Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Effects of diazoxide preconditioning on mitochondrial respiratory function and enzyme activity in rats

AIM: To investigate the effects of the selective mitochondrial ATP-sensitive K+ channels opener diazoxide on mitochondrial respiratory function and enzyme activity in isolated rat myocardium under ischemia/reperfusion.METHODS: Observation was made on rat hearts perfused with Langendorff apparatus.72 Sprague-Dawley (SD) rats were randomly divided into 4 groups: normal group (NOR),ischemia reperfusion (IR),diazoxide group (DIA) and 5-hydroxydecanoate (5-HD) antagonized diazoxide group (5HD-DIA).Hearts isolated from SD rats were mounted on a Langendorff apparatus and started with a 20 min perfusion for equilibration.NOR went on perfusion for another 100 min after equilibration.IR underwent 40 min global ischemia and followed by 30 min reperfusion after 30 min stabilization.DIA was administered with K-H solution containing diazoxide at concentration of 50 μmol/L for 10 min before ischemia and reperfusion.5HD-DIA was infused with 100 μmol/L 5-HD (a specific mitochondrial ATP sensitive K+ channel blocker) and the same procedure was carried out as DIA group.Hearts were taken down to extract mitochondrial at the end-equation,before ischemia and at the end-reperfusion for determination of mitochondrial respiratory function and the enzyme activity of mitochondria.RESULTS: At the end of reperfusion,mitochondrial respiratory function (mitochondrial respiratory control rate,P/O ratio and state 3 respiration) and mitochondrial enzyme activity (NADH oxidase,succinate oxidase and cytochrome C oxidase) in DIA group were better than those in IR group and 5HD-DIA group (P<0.05),but worse than those NOR group (P<0.01).No significant difference in all parameters was observed between IR and 5HD-DIA (P>0.05).CONCLUSION: Preconditioning with mitochondrial ATP sensitive potassium channel opener,diazoxide,protects rat heart mitochondria against ischemia-reperfusion injury.The mechanisms are involved in the safeguarding of respiratory function and activity of enzymes of respiratory chain.     

Effects of olaquindox on PLA2 activity and phospholipid metabolism of biomembrane in chicks

AIM: To study the effect of olaquindox on phospholipase A₂ (PLA₂) activity and phospholipid metabolism in biomembrane.METHODS: 150 healthy avin chicks were divided into normal control group and olquindox group.The two groups were fed with same feedstuff without olaquindox.The animals in olquindox group were fed with olquindox at dose of 15mg/kg body weight everyday.The experimental period lasted six weeks.Every week 6 vein blood samples were collected randomly from every group for preparing erythrocyte membrane (ECM) and hepatic mitochondria membrane (MiM).RESULTS: PLA₂ activities of ECM and MiM in olquindox group were significant higher than those in control group from 3 to 6 weeks of experiment (P<0.05，P<0.01).The level of major membrane phospholipids in olquindox group was lower than that in control group (P<0.05,P<0.01).CONCLUSION: Olaquindox affects PLA₂ activity and major phospholipid concentration in ECM and MiM,indicating that olaquindox may induce biomembrane injury.     

丝瓜多酚氧化酶的酶学特性初步研究

以邻苯二酚为底物,应用分光光度法对丝瓜多酚氧化酶（PPO）的酶学特性进行研究。结果表明,丝瓜多酚氧化酶的最适反应温度是20 ℃,最适pH值是6.8。反应体系在15～25 ℃、pH值6.4～7.2之间有较高的活性。丝瓜多酚氧化酶的热稳定性试验表明,在沸水中处理3 min,残留的酶活性仅为处理前的4.81 %,而在60 ℃处理3 min,残留的酶活性还有38.08 %。动力学研究结果表明,以邻苯二酚为底物时,米氏方程K_m=0.067 56 mol·L^-1,V_max=0.238 5 A₄₁₀·min^-1。单一的抑制剂VC和硫代硫酸钠对丝瓜多酚氧化酶活性有抑制作用,但有效抑制浓度不同,VC的有效抑制浓度最低,为50.0 mg·L^-1,其次是硫代硫酸钠,为100 mg·L^-1,柠檬酸的抑制效果最弱。     

