巨噬细胞对低密度脂蛋白的修饰和PGE2抗AS作用的实验研究

目的研究巨噬细胞对低密度脂蛋白(LDL)的修饰和前列腺素E2(PGE2)对动脉粥样硬化形成的抑制作用。方法以巨噬细胞(Mp)作用LDL,并设PGE2(20mg/L)组,检测每组过氧化脂质含量,谷胱甘肽过氧化物酶活性,及形态计量学测定细胞变化和含脂量。结果细胞修饰组的脂质过氧化物含量高于给PGE2组,而谷胱甘肽过氧化物酶活性显著低于给药组。作用24小时的修饰组细胞面积明显增大,摄脂增加,均分别显著高于给药组和细胞对照组。结论提示Mp能够氧化修饰LDL并能摄取脂质,导致泡沫细胞形成。大剂量PGE2有抗LDL氧化修饰,抑制Mp细胞摄脂,和泡沫细胞形成,从而抑制动脉粥样硬化发生的作用。     

脂多糖通过LDLr诱导巨噬细胞泡沫化:炎症应激下巨噬细胞泡沫化的另一条通路

目的研究炎症介质-脂多糖(LPS)诱导的炎症应激是否通过扰乱固醇调节元件结合蛋白裂解激活蛋白-固醇调节元件结合蛋白2(SCAP-SREBP2)复合物介导的低密度脂蛋白受体(LDLr)负反馈调控,增加THP-1巨噬细胞对非修饰低密度脂蛋白胆固醇(LDL)摄取,导致泡沫细胞形成。方法诱导分化成功的THP-1巨噬细胞在无血清培养基中培养4 h后,分为对照组(继续无血清培养),高脂组(加入LDL负荷,终浓度为25 mg·L-1),高脂加炎症刺激组(加入LDL负荷,终浓度为25 mg·L-1,同时加入炎症介质LPS,终浓度200μg·L-1),单纯炎症刺激组(加入炎症介质LPS,终浓度200μg·L-1),以上各组细胞培养24 h后收获。ELISA法检测细胞培养基中TNF-α浓度,酶化学法检测细胞内胆固醇浓度,琼脂糖电泳检测LDL氧化程度,Real time PCR法检测LDLr、SREBP2和SCAP mRNA表达水平,Western blot法检测LDLr、SREBP2和SCAP蛋白表达,激光共聚焦检测SCAP在内质网与高尔基体间的转位情况。结果细胞内胆固醇测定发现,炎症介质LPS通过增加THP-1巨噬细胞对非修饰LDL摄取,导致巨噬细胞转变为泡沫细胞。在无炎症介质LPS时,25 mg·L-1LDL减少LDLr mRNA和蛋白表达(P<0.05)。然而,LPS增加LDLr mRNA和蛋白表达,逆转25 mg·L-1LDL对LDLr的抑制效应,不恰当的增加了巨噬细胞对LDL摄取(P<0.05),LPS刺激也导致了SCAP和SREBP2的mRNA和蛋白过表达(P<0.05),同时促进了SCAP从内质网转移到高尔基体。这些结果提示LPS干扰了胆固醇介导的LDLr负反馈调控,使非修饰LDL在巨噬细胞内堆积,导致泡沫细胞形成。结论本研究提示,在LPS诱导的炎症应激状态条件下,天然LDL可以通过LDLr被巨噬细胞大量摄取入细胞内,导致泡沫细胞形成,这可能是巨噬细胞泡沫化的另一条通路。     

巨噬细胞对低密度脂蛋白的修饰和PGE2抗AS作用的实验研究

目的：研究巨噬细胞对低密度脂蛋白（LDL）的修饰和前列腺素E2（PGE2）对动脉粥样硬化形成的抑制作用。方法：以巨噬细胞（Mp）作用LDL,并没PGE2（20mg/L组）,检测每组过氧化脂质含量,谷胱甘肽过氧化物酶活性,及形态计量学测定细胞变化和含脂量。结果：细胞修饰组的脂质过氧化物含量高于给PGE2组,而谷胱甘肽过氧化物酶活性显著低于给药组。作用24小时的修饰组细胞面积明显增大,摄取增加,均分别显著高于给药组和细胞对照组。结论：提示Mp能够氧化修饰LDL并能摄取脂质,导致泡沫细胞形成。大剂量PGE2有抗LDL氧化修饰,抑制Mp细胞摄脂,和泡沫细胞形成,从而抑制动脉样硬化发生的作用。     

