Abstract:Aim To clone the upregulated expressed gene during rat myocardium ischemic preconditioning. Methods New gene was in silicon cloned using exon finding, blast alignment, assembling program and so on. Reverse transcriptase polymerase chain reaction (RT-PCR) assay confirmed the open reading frame (ORF) of new gene. Results Rat heterogeneous nuclear ribonucleoproteins (hnRNP) A2/B1 gene was cloned by bioinformatics methods. The product of RT-PCR was 1.1 kb, which was identical to the predicted length. The ORF of rat hnRNPA2/Bl gene was 1 026 bp, which was composed of ten exons and encoded 341 amino acids. The coding sequence showed 92% and 95% identity respectively in 1 026 bp overlap with that of human and mouse, and the predicted polypeptide showed almost 100% identity with that of human and mouse. Conclusions The results showed that hnRNPA2/Bl gene was highly conserved among various species. It maybe plays an important role in endogenous protection of ischemic preconditioning.

Abstract:Aim To explore the production of adrenomedullin (ADM), changes and significance of ADM, ADM receptor system-calcitonin receptor like receptor (CRLR) and receptor activity modifying proteins (RAMP) mRNA in calcified vascular smooth muscle cells (VSMC). Methods Calcification of cultured rat VSMC was produced by incubation with β-glycerophos phate. Content of ADM released by VSMC was measured by radioimmunoassay (RIA). The amount of ADM, CRLR and RMAP mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The intracellular calcium content, alkaline phosphatase activity and cellular 45 Ca²⁺ uptake were determined. Results The content of calcium, 45 Ca²⁺ uptake and alkaline phosphatase activity in calcified VSMC increased by 118% , 174% and 7-fold (all P< 0.01), respectively, compared with control VSMC. Content of ADM in medium increased by 99% (P< 0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP 2 and RAMP 3 mRNA in calcified cells were elevated by 78% , 93.7% , 91.8% and 109.5 % (all P< 0.01), respectively, compared with control. The elevated levels of CRLR, RAMP 2 and RAMP 3 mRNA were positively correlated with ADM mRNA ( r = 0.83, 0.92 and 0.93, respectively, P< 0.01) in calcified VSMC. Conclusion Calcified VSMC generated an increased amount of ADM, up-regulated gene expressions of ADM, CRLR, RAMP 2 and RAMP 3, which suggested that changes of autocrine/paracrine function of vessel might be involved in the regulation of vascular calcification .

Abstract:Aim To explore the role of nuclear factor-κB (NF-κB) in high doses of glucose (HG) induced impairment of ECV304 cells, a vascular endothelial cell line. Methods Recombinant adenovirus mediated nuclear factor-KB supper-repres sor IκBαM with mutant IκBα was constructed. Western blot, electrophoretic mobility shift assay (EMSA) and reverse tran scriptase-polymerase chain reaction(RT-PCR) assay were applied in this study. Results HG induced IκBα degradation and nuclear factor-KB activation were found in ECV304 cells but not in ECV304/IκBαM cells infected with IκBαM recombinant adenovirus . The expression of inflammatory factors such as monocyte chemotactic protein-1 (MCP-1), endothelin-1 (ET-1) and intercellular adhesion molecule-1 (ICAM-1) of ECV304 cells incubated with HG was increased obviously, but ECV304/IκBαM cells could resist HG induced increase of the expression of inflammatory factors. Conclusions HG increase the expression of inflammatory factors in vascular endothelial cells. Inhibition of nuclear factor-κB activation can protect vascular endothelial cells from the cytotoxicity of HG.

Abstract:Aim To construct an adenovirus expression vector which can express hepatocyte growth factor (HGF) in vascular smooth muscle cells (SMC). Methods The plasmid containing HGF fragment was cleaved by restriction enzyme digestion, and the resultant fragment was inserted directionally into adenoviral shuttle plasmid. The linearized recombinant adeno viral shuttle plasmid and adenovirus expression vector were cotransformed into Escherichia coli BJ5183 cells for homologous recombination . The resultant recombinant plasmid, pAd-HGF, then was transfected into HEK293 cells with liposome for packaging. The recombinant adenoviral shuttle plasmid and pAd-HGF were identified by enzyme digestion and sequencing. The package of pAd-HGF in HEK293 cells was tracked by fluorescent microscope, and was observed by electronic microscope. The expression of packaged pAd-HGF in abdominal aortic SMCs of rat was identified by RT-PCR and Western blotting. Results HGF fragment was inserted correctly into the adenoviral shuttle plasmid and adenovirus expression vector. High-liter packaged adenovirus vector was produced and expressed in abdominal aortic SMCs of rat. Conclusions A recombinant adenovirus expression vector of HGF was constructed successfully. This study suggested that HGF may be a potential target for the gene therapy of vascular diseases and established a foundation for further study.

