The Ca^2＋ Antagon i z i ng Effect of Oh i nese Cobra Venom Factor on Formation of Macrophage-derived Foam Cells

Purpose CCVF was isolated from Chinese cobra （Naja naja） venom, its Ca^2＋ antagonizing effect on formation of macrophage-derived foam cells was explored in these studies. Methods Foam cell models were induced with C57BL/6J mouse peritoneal macrophages incubated in 10mg/L oxidized low density lipoprotein （OLDL）, and their intracellular Ca^2＋ levels influenced both slowly and transiently by CCVF were determined with the technique of Ca^2＋ fluorescent indicator. Results The intracellular Ca^2＋ level with the macrophages incubated in 10mg/L OLDL and lOmg/L CCVF was 40.2%of the macrophages incubated in 10mg/L OLDL （P〈0.05）; While the transient influence of CCVF on the intracellular Ca^2＋ levels were not significant. Conclusion CCVF exerted a long-lasting antagonizing role on the enhancement of intracellular Ca^2＋ levels, thus inhibited the formation of macrophage-derived foam cell.     

8.METABOLISM

8.3 Protein,carbohydrate and lipid metabolism980213 Metabolism of oxidized low density lipopro-teins by human peripheral monocyte-macrophages.LUO Jun(罗骏),et al.Cardiovasc Res Lab,NanjingRailway Med Coll,Nanjing,210009.Chin J Arte-rioscler 1997;5(4):287-290.Objective:To elucidate the role of oxidized low den-sity lipoproteins(OLDL),the metabolism of OLDL inmacrophages and the effect of OLDL on the totalcholesterol content in macrophages were examined.Methods:The metabolisms of low density lipoprotein(LDL)and OLDL in macrophages were assessed with     

Thymic Stromal Lmphopoietin Pomotes Macrophage-derived Foam Cell Formation简

The effect of thymic stromal lymphopoietin（TSLP） on macrophage-derived foam cell formation and the underlying mechanism were studied. Macrophages isolated from C57BL/6 mice were co-cultured in vitro with different concentrations of TSLP or TSLPR-antibody in the presence of oxidized low density lipoprotein（ox-LDL）. The effects of TSLP on macrophage-derived foam cell formation were observed by using oil red O staining and intracellular lipid determination. The expression levels of foam cell scavenger receptors（CD36 and SRA） as well as ABCA1 and TSLPR were detected by using RT-PCR and Western blotting. As compared with the control group, TSLP treatment significantly promoted lipid accumulation in macrophages, significantly increased protein expression of CD36 and TSLPR in a dose-dependent manner, and significantly reduced the expression of ABCA1 protein in a dose-dependent manner. No significant differences were noted between the TSLPR-antibody group and the control group. TSLP may down-regulate the expression of cholesterol efflux receptor ABCA1 and up-regulate scavenger receptor expression via the TSLPR signaling pathway, thereby promoting macrophage-derived foam cell formation.     

Effects of Metallothionein on Isolated Rat Heart

To investigate the effects of metallothionein （MT） on isolated rat heart, 16 Wistar rats were randomly divided into 2 groups. In control group （group C）, distilled water was injected intraperitoneally and 24 h later isolated hearts were perfused with Langendorff and stored at 4℃ for 3 h with histidine-tryptophan-ketoglutarate （HTK） solutions, and then isolated hearts were perfused for 2 h by Langendorff. In experimental group （group E）, 3.6% ZnSO4 was injected intraperitoneally, 24 h later isolated hearts were perfused by Langendorff and stored at 4℃ for 3 h with HTK solutions, and then the isolated hearts were perfused for 2 h with Langendorff. MT content, the recovery of hemodynamics, myocardial water content （MWC）, lactate dehydrogenase （LDH） and creatine kinase （CK） leakage, adenosine triphosphate （ATP） and malondialdehyde （MDA） content, superoxide dismutase （SOD） activity, myocardial cell Ca^2＋ content, Ca^2＋-ATPase activity of mitochondria （[Ca^2＋-ATPase]m） and its Ca^2＋ content （[Ca^2＋]m）, synthesizing ATP activity of mitochondria （[ATP]m）, and the ultrastructure of cells were examined. There were a significant increase in group E in hemodynamic recovery, ATP content, SOD activity, [Ca^2＋-ATPase]m activity, [ATP]m activity, and substantial reduction in MWC, LDH and CK leakage, MDA content, myocardial cell Ca^2＋ content, [Ca^2＋]m content, and the ultrastructural injury were obviously milder than that of group C. This study demonstrated that MT has protective effects on isolated rat heart.     

