Abstract:Aim To explore the effect of lysophosphatidylcholine on cholesterol efflux from macrophage foam cells, and the function of calcium signaling pathway on this effect. Methods Macrophage foam cells were harvested from female mice weighing 25～30 g. Human LDL was separated from healthy subjects and isolated by differential ultracentrifugation. LDL was acytylated to ac-LDL to convert macrophage to foam cells. Cholesterol mass of medium and cells were quantified by enzymetic fluorometry. Intracellular calcium concentration was measured by fluorometry. Involvement of intracellular calcium and PKC on effect of cholesterol efflux from macrophages by LPC was measured by applying EGTA and H 7 to adduct extracellular calcium and to inhibit the activity of PKC, then observed whether cholesterol efflux could occur. Results Within the dosage of 10～80 μmol/L, LPC obviously promoted cholesterol efflux from macrophage foam cells, and LPC increased intracellular calcium concentration. When extracellular calcium was cleared by EGTA or PKC activity was inhibited by H 7, LPC could not promote cholesterol efflux from macrophage foam cells. Conclusions LPC could promote cholesterol efflux from macrophage foam cells within the dosage of 10～80 μmol/L, this effect is related to influx of intracellular calcium. Calcium-PKC signaling pathway induced LPC's effect on cholesterol efflux from macrophage foam cells.

Abstract:Aim To investigate the effects and mechanisms of anisodamine on plasminogen activator inhibitor type 1 (PAI 1) expression in normal endothelial cells (EC) and lipopolicaccharide (LPS) treated EC. Methods Human umbilical vein endothelial cells (hUVEC) were cultured by trypsin digestion method. PAI 1 and tissue plasminogen activator (tPA) proteins in hUVEC conditioned medium were measured by sandwich enzyme linked immunosorbent assay (ELISA), and their mRNA expressions were determined by Northern blot analysis. Using electrophoretic mobility shift assay (EMSA), we assessed hUVEC nuclear factor kappa B (NF κB) nuclear translocation. Results LPS treatment of cultured hUVEC resulted in a significant increase in PAI 1 protein as well as mRNA expression by these cells. However, when hUVEC were incubated with LPS plus anisodamine, the upregulation of PAI 1 by LPS was abated. Moreover anisodamine was able to decrease the basal level of PAI 1 protein and mRNA as compared to control. Nuclear extracts prepared from LPS stimulated hUVEC demonstrated increased binding to the NF κB oligonucleotide as compared to unstimulated cells, and anisodamine could block those binding in the presence of LPS. Conclusion Anisodamine downregulated both basal and LPS induced PAI 1 protein and mRNA expression in EC, and the mechanism of the modulation might be via NF κB pathway.

Abstract:Aim To study the expression of macrophage inflammatery protein (MIP) 1α in atherosclerotic lesions and to examine the effects of native and oxidized low density (LDL, oxLDL) and very low density lipoproteins (VLDL,ox VLDL) on the expression of MIP 1α in human peripheral blood monocytes. Methods The expression of MIP 1α mRNA in dietary atherosclerotic lesions in rabbits was detected by in situ hybridization. The effects of LDL, ox LDL, VLDL and ox VLDL on the expression of MIP 1α mRNA and protein in human blood monocytes were determined by RT PCR and cell ELISA respectively. Results In situ hybridization showed that the cytoplasm of the foam cells was stained brownish purple, and the lipid droplets in the cytoplasm were not stained so that the whole foam cell area looked like a brownish purple network. The cytoplasm of endothelial cells was stained as well. RT PCR demonstrated that ox LDL and ox VLDL induced a 2.4 fold and a 1.6 fold increase in MIP 1α mRNA expression in human blood monocytes respectively compared with the control group, and cell ELISA also showed that ox LDL and ox VLDL induced a 2.3 fold and a 2.0 fold increase in MIP 1α protein expression in monocytes respectively, while LDL and VLDL didn't. Conclusions The Foam cells and endothelials in the atherosclerotic plaques can express MIP 1α. ox LDL and ox VLDL might be able to induce increased expression of MIP 1a mRNA and protein in human blood monocytes, and could, therefore, enhance the atherogenesis through increased recruitment of more and more monocytes into the arterial intima.

Abstract:Aim To study the effect of lysophosphatidylcholine (LPC) on the expression of vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cell line (ECV304) and the inhibitory effect of salvianolic acid B(Sal B) in vitro. Methods Exposured to 5 mg/L LPC or LPC +Sal B for 24 h, VEGF protein in ECV304 cells conditioned media of each group were determined by enzyme linked immunosorbent assay (ELISA). Meanwhile, VEGF mRNA expression in ECV304 was examined by in situ hybridization. Results LPC upregulated VEGF protein and VEGF mRNA expression in the ECV304 cells. Sal B could markedly inhibit the LPC-induced increasing of VEGF(P<0.001). Conclusions LPC could induce a strong expression of VEGF in ECV304 cells and Sal B could inhibit it.