蛋白激酶C激活剂和抑制剂对肿瘤坏死因子杀瘤活性的影响

以小鼠成纤维细胞瘤L929细胞为靶细胞,研究蛋白激酶C(PKC)的激活剂和抑制剂对人重组肿瘤坏死因子(rHuTNF)杀瘤活性的影响.各种药物与rHuTNF 10 ng·ml~(-1)和放线菌素D 1 μg·ml~(-1)温育18 h,结果表明PKC的激活剂佛波醇-12-肉豆蔻酸盐-13-乙酸盐(PMA)2.5～160ng·ml~(-1)可浓度依赖性地抑制rHuTNF的杀瘤活性.Sc-10(1～16μg·ml~(-1))单独作用很弱,但可浓度依赖地增强PMA 10 ng·ml~(-1)或A23187 0.5μg·ml~(-1)的抑制作用,PKC抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪(H-7)单独作用对rHuTNF杀瘤活性无影响,但可减弱PMA 50ng·ml~(-1)的抑制作用.槲皮囊2～16μg·ml~(-1)则可直接抑制rHuTNF的杀瘤活性,钙调蛋白抑制剂N-(6-氨己基)-5-氯-1-萘磺酰胺(W-7)及其同系物也有微弱的抑制作用.结果提示PKC在rHuTNF杀瘤作用中起着重要作用.     

286例心血管疾病患者氧化低密度脂蛋白测定分析

近年研究表明 ,内皮细胞损伤和功能失调是冠状动脉粥样硬化发生的始动因素 ,低密度脂蛋白发生氧化修饰形成的氧化低密度脂蛋白 (OX -LDL)是致动脉粥样硬化的关键步骤。OX -LDL可被内皮下巨噬细胞摄取 ,转化为脂质泡沫细胞 ,逐渐形成动脉壁粥样斑块。因此 ,OX -LDL测定在心血管疾病防治中愈显其重要性。两年多来 ,我们分别检测了 86例冠心病患者、 10 0例高血压患者、 10 0例高脂血症患者及 5 0例非以上疾患对照病例血浆OX -LDL浓度 ,以探讨其在心血管疾病防治中的意义。1　对象和方法1 1　对象 :1999年 2月至 2 0 0 1年 8月某院门诊…     

三七中人参三醇甙对大鼠离体心脏缺血再灌注损伤及心律失常的保护作用

三七中人参三醇甙(PTS)5,50μg·ml~(-1)灌流对离体大鼠Langendorff心脏缺血再灌注所致心律失常具有明显的保护作用,可降低室颤发生率,并可保护心肌组织中超氧化物歧化酶的降低,PTS 50μg·ml~(-1)还可抑制心肌丙二醛的产生,对缺血再灌注所致心肌乳酸脱氩酶和磷酸肌酸激酶的漏出亦有明显的抑制作用。     

老山云芝多糖P对巨噬细胞代谢乙酰低密度脂蛋白的影响

老山云芝多糖P可使小鼠腹腔巨噬细胞乙酰LDL(acLDL)受体数目增加。提高巨噬细胞对acLDL的结合、内移和降解。其对巨噬细胞降解acLDL的影响呈浓度效应关系,该效应在云芝多糖100μg/ml时最大,可使巨噬细胞对(~(125)Ⅰ)ac LDL的降解增加77%。实验结果提示,老山云芝多糖P可能通过刺激清道夫受体途径在整体发挥降脂、抗动脉粥样硬化的作用。     