The relationship between LDL oxidation and macrophage myeloperoxidase activity

Objective To explore low density lipoprotein (LDL) oxidation by macrophage myeloperoxidase(MPO) at molecular level.Methods Using a mouse macrophage model, we examined the relationship between LDL oxidation and macrophage MPO by measuring macrophage MPO activity, LDL oxidation products, MPO gene expression and cellular orientation of LDL oxidation.Results MPO gene expression increased to its maximum gradually when the concentration of LDL was increased, and then maintained at that level. NaN3 inhibied the elevation of MPO activity and LDL oxidation, which was LDL concentration-dependent. After the composition of macrophage membrane was roughly analyzed, it was determined that the contents of MPO and LDL in 5% sucrose were 7. 667 and 21 times higher than those in 10% sucrose, respectively.Conclusion LDL is attached to the “microdomain” of the macrophage membrane in which LDL is oxidized by MPO.     

Effect of HDL and apoAI on PGE2 Production by Monocyte—Derived Macrophages

Effect of antiatherogenic high density lipoprotein(HDL) and apolipoprotein AI (apoAI) on production of prostaglandin E2(PGE2) by human monocyte-derived macrophages was investigated.Macrophages were loaded with acetylated low density lipoprotein followed by incubation with HDL3 or apoAI.PGE2 produced and secreted in culture supernatant was quantified by enzyme immunoassay.HDL3 induced production of PGE2 by macrophages in a time-dependent manner.24h after incubation.PGE2 production by HDL3-treated macrophages increased 3.7-fold of that by control cells.ApoAI also induced PGE2 secretion to 2.1-fold,which was significantly less than HDL3.The data indicate that both HDL3 and lipid-free apoAI enhance PGE2 synthesis and secetion by human macrophages and this may further contribute to the protection from atherosclerosis.     

EFFECTS OF CALDESMON, CALPONIN, AND TROPOMYOSIN ON THE MG^2＋ -ATPase ACTIVITIES OF SMOOTH MUSCLE MYOSIN

Objective To test whether in the absence of actin, actin-binding proteins such as caldesmon, calponin, and tropomyosin interact with the myosin of unphosphorylation, Ca^2 -dependent phosphorylation (CDP), and Ca^2 -independent phosphorylation (CIP) and stimulate myosin Mg^2 -ATPase activities. Methods Mg^2 -ATPase activities were measured to evaluate the effects of caldesmon, calponin, and tropomyosin on the myosin in unphosphorylation, CDP by myosin light chain kinase (MLCK), and CIP by MLCK. (1) At different incubation-time, i.e., 5, 10, 20, 40, and 60 minutes, the highest Mg^2 -ATPase activity was observed when myosin was in the state of CDP, the medium was CIP of myosin, and the lowest was the unphosphorylated myosin. (2) In the absence of caldesmon, calponin, and tropomyosin, the Mg^2 -ATPase activities from high to low were in the following order: CDP, CIP, and unphosphorylated myosin. However, in the presence of caldesmon, calponin, and tropomyosin, the order of relative value of Mg^2 -ATPase activities from high to low was unphosphorylated, CIP, and CDP of myosin respectively compared to the corresponding controls. Conelusions The results propose that caldesmon, calponin, and tropomyosin are capable of stimulating Mg^2 -ATPase activity of smooth muscle myosin in Ca^2 -independent manner, since Ca^2 is not obligating for the stimulating effects of the three proteins. The common characteristic of the three proteins is that when myosin activities are low, their activations are relatively strong and this property might be involved in smooth muscle tension keeping.     

Serum paraoxonase activity and protein thiols in patients with hyperlipidemia

Objective： In the present study we evaluated the paraoxonase activity and protein thiols level in south Indian population with newly diagnosed hyperlipidemia. Methods： The study was conducted on 55 newly diagnosed hyplerlipidemic patients and 57 healthy controls. Serum paraoxonase activity and protein thiols were estimated by spectrophotometeric method and lipid profile by enzymatic kinetic assay method. Results： Serum paraoxonase activity, protein thiols and high density liPopretein levels were low and total cholesterol, triglycerides and low density lipoprotein levels were high in patients with hyperlipidemia compared to healthy controls （P〈 0.01 ）. Serum paraoxonase activity correlated positively with protein thiols and high density lipoprotein （P〈0.01）. Conclusion： Decreased paraoxonase activity and protein thiols were found in patients with hyperlipi- demia. This may indicate the susceptibility of this population to accelerated atherogenesis and protein oxidation.     