Effect of bitter melon on liver fibrosis induced by carbon tetrachloride

AIM: To investigate the effects of bitter melon (BM) on liver fibrosis induced by CCl₄ in Wistar rats. METHODS: Healthy male Wistar rats were randomly divided into 4 groups (with 8 each): olive oil control group (group C), olive oil CCl₄ model group (group M), CCl₄+BM at low concentration (BM 100 g/kg, group BM-L), CCl₄+ BM at high concentration (BM 200 g/kg, group BM-H). All rats except those in group C were subcutaneously injected with CCl₄ twice a week for 8 weeks to induce liver fibrosis. After injection of CCl₄ for 8 weeks, all rats were sacrificed and the samples of blood and livers were collected. The weight ratio of liver to body was measured. The serum level of MDA and the activity of SOD were tested. The contents of total protein and albumin, the activity of GSH-Px, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were determined. Hepatic inflammation and collagen deposition were observed under microscope with Masson staining. RESULTS: In the rats treated with BM, the weight ratio of liver to body, the serum level of MDA, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were lower than those in group M (P<0.01). The serum activity of SOD, the contents of total protein and albumin, and the activity of GSH-Px in the liver homogenate were enhanced (P<0.01). The livers of the model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis. The rats in BM-treated group showed slighter hepatic injury and collagen deposition, and the liver functions were much better than those in the model group. High dose of BM showed more obvious liver-protective effects. CONCLUSION: BM attenuates liver fibrosis by its antioxidant effect and the mechanisms of reducing hydroxyproline content and monoamine oxidase activity.     

Adipophilin antisense oligonucleotides decrease ACAT activity

AIM: Based on the finding of adipophilin expression with the increase in cellular cholesterol, the aim of the present study was to look for the active site of adipophilin in cellular cholesteryl metabolism. METHODS: Mouse peritoneal macrophages were incubated with 80 mg/L Ox-LDL (Ox-LDL group) or 80 mg/L Ox-LDL plus 1 mmol/L adipophilin antisense oligonucleotides (Ox-LDL+antisense group), respectively. At the various time points, the incubated cell samples were observed with adipophilin immunofluorescence staining, flow cytometric analysis and cellular cholesterol analysis. RESULTS: The Ox-LDL+antisense group cells contained significantly lower cholesteryl ester (19.9±1.9) mg/g (protein) than that of cells in Ox-LDL group (46.6±3.4) mg/g (protein) at 4 days. From 12 h, expression of adipophilin in Ox-LDL group increased more quickly than that of the cells in Ox-LDL+antisense group. At day 4, the level of adipophilin expression in Ox-LDL group was significantly higher than that in Ox-LDL+antisense group. During the observation, the amount of Ox-r［CL-3H］ LDL taking up increased gradually in both groups, however, from day 1 the taking up amount in Ox-LDL+antisense group was less than that in Ox-LDL group. There was a statistical difference between the two groups from day 2 to day 4. From 6 h to day 2, the relative ACAT activity increased in both groups. The relative ACAT activity kept unchanged from day 2 to day 4 in the two groups. At day 2, the relative ACAT activity in Ox-LDL+antisense group was significantly lower than that in Ox-LDL group. Correlative analysis between activity of ACAT and adipophilin expression showed than R2 were 0.6176 and 0.8212 (P<0.05) in Ox-LDL group and Ox-LDL+antisense group, respectively. CONCLUSION: At least partly, expression of adipophilin is related to cellular taking up of lipoproteins and to ACAT activity. Therefore, the results indicate that adipophilin has a role in metabolism of cellular lipid droplets and the effective point may be ACAT.     