舒胸微丸中三种成分含量测定

目的:建立舒胸微丸中三种有效成分阿魏酸、川芎嗪和红花黄色素的含量测定方法。方法:采用 UV-Vis法测定红花黄色素的含量,HPLC 法测定阿魏酸与川芎嗪的含量。结果:阿魏酸与川芎嗪在1μg·mL~(-1)-150μg·mL~(-1)范围内线性关系良好,分别为0.9996和O.9998,平均回收率分别为96.3%(RSD=3.0%)和93.9%(RSD=2.3%);红花黄色素在1μpg·mL~(-1)—120μg·ml~(-1)范围内线性关系良好,r 为0.9999,平均回收率为101.1%(RSD=2.4%)。结论:本文所采用的含量测定方法准确,简便,灵敏可靠。     

西替利嗪中氯苯残留量检查方法的研究

目的 建立西替利嗪中氯苯的HPLC检查法。方法 选用Kromasil C_(18)(150×4.6mm,5μm)色谱柱,乙腈-0.01mol·L~(_1)磷酸二氢钠-0.02mol·L~(-1)庚烷磺酸钠(46:54:10)为流动相,检测波长:263nm,流速为1.0ml·ml~(-1),进样量20μl。结果 氯苯在浓度为10.63～212.64μg·ml~(-1)范围内线性关系良好。回归方程为A=20118+×1803.91C,r=0.9999。结论 回收率为99.76％,精密度及稳定性均较佳,RSD分别为1.09％及0.88％,最低检测限为1μg·ml~(-1)。本法简便易行,重现性好,可用于西替利嗪中残留氯苯的限度检查。     

HPLC法测定复方磺胺甲噁唑片中磺胺甲噁唑与甲氧苄啶的含量

目的:建立了HPLC法测定复方磺胺甲噁唑片中磺胺甲噁唑与甲氧苄啶的含量。方法:采用ODS柱,以0.025mol·ml~(-1)磷酸溶液(用三乙胺调pH至3.0)—乙腈(80:20)为流动相,流速为1.Oml·min~(-1),检测波长为246nm。结果:磺胺甲噁唑与甲氧苄啶的线性范围分别为40.0～360.0μg·ml~(-1)和8.0～72.0μg·ml~(-1),平均回收率分别为99.8％(RSD=0.45％)和100.5％(RSD=0.26)。结论:本方法准确,快速,简便。     

Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages

Low density lipoproteins (LDL) can be oxidatively modified in vitro by macrophages and certain other cell types so that macrophages will take them up much faster. This process may be important in the formation of cholesterol-laden foam cells derived from macrophages in atherosclerotic lesions. In this study, we have shown that certain flavonoids, plant constituents found in the diet, are potent inhibitors of the modification of 125I-labelled LDL by macrophages, with IC50 values in the micromolar range (e.g. morin and fisetin 1 microM; quercetin and gossypetin 2 microM). The potencies of individual flavonoids in inhibiting LDL modification did not correlate with their previously determined potencies as inhibitors of 5-lipoxygenase and cyclo-oxygenase. The modification of LDL by macrophages exhibits a lag period of about 4-6 hr before enhanced uptake is detected. During this time, there is a rapid depletion in its content of alpha-tocopherol (an endogenous antioxidant found in lipoproteins) followed by a large increase in the level of hydroperoxides. The flavonoids conserved the alpha-tocopherol content of LDL and delayed the onset of detectable lipid peroxidation. Flavonoids also inhibited the cell-free oxidation of LDL mediated by CuSO4. These findings raise the possibility that flavonoids may protect LDL against oxidation in atherosclerotic lesions and may therefore be natural anti-atherosclerotic components of the diet, although this will depend to a large extent on their pharmacokinetics.     

Pravastatin inhibits cellular cholesterol synthesis and increases low density lipoprotein receptor activity in macrophages: in vitro and in vivo studies.

1. Pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) inhibitor, is a highly selective inhibitor of hepatic cholesterol synthesis. We studied the in vivo and in vitro effects of pravastatin on macrophage cholesterol metabolism. 2. The effects of incubating pravastatin with human monocyte derived macrophages (HMDM), mouse peritoneal macrophages (MPM) and a J-774 A.1 macrophage-like cell line, on macrophage cholesterol synthesis, cellular degradation of native low density lipoprotein (LDL) and modified LDL, cholesterol efflux from these cells and the cholesterol esterification rate were determined. 3. Pravastatin was administered either as one 40 mg dose or 40 mg daily for 8 weeks to normocholesterolaemic and hypercholesterolaemic individuals. The effects on cholesterol synthesis and degradation in monocytes derived from these subjects were studied. 4. In vitro, pravastatin resulted in a dose-dependent inhibition of macrophage cholesterol synthesis. Cellular degradation of native LDL increased by 119% in the presence of 0.1 mg ml-1 pravastatin. Degradation of both acetyl LDL and oxidized LDL was unaffected. Small concentrations of pravastatin (up to 0.19 micrograms ml-1) increased the cellular cholesterol esterification rate after incubation with LDL, but higher concentrations resulted in an inhibition of the esterification. 5. Single dose pravastatin administration caused a reduction in cholesterol synthesis by the subjects own HMDM by 62% and 47% in normocholesterolaemic and hypercholesterolaemic individuals, respectively. Chronic administration resulted in a 55% inhibition of cholesterol synthesis and a 57% increase in LDL degradation. 6. The results indicate that the selective uptake of pravastatin shown for hepatocytes can be extended to macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of a complex of dexamethasone palmitate-low density lipoprotein and its effect on foam cell formation of murine peritoneal macrophages.

In the early progression of atherosclerosis, LDL migrates in the subendothelial space of the artery and plays an important role in foam cell formations of macrophages. LDL may serve as a carrier of site-specific delivery of drugs to atherosclerotic lesions. In this exploratory study, dexamethasone palmitate (DP) was incorporated in LDL, and an inhibitory effect of this complex on foam cell formations was examined. LDL was isolated from human plasma, and the DP-LDL complex was prepared by incubation in the presence of Celite 545. No degradation nor modification of LDL was observed. The DP/LDL molar ratio of the complex was 35-50:1. Foam cell formations of murine macrophages were induced by incubation with oxidized LDL. When macrophages were pretreated with the DP-LDL complex, accumulation of cholesterol ester in the macrophages induced by oxidized LDL, i.e., an index of foam cell formation, was decreased. These findings indicated that the DP-LDL complex showed similar characteristics to LDL, and the DP-LDL complex inhibited foam cell formations of macrophages in vitro. This study provides the basis for further study of the DP-LDL complex as a drug-carrier complex for treatment of atherosclerosis.     

Captopril inhibits the oxidative modification of apolipoprotein B-100 caused by myeloperoxydase in a comparative in vitro assay of angiotensin converting enzyme inhibitors

The oxidative modification of low-density lipoproteins (LDL) is a key event in the formation of atheromatous lesions. Indeed, oxidized derivatives accumulate in the vascular wall and promote a local inflammatory process which triggers the progression of the atheromatous plaque. Myeloperoxidase (MPO) has been mentioned as a major contributor to this oxidative process. It takes part in the oxidation both of lipids by chlorination and peroxidation and of apolipoprotein B-100. Based on recent observations with several anti-inflammatory and thiol-containing drugs, the present study was designed to test the hypothesis that anti-hypertensive agents from the angiotensin converting enzyme (ACE) inhibitors group inhibit the oxidative modifications of Apo B-100 caused by MPO. Captopril, ramipril, enalapril, lisinopril and fosinopril were assessed by measuring: their inhibiting effect on the MPO/H(2)O(2)/Cl(-) system, the accumulation of compound II, which reflects the inhibition of the synthesis of HOCl and the LDL oxidation by MPO in presence of several concentrations of ACE inhibitors. Only captopril, a thiol-containing ACE inhibitor, was able to significantly decrease the oxidative modification of LDL in a dose dependent manner and this by scavenging HOCl. This efficient anti-hypertensive drug therefore appears to also protect against the atherosclerotic process by this newly documented mechanism.     