REGULATION OF ANTI-SRBC ANTIBODY PRODUCTION BY OPIOIDS AND THEIR MECHANISMS

This study focused on the influences of opioids on the generation of antibody againse sheep erythrocyte in vitro.It was found that morphine,a-CAO,DADLE,MENK were able to inhibit the capacity of murine spleen cells to generate antibody and leukotriene C4 and conversely,dynorphin was able to stimulate the capacity of murine spleen cells to generate antibody and leukotriene C4. Morphine,a-CAO,MENK,DA-DLE,dynorphin decreased intracellular cAMP level,increased [Ca^2 ]i and calmodulin activity.The effects were completely blocked by naloxone,the specific opioid antagonist.Our results showed that opioids regulate the production of antibody in murine spleen cells,and alter intracellular cAMP,[Ca^2 ]i calmodulin activity,and leukotriene C4 production by way of binding to different receptor types.     

The Protective Effect of Propofol on Erythrocytes during Cardiopulmonary Bypass

To evaluate the relationship between erythrocyte injury and intracellular calcium ion overload, and the protective effect of propofol on erythrocytes during cardiopulmonary bypass (CPB). 40 children with congenital heart diseases who underwent surgical repair under CPB were included. The patients were randomly divided into two groups: control group (group C) and propofol group (group P). Anesthesia was maintained in the patients with 6 mg/kg/h propofol in Group P, and those in the Group C inhaled 1%—2% isoflurane. The blood samples were taken before CPB, 30 min after CPB, at the end of CPB, and 2 h and 24 h after CPB to measure the content of e-rythrocyte intracellular calcium ion (E-Ca^2 ), Ca^2 -Mg^2 -ATPase and Na^ - K^ - ATPase activi-ties, index filtration of erythrocytes (IF), mean corpuscular volume (MCV) and the concentration of plasma free hemoglobin (F-Hb). Results showed that in the control group, E-Ca^2 , IF, MCV and F-Hb were gradually increased and Ca^2 - Mg^2 - ATPase and Na^ - K^ - ATPase activities were decreased. The increase of E-Ca^2 was linearly paralleled to IF, MCV and F-Hb. In propofol group, all the above-mentioned parameters were significantly improved (P＜0. 05). This study suggests that erythrocyte injury is related to elevation of intracellular calcium during CPB and propofol has a protective effect on erythrocyte injury.     

Effects of Cyclosporine A on Angiotensin Ⅱ-induced Cardiomyocyte Hypertrophy in Neonatal Rats

Summary： The effects of cyclosporine A （CsA） on Angiontensin Ⅱ （Ang Ⅱ ）-induced protein contents, c los protein levels and cytosolic Ca^2＋ level （[Ca^2＋]i） in cultured eardiomyocytes of neonatal rats were observed. Total protein contents were determined by Bradford method. The expression of c-fos protein was detected by Western blot. （[Ca^2＋]i） labeled with fluorescent probe Fluo-3/AM was measured under a laser scanning confoeal microscope. The results revealed that as compared with control, the total protein contents were increased in cardiomyocytes treated with Ang Ⅱ （10-1 mol/ L）, which could be inhibited by CsA in a dose-dependent manner. It was found that Ang Ⅱ could increase the c-los protein expression, which could be inhibited by CsA in a dose-dependent manner. Ang Ⅱ induced the [Ca^2＋]i elevation in cardiomyocytes. CsA did not influence the resting intracellular Ca^2＋ , but inhibited significantly the Ang Ⅱ-induced [Ca^2＋]i elevation. It was concluded that CsA can suppress the Ang Ⅱ-induced c-fos protein expression and [Ca^2＋]i elevation in single cardiomyocyte, which might play a role in the prevention of Ang Ⅱ-induced cardiomyocyte hypertrophy by CsA.     