Substance P enhances pump function of isolated lymphatics from hemorrhagic shock rats

AIM: To establish a technique of lymphatic perfusion in vitro and to determine the effect of substance P (SP) on lymphatic contractility during the process of hemorrhagic shock (HS). METHODS: Male Wistar rats were randomly divided into control group (surgical procedure only) and HS group . Thoracic ducts were isolated from HS rats at the corresponding time points. The segment of thoracic duct was prepared, perfused in vitro at the transmural pressure of 3 cmH₂O and stimulated with gradient concentrations of SP to measure the lymphatic contractile activity. The end-systolic diameter, end-diastolic diameter, contraction frequency (CF) and passive diameter of isolated lymphatics were measured. The contraction amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were also calculated.RESULTS: SP increased lymphatic CF, TI and FPF, and the effects were enhanced with the increase in the concentration of SP. The CF, TI and FPF of 2 h- and 3 h- shocked lymphatics were elevated to/over the value of baseline levels before the experiment by SP at the concentration starting from 3×10^-8 mol/L. At the same concentration of SP, the CA of lymphatics showed no significant statistical difference among the groups. However, with the increase in SP concentration, the lymphatic CA had a downward trend in all groups.CONCLUSION: SP enhances the pump function of lymphatics not only under physiological condition, but also in shock during different stages.     

Effects of glycated serum albumin on expression of MCP-1 in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of glycated serum albumin (GSA) on the expression of monocyte chemoattratant protein-1 (MCP-1) in endothelial cells.METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with GSA at different concentrations in the presence or absence of glycosylation-product inhibitor aminoguanidine (AG) and anti-oxidant N-acetylcysteine (NAC). The expression of MCP-1 was evaluated by the methods of immunocytochemistry and sandwich ELISA.Malondialdehyde(MDA) content and superoxide dismutase(SOD) activity were determined by the technique of thiobarbituric acid(TBA) and xanthine oxidase(XOD),respectively. RESULTS: GSA stimulated HUVECs to produce and release MCP-1. After HUVECs were treated with 50 mg/L GSA, the expression of MCP-1 at 4 h, 8 h and 12 h was 1.3, 1.9 and 2.8 folds higher than that in control group (P<0.01), respectiuely.The significant difference among the experiment groups (P<0.01) was observed, indicating that GSA took effect in a concentration-dependent manner. The release of MCP-1 in cultured supernatants in the experiment groups with 3 different concentrations of GSA was 1.6, 2.4 and 3.0 folds as much as that in control group (P<0.01), and the significant difference among the experiment groups (P<0.01) was also observed. GSA decreased the activity of SOD (P<0.05) and increased the content of MDA (P<0.01). AG and NAC obviously inhibited the upregulation of MCP-1 expression in HUVECs by GSA (P<0.01). NAC also inhibited the effect of GSA on SOD activity and MDA content in HUVECs (P<0.05). CONCLUSION: GSA stimulates the expression of MCP-1 by inducing oxidative stress in endothelial cells.     

Effect of vitamin C on expansion of CD4+ effector memory T cells in vitro

AIM：To investigate the effect of vitamin C (VC), a small-molecule compound, on the expansion of CD4+ effector memory T cells (TEM) in vitro and to improve the effect of adoptive immunotherapy. METHODS：Normal human peripheral blood CD4+ T-lymphocytes were isolated and randomly divided into 2 groups: control group and experiment group. VC was added into experimental group. Thereafter, the expansion of CD4+ TEM in the 2 groups was detected by cell counter and flow cytometry. RESULTS：VC did not significantly affect the total number of CD4+ T cells, while it raised the ratio of TEM in CD4+ T cells at an optimal concentration of 100 mg/L. After 10 days of the expansion of CD4+ T cells, the number of CD4+ TEM in experiment group was (3.56±0.35)×106, significantly larger than that in control group ［(1.22±0.15)×106, P<0.01］. CONCLUSION: VC can effectively promote the expansion of CD4+ TEM in vitro, which provides a simple, safe and effective expansion method for adoptive immunotherapy.     