Abstract:Aim To elucidate the intracellular signaling pathways for β-very low density lipoprotein (VLDL) -induced VLDLR transcription and their effects on lipid accumulation in macrophages. Methods Phosphorylated extracelcular signal regucated kinase (ERK1/2) protein was detected with Western blot. mRNA level of VLDL receptor was assayed by RT-PCR.Results We found that β-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. Studies using different protein kinases inhibitors or activators showed that the effect of β-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of P38 kinase and cAMP analog, but extremely abolished by pretreating cells with an inhibitor of ERK and an inhibitor of PKC. Studies on effects of PD98059 (the inhibitor of ERK) on β-VLDL-induced cellular lipid accumulation, which was assessed by cholesterol and triglycerid assay kit showed that ERK inhibitor decreased cellular cholestorol and triglycerid increase exposed to β-VLDL in a dose-dependent manner. Conclusions These results demonstrated that the PKC-ERK1/2 cascade was the essential signaling pathway by which β-VLDL activated VLDL receptor mRNA expression and inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by β-VLDL in macrophages.

Abstract:Aim Ubiquitin-proteasome pathway is an important pathway of protein degradation in cells, it is involved in many physiological processes including cell cycle regulation, DNA repair and cell apoptosis. To study the effect of ubiquitin-pro teasome pathway on apoptosis in THP-1 cells and the possible mechanism of apoptosis, THP-1 cells were treated and the related indexes were detected. Methods THP-1 cells were treated with MG132 (5 μmol/L) for 24 h. Ubiquitin, ubiquitin-activat ing enzyme (E1), ubiquitin-conjugating enzyme (E₂), ubiquitin-protein ligase (E3) and 26S proteasome genes expression were detected by reverse transcription-polymerase chain reaction (RT-PCR). Apolipoprotein B (ApoB) concentration in THP-1 cells and cell apoptosis rate were detected by flow cytometric analysis (FCA). Results RT-PCR showed that E1, E₂, E3 genes expression were decreased in treatment group and the concentration of ApoB and apoptosis rate were increased obviusly. Conclusions Protesome inhibitr MG132 could inhibit the expression of E1, E₂ and E3, UPP activity was inhibited. Degradation of ApoB decreased and so cell apoptosis rate increased. The apoptosis of macrophages in the early stage of atherosclerosis could delay the atherogenesis. This experiment is to detect the novel mechanism of macrophages from the regulation of ubiquitin-proteasome pathway, it could be a new target to prevent the atherogenesis.

Abstract:Aim The study aims to observe how lipoprotein lipase activator NO-1886 affect the content of vanadium and chromium in pig pancreas with high fat and high sugar. Methods Guizhou minipigs were divided into three groups randomly: control group fed with basic feedstuff, sugar fat group of fed with high fat and high sugar feedstuff, and NO-1886 group fed with high fat and high sugar feedstuff in the first 3 mouths and then added 1 % NO-1886 since the 4th mouth. The pigs were fed separately. Theirs sugar, fat and insulin of blood plasma were observed. The pigs were killed to get the pancreas at the end of the experiment and the tissue were digested with acid. The content of vanadium and chromium were tested Atomic Emission Spec trometry. Resutls The sugar fat group and NO-1886 group are as the followings: before the NO-1886 was added, the blood insulin in each group is 11.4±2.7 mU/L, 21.0± 4.8 mU/L, and 21.9 ± 6.6 mU/L, and compared with the control group, the blood insulin of the later two groups rises ( P< 0.05); then adding the NO-1886, at the end of the experiment the blood insulin in each group is 11.4 ± 6.2 mU/L, 20.4±2.3 mU/L, and 15.4± 1.8 mU/L; the content of insulin has no obvious difference between control group and NO-1886 group, while there is some difference between control group and sugar fat group ( P< 0.05) . Through insulin sensitive experiment, it was found that before injection blood insulin in each group is 19.3 ± 6.5 mU/L, 11.6 ±2.9mU/L, 19.3±7.1 mU/L, 30 min after injection the data is 123.6 ± 32.9 mU/L, 71.7 ±21.7 mU/L, and 141.5 ±29.4 mU/L, and 90 min after injection the data is 45.9±5.6 mU/L, 17.9± 12.4 mU/L, and 32.9± 12.9 mU/L, and compared with the control group, the content of insulin in the sugar fat group reduced (in the 30th min p< 0.05, in the 90th min P< 0.01). At the end of experiment, the pancreas' vanadium content in each group is0.8±0.8ng/g,0.7±0.1 ng/g, and 0.8 ± 0.3 ng/g, the chromium content is 2.3 ± 1.2 ng/g, 1.9± 0.6 ng/g, and 2.1 ± 0.9 ng/g, and compar with NO-1886 group, the vanadium and chromium content in the sugar fat group reduced( P< 0.05), while there is no obvious difference between control group and NO-1886 group. Conclusion NO-1886 can improve the content levels of vanadium and chromium and the sensitivity of the insulin in the pigs' pancreas with high fat and high sugar.