Abstract:Aim To check the effect of oxidative stress on inductive nitric oxide synthase (iNOS) mRNA expression in macrophages. Methods The effect of tert butyl hydroperoxide (tbOOH) on iNOS mRNA expression in RAW264.7 cells was in vestigated by RT PCR method. Results It showed that, 1.5×10 -4 mol/L tbOOH could induce iNOS mRNA expression in RAW264.7 cells; and the induction could be attenuated by actinomycin D or cycloheximide or acetovanilone added in the medium. Transcription factor NF κB specific inhibitor PTDC could also reduce the induction. Conclusions It indicated that, tbOOH could transcriptionally induce iNOS expression in RAW264.7 cells, de novo protein synthesis and intracellular superoxide (O 2 -·) production might be involved in the process. The activation of NF κB was also one part of the process.

Abstract:Aim To evaluate the mechanism of vascular endothelial growth factor (VEGF) on prevention of restenosis after angioplasty. Methods The recombinant adenovirus containing the cDNA for hVEGF165 was constructed and infected vascular smooth muscle cell(VSMC)in vitro. 72 hours after the infection the conditioned medium containing VEGF was collected. Then the VSMC and human umbilical vein endothelial cell(hUVEC) were divided into control group,H 2O 2 treated group and H 2O 2+VEGF treated group to observe the proliferation and apoptosis by WST-1 method,TUNEL and FCM. Results To hUVEC,the OD value wasless frequent in the H 2O 2 treated group than that in the control and H 2O 2+VEGF treated group,the apoptosis was markedly superior in H 2O 2 treated group compared with control and H 2O 2 + VEGF-treated group. To VSMC,the changes of OD value and apoptosis were contrary to hUVEC. Conclusions H 2O 2 stimulated the proliferation of VSMC and induced the apoptosis of hUVEC, inhibited the proliferation of hUVEC and the apoptosis of VSMC, improved the restenosis. VEGF inhibited the effect of H 2O 2 on hUVEC and VSMC, and prevented restenosis. These results offered further theoretical evidence for VEGF on the prevention of restenosis after angioplasty.

Abstract:Aim To investigate the possible signal transduction pathway by which IL 10 inhibits the proliferation of vascular smooth muscle cells (VSMC) induced by angiotensin Ⅱ (AngⅡ). Methods 3 H TdR and 32 P ATP incorporation were used to detect the proliferation of VSMC and the activity of mitogen activity proteinkinase(MAPK) and protein kinase C (PKC), respectively. Western blot and immunoprecipitation were applied to assay the expression and activity of focal adhesion kinase (FAK), respectively. Results IL 10 inhibited VSMC proliferation induced by AngⅡ, and down regulated AngⅡ induced activition of MAPK, PKC and FAK(P<0.05 orP<0.01). Conclusions The results suggest that the inhibiting effect of IL 10 on AngⅡ stimulated VSMC proliferation may be mediated by down regulating MAPK, PKC and FAK activity stimulated by AngⅡ.

Abstract:Aim Snake venom polypeptide X was isolated from Naja naja atra snake venom, its Ca 2+ antagonizing effect on formation of foamy macrophages was explored in these studies. Methods Foam like cells were induced by C57BL/6J mouse peritoneal macrophages incubated with 10 mg/L oxidized low density lipoprotein, and their intracellular Ca 2+ levels influenced both slowly and instantly by snake venom polypeptide X were determined with the Ca 2+ fluorescent indicator technique. Results The average intracellular Ca 2+ level of the foam like cells in 10 mg/L snake venom polypeptide X was 40.2% of the control(P<0.05); While the instant influence of snake venom polypeptide X on the Ca 2+ level was not considerable. Conclusions Snake venom polypeptide X exerted an antagonizing role to the increasing intracellular Ca 2+ level, thus prevented the macrophage derived foam cell formation.

Abstract:Aim To study the association of angiotensin Ⅱ type 1 receptor (AT1R) gene A1166/C polymorphisms and coronary heart disease (CHD) and to evaluate the effect of AT1R gene A1166/C polymorphism on plasma lipid levels. Methods By Polymerase chain reaction-Restriction fragment length polym orphism (PCR RFLP) methods, AT1R gene A1166/C genotype of 161 CHD patients and 167 controls were determined. Plasma lipid levels are measured by routine way. Results There are no statistically differences in AT1R genotype AC and AA frequencies between aged CHD patients and controls, also no differences are found in plasma lipid levels. Conclusion AT1R gene A1166/C polymorphism play no role on the occurrence of CHD and has no effects on plasma lipid levels.