Natural Antioxidants for the Prevention of Atherosclerosis

Hypercholesterolemia, cigarette smoking, hypertension, and obesity are known contributing risk factors for the development of atherosclerotic coronary artery disease (CAD). However, they account for only half of all cases of CAD, and the complete pathologic process underlying atherosclerosis remains unknown. Growing evidence suggests that oxidative modification of low-density lipoprotein (LDL) may be of particular importance in the pathogenesis. Oxidized LDL exhibits proatherogenic effects. Therefore, current research has focused on inhibiting the oxidation of LDL as a means of inhibiting the atherosclerotic process. One such approach is to enhance the endogenous antioxidant defense systems within the LDL particle with lipophilic antioxidants such as α-tocopherol and β-carotene, or by supplementing the aqueous-phase antioxidant capacity with ascorbic acid. Observational data suggest a protective effect of antioxidant supplementation on the incidence of CAD; however, specific doses cannot be recommended since the data are inconclusive.     

丹参酮ⅡA对低密度脂蛋白引起牛血管内皮细胞损伤的影响

<正> 动脉内皮细胞(endothelial cells,EC)损伤在动脉粥样硬化(AS)的发生,发展中极为重要。血液中胆固醇浓度增高,特别是低密度脂蛋白(low densitylipoprotein,LDL)含量增加可引起动脉EC损伤,导致前列腺环素(prostacyclin,PGI_2)合成障碍,从而与血栓素A_2(TXA_2)平衡失调,促进AS的发生发展。本文的研究目的是评价丹参酮ⅡA(tanshinone ⅡA,TN ⅡA)对LDL引起的EC损伤的影响。     

Morin attenuates oxidized low‐density lipoprotein‐mediated injury by inducing autophagy via activating AMPK signalling in HUVECs

Endothelial dysfunction is a precursor of cardiovascular disease, and oxidized low‐density lipoprotein (ox‐LDL) has been implicated in the development of atherosclerosis by directly targeting endothelial cells. Morin, a natural flavonol, has been shown to protect endothelial cells from dysfunction. The present study was designed to evaluate the effect of morin on ox‐LDL‐induced injury and to investigate the underlying molecular mechanisms in human umbilical vein endothelial cells (HUVECs). The results showed that morin alleviated ox‐LDL‐induced endothelial injury and promoted the viability of HUVECs exposed to ox‐LDL. Morin significantly inhibited the oxidative stress induced by ox‐LDL by inhibiting the production of reactive oxygen species and malondialdehyde, and downregulating the level of superoxide dismutase. Moreover, morin markedly attenuated the overexpressed mRNA levels of the inflammatory factors interleukin (IL)‐1β, IL‐6, and the adhesion molecules ICAM‐1 and VCAM‐1 induced by exposure to ox‐LDL. We found that morin attenuated ox‐LDL‐induced injury in HUVECs by inducing autophagy. The protective effects of morin against ox‐LDL‐induced injury were dramatically reversed by chloroquine phosphate (CQ) treatment. Furthermore, morin up‐regulated the expression of p‐AMPK and down‐regulated the level of p‐mTOR in HUVECs exposed to ox‐LDL, and this was significantly reversed by the AMPK inhibitor Compound C (CC). Taken together, our results demonstrated that morin attenuates ox‐LDL‐mediated injury by inducing autophagy via activating AMPK signalling in HUVECs.     

反相高效液相色谱法同时测定人血浆中对乙酰氨基酚/曲马朵及其代谢物的含量

目的:建立一种用高效液相色谱法(HPLC)同时测定人血浆中对乙酰氨基酚/曲马朵及其代谢物氧去甲基曲马朵(M1)浓度的方法。方法:以间乙酰氨基酚为内标,血浆样品经碱化后,用乙酸乙酯提取。HPLC-UV测定采用DiamonsilTM C18色谱柱(250mm×4.6mm),流动相为0.01mol·L-1磷酸二氢钾缓冲液(含三乙胺0.005mol·L-1,磷酸调pH3.0)-乙腈(79:21),流速:1.0ml·min-1,紫外检测波长:218nm。结果:本实验条件下各物质分离良好,对乙酰氨基酚在0.2-21.6μg·ml-1范围呈线性,曲马朵及其代谢物M1分别在10-720和5-160ng·ml-1范围呈线性。对乙酰氨基酚的最低检测限为0.2μg·ml-1,曲马朵和M1的最低检测限分别为10和5ng·ml-1。三种物质的方法回收率均在80%-120%范围内,提取回收率均在75%以上。结论:本法简单、快速,可用于人血浆中对乙酰氨基酚/曲马朵及M1的同时检测。     