Abstract:Aim To compare the liver protein profiles of hypercholesterolemic and normal mice by using the technology of proteomics. Methods The hypercholesterolemic mice were obtained after feeding atherogenic diet for 14 weeks. Protein extractions from mice livers were separated by two-dimensional electrophoresis and the gels were analysed with image analysis software. The differentially expressed proteins were identified primarily by mass spectrometry and then confirmed by comparing with the nice gel image in protein database Swiss Prot. Results The hypercholesterolemic mouse model has been successfully prepared. The total cholesterol levels in plasma and liver of hypercholesterolemic mice increased significantly. The protein extractions separated by two-dimensional electrophoresis have got high resolution and reproducibility. Sixteen differentially expressed protein spots( > 2 fold) have been found and 8 of which were identified as major urinary proteins (MUPs), carbonic anhydrase Ⅲ and Glutathione S-transferase P2. Conclusion The under-expression of MUPs, carbonic anhydrase Ⅲ and Glutathione S-trans ferase P2, which regulated by androgens, may be related to diet-induced hypercholesterolemia.

Abstract:Aim To study the protective effect of onychin against vascular endothelial cell apoptosis induced by oxida tive stress. Methods Cultured human umbilical vein endothelial cells (ECV304) were incubated for 30 min with either vehicle (DMSO), genistein or onychin before being challenged with H₂O₂ . Cell viability were measured by the MTT assay, and cell apoptosis was determined by flow cytometric analysis and terminal deoxynucleotidyl transfersas-mediated dUTP nick end-labeling (Tunel) respectively. Meanwhile, Western blot was used to measure the activation of caspase-3. Results H₂O₂ treatment for 24 h evoked endolhelium apoptosis, and onychin (0.3, 1 and 3 μmol/L.) decreased the rate of apoptoic endothelial cells (15. 5%±1.2%, 12.6% ±0.9% and 8.2% ±0.8% vs 22.7% ± 3.9% )in a concentration-dependent manner. Meanwhile, Onychin decreased the expression of active caspase-3 induced by H₂O₂ as genistein. Conclusion onychin prevents H₂O₂-in-duced endothelium apoptosis which correlated with inhibition of caspase-3 activation.

Abstract:Aim To study the effect of TNFαon L-type cacium channel current (ICa-L) in isolated guinea pig ventricular myocytes in normal Tyrode solution and with imitating acute ischemia. Methods Using the whole cell patch clamp technique in isolated guonea pig ventricular myocates both normal Tyrode solution or simulation of acute ischemia. Results Under normal Tyrode solution, the application of TNFα (100 kU/L, 300 kU/L and 500 kU/L) did not produce any detectable alterations on peak of ICa-L, but inhibitory rate on peaks of ICa-L were decreased markedly by 46.5% ± 3.5% , 62.7%±3.0% and 80.7% ±3.7% respectivelyby TNFα at the same concentration accompany simulation of acute ischemia and showed dose-dependence; there are significant difference compare with control group or simulate of acute ischemia alone group ( P< 0.05, respectivelyby) . Conclusion TNFαrwould not alter peak of ICa-L in guinea pig ventricular cells under normal Tyrode solution; but increased inhibitory effects of ischemia on peaks of ICa-L accompany imitation of acute ischemia and showed concentration-dependence .

Abstract:Aim To explore the mechanism of anoxia-reoxygenation injury (H/R) and the late protective effect of cato pril on endothelial cells (EC) . Methods To establish anoxia-reoxygenation model on human unbilical veins endothelial cell line ECV304, and observe the injury degree of anoxia and reoxygenationg at the different time. Then these ECs were randomly divided into 5 groups: catopril, catopril + bradykinin β₂ receptor inhibitor, catopril + PKC inhibitor, catopril + NOS inhibitor, catopril + NF-κB inhibitor, and obsene the cell morphologic changes, mortality and the activities of malondialdehyde (MDA), su peroxide dismutase (SOD), glutathione peroxidase (GSH-PX) . Results After treatment with anoxia and anoxia-reoxygen-ation, the ECs' morphologic changes were observed. The mortality increased and MDA concentration became higher, the concentration of SOD and CSH-PX became lower( P< 0.01). Meanwhile, the changes became more obviously with the time extending. The late preconditioning with catopril, the cell morphologic, MDA, SOD and CSH-PX keep normal. The protective effect became strenghen along with the catopril concentration increasing. However, culturing with above four inhibitors, the protective effect was partly diminished. Conclusion Anoxia and anoxia-reperfusion induce lipid peroxidation and the weakening of an tioxidation ability in a time-dependented manner. The late preconditioning with catopril can weaken anoxia-reperfusion injury in a concentration- dependented manner. Bradykinin β₂ receptor, PKC activating, nitric oxide and nucleus factor are all involved in the protective effect.