Effects of emodin on activity of sphingomylinase and content of ceramide in rabbit aorta of experimental atherosclerosis

AIM: To study the changes of the sphingomylinase activity and ceramide content in rabbit aorta of experimental atherosclerosis and investigate the effects of emodin on them. METHODS: The qualified rabbits were fed with food containing 1% cholesterol and 5% lard for 10 weeks to establish the animal models. The concentration of cholesterol (TC) was assayed by a enzyme method. Trace-fast-test method was used to test the activity of superoxide dismutase (SOD) and motified- BAMuGuoFu methods was employed to assay the content of myocardial malondialdehyde (MDA). Radiolabeled -enzyme-tracing was used to detect the activity of the sphingomyelinase,and thin-layered scanning was conducted to analyze the content of the ceramide in aorta. RESULTS: The ceramide content in aorta and the sphingomyelinase activity were markedly increased in the rabbits with experimental atherosclerosis. The increase was positively correlated with the content of TC and MDA and negatively correlated with the activity of SOD in blood. Compared to the model animals, emodin at concentration of 5 mg·kg^-1, 10 mg·kg^-1 and 20 mg·kg^-1 respectively reduced the area of plague on endothelium in rabbit's aortic artery and elevated the activity of SOD (P<0.05). The activity of sphingomylinase and the content of ceramide were decreased at the same time (P<0.05). 10 mg·kg^-1 emodin proved to be more effective than 5 mg·kg^-1 and 20 mg·kg^-1 emodin (P<0.01). CONCLUSIONS: Our data suggest that atherosclerosis is related to ceramide signal transduction initiated by factors such as oxidative stress in hypercholesterolemia. The emodin prevents the development of atherosclerosis probably by interfering with the above pathway.     

DNCB induces colitis and its relation with LMIF activity

AIM: In order to explore the pathogenesis of ulcerative colitis (UC), an experimental colitis in mouse was induced by the hapten dinitrochlorobenzene (DNCB), and the activity of leukocyte migration inhibitory factor (LMIF) was measured at the same time. METHODS: 67 BALB/c mice were randomly divided into control (60% ethanol) and DNCB groups. After they were sensitized by smearing 3.3% DNCB on the abdominal skin, they were challenged with DNCB at concentration of 0.1%, 0.2% and 0.4% respectively by instillation once a day. The weight, stool viscosity and hematochezia were observed and accumulated as disease active index (DAI) score. The pathological changes in colon tissue were judged macropathologically and by means of microscope. LMIF activity was determined by the absorbance (A) of migrated leukocytes. RESULTS: Compared to control group, the increases in DAI accumulate score, pathologic score, and LMIF activity in DNCB groups were observed. CONCLUSION: Mouse colitis was induced by DNCB, which was accompanied by an increase in LMIF activity.     

Correlation between metallothionein and matrix metalloproteinase-2 expression and activity in rats during CC14-induced hepatic fibrogenesis

AIM: To investigate the relevance between metallothionein(MT) and matrix metalloproteinase-2 (MMP-2) expression and activity in hepatic stellate cells (HSCs). METHODS: Fifty Kunming male mice, weighing 25 g±5 g, were randomly divided into two groups: experiment group (40 mice) and control group (10 mice). CCl4 was used to induce the hepatic fibrosis model in the experiment group. Conditioned medium of HSCs (containing MMP-2) was added at different concentrations of MT, then MMP-2 activity was detected. Liver tissue microarray was used. Collagen fibers was detected by Sirius red staining. The expressions of MMP-2 and MT proteins in liver tissues were detected by immunohistochemistry staining. Gel zymography was used to confirm the activity of MMP-2 in liver tissue homogenate and in the conditioned medium. RESULTS: The expressions of both MMP-2 and MT proteins in liver tissues increased fluctuating with the process of fibrogenesis in the model mice alternately, but the proteins showed out of phase changes. The activity of MMP-2 in the liver tissues also increased gradually and fluctuated with the development of fibrosis. Apart from individual time points, the activity of MMP-2 and MT expression were negatively correlated. The activity of MMP-2 in the conditioned medium treated by MT declined in a dose-dependent manner (r=-0.9990, P<0.01). CONCLUSION: The results suggest that there is an upward tendency in the expression of MMP-2 and MT proteins in the liver fibrogenesis, but interaction between them may be exist, and MT inhibits the activity of MMP-2.     

