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AIM: To investigate the effect of vasodilator-stimulated phosphoprotein (VASP) phosphorylation on the cell migration induced by platelet-derived growth factor BB (PDGF-BB), and to identify which phosphorylation site was more important among the three phosphorylation sites, namely Ser157,Ser239 and Thr278. METHODS: Two phosphorylation mutants of VASP, pcDNA3.1(+)/VASP-S157A and pcDNA3.1(+)/VASP-S239A, were constructed and respectively transfected into the cultured ECV304 cells by means of liposome. The stable expression cells were screened by using antibiotic G418. Protein expression of VASP was measured by Western blotting. The ECV304 cell migration was evaluated using Transwell chamber. RESULTS: Compared to control group, the cell migration was significantly inhibited in ECV304 transfected with VASP-S157A and VASP-S239A (P<0.05), although slight differences existed between VASP-S157A and VASP-S239A transfected cells (P>0.05). CONCLUSION: VASP mutation on the phosphorylation sites of Ser157 and Ser239 inhibits cell migration, and the phosphorylation sites of Ser157 and Ser239 both greatly affect the function of VASP.
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Objective To sequence follicle stimulating hormone receptro (FSHR) promoter of the ovarian granulocyte and initially research the molecular mechanism of the poor ovarian response. Methods To study the relationship between FSHR promoter mutation of ovarian granulocyte and ovarian respone. The 263 bp DNA fragments before FSHR 5'initiation site in 70 cases of patients with poor ovarian respone and 88 cases of patients with ovarian normal respone who were in the cycle of IVF-ET were sequenced, Results There were 63 cases which occurred 29th site G → A point mutation in 158 women and the mutation rate was 40. 0%. Mutation rate [ 60. 0% ( 42/70 ) ] of 29th site G → A in group of poor ovarian respone was significantly higher(χ2 = 21. 450,P < 0. 01 ) than normal response group [ 23.9% ( 21/88 ) ]. There was no obviously variability ( t = 0. 457, P 0. 05 ) of basic FSH values between two groups [ G/G group was (7.2 ± 2. 3) U/L, G/A & A/A group was (7. 1±2. 0) U/L];there was obviously variability (t = 35. 81 ,P < 0. 05 ) in the number of follicles sinus between two groups ( G/G group was 14. 2±1.3, G/A & A/A group was 4. 5±0. 8 ) ;there was obviously variability ( t = 40. 35, P < 0. 05 ) in the number of ovum pick-up between two groups ( G/G group was 14. 0±1.2, G/A & A/A group was 4. 5±1.1 ) ;there was obviously variability (t =25. 80,P <0.05) of FE2-peak value between two groups [G/G group was (2 865±557) pmol/L, G/A & A/A group was (880±211 ) pmol/L] ;there was obviously variability (t =40. 22 ,P <0. 05) in the number of mature eggs ( G/G group was 13.6±1.2, G/A&A/Agroupwas4.3±0. 9).Conclusion The 29th site of FSHR promoter significantly affect the activity of FSHR promoter. Mutation of G→A can weaken promoter activity, so that ovarian granulocyte poor respone to FSH.
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BACKGROUND: The structural and functional impairment of endothelium cells were mainly presented by lowered endothelium activity, reduced nitrogen monoxide production, as well as increased endothelium vasoconstrictor peptide (EVCP).OBJECTIVE: To study the protective role of astragalus polysaccharide on atherosclerosis induced by eudothelium cell injury, which was compared with that of Captopril.DESIGN: Randomly controlled observation.SETTING: Department of Physiology, Hubei College of Traditional Chinese Medicine; Department of Pathophysiology, Medical College of Wuhan University;Department of Surgery,Hetang Hospital of Guangdong Province;Department of Endocrinopathic Sciences,Renmin Hospital,Wuhan University.MATERIALS: The study was carried out at the Organic Function Laboratory of Hubei College of Traditional Chinese Medicine and the Pathophysiological Department of Wuhan Medical University from July 2001 to December 2002. Forty healthy male rabbits provided by the experimental animal center of Wuhan medical university, weighed of 2.4-3.0 kg, were randomly divided into blank group,model control group,astragalus polysaccharide group and captopril group with 10 rabbits in each group.Astragalus polysaccharide was extracted from Shanxi produced astragalus root and made into injection powder that should be freshly composed with physical saline before usage.METHODS: Rabbits in blank group were raised with granular feed, while rabbits in other three groups were given hyperlipid feed (80% basal feed mixed with 