| BackgroundCardiovascular and cerebrovascular diseases which mainly including hypertension, coronary heart disease (CHD), stroke and so on are the main diseases, which are harmful to health and life of human (especially older than50) in today’s society[1]. Because cardiovascular disease has the characteristics of high incidence, high disability rate, high death rate, high relapse rate, more complications. Both the incidence and mortality rate of cardiovascular disease are on the rise in recent years. The domestic and foreign researches found that many cardiovascular diseases and oxidative stress caused by oxygen free radical are closely related.Free radicals of human body originate from two aspects:the exogenous FR and the endogenous FR. The exogenous FR is mainly caused by atmospheric pollutants, ioniz radiation, alcohol, high pressure oxygen poisoning and some drugs. The endogenous FR is produced from:①the process of the normal metabolism in human body;②when the inflammation is happened in the body [2];③the metal ion as a catalyst in the oxidation and reduction reaction;④tissue is subject to ischemia and hypoxia. There are many kinds of free radicals and the most of them are oxygen free radicals (OFR), including superoxide anion (O-2), hydroxyl radical (-OH), organic peroxyradicals (ROO·), hydrogen peroxide (H-2), nitrogen and oxygen free radical (NO-) etc, which are collectively referred to as reactive oxygen species (ROS). Pre oxidation system to generate ROS mainly in mitochondria, cytochrome P450, iron and other metal ions, neutrophils and macrophages etc. Antioxidant defense system to generated ROS are①the first class of antioxidant defense system, Including catalase, superoxide dismutase, glutathione system, otherantioxidant system etc;②the second class of antioxidant defense system, Including DNA repair, protein repair and degradation, detoxification system, membrane repair, regeneration system. Oxidative stress is the balance disorder result from ROS produces too much or eliminated less in vivo between pre oxidation system and antioxidant defense system.Antioxidant enzyme system and non enzymatic antioxidants constitute the antioxidant systems in the body. The former is the first line of defense against FR, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) etc. The latter includes Vitamin C (VitC), vitamin E (VitE), melatonin (MLT) and glutathione (GSH) etc. They can remove FR, inhibit the damage of tissue and cells from FR, and protect cells and body from abnormal physiological function.Antioxidants have common characteristics as follows:(1) They can effectively remove O2", H2O2, O2and other active oxygen;(2) They can remove02-OH. ROO.(lipid free radical) and other free radicals;(3) They can remove the lipid peroxide, and reduce oxidative damage to tissue cells from ROS, reduce the formation of lipid peroxidation;(4) They can prevent LDL from forming oxidized LDL;(5) They can effectively reduce the content of lipid peroxide MDA in the human body, and improve the two major antioxidant enzymes SOD and GSH-Px activity.VitC as an water soluble antioxidant, which exists in the intracellular and extracellular fluid, is the major antioxidant of extracellular fluid. It constitutes the first line of antioxidant defense, can effectively remove O2-,H2O2, O2and other active oxygen when human plasma suffer from all kinds of oxidative damage. Yallampalli etal [3] found that VitC could inhibit the lipid peroxidation produced by arterial endothelial cells, inhibit apoptosis and increase antioxidant activity in cells. Some studies showed that VitC reduce the major coronary events, in patients with dilated cardiomyopathy treated with high dose VitC treatment, patients have a remission and a long stable duration and a decreased arrhythmia[4]. The peak value of myocardial enzyme was significantly reduced when patients with acute myocardial infarction thrombolytic therapy with urokinasefor VitC10g treatment, which indicated that VitC could reduce the ischemia-reperfusion injury of myocardium.108cases of hypertension were divided into treatment group and control group with54cases in each group by wang [5], The control group was treated with conventional drug, the treatment group take a large dose of VitC on the basis of control group. As a result, the treatment group was better than control group in the follow-up effect. The blood pressure of patients can be stabilized in120/80mmHg basically, all kinds of hypertension symptoms in patients were disappeared. Those results indicated that high-dose VitC is helpful to reduce hypertension.VitE also called tocopherol, is a small molecule strong antioxidant found in cells, which can remove O2", OH, ROO (lipid free radical) and other free radicals. VitE could increase the expression of nitric oxide synthase in endothelial, reduce the expression of inducible nitric oxide synthase [6]. In patients with acute myocardial infarction were treated with VitC (1200mg/d x30d)and VitE (1000mg/d x30d). We found that incidence of cardiovascular events in patients was significantly reduced [7]. Healthy subjects were treated with VitE100mg/d. We found that LDL cholesterol, mean platelet volume, platelet aggregation were reduced [8], this effect may be related to the reduced thromboembolic events.Beta carotene as the precursor of VitA, is a lipid soluble antioxidant and effective singleelectron bond oxidation inhibitor, also can inhibit lipid peroxidation. Beta carotene can prevent the LDL from oxidating to oxidized LDL, which can damage the vascular epithelial cells, accelerate the deposition of lipid plaque, vascular obstruction, causing obstructive diseases such as atherosclerosis, In patients with coronary heart disease were treated with50mg β-carotene capsule orally every other day, leading to the onset frequency reduced by50%. In addition, beta carotene can reduced mortality risk of44%of coronary artery disease in patients with chronic angina pectoris. Carotene concentrations in plasma increased was associated with the decrease of blood pressure [9]. Myocardial cells pretreated with beta carotene were exposed tohypoxia. We found that the antioxidant mechanism was