CHANGES OF POLYAMINE METABOLISM IN HL-60 CELLS DURING THE INDUCTION OF DIFFERENTIATION BY RETINOIC ACID AND DIMETHYLSULFOXIDE

The polyamines putrescine, spermidine and spermine have been implicated in the regulation of cell proliferation and differentiation. In this study, the changes of intracellular polyamine contents and activity of ornithine decarboxylase, a rate-limiting enzyme in the polyamine synthetic pathway, were studied. The results showed that both retinoic acid (RA) and dimethylsulfoxide (DMSO) could elevate intracellular putrescine level by more than 2-fold over control value, then it declined gradually. In RA-treated cells, transient increase in spermidine and spermine levels was noted. In contrast, the spermidine and spermine levels in DMSO-treated cells declined to about 50% of the level of control cells at 96 h. The measurement of ornithine decarboxylase activity demonstrated that the increase of intracellular putrescine in RA and DMSO treated cells was due to the polyamine synthesis by inducing ornithine decarboxylase which reached 2 to 4-fold higher over basic level at 2 h, and above 6-fold at 16 h. These results suggest that the polyamine metabolism may be involved in RA and DMSO-induced granulocytic differentiation of HL-60 promyelocytic leukemia cells.     

Analysis of the inhibitory effect of gypenoside on Na+, K+-ATPase in rats’ heart and brain and its kinetics

Objective： To study the effects of gypenoside （Gyp） on the activity of microsomalNa^＋, K^＋-ATPase in rat＇s heart and brain in vitro. Methods： The microsomal Na^＋, K^＋-ATPase was prepared from rat＇s heart and brain by differential centrifugation. The activity of microsomal Na^＋, K^＋-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na^＋, K^＋-ATPase of rats. Results： Gyp reversibly inhibited the brain and heart＇s microsomal Na^＋, K^＋-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07± 6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na^＋, or K^＋-concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na^＋, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K^＋. Cenclusien： Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain＇s microsomal Na^＋, K^＋-ATPase activities in rats.     

Effects of the Drug(BSZGC)--containing Serum on Proliferation of Rat''s Osteoclasts and TRACP Activity in vitro

Objective:To observe effects of the drug-containing serum of Bu Shen Zhuang Gu Capsule(BSZGC 补肾壮骨胶囊 Capsule for Tonilying the Kidney to Strengthen the Bones)on proliferation of the rat's osteoclasts and tartrate-resistant acid phosphatase (TRACP)activity in vitro SO as to delve into the mechanisms of its preventive and therapeutic actions on osteoporosis.Methods:Forty female Sprague.Dawley rats aged three months were randomly divided into high dosage BSZGC group,medium dosage BSZGC group,low dosage BSZGC group,and the control group.BSZGC was orally administered into the rats of high,medium,and low dosage groups at difierent dosages for 12 days.and isometric normal saline was orally administered to the rats of the Control group.The drug-containing serum and control serum were prepared.Osteoclasts isolated mechanically from the femur and tibia of Sprague-Dawley rats aged one week were cultured wim medium added with different drug-containing sera and control serum.The growth of osteoclasts was observed under an inverted phase-contrast microscope,and optic density(OD)value of osteoclasts was determined by MTT colorimetric assay.TRACP activity was measured by the diazol method.Results:OD value of osteoclasts in the high dosage drug-containing serum group,medium dosage drug-containing serum group,and low dosage drug-containing serum group was significantly lower than that in the control serum group(P＜0.05)with a dose-effect correlation.TRACP activity in high dosage drug-containing serum group,medium dosage drug-containing serum group,low dosage drug-containing serum group was significantly lower than that of the control serum group(P＜0.01),and no significant differences in TRACP activity were not found among the difierent dosages drug-containing serum groups.Conclusions:BSZGC can inhibit the proliferation of the osteoclasts and reduce TRACP activity,which may be one of the mechanisms of its preventive and therapeutic actions on osteoporosis.     

Effects of remifentanil on intracellular Ca2+ and its transients induced by electrical stimulation and caffeine in rat ventricular myocytes

Background Preconditioning with remifentanil confers cardioprotection. Since Ca^2＋ overload is a precipitating factor of injury, we determined the effects of remefentanil on intracellular Ca^2＋ （[Ca^2＋]i） and its transients induced by electrical stimulation and caffeine, which reflects Ca^2＋ handling by Ca^2＋ handling proteins, in rat ventricular myocytes. Methods Freshly isolated adult male Sprague-Dawley rat myocytes were loaded with Fura-2/AM and [Ca]i was determined by spectrofluorometry. Remifentanil at 0.1-1000 μg/L was administered. Ten minutes after administration, either 0.2 Hz electrical stimulation was applied or 10 mmol/L caffeine was added. The [Ca^2＋]i, and the amplitude, time resting and 50% decay （t50） of both transients induced by electrical stimulation （E[Ca^2＋]i） and caffeine （C[Ca^2＋]i） were determined. Results Remifentanil （0.1-1000.0 μg/L） decreased the [Ca^2＋]i in a dose-dependent manner. It also decreased the amplitude of both transients dose-dependently. Furthermore, it increased the time to peak and tso of both transients dose-dependently. Conclusion Remifentanil reduced the [Ca^2＋]i and suppressed the transients induced by electrical stimulation and caffeine in rat ventricular myocytes.     