15% yolk powder, 0.5% cholesterin and 5% lard), in addition with venous injection of bovine serum by 1 mL/kg once, atherosclerosis induced endothelium injury model was established on rabbit by hyperlipid feed combined with immune injury. Rabbits in astragalus polysaccharide group received intraperitoneal injection of polysaccharide of 500 mg/kg once a day; which replaced by 5 mg/kg captopril in captopril group that equals to 5 times clinical dosage; While rabbits in blank group and model control group were given the same volume physical saline of 4 mL/kg for totally 50 days. Blood were collected from SVC 24 after the last medication and then rabbits were put to death, the morphological changes of abdominal aorta were observed under optical microscope, meanwhile the changes of serum total cholesterol, triglycerides, nitrogen monoxide, EVCP, superoxide dismutase, malonaldehyde and total antioxidation activity were examined.MAIN OUTCOME MEASURES: ① Morphological changesof abdominal aorta. ② Changes of serum parameters.RESULTS: All 40 rabbits complete the experiment without loss. ① In contrast with model control group group, the total serum total cholesterol and triglycerides in astragalus polysaccharide group and captopril group obviously decreased [(9.33±1.13), (6.60±0.61), (7.09±0.74) mmol/L, P < 0.05;(3.05±0.44), (1.26±0.16), (2.17±0.46) mmol/L, P < 0.01, P< 0.05],malonaldehyde and EVCP markedly decreased [(9.98 ± 1.11 ), (7.10 ±0.68),(9.46±1.27) μmol/L, P < 0.01; (741.90±34.98), (632.62±26.95),(600.74±32.59) ng/L, P < 0.01]. ② Comparing to model control group group,the serum nitrogen monoxide and superoxide dismutase were obviously increased in astragalus polysaccharide group and captopril group ·[(11.04±1.68),(19.96±6.05), (18.35±3.52) μmol/L, P < 0.01, P < 0.05; (159.32±5.26),(207.54±16.98), (197.59±28.41) NU/mL, P < 0.0l, P < 0.05], the total antioxidation activity also increased [(23.8±3.5), (34.7±5.6), (30.7±6.8)%,P < 0.01, P < 0.05]. ③ Either the decrement of serum triglycerides, total cholesterol and malonaldehyde or the increment of nitrogen monoxide, superoxide dismutase and total antioxidation activity in astragalus polysaccharide group was greater than captopril group (P < 0.01). ④ Morphological changes of abdominal aorta: The aorta intima was smooth and endothelium cells were continuous with small intervals between cells in blank control group,endothelium cells presented normal configuration without edema;while intima in model control group became thick and upheaved, part of endothelinm cells detached with widened intervals. The media became thickened with leiomyocyte displaying hyperplasic and infiltering into endothelium, foaming cells could also be observed; the aorta intima was smooth and endothelium was closely connected in astragalus polysaccharide group, the hyperplasia of leiomyocyte was not active and foaming cells seldom observed; while in captopril group, the aorta intima was smooth without obvious detachment of endothelium cells and infiltration of leiomyocyte, leiomyocytes were normal and ranked orderly.CONCLUSION:Astragalus polysaccharide could markedly eliminate serum triglycerides, total cholesterol, malonaldehyde and EVCP, thereby alleviate vascular impairment induced by EVCP, meanwhile markedly increased serum nitrogen monoxide, superoxide dismutase and total antioxidation activity, the intima surface of abdominal aorta could be smooth due to the administration of AP, endothelium configuration would be basically complete, implying that it has better antioxidation property and protective role for endothelium cells.
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This article deals with the influence of shear stress on endothelial NO synthesis, and the role of caveolae in shear stress-induced eNOS activation. Human umbilical vascular endothelial cells (HUVEC) were cultured and exposed to different levels of laminal shear stress and Filipin, the perfused cultures were collected, and NO(2-)/NO(3-) was detected using nitrate reduction method. The structure of caveolae was observed through transmission electron microscopy (TEM). The level of NO(2-)-/NO(3-) was found to increase with the elevation of shear stress level (P < 0.01). It was the highest at 1.5 N/m2. After treatment with Filipin, the level of NO produced by HUVEC decreased significantly (P < 0.01), but after recovery and shear without Filipin, the level of NO synthesis bounded back (P < 0.01). It was then concluded that shear stress can induce endothelial NO synthesis and caveolae plays a key role in shear stress-induced eNOS activation.