Inhibitory effect of ginkgo-dipyridamole injection on ischemia/reperfusion injury in rat hearts in vitro

AIM：To investigate the effect of ginkgo-dipyridamole injection (GD) on ischemia/reperfusion (I/R) injury in rat hearts in vitro and its possible mechanism. METHODS：Forty male Sprague-Dawley rats were randomly divided into 5 groups (n=8): normal control (NC) group, I/R group, ischemic preconditioning (IPC)+I/R group, GD+I/R group and GD+LaCl3+I/R group. Cardiac function indexes, including heart rate (HR), left ventricular systolic pressure (LVSP) and the maximal rise/fall rate of left ventricular pressure (±dp/dtmax), were detected at 5 time points, including stabilizing point, 30 min after ischemia, and 5, 30 and 60 min after reperfusion. The activity of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent at the five time points was assayed. The concentration of Ca2+ and the content of α-ketoglutarate dehydrogenase (α-OGDH) in myocardial mitochondria were determined at the end of the whole experiment. RESULTS：Compared with I/R group, the cardiac function indexes in IPC+I/R and GD+I/R groups were improved at the reperfusion period (P<0.05), the activity of LDH and CK in coronary effluent and the concentration of Ca2+ in mitochondria were significant reduced (P<0.01), and the content of α-OGDH was increased (P<0.05). However, the protective effect of GD was inhibited by LaCl3 (P<0.05). CONCLUSION：GD protects rat hearts against I/R injury by inhibiting calcium overload and improving mitochondrial enzyme activity to stabilize mitochondrial energy metabolism.     

Changes of hydrogen sulfide and heme oxygenase-1 in blood of children with congenital heart disease and pulmonary hypertension before and after surgery

AIM: To evaluate the changes and clinical significance of hydrogen sulfide (H2S) and heme oxygenase-1 (HO-1) in patients with congenital heart disease (CHD) and pulmonary hypertension (PH). METHODS: Forty-eight patients with CHD and left-to-right shunt were selected randomly from cardiac surgery in the Fourth Hospital of Hebei Medical University. Forty-eight patients were divided into 3 groups according to the pulmonary artery systolic pressure (PASP) by Doppler echocardiography before operation: group A was normal (PASP<30 mmHg, n=15); group B was mild PH (PASP 30-49 mmHg, n=15); group C was moderate-severe PH (PASP≥50 mmHg, n=18). The radical operation was performed successfully under complex intravenous anesthesia in all 48 patients. The radial arterial blood was collected immediately before operation, 1 h and 24 h after operation. The concentration of plasma H2S was measured by optical absorbance method at 670 nm and HO-1 activity in serum was detected by dual wavelength spectrophotometer at different time points. RESULTS: Plasma level of H2S in all groups after operation was higher than that in the same group before operation. Significant difference of the plasma H2S level was observed between pre-operation and 24 h post-operation in the same group (P<0.05). The plasma level of H2S in group C and group B at time points of pre-operation, 1 h post-operation or 24 h post-operation was significantly lower than that in group A, the plasma level of H2S in group C was significantly lower than that in group B. Compared to the values in each group before operation, the activity of serum HO-1 among groups after operation was not obviously and statistically different (P>0.05). No obvious difference of HO-1 activity in blood among 3 groups was observed (P>0.05). There was a negative correlation between PASP and the level of H2S in 3 groups at time points of pre-operation, 1 h and 24 h post-operation (pre-operation, r=-0.66, P<0.01; 1 h post-operation, r=-0.458, P<0.01; 24 h post-operation, r=-0.730, P<0.01). No correlation between PASP and HO-1 activity in blood serum was found. CONCLUSION: H2S plays an important role in the formation of PH and remodeling of pulmonary vasculature. There is no correlation between PASP and HO-1 activities in blood, but HO-1 may play an indirect action in the formation of PH and remodeling of pulmonary vasculature. Measuring the level of H2S may be a reliable method to follow the change of pulmonary pressure and worsened PH.     