Serum 25（OH）D and Lipid Levels in Chinese Obese and Normal Weight Males before and after Oral Vitamin D Supplementation

Objective To study the effect of oral vitamin D （VD） supplementation on VD status and serum lipid in Chinese obese and healthy normal-weight men. Methods Twenty-one obese men with their body mass index （BMI）〉28 kg/m2 served as an obese group and 22 healthy normal-weight men with their BMI〈24 kg/m2 served as a control group in this study. After they were given 50 000 IU of oral VD, once a week for 8 weeks, the serum 25-hydroxyvitamin D [25（OH）D] concentration was measured with an enzyme-immunoassay kit. Results After oral VD supplementation, the serum 25（OH）D concentration significantly increased from 46.1＋9.1 nmol/L to 116.7_＋20.3 nmol/L in the obese subjects （P〈O.01） and from 52.8_＋17.8 nmol/L to 181.3_＋30.2 nmol/L in the control ones （P=0.13）. The serum high-density lipoprotein cholesterol （HDL-C） level was reduced within the normal reference range in the obese group. However, no significant change was observed in the level of other serum lipids （triglycerides, total cholesterol, and low-density lipoprotein cholesterol） in either of the two groups. Conclusion The effect of high-dose oral VD supplementation is weaker on VD status in the obese group than in the control group. High-dose oral VD supplementation has no side effect on serum lipid level in obese and control groups.     

Comparison between the Effects of Intraperitoneal Injection of LDL and Intravenous Injection of LDL on Arterial Endothelial Cells Apoptosis

To observe the effect of oxidized low density lipoprotein (OxLDL) on arterial endothelialcells apoptosis in vivo, we established a model in which Sprague-Dawley rats were given intraperi-toneal and intravenous injection of unmodified LDL (8 mg/kg every day) via the tail vein. Sevendays after the injection, the aortic endothelial cells specimens were prepared by an en face preparationof rat aorta. The apoptotic cells were identified and counted by in situ nick and labelling (TUNEL)method and light microscopy. The numbers of the apoptotic cells were 12.52±4.71/field in the in-traperitoneal injection control group, 11.41±2.94/field in the intravenous injection control group,22.98±8.01/field in the intraperitoneal injection LDL group and 103.8±11.5/field in the intra-venous injection LDL group, respectively. The difference was significant between injection LDLgroup and control (P<0.01), and the difference was also significant between two LDL injectiongroups (P<0.01). These findings suggest that injectio     

EFFECT OF OXIDIZED-LDL ON NF-κB NUCLEAR TRANSLOCATION IN AORTIC SMOOTH MUSCLE CELLS ORIGINATED FROM RATS OF DIFFERENT AGES

Objective To investigate the molecular mechanism of atherosclerosis that related to age. Methods Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kappa B (NF-κB) and expression of platelet-derived growth factor B (PDGF-B) in smooth muscle cells(SMCs) co-cultured with low density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL high density lipoprotein(HDL) originated fi‘om rats of 2 and l0 months old respectively. Fat stain was used to identify the lipid intake in SMCs. Results The optimal stimulation time ofox-LDL to SMCs was 12 hours. NF-KB intensity increased in most nuclei of SMCs that originated fi‘om rats of either 2 or l0 months old co-cultured with ox-LDL. The intensity of NF-KB and the amount of intracellular lipid taken in SMCs were more obvious in cells fi‘om 10-month-old rats than fi‘om the younger ones.Change of PDGF-B expression in SMCs was not remarkable in each group of rats. Conclusions The 10-month-old rats are more susceptive to ox-LDL than 2-month-old rats in activating nuclear translocation of NF-KB. Maybe this is one of the important reasons contributing to the difference between the older and younger rats on the initiation and development of atherosclerosis lesion. Expression of PDGF-B is not associated with the activity of nuclear translocation of NF-κB.