Abstract:Aim To prepare catalase-contained recombinant adenovirus. Methods Catalase gene was digested from plasmid of pZeoSV2-Cat, subcloned into plasmid of pBluescript Ⅱ sk( + ) and formed plasmid of pBluescript Ⅱ sk( + )-Cat. Then Catalase gene was digested from plasmid of pBluescript Ⅱ sk( + ) -Cat, subcloned into shuttle plasmid of pShuttle-CMV and formed transfer plasmid of pShuttle-CMV-Cat. Adenovirus genomic plasmid of pAdEasy-1 was transformed into BJ5183 bacteria and prepared ultracompletent BJ5183 containing pAdEasy-1. pShuttle-CMV-Cat was linealinzed with Pme I and transformed into ultracompletent BJ5183 containing pAdEasy-1. The identified recombinant adenovirus plasmid of pAdEasy-1-Cat was linealinzed with Pac I and transfected into Ad-293 cells to package recombinant adenovirus particles. The target gene was decteted by PCR. Results The hneahnzed pShuttle-CMV-Cat was transformed into ultracompletent BJ5183 containing pAdEasy-1 .There were 30% positive recombinant plasmid. After pAdEasy-1-Cat was transfected into Ad-293 cells, recombinant adenovirus particles were produced and further amplified. PCR test indicated that the recombinant adenovirus AdCat contained Catalase gene. The liter of the purified AdCat was 4.12 × 1015 OPU/L. Conclusion The homologous recombination in bacteria is a convenient and high efficient method to prepare AdCat. This affords a good gene transfer vector for the gene therapy in restenosis.

Abstract:Aim To study effects of the Bu-shen-ning-xin recipe on atherosclerosis of ovariectomized rabbits and its possible mechanism. Methods Twenty-six female New Zealand white rabbits of three months old were randomly divided into normal group, sham operation group, ovariectomized group and treatment group in which the rabbits were ovariectomized and afforded with Bu-shen-ning-xin decoction. All of rabbits, except normal control, were given high-cholesterol diet two weeks after operation, the treatment group was given additionally by the medication. Twelve weeks later, serum total cholesterol (TC), trig lyceride (TG), high density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDLC) levels were measured in routine. The aortas were collected for histomorphometrical evaluation with light and scanning electron microscope. And the expression of vascular cell adhesion molecule-1 (VCAM-1) was analyzed by immunohistochemical techniques. Results Following treatment for 12 weeks, no significant difference was found in serum TC, TG, HDLC and LDLC levels among three groups except the normal. The recipe could lower the area ratios of atherosclerotic lesion to endothelium and the thickness ratios of en-dothelium to the medium in treatment group compared with ovariectomized group. The pathologic damage of aortas were relieved and the expression of VCAM-1 protein in aortas were also significantly reduced following the recipe treatment. Conclusion The Bu-shen-ning-xin recipe could inhibit the VCAM-1 expression of aorta, which contributed to alleviate the pathologic progress of atherosclerosis of the ovariectomized rabbit.

Abstract:Aim To investigate the simultaneous effects of antisense and decoy oligonucleotides of nuclear factor (NF) κB on expression of adhesion molecules induced by tumor necrosis factor(TNF)-α in vitro. Methods Intracellular uptake of fluorescein isothiocyanate (F1TC) -labeled oligonucleotides was detected by fluorescence active cell sort (FACS) ; Adhesion molecules expression was presented by reverse transcription-polumerase chain reaction (RT-PCR) at RNA level, FACS at protein level. Results Simultaneous incubation with antisense P65 and decoy oligonucleotides (ODN) of NF-κB, the inhibitory effect of VGAM-1 was 59.0%, 41.1% and 35.2% at mRNA level, 85.5%, 44.3% and 49.2% on human vascular endothclial cells (hUVEC) surface compared with control group, decoy group and antisense group. The inhibitory effect of simultaneous incubation with antisense and decoy oligonucleotides was stronger than the effect of incubation with one of them. Conclusion It was demonstrated that a simultaneous administration of antiscnse and decoy oligonucleotides of NF-κB was effective and necessary if a sufficient inhibition of NF-κB mediated activities should be achieved.

Abstract:Aim To investigate the simultaneous effects of antisense and decoy oligonucleotides of nuclear factor (NF) κB on expression of adhesion molecules induced by tumor necrosis factor(TNF)-α in vitro. Methods Intracellular uptake of fluorescein isothiocyanate (F1TC) -labeled oligonucleotides was detected by fluorescence active cell sort (FACS) ; Adhesion molecules expression was presented by reverse transcription-polumerase chain reaction (RT-PCR) at RNA level, FACS at protein level. Results Simultaneous incubation with antisense P65 and decoy oligonucleotides (ODN) of NF-κB, the inhibitory effect of VGAM-1 was 59.0%, 41.1% and 35.2% at mRNA level, 85.5%, 44.3% and 49.2% on human vascular endothclial cells (hUVEC) surface compared with control group, decoy group and antisense group. The inhibitory effect of simultaneous incubation with antisense and decoy oligonucleotides was stronger than the effect of incubation with one of them. Conclusion It was demonstrated that a simultaneous administration of antiscnse and decoy oligonucleotides of NF-κB was effective and necessary if a sufficient inhibition of NF-κB mediated activities should be achieved.