Effects of atorvastatin on expression of PAPP-A induced by TNF-α and IL-1β in rat aortic endothelial cells

AIM: To investigate the effects of atorvastatin on the expression of pregnancy-associated plasma protein A(PAPP-A)induced by TNF-α and IL-1β in endothelial cells. METHODS: The rat aortic endothelial cells were isolated from thoracic aortas and cultured by the tissue explant method. The cells in passage 3-4 were used in the experiment and were randomly divided into 4 groups: blank control group: the cells were treated without any intervention; atorvastatin concentration groups: the cells were incubated with atorvastatin at the concentrations of 0.1, 1 and 10 μmol/L for 24 h; atorvastatin time groups: the cells were incubated with atorvastatin at the concentration of 10 μmol/L for 6 h,12 h and 24 h; atorvastatin+inflammatory factors groups: the cells were pre-incubated with 60 μg/L TNF-α or 20 μg/L IL-1β for 1 h, then different concentrations of atorvastatin (0.1, 1.0, 10 μmol/L) were added for 6 h,12 h and 24 h. MTT reduction assay was used to observe the cell proliferation. The mRNA expression of PAPP-A was detected by RT-PCR. The protein level of PAPP-A in the supernatants of cultured cells was measured by ELISA. RESULTS: Compared with blank control group, no significant change of cell proliferation was observed after the intervention of atorvastatin and TNF-α/IL-1β for 3 h, 6 h, 12 h, 24 h and 48 h, indicating that the drugs had no toxic effects on the cells. No significant difference of PAPP-A expression between atorvastatin groups and blank control groups was found. Compared with TNF-α groups and IL-1β groups, PAPP-A expressions in atorvastatin intervention groups significantly decreased. The protein level of PAPP-A was gradually decreased with the raised concentration of atorvastatin and the prolonged time in a concentration- and time-dependent manner. CONCLUSION: Atorvastatin doesn't influence the PAPP-A expression, but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration- and time-dependent manner in primary cultured rat aortic endothelial cells.     

Changes in monoamine oxidase activity during thrombotic cerebral ischemia and protection mechanisms of ginkgolide B in tree shrews

AIM:The present study was designed to examine changes in monoamine oxidase (MAO) activity during cerebral ischemia and whether ginkgolide B's brain protection challenges with inhibiting monoamine oxidase. METHODS: The focal thrombotic cerebral ischemia was formed by photochemistry-induced in tree shews.MAO activities in different areas which include ischemic,core, penumbra and contralater and serum, were tested by enzyme color-compared way. The protein contents in different area above was examined by amino acid autoanalytic apparatus. RESULTS:MAO activities in ischemic core in different group were much lower than that in the sham operation group and contralatetral areas, with its peak at seventy-two hours after occlusion, but that in penumbra and serum ascended. There were significant differences in MAO activities between ischemic group and control (P＜0.01). In ginkgolide B(GB) group, the MAO activities in all areas but not in core descended, significant differences(P＜0.01) between in GB group and in twenty-four hours after occlusion. Changes in MAO activity was consistent with alterations of brain proein content(r=0.81,P＜0.05). CONCLUSION:The changes in monoamine neurotransmitters in core and penumbra considerably depend on the alterations of MAO activities after thrombotically cerebral ischemia. Probably, protective effects of GB on ischemic neurons is related to its acting as antagonist of platelet activating factor and regulator of monoamine oxidase.     

Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human umbilical vein endothelial cells

AIM:To explore the effect of atorvastatin on high glucose-induced oxidative stress and underlying mechanisms in human endothelial cells. METHODS:Human umbilical vein endothelial cells(HUVECs) were cultured in medium 199 containing normal concentration of glucose(5.5 mmol/L). For high glucose treatment, glucose solution was added to the final concentration of 25 mmol/L. Reactive oxygen species(ROS) were detected by flow cytometry and confocal microscopy. The activity of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase was measured by lucigenin assay. Phosphorylated protein kinase C(PKC) and the expression levels of NADPH oxidase subunits Nox4 and Nox2/gp91^phox were determined by quantitative real-time PCR and immunoblotting. RESULTS:High glucose increased ROS production, NADPH oxidase activity and the expression of Nox4 and Nox2/gp91^phox subunits. Treatment of endothelial cells with atorvastatin resulted in significant inhibition(in a concentration-dependent manner) of high glucose-induced ROS production, NADPH oxidase activation and the expression of Nox4 and Nox2/gp91^phox subunits. PKC inhibitor showed a similar effect to that of atorvastatin on high glucose-induced oxidative stress. Furthermore, atorvastatin rapidly inhibited high glucose-induced activation of protein kinase C, an upstream activator of NADPH oxidase. CONCLUSION:PKC is involved in high glucose-induced oxidative stress in HUVECs. Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human endothelial cells.     