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Humains , Cavéoles , Physiologie , Cellules cultivées , Endothélium vasculaire , Biologie cellulaire , Filipine , Pharmacologie , Nitric oxide synthase type III , Métabolisme , Résistance au cisaillement , Veines ombilicales , Biologie cellulaireRésumé
To investigate the effects of physiological shear stress on the vasodilator-stimulated phosphoprotein (VASP) location and expression changes associated with actin remodeling, we isolated and cultured human umbilical endothelial cells(HUVECs) with trypsin digestion. A parallel-plated flow chamber device was used to create laminar shear stress in vitro. The distributions of VASP and microfilaments in cells were observed by double staining with Alexa488 and rhodamine-phalloidin. Changes of VASP expression and phosphorylation were analyzed quantitatively with Western blot before and after exposure to shear flow for different times. We found that, under a shear stress of 10 dyn/cm2, HUVECs were elongated and oriented gradually to the flow direction. Microfilaments were recruited and oriented also to the flow direction with thicker VASP, specially targeted to their extremities. Western blotting data showed a rapid phosphorylation of VASP, and an increase of total VASP expression which peaked at 2 h (2 folds), then recovered until 8 h, followed by a slow increase again. These results suggest that VASP is a potential component which participates in the regulation of cell actin remodelling induced by shear flow.
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Humains , Molécules d'adhérence cellulaire , Cellules cultivées , Endothélium vasculaire , Biologie cellulaire , Métabolisme , Protéines des microfilaments , Phosphoprotéines , Contrainte mécanique , Veines ombilicales , Biologie cellulaire , MétabolismeRésumé
Objective:To observe the affection of angiotensin Ⅱ antagonists on the cultured subtype 2 receptor of angiotensin II transfected aortic vascular smooth muscle cells of rat.Methods:After transfected the plasmid that contained the cDNA of subtype 2 receptor of angiotensin II into cultured rat vascular smooth muscle cells, the cells were divided into three groups:cells of group 1 were treated with angiotensinⅡ,cells of group 2 were treated with angiotensinⅡand losartan,cells of group 3 were treated with angiotensinⅡ and PD123319 .After experiments,the expression of PCNA, NOS and the cell number was tested, respectively.Results:After treated with Losartan,the cell number of group 2 was(4.17±0.15)×105,the OD value of PCNA was 0.202 6±0.007 6,both of which were less than that of cells of group 3;the OD value of NOS of cells was more in group 2(0.027 5±0.002 1 ) than that in group 3 (0.016 9±0.002 0) (P<0.01).Conclusions:It suggests that when being activated,subtype 2 receptor of angiotensin Ⅱ could inhibit the proliferation of vascular smooth muscle cells and antagonist the effect of subtype 1 receptor of angiotensin Ⅱ,such an effect may be related to the activation of NOS.
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AIM: To investigate the effect and mechanism of Astragalus polysaccharide (APS) on the amelioration of hepatic insulin resistance in high fat-fed mouse model.METHODS: C57BL/6J mice (n=26) were divided into three groups randomly: C group (an animal model for control, n=10); IR group ( an animal model of insulin resistance, n=8) and IA group (an animal model in high-fat diet with APS treatment for12 weeks, 700mg?kg-1?d-1, ig). High-fat diet was used to induce the formation of insulin resistant. The parameters and insulin sensitivity of the animals were observed. The pathological features of the liver were presented through microscope and TEM. The expression changes of hepatic GSK3? were measured by Western blotting.RESULTS: In this study, the fat-fed mouse model of insulin resistance was established successfully. The mice in IA group responded to the 12-week APS therapy with a significant decrease in the level of blood glucose, plasma insulin, body weight, hepatic TG/FFA and improved glucose tolerance compared with those in IR group. In addition, the expression and the activity of GSK3? were lower in IA group (vs IR group,P
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AIM: To study the effect of thyroid hormone on the expressional change of myosin heavy chain(MHC) gene in cardiomyocyte induced by angiotensinⅡ(AngⅡ) and its potential mechanism. METHODS: Cardiac myocyte was cultured according to the method of Simpson. 10 -8 mol/L T_3 and 10 -7 mol/L AngⅡ were added to the culture medium,respectively or synchronously. After 48 h,the expression of ? and ?-MHC mRNA in myocytes were detected by RT-PCR. The protein kinase C activation were detected by PepTag non-radioactive PKC assay. The incorporation of -Leucine and -thymine to test the protein and DNA synthesis in myocytes were also performed. RESULTS: AngⅡalone increased the incorporation of -Leucine of myocytes while it had no effect on the incorporation of -thy mine. The expression of ?-MHC mRNA was increased and the expression of ?-MHC mRNA was decreased significantly at the condition of AngⅡ. The enhanced PKC activation was induced by AngⅡalso. When AngⅡand T_3 were added to the culture medium synchronously,though the incorporation of -leucine and -thymine were not changed compared with AngⅡ treated alone. The ?-MHC mRNA expression was increased and the ?-MHC mRNA expression was decreased significantly. The PKC activation of the myocytes also was decreased. CONCLUSIONS: T_3 inhibited the expressional change of myosin heavy chain gene in cardiac myocytes induced by AngⅡ. The effect of T_3 on the change of PKC activation in cardiac myocytes may be one of its mechanisms.