Functional evidence for anti-oxidant action of fluvastatin on low-density lipoprotein using isolated macrophages and aorta

1. Fluvastatin has been reported to have not only a hypocholesterolaemic effect, but also a protective effect on low-density lipoprotein (LDL) from oxidation. We functionally evaluated the anti-oxidant effect of fluvastatin on oxidation of LDL by copper ions in vitro using mouse macrophages and rabbit aorta preparations. 2. After native LDL (N-LDL) from rabbit plasma had been pre-incubated in the presence or absence of fluvastatin (10 micromol/L) for 4 h, the N-LDL was mildly oxidized by incubation with 5 micromol/L CuCl2 for 5 h and two oxidized LDL, fluvastatin-pretreated (Flu-OxLDL) and -non-treated (OxLDL), were prepared. The level of thiobarbituric acid-reactive substances (TBARS) in Flu-OxLDL and OxLDL markedly increased compared with N-LDL. The degree of increment was significantly less in Flu-OxLDL than OxLDL. 3. When macrophages were incubated with Flu-OxLDL or OxLDL, the amount of cholesteryl ester that accumulated in the macrophages markedly increased compared with N-LDL. The degree of increment was significantly less in Flu-OxLDL than OxLDL. 4. Acetylcholine-induced endothelium-dependent relaxations in rabbit aortic rings were impaired in the presence of either Flu-OxLDL or OxLDL. The degree of impairment was significantly less in Flu-OxLDL. 5. The increased TBARS level, facilitated cholesteryl ester accumulation in macrophages and impaired endothelium-dependent relaxation elicited by OxLDL were not affected by simultaneous treatment with fluvastatin (10 micromol/L). 6. These findings indicate that fluvastatin can protect plasma LDL from oxidative modification and, thereby, prevent cholesterol accumulation in macrophages and endothelial dysfunction in blood vessels. This additional anti-oxidative effect of fluvastatin may be beneficial for preventing the progression of atherosclerosis.     

Glibenclamide inhibits accumulation of cholesteryl ester in THP-1 human macrophages

Glibenclamide is an adenosine triphosphate (ATP)-sensitive potassium channel inhibitor that is widely used in treating diabetes mellitus. However, the effects of this drug on cholesterol metabolism and atherogenesis are not well known. We investigated the effects of this agent on the cellular cholesterol metabolism in cultured human macrophages. The effect of glibenclamide was evaluated by the measurement of the cellular contents of total cholesterol, free cholesterol, and cholesteryl ester in the presence of low-density lipoprotein (LDL). The effect on the degradation and association of 125I-labeled LDL (125I-LDL) also were determined. Cholesterol efflux was measured in the absence and the presence of high-density lipoprotein (HDL). The secretion of apolipoprotein E also was determined. The synthesis and hydrolysis of cholesteryl ester were evaluated. Glibenclamide stimulated both synthesis and hydrolysis of cholesteryl ester, and inhibited the net accumulation of cholesteryl ester by LDL in a concentration-dependent manner and even decreased its content compared with time 0 control. This drug had no effect on the degradation or association of 125I-LDL. Glibenclamide promoted the HDL-independent cholesterol efflux by decreasing esterified cholesterol and increasing the release of free cholesterol and secretion of apolipoprotein E into the medium. The other potassium channel inhibitors or openers had no effect on the cellular cholesterol levels. These results suggest that glibenclamide inhibits the accumulation of cholesteryl ester in macrophages by enhancing the hydrolysis of cholesteryl ester as well as by increasing cholesterol efflux, and possibly, by increasing the secretion of apolipoprotein E. These effects appeared to be unrelated to an effect on the potassium channel. Inhibition of accumulation of cellular cholesterol by glibenclamide might be favorable for the prevention of atherosclerotic disease.