Abstract:Aim This report presents the studies of sera levels of lipoprotein (Lp)(a) and phenotypes of apolipoprotein (a) in type 2 diabetes mellitus. The aims of this study were to explore the relationship between aged patients with type 2 diabetes mellitus and sera levels of Lp (a) and the distributive characteristics of genetic phenotypes of apo (a). Moreover the report present the studies the relationship sera levels of Lp (a) and the complications of the aged type 2 diabetic patients with hypertension or retinopathy or nephropathy. Methods Enzyme linked immunosorbent assay (ELLSA) was used to measure sera levels of Lp (a). The phenotypes of apo (a) were determined by SDS polyacrylamide gel electrophoresis (SDS PAGE) and Western blot analysis. Results and Conclusion 1. There was no significant difference in serum Lp (a) concentration among the three groups (P>0.05,respectively). Serum Lp (a) concentration did not significantly correlate with the levels of serum TC,TG,LDLC,HDLC,apo A,apo B (P>0.1,respectively). 2. In the aged type 2 diabetic patients with hypertension or retinopathy or nephropathy, the level of serum Lp (a) concentration was higher than that in patients without these complications (P<0.05 andP<0.001). 3. A total of 7 different phenotypes of apo (a) were determined among the three groups. The aged type 2 diabetic patient with complications mostly had S1 and S2 phenotypes than other groups and the aged type 2 diabetics without the complications.

Abstract:Aim To explore the effect of lipid metabolism after fat loading on the serum concentration of nitric oxide(NO) and plasm endothelin 1(ET 1) level. Methods 20 healthy subjects were given fat loading after fast for 12～14 h. Peripheral blood examples were drawn before and 2, 4, 6, 8 h after fat loading to determinate the concentration of triglyceride(TG), total cholesterol(TC), low density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), apolipoprotein A_Ⅰ(ApoA_Ⅰ), apolipoprotein B(ApoB) and glucose(Glu). Concentration of NO and ET 1 at different time point above were detected through colorimetry and radio immune assay respectively. The ratio of NO/ET 1 was calculated. Results TG concentration began to ascent at 2 h(2.19±0.16 vs 1.16±0.11 mmol/L,P<0.05) and reach the peak at 4 h(3.34±0.37 vs 1.16±0.11 mmol/L,P<0.01) after fat loading. During the course, serum level of NO was highest(61.58±5.97 vs 42.50±7.42 ng/L,P<0.05)and ET 1 plasma level was lowest at 2 h (99.08±16.55 vs 114.22±16.45 ng/L,P<0.05). 4 h later, with the descendent of TG concentration, NO and ET 1 concentration came back to the basic level. There was a peak of NO/ET 1 ratio at the 2 h. Conclusions Transient triglyceridemia affects the production of NO and ET 1, which leads to a compensatory increasing production of NO and momently descendent of ET 1 level.

Abstract:Aim To explore the correlation between TTTTA Pentanucleotide repeats(PNR) polymorphism of the apolipoprotein(a) gene and atherosclerosis cerebral infarction(ACI) in Chinese Han nationality,235 subjects with no blood links in Hubei region, including 82 patients with ACI and 153 healthy controls were studied. Methods Polymerase chain reaction (PCR) denature polyacrylamide gel electrophoresis silver stain technique was employed to determine the TTTTA PNR polymorphism of apo(a) gene. And (TTTTA) 5 allele of the apo(a) PNR was cloned into a plasmid T vector. The recombinant PGEM apo(a) was sequenced. Results The Chinese Han nationality in Hubei region has apo(a) gene polymorphism,7 alleles with 4,5,7,8,9,10,11 TTTTA PNR and 11 genotypes with 4/8, 4/9, 5/8, 5/9, 7/8, 8/8, 8/9, 8/10, 8/11, 9/9 and 9/10 was detected; the allele frequency in ACI with 5 repeats was remarkably higher than that in controls(P<0.05),the frequency in ACI with 9 repeats was remarkably lower than that in controls(P<0.01); sequence mutation of the apo(a) PNR(TTTTA) 5 was not found. Conclusions It was possible that the copy number of the apo(a) PNR (TTTTA) was associated with the susceptibility to ACI .

Abstract:Aim To study the improved effect of Xuezhikang on endothelial function in patients with hyperlipoidemia. Methods Patients with hyperlipoidemia were randomized to placebo(n=28)or Xuezhikang 0.06 g daily(n=30) for 8 weeks.Brachial ultrasound was used to measure endothelium dependent flow mediated dilatation (FMD)and response to endothelium independent nitroglycerin. Serum lipid profile was determined enzymatically. Results Total and LDL cholesterol and TG levels were similar before randomization in both groups. With Xuezhikang,not with placebo, they decreased by 21.5%(P<0 01)and 28.2%(P<0 01)and 16.2%(P<0 01) respectively. FMD was unchanged with placebo,2.98±2.16 to 3.23±1.89,but increased with Xuezhikang, 2.86±1.59 to 6.47±2.72 (P<0 01). Responses to nitroglycerin were similar in the two groups. Conclusions Xuezhikang could markedly decrease serum TC and LDLC and TG concentration and improve endothelial function in patients with hyperlipoidemia.