the high dosage (40 mg/kg) significantly enhanced the activity of MAO-A in hippocampus (P<0.01). (4) With acute administration, MC in dosages of 10 mg/kg and 20 mg/kg significantly inhibited the activity of MAO-B in cortex (P>0.05), and MC in dosage of 10 mg/kg significantly inhibited the activity of MAO-B in hypothalamus (P<0.05), and MC in dosage of 20 mg/kg significantly enhanced the activity of MAO-B in hippocampus (P<0.01). With chronic administration, MC of 3 dosages produced no significant effects on the activities of MAO-B in 3 different rat brain regions (P>0.05). CONCLUSION: These results support the idea that MC produces antidepressant effects through MAO inhibition in rat brain, which seems stronger with acute administration than chronic administration, while its mechanism remains to be further studied.