Abstract:Aim To investigate the effect of angiotensin Ⅱ (AngⅡ ) on the expression of monocyte chemoattractant protein-1 (MCP-1 ) and the activation of nuclear factor κB {NF-κB) in the endothelial cells under high glucose conditions, and the influence of losartan on MCP-1 and NF-KB. Methods MCP-1 mRNA was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR), and the level of MCP-1 in culture media was measured by means of sandwich ELISA method. NF-κB activation in endothelial cells was determined with laser scanning confocal microscope. Results Ang Ⅱ significantly increased the levels of MCP-1 mRNA and protein expression and NF-KB activation in the cultured endothelial cells. High glucose (25 mmol/L) could significantly increase the activation of NF-κB and the expression of MCP-1 in endothelial cells, while normal glu cose(5 mmol/L) could not. It was also shown that high glucose promoted AngⅡ -induced NF-KB activation and MCP-1 expression in endothelial cells. Losartan significantly inhibited MCP-1 mRNA and protein expression and markedly reduced the contents of MCP-1. Conclusion High glucose aggravate AngⅡ -induced NF-κB activation and MCP-1 expression in endothelial cell, which can be significantly inhibited by Losartan.

Abstract:Aim To investigate the effect of angiotensin Ⅱ (AngⅡ ) on the expression of monocyte chemoattractant protein-1 (MCP-1 ) and the activation of nuclear factor κB {NF-κB) in the endothelial cells under high glucose conditions, and the influence of losartan on MCP-1 and NF-KB. Methods MCP-1 mRNA was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR), and the level of MCP-1 in culture media was measured by means of sandwich ELISA method. NF-κB activation in endothelial cells was determined with laser scanning confocal microscope. Results Ang Ⅱ significantly increased the levels of MCP-1 mRNA and protein expression and NF-KB activation in the cultured endothelial cells. High glucose (25 mmol/L) could significantly increase the activation of NF-κB and the expression of MCP-1 in endothelial cells, while normal glu cose(5 mmol/L) could not. It was also shown that high glucose promoted AngⅡ -induced NF-KB activation and MCP-1 expression in endothelial cells. Losartan significantly inhibited MCP-1 mRNA and protein expression and markedly reduced the contents of MCP-1. Conclusion High glucose aggravate AngⅡ -induced NF-κB activation and MCP-1 expression in endothelial cell, which can be significantly inhibited by Losartan.

Abstract:Aim To investigate the antihypertrophic effects of pioglitazone and its effects on proinflammatory cytokines. Methods Cardiac hypertrophy was produced by abdominal aortic banding(AB) . Pioglitazone(20mg/kg-1 day-1 )was given orally 1 week before operation and continued till 4 weeks after operation, then the rats were killed, part of the heart was used for measurements of left ventricular wall thickness, and the mvocyte diameter, the other part of the heart was used for testing the mRNA expression of proinflammatory cytokines. Result The mRNA expression of proinflammation cytokines Interleukin-1β, Cardiotropin-1 and brain natriuretic peptide (a molecular marker for cardiac hypertrophy) was markedly enhanced in the hypertrophic myocardium of AB rats. Compared with control group, treatment of AB rats with pioglitazone significantly inhibited the mRNA expression of the gene mentioned above,and reduced the increases in the heart weight-to-body weight ratio (4.77 ± 0.25 mg/g vs 4. 23 ± 0.22 mg/g, p< 0.05), myocyte diameter (11.61 ± 1.34 μm vs 10.07± 1.07 μm, p<0.05) and left ventricular wall tliickness. Conclusion Pioglitazone may attenuate cardiac hypertrophy through inhibition of proinflammatory cytokines.

Abstract:Aim To investigate the antihypertrophic effects of pioglitazone and its effects on proinflammatory cytokines. Methods Cardiac hypertrophy was produced by abdominal aortic banding(AB) . Pioglitazone(20mg/kg-1 day-1 )was given orally 1 week before operation and continued till 4 weeks after operation, then the rats were killed, part of the heart was used for measurements of left ventricular wall thickness, and the mvocyte diameter, the other part of the heart was used for testing the mRNA expression of proinflammatory cytokines. Result The mRNA expression of proinflammation cytokines Interleukin-1β, Cardiotropin-1 and brain natriuretic peptide (a molecular marker for cardiac hypertrophy) was markedly enhanced in the hypertrophic myocardium of AB rats. Compared with control group, treatment of AB rats with pioglitazone significantly inhibited the mRNA expression of the gene mentioned above,and reduced the increases in the heart weight-to-body weight ratio (4.77 ± 0.25 mg/g vs 4. 23 ± 0.22 mg/g, p< 0.05), myocyte diameter (11.61 ± 1.34 μm vs 10.07± 1.07 μm, p<0.05) and left ventricular wall tliickness. Conclusion Pioglitazone may attenuate cardiac hypertrophy through inhibition of proinflammatory cytokines.