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AIM: To study the role of calcineurin (CaN)-dependent signaling pathway in proliferation of vascular smooth muscle cells (VSMCs) by observing the effect of cyclosporin A (CsA) on proliferation of neuropeptide Y (NPY)-induced rat VSMCs. METHODS: Upon the model of cultured rat VSMCs, the study consisted of three groups: NPYgroup,CsA+NPY group and control group. CaN activity was determinated by enzyme reaction phosphorus measurement. The methods of biochemistry (MTT) and quantitative immunocytochemistry were applied to investigate the proliferation of VSMC and the expression of proliferation cell nuclear antigen (PCNA) in cultured rat VSMCs. (RESULTS:) (Compared) with the control group, the VSMC's CaN activity, proliferation activity and expression of PCNA (by photo densitometry A_(PCNA)) were obviously increased in NPY group (P
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AIM and METHODS: The protective effects of multi-enzyme Ⅱ was studied on cultured endothelial cells which was injuried by hyperlipidemia serum. RESULTS: Hyperlipidemia serum increased ICAM-1 expression on the surface of endothelial cells, and decreased NO- 2 release significantly (P
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AIM: To investigate the effects of sodium ferulate on cholesterol and triglyceride metabolism in atherosclerosis with hyperlipidemia.METHODS: The rabbit model of atherosclerosis was produced by feeding high lipid forages.RAW264.7 foam cell and HepG2 injured cell models were established by incubation with oxidized low density lipoprotein(ox-LDL).The atherosclerotic plaque area was measured,and serum lipids were detected.The cellular lipid accumulation was examined by oil red O staining.The cellular contents of total cholesterol and cholesterol ester were quantified by high performance liquid chromatography.The hepatic lipase(HL) mRNA expression was determined by RT-PCR.RESULTS:(1) Compared with hyperlipid group,the aorta atherosclerosis plaque area and the serum triglyceride level were significantly decreased in sodium ferulate-treated rabbits,but the serum cholesterol level showed little change.(2) Compared with ox-LDL group,the HL mRNA expression in HepG2 cells was enhanced significantly in sodium ferulate-treated group,but the cellular contents of total cholesterol and cholesterol ester in RAW264.7 foam cells showed little change.CONCLUSION: Sodium ferulate inhibits the formation of atherosclerotic plaque in high-cholesterol-fed rabbits aorta.This antiatherosclerotic function may reduce serum triglyceride level through enhancing the expression of HL mRNA without influencing serum cholesterol level and foam cell formation.
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Objective To investigate the ameliorative effect and a mechanism of APS on the hepatic steatosis in diabetic KKAy mice.Methods KKAy and C57BL/6J mice were respectively seperated into KK and K+A(n=8)as well as C and C+A(n=10)groups.Blood and hepatic biochemical parameters were observed.Insulin sensitivity was analyzed by OGTT & HOMA-IR.Hepatic pathological changes were presented through microscope and TEM.The expressions of hepatic GSK3? and insulin-induced Ser9GSK3? were performed by Western blot.Results With 8-week APS therapy in K+A group,the levels of blood glucose and hepatic TG and FFA were decreased,insulin sensitivity was improved,the hepatic steatosis was significantly alleviated(P
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AIM To study the anti atherogenesis action of sodium ferulate and its mechanisms. METHODS Atherosclerotic rabbit models were duplicated by feeding high lipid forage and ECV304 were cultured with the hyperlipidemic serum. The atherosclerotic plaque area was measured. Scanning electron microscope, spectrophotometer and immunocytochemical methods were used to detected the microstructures of endothelial cell, the content of NO in suspension and the expressions of TGF? 1, bFGF on the cell surfaces. RESULTS Sodium ferulate could decrease the plaque area, lessen the damnification of endothelial cell induced by HLS, enhance the expression of TGF? 1 and the release of NO from ECs, and reduce the expressions of bFGF in ECs, significantly. CONCLUSION Sodium ferulate can decrease the atherosclerotic plaque area induced by hypercholesterol, which may be relate to the expression change of cytokines.