existed in cells [10]. Therefore, a moderate supplement of Beta carotene reduced the occurrence of cardiovascular disease [11].Coenzyme Q10as a lipid soluble antioxidant, is cell mitochondria energy conversion agent, which not only protecs cells from apoptosis triggered by oxidative stress in mitochondrial of mammalian, but also has the effect of resisting lipid peroxidation. In addition, Coenzyme Q10can effectively reduce the content of MDA, and increase SOD and GSH-Px activity. The hypertensive patients were treated with coenzyme Q10in the use of antihypertensive drugs at the same time, which not only relieve hypertension patients lack of coenzyme Q10caused by antihypertensive drugs, enhanced hypotensive effect, also can prevent side effect of drugs. Patients with myocardial ischemia were treated with coenzyme Q10based on conventional treatments. We found angina attack frequency was significantly reduced [12]. In a word, coenzyme Q10can improve myocardial energy production in cardiovascular disease, with the characteristics of antioxidant activity and stability of biological membranes. In addition, coenzyme Q10is safety and has no toxic and side effects [13]. MLT protects the cell structure, prevents DNA damage and reduces the content of peroxide in vivo through scavenging free radical, antioxidative and inhibition of lipid peroxidation. MLT play a powerful antioxidant in the cell membrane, cytoplasm and nucleus. Both MLT and its metabolites are effective scavengers of free radicals. Brugger et al found that MLT concentration in patients with coronary heart disease was significantly lower than normal. If the patients were given MLT orally, incidence of coronary heart disease at night was decreased [14]. MLT is closely associated with primary hypertension [15]. Clinical also found that along with the circadian rhythm changes, there is a concentration and time relationship between MLT and blood pressure. The lowest MLT was at6-9o’clock in the morning, while blood pressure was highest at the same time, prone to hypertension crisis. Patients with primary hypertension treated with MLT for a month, the blood pressure was decreased in different degree [16].The structure of GSH contains an active thiol-SH, which was susceptible to oxidative dehydrogenation. This special structure makes it become the main free radical scavenger in vivo. GSH can resist on oxidative stress obviously, reduce oxidative stress injury, protect myocardial cells, relieve myocardial stunning, narrow the enlargement of ventricles, inhibit left ventricular remodeling and improve heart function in patients with acute myocardial infarction after reperfusion. Therefore, Adding GSH on the basic of conventional therapy is one of the effective methods in treating acute myocardial infarction. GSH can be combined with free radicals in the body by sulfhydryl, so that accelerate the excretion of free radical [17]. When Shuxuening and reduced glutathione combined in the treatment of chronic heart failure, we found that free radicals were removed effectively, hemodynamics was improved well, the coronary vessels were expended, blood viscosity was reduced, the micro thrombosis was reduced, so that speed up recovery of the chronic heart failure patients, improve the therapeutic effect, shorten treatment time, improve the life quality of the patients.SOD increased the dismutation rate of02-by104under physiological PH, which is a very important way to remove free radicals of the body. ROS participated in the whole process of cardiovascular disease [18]. SOD could scavenge lipid peroxide, reduce the tissue and cells oxidative damage result from ROS, reduce lipid peroxide formation, so as to protect endothelial cells against oxygen free radical damage and prevent the formation of atherosclerosis. Reperfusion injury was prevented in surgical operation by use of rh-SOD.A-lipoic acid (alpha liPoic acid, ALA) is an ideal biological antioxidant, which is a coenzyme in mitochondria, with characteristics of lipid and water-soluble [19]. ALA can remove FR and ROS, chelate metal ions, regenerate glutathione, vitamin C, vitamin E and other antioxidants. ALA is widely used in the treatment of diabetic peripheral neuropathy, AIDS [20] in clinical because of little clinical side effect. ALA also takes the protective effect on oxidative stress of endothelial cells induced by multiple pathways. Therefore, ALA is a very promising drug of treating cardiovascular disease, if we can clear the role of ALA and elucidate the molecular mechanism of action.Part I Alpha lipoic acid protects against oxidative stress injury of HUVEC ObjectiveTo establish anoxia/reoxygenation model of HUVEC, explore the most effect concentration of ALA for HUVEC, observe whether ALA have protective effect on hypoxia/reoxygenation induced oxidative damage of HUVEC. Method1. The establishment of HUVEC anoxia/reoxygenation model Cells were subjected to hypoxia for2,4,8,12h and reoxygenation for4,6,12,18,24h respectively. Cell vitality was determined by MTT test. 2. The choice of the ALA concentration. HUVEC was pre-or post-treatment with different concentrations of ALA (0.25,0.50,0.25mM/L). Cell vitality was determined by MTT test.3. ALA pre-or post-treatment has protective effect on hypoxia/reoxygenation induced oxidative damage of HUVEC. We detected cell culture supernatant of LDH, GSH content and TAOC, SOD and MDA level in cultured cells of each group according to the corresponding kit instructions.ResultCells were exposed to various time of hypoxia (2,4,8and12h), cell viability was assessed by MTT assay. Cell viability was significantly decreased in a time-dependent manner compared with the control group. Exposure to2h hypoxia reduced cell viability to95.0%of the control (P<0.05), while4h,8h, and12h of hypoxia reduced cell viability to87.7%,60.7%,45.3%relative to the control respectively (P<0.01). In addition, after4h hypoxia, the cells underwent different time period of reoxygenation (2,4,6,12,18and24h) respectively. Cell viability was significantly reduced to48.3%after12h of reoxygenation (P<0.01) compared with the0h reoxygenation group. However, cell viability was increased to52.