Abstract:Aim To investigate the dynamic changes of adrenomedullin and its specific receptors sys tem-calcitonin receptor like receptor (CRLR) mRNA expression in the stress-induced-hypertensive rats. Methods Distribute 48 rats to control group with 18 of them and stress group with 30 of the total randomly. Inflict noise or noise with eletric foot shock upon the stress group. Excute all the rats and then separate their hypothalamus to isolate the total RNA indiridually at stress 1,5, 10 and 15 days and poststress 5 and 10 days. Determine the changes of adrenomedullin and calcitonin receptor-like receptor mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) . Results Compared with control group at the same time point, ADM and CRLR mRNA expression in the hypothalamus upregulated within foot-shock and sound stress 10 days (stress 5 days: ADM mRNA expression 0.92 ± 0.04 vs 0.99 ± 0.05, p< 0.05; stress 10 days: 1.26 ± 0.04 vs 0.92 ± 0.04, P<0. 01, and then downregulated, but still kept at a high level at stress 15 days (1.00 ± 0.04 vs 0.92 ± 0.04, P< 0.05) and posts tress 5 days ( P> 0.05) . Conclusion According to the results described above, at the gene level, the expression of ADM and its specific receptor components CRLR mRNA had changed to some degree in the process of chronic stress-induced-hyperten sion, indicating that ADM and its specific receptor maybe involve in the stress-induced-hypertension.

Abstract:Aim To evaluate the changes of nitric oxide synthase-nitric oxide system activity in rat cardiac fibroblasts (CF) derived from spontaneously hypertensive rat (SHR) and Wistar rat with and without carvedilol. Methods CF were iso-lated by trypsin digestion method. Nitric acid reductase method and spectrophotometry were used to detect the NO contents and NOS activity respectively with or without carvedilol. Results Under basal condition (72 h), NO content (66.6 ± 3.5 μmol/ L)ancl NOS activity (16.7 ± 0.7 kU/L)of CF derived from SHR were siginificantly lower than those of Wistar rats (80. 8 ± 6.2 μmol/L, 29.1 ± 2. 1 kU/L) ( P< 0.01) . Carvedilol could significantly increase NOS activity and NO content of CF derived from two groups, which showed dose-dependent effects. Moreover, NO contents increased with the enhancement of NOS activity, and there was significant positive relevance between them ( r = 0.911, P< 0.01) . Conclusion The NOS-NO system of CFin SHR was abnormal, which showed that NOS activity and NO content of CF in SHR were lower. Carvedilol could significantly increase NOS activity and NO content of CF derived from two groups, but the effect on CF of SHR was much more powerful than that of Wistar rats. The effect may be an important mechanism to control blood pressure.

Abstract:Aim To investigate whether thrombin could activate the signaling pathway-nuclear faclor-κB (NF-κB) and upregulate the expression of matrix metalloproteinase-2 (MMP-2) on human umbilical vein endothelical cellls (hUVEC) in vitro. Methods hUVEC was stimulated with thrombin, adding to hirudin and heparin according to separate groups. Immunocy tochemistry and Western blot analyses were used to detect the activation of NF-κB, which means the transfer of NF-KB from cyto plasina to nucleus. The expression of MMP-2 was determined with reverse transcriptase-polymerase chain reactin (RT-PCR).Results It was found that tlirombin could rapidly induce the activation of NF-KB in hUVEC. Baseline NF-icB nuclear binding in hUVEC increase by more than 14. 13% and 31.29% at 15 min and 30 min respectively. Thrombin significantly increased MMP-2 mRNA expression and production in hUVEC with time-dependent pattern (1-48 h). Hirudin was able to inhibit the activation of NF-κB and expression of MMP-2 in hUVEC. Conclusion Thrombin could activate the signal pathway-NF-κB and had remarkable effect on hUVEC expression of MMP-2, which may thereby influence the pathogenesis of atherosclerosis.

Abstract:Aim To investigate the influence of lipopolysaccharide (LPS)-induced adventitial inflammation on artery in timal proliferation and its mechanism. Methods 24 New Zealand rabbits were fed with high cholesterol diet for 2 weeks, two sides of iliac artery were randomly divided into two groups: LPS group ( n = 24) and control group ( n = 24). A nonconstrictive polyethylene cuff was placed loosely around the iliac artery of New Zealand rabbits, between the artery and the cuff, and a sterilized cotton thread was placed, which was previously soaked overnight in 6 mg/L LPS in saline. Animals were killed at intervals of 0 day ( n = 8) ,3 days { n = 8), 2 weeks ( n = 8) after surgery. The morphology of the artery and the immunochemistory expression of nuclear factor (NF) κB p65 in the artery was investigated. Results In LPS group, 3 days after surgery, inflammation cells migrated into artery from both the luminal and adventitial side, and 2 weeks after surgery, inflammation cells could only be seen in adventitia; NFκB p65 was expressed in endothelium and adventitial cell in LPS group 3 days after surgery; 2 weeks after surgery, LPS group have increasd intimal area (0.93 ± 0.14 mm2), compared with control group(0.75 ± 0.15 mm2) (P< 0.05). Conclusions LPS-induced adventitial inflammation contributes to intimal proliferation, and the activation of NFκB in endothelium and adventital cells may be the mechanism of it.