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AIM: To observe the effect of angiotensinⅡ subtype 2 receptor (AT_2 receptor) on the cultured rat aortic vascular smooth muscle cells. METHODS: The plasmid contained the cDNA of AT_2 receptor was transfected into cultured rat vascular smooth muscle cells. The effects of AngⅡ, Ang Ⅱ+losartan, Ang Ⅱ+PD123319 on the expression of PCNA, the NOS activity and the cell number were observed. RESULTS: The cell number and the expression of PCNA decreased after the cells were treated with losartan. When treated with PD123319, the cell number and the expression of PCNA increased, but the expression of NOS decreased. CONCLUSIONS: These data suggest that when being activated, AT_2 receptor inhibits the proliferation of vascular smooth muscle cells and antagonizes the effect of AT_1 receptor, such an effect may be related to the activation of NOS. [
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Blood viscosity, plasma viscosity, haematocrit and electrophoresis time of red blood cell and platelet were raeasured before and 24h after operation, in which 8 rabbits were AMI, and 8 with the coronary arteria not ligated as control. The AMI group had significantly higher adjusted viscosity and electrophoresis time of red cell and platelet than the control group (P
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The effects of DMSO and nifedipine on experimental myocardial infarction induced by isoproterenol(ISP)were investigated.The results showed a significant reduction in myocardial FFA and MDA contents in DMSO and nifedipine-treated groups, as compared with the ischemic group, and a beneficial effect on activity of SOD,GSH-px and Ca~(2+)-Mg~(2+)-ATPase. The effects of OH. and Ca~(2+) in myocardialg infarction were further analysed in this study.
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The changes of lipid peroxidation in the ischemic (renal artery occlusion for 75 min) and ischemie/reperfusion (renal artery occlusion 60 min plus 15 min reflow) kidney was studied in 19 male SD rats. The results showed that malondialdehydc of renal cortex and medulla was increased, but in both groups SOD and GSH-Px were not changed significantly. Activity of xanthine oxidase activity in the ischemic/reperfusion kidney were increased as compared with normal. These results suggest that lipid pcroxidation was involved in the ischemic and ischemic/reperfusion renal injury, and xanthine-xanthine oxidase might be one of the major sources of oxygen free radical when oxygen supply to ischemic kidney was restored.
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AIM To compare the protective effects of norepinephrine preconditioning(NEPC)and ischemic preconditioning(IPC)on myocardial ischemic/reperfused injury in rats in vivo . METHODS Rats were divided into control, ischemia/ reperfusion, classic IPC and NEPC groups. After 20 min reperfusion, several indexes including cardiac function indexes, MDA were tested, the myocardial ultrastr ucture and the injury reaction of catalase observed. RESULTS Both IPC and NEPC could protect myocardial ultra structure, ameliorate left heart function, decrease cardiac MDA content and protect the activity of catalase. CONCLUSION Extra micro norepinephrine preconditioning as a non injury method can mimic the protective effects of classic IPC and shows its clinical values.
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AIM: The effects of BDM on isolated rat heart in cold cardioplegia were studied METHODS: Rat heart were subjected to cold cardioplegia at 4℃ for 8, 18 and 24 h Then each heart was perfused (90 cm H 2O) in Langendorff model at 37℃ for 40 min In the high K + group( n =24) the hearts were preserved in St Thomas cardioplegic solution, in BDM group( n =24) hearts were preserved in K-H solution with BDM 30 mmoL/L RESULTS: After 18 h, heart rate and the coronary flow in BDM group were significantly higher than in high K + group( P
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AIM:To investigate the injurious effects of oxidized low-density(OLDL) on human endothelial cells(ECs) and protective effects of Angelica Sinensis in vitro. METHODS: The effect of OLDL on nitric oxide (NO) release from EC and intercellular adhesion molecule-1 (ICAM-1) expression on EC surface were studied, and the protective effects of Angelica Sinensis on normal and abnormal EC were investigated using cultured EC with or without OLDL and immunocytochemical staining technique. RESULTS: OLDL led to a decrease of NO release and an increase of ICAM-1 expression, which can be reversed by Angelica Sinensis and atropine can block this role of Angelica Sinensis. CONCLUSION: Angelica Sinensis has antagonistic effect on OLDL-induced decreasing of NO released from ECs and increasing of ICAM-1 expression on ECs surface . These effects of Angelica Sinensis may be exerted by the excitation of muscarinic receptor.