Effect of amiodarone and metoprolol on platelet activation,fibrinolytic activity and vasoactive mediators in acute myocardial infarction in rabbits

AIM:To explore the significance of platelet activation, fibrinolytic activity and the changes of vasoactive mediators in acute myocardial infarction in rabbits and the intervention of amiodarone and metoprolol.METHODS:Fifty New Zealand white rabbits were randomly assigned to five groups, ten for each. Group Ⅰ: sham group, group Ⅱ: acute myocardial infarction(AMI) group, group Ⅲ: AMI and lidocaine group, group Ⅳ: AMI and amiodarone group, group Ⅴ: AMI and metoprolol group.The middle point of left ventricular coronary artery was ligated (groupⅡ,Ⅲ, Ⅳ and Ⅴ ) or a sham ligation(group Ⅰ). Four hours later, blood was collected for measuring plasma concentration of TXB₂, 6-Keto-PGF_1α, ET, NO, plasma activity of t-Pa and PAI.After that, the heart was taken out to evaluate the infarction size(IS).RESULTS:Plasma concentration of TXB₂, ET, NO and plasma activity of PAI were significantly higher in groupⅡ,Ⅲ, Ⅳ and Ⅴ than those in group Ⅰ(P＜0.01), but the plasma concentration of 6-Keto-PGF_1α and plasma activity of t-Pa were remarkably lower in groupⅡ,Ⅲ, Ⅳ and Ⅴ than those in group Ⅰ(P＜0.01). There were no difference in plasma concentration of TXB₂, 6-Keto-PGF_1α, t-Pa activity and infarction size in group Ⅱ,Ⅲ and Ⅳ(P＞0.05).Compared to group Ⅱ, plasma concentration of ET, NO and PAI activity were significantly decresed (P＜0.01)in group Ⅳ. Plasma concentration of TXB₂, ET, NO and plasma activity of PAI were significantly lower in groupⅤ than those in group Ⅱ(P＜0.01). Conversely, plasma concentration of 6-Keto-PGF1 and plasma activity of t-Pa were remarkably higher in group Ⅴ than those in group Ⅱ(P＜0.01). The infarction size was remarkly decrease(P＜0.01)in group Ⅴ.CONCLUSIONS:Amiodarone inhibited PAI avtivity, decreased release of ET and NO in AMI in rabbits. Metoprolol inhibited platelet activation, improved fibrinolytic, decreased release of ET and NO, and reduced myocardial infarction size in AMI in rabbits; Lidocaine had no effect above.     

Effects of diosmin on activity of myeloperoxidase in renal tissues and expression of CD11b,CD54 and CD62L on neutrophils in rats with kidney ischemia and reperfusion

AIM：To observe the effects of diosmin on the activity of myeloperoxidase (MPO) in renal tissues and the expression of CD11b, CD54 and CD62L on neutrophils in rats with kidney ischemia and reperfusion. METHODS：One hundred and eighty Sprague-Dawley rats were randomly divided into 3 groups: sham operation (SO) group, ischemia and reperfusion (I/R) group and diosmin+I/R (DOSM+I/R) group. At the end of the experiment, renal tissues were obtained and the activity of MPO was detected by ELISA. The expression of CD11b, CD54 and CD62L on polymorphonuclear leukocytes was determined by flow cytometry after monoclonal antibody labeling. RESULTS：The renal MPO activity at different time points in DOSM+I/R group was significantly lower than that in I/R group (P<0.01 or P<0.05). The expression levels of CD11b, CD54 and CD62L on polymorphonuclear leukocytes in I/R group were significantly higher than those in SO group (P<0.01 or P<0.05), while those in DOSM+I/R group were lower than those in I/R group (P<001 or P<0.05). CONCLUSION: Diosmin not only decreases the activity of MPO in renal tissues, but also reduces the expression of CD11b, CD54 and CD62L on polymorphonuclear leukocytes, suggesting that its protective effect against kidney ischemia-reperfusion injury through anti-inflammatory mechanism.