Abstract:Aim To observe the effect of probucol on endothelium-dependent vasodilator activity in high cholesterol plus high fat diet (HCHF) rabbits. Methods 26 New Zealand white rabbits (1.5-2.5 kg) were randomly divided into three groups, that is, control group ( n = 6, normal diet); hyperlipidemic group ( n = 10, HCHF-diet); probucol group ( n = 10, HCHF-diet plus probucol 200 mg/kg everyday). There blood was drawn from the central ear artery to analyze plasma total cholesterol (TC) at the pre-experiment and in the 12th week of post experiment. Then all rabbits were killed in the 12 th week and thoracic aortas were isolated to analyze vascular endothelial function. Results Probucol could remarkably lower plasma TC lever,compared to hyperlipidemic group, TC level decreased 39.1 % in probucol group ( P< 0.01). The HCHF diet significantly attenuated endothelium-dependent relaxation (EDR) of rabbit aortic rings to acetylcholine (Ach), but probucol could significantly protected against HCHF diet-injured EDR of rabbit aortic rings. The HCHF diet and probucol did not significantly influence the endothelium-independent relaxation of the rabbit artery elicited by sodium nitroprusside (SNP). Conclusion Probucol can protect against injuries of vascular endothelial function induced by hypercholesterolemia through lowering plasma TC and anti-oxidant.

Abstract:Aim To investigate the levels of plasma adiponectin and resistin in type 2 diabetes and to explore their roles in this disease. Methods According to the body mass index (BMI), 60 type 2 diabetes patients were divided into two groups, one group was non-obese diabetes patients with BMI < 25 kg/m2 (30 cases) and the other group was obese diabetes patients with BMI > 25 kg/m2 (30 cases). There were 28 healthy persons in the control group. ELISA technique were employed to determine the plasma adiponectin and resistin concentration; at the same time the fasting blood glucose, insulin and blood lipid were detected; insulin resistance index and insulin sensitive index were calculated by the HOMA. Results ③The levels of plasma adiponectin were decreased significantly in diabetes group compared with that in control group ( P< 0.01); moreover, the adiponectin concentration in obese diabetes group were significantly decreased compared with that in non-obese diabetes group (P< 0.01). ②The levels of plasma resistin were increased significantly in diabetes group compared with that in control group ( P< 0.05, p<0.01); furthermore, the levels of resistin in obese diabetes group were increased significantly compared with that in non-obese diabetes group ( P< 0.01). ③Plasma adiponectin correlated negatively with BMI, blood glucose, insulin resistance index and triglyceride (respectively r= -0.55, p<0.01; r = -0.51, p<0.01; r= -0.52, p<0.01; r= -0.39, p< 0.05). It was positively correlated with insulin sensitive index (r=0.45, P< 0.01). Oppositely plasma resistin correlated positively with BMI, blood glucose, triglyceride and insulin resistance index (respectively r = 0.40, p<0.01; r = 0.52, P< 0.01; r = 0.46, p<0.01; r = 0.27, p<0.05), but it was negatively correlated with insulin sensitive index ( r = -0.32, P< 0.01). Conclusion Plasma adiponectin and resistin take apart in the development of insulin resistance and they may be associated with the disorder of glucose and lipid in type 2 diabetes.

Abstract:Aim To explore the dynamic changes of the plasmatic activities of tPA and PAI-1 and its clinical significance by observing the plasmatic activity of tPA and PAI-1 in acute and recovery phases of the patients with arteriosclerotic cerebral infarction (ACI). Methods The plasmatic activities of tPA and PAI-1 were determined by Chromgenic Substrate methods in 91 patients with ACI and 40 normal old ages as control, while the infarction volume and the neurological damage score in those with ACI were measured. Results The plasmatic activities of tPA in acute and recovery periods of the patients with ACI was 0.26±0.14and0.21±0.11 kIU/L respectively and was significantly lower than those of the control subjects ( P< 0.01), and the plasmatic activities of PAI-1 in acute and recovery periods of the patients with ACI were 0.90 ± 0.25 and 0.98 ± 0.12 kAU/ L respectively and were significantly higher than those of the control subjects ( P< 0.01), while the infarction volume and the neurological damage score in those with ACI were 8.75 ± 1.21 cm and 18.56 ± 3.62 respectively, and the plasmatic activity of tPA was negatively related to the infarction volume and the neurological damage score in those with ACI (r = - 0.5133, P< 0. 05 and r = - 0.4914, P< 0.05 respectively ), and the plasmatic activity of PAI-1 was positively related to the infarction volume and the neurological damage score in those with ACI(r = 0.5621, p< 0.05 and r = 0.5342, p< 0.05 respectively). Conclusions The fibrinolysis activities of the acute and recovery periods of the patients with ACI decline significantly, which suggests that tPA and PAI-1 play an important role in the pathological process of arteriosclerotic cerebral infarction.

Abstract:Aim To explore frequency of angiotensin converting enzyme (ACE) gene insertion/deletion( I/D ) polymorphism and effect of ACE gene polymorphism on level of serum ACE and blood lipid in patient with essential hypertension and healthy subjects. Methods 216 subjects (98 heathy subjects and 118 hypertensives) were collected for the determination of the I/D genotype in intron 16 of ACE gene by polymerase chain reaction(PCR) and the level of serum ACE, total cholesterol (TC), triglycerides(TG), low density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), lipoprotein (a) [ Lp( a) ] , apolipoprotein AI (ApoAI), apolipoprotein B (ApoB). Results The frequency of I/D genotypes and D and I alleles of the ACE gene had no significant difference between hypertensives and healthy subjects (X2 = 0.468, P = 0.791; X2 = 0. 379, P = 0.538, respectively) . The level of serum ACE activity in deletion homozygotes (genotype DD) was the highest among three genotypes, and the lowest in insertion homozygotes (genotype II ), and intermediate in heterozygotes( genotype ID) (F= 17. 107, P = 0.000). Hypertensives had significantly higher TC, LDLC, Lp(a) than those of control group (p<0.0l). The level of blood lipid was significantly different among genotype DD, genotype ID, and genotype II in hypertensives group ( P< 0. 01). The level of TC, LDLC, HDLC, ApoB was significantly different among three genotypes in control group ( P< 0.05).Conclusions ACE gene polymorphism is associated with level of serum ACE and blood lipid. The level of serum ACE and blood lipid is higher in genotype DD than in genotype ID and genotype II .

Abstract:Aim To observe therapeutic effects and safety of Die-mai-ling injection (DMLI) on acute cerebral infarction. Methods 110 cases with acute cerebral infarction were randomly divided into three groups. 34 cases were given low molecular heparin (LMWH), 36 cases were given DMLI, and 40 cases were given DMLI + LMWH. Supporting treatment and heteropathy were employed in three groups. Neurological scales, therapeutic assessment, fibroprotein and the largest infarction area in CT of this attack were compared in three groups before and after treatment. Liver and renal function, blood routine and ECG were observed simultaneously. Results On the 14 th and 28 th day after treatment, neurological scales decreased significantly in the LMWH + DMLI group compared with LMWH group and DMLI group ( P< 0.05 or P< 0.01), there was no significant difference between LMWH group and DMLI group ( P> 0.05). On the 14th and 28 th day, the total effective rate of LMWH group, DMLI group and LMWH + DMLI group was 50% , 52.7%, 67.5% and 70.6% , 72% , 87.5%, LMWH+DMLI group was significantly higher than LMWH group and DMLI group ( P< 0.01), but no significant difference was found between LMWH group and DMLI group (P> 0.05). Rates of healing in LMWH + DMLI group was higher than LMWH group and DMLI group. The recovery ratio of DMLI group and LMWH + DMLI group was higer than that of LMWH group ( P< 0.01). There was no significant difference on the fibroprotein levels and the largest infarction area in the three groups before and after treatment ( P> 0.05). The treatment of DMLI was well toloranced, no adverse effects on liver and renal function and blood routine were observed. Conclusions DMLI is effective for the treatment of acute cerebral infarction, which curative effect is similar to LMWH. DMLI conjoined with LMWH in the treatment of acute cerebral infarction can improve effective rates significantly. The safety and tolerance of DMLI were comparatively good.

Abstract:Aim To observe the change of plasma nitric oxide (NO) of acute cerebral infracted patients and the influence of simvastatin on the level of plasma NO. Methods Using HNO3 deoxidize enzyme method, we examined the levels of plasma NO in 54 patients with cerebral infarction (They were devided into two groups at random: 31 patients in simvastatin therapy group and 23 patients in ordinary therapy group) and 37 normal controls. At the same time, we scored the neurologic impairment of the patients before and after the therapy. Results In cerebral infarction group, the level of plasma NO was significantly higher than the controls at 3d (P< 0.05). After 3 weeks of therapy, the level of plasma NO was not notably different between ordinary therapy group and controls (P> 0.05). While, in simvastatin therapy group, the level of plasma NO was higher than the ordinary therapy group ( P< 0.05). It was significantly different between the two therapy groups about neurologic impairment score after therapy ( P< 0.01). Conclusion The level of plasma NO in patients with acute cerebral infarction might be a reference index for cerebral ischemic damage. Simvastatin can raise the level of plasma NO and improve cerebral infarction prognosis.

Abstract:Aim To explore the relation between the levels of serum vascular endothelial growth factor (VEGF) and different area of cerebral infarction. Methods 50 acute cerebral infarction patients had been tried. Each of them would be drawed 2 mL of blood and analyzed at the admission time, 1,3,7 and 14 day after acute ischemic stroke. After centrifugated the sample was stored in - 70℃ According to CT (the 3rd day after stroke), all patients were classified into three groups as having a large infarct or a moderate infarct or a small infarct. And then, the serum levels of VEGF would be investigated. Results There were difference in the serum levels of VEGF of the three groups at the time when they were admissioned ( P< 0. 05), which were the same at other periods. Conclusions The more size of the infarction, the higher the serum level ofVEGF. This phenomenoncan be discovered in the earlier period (less than 16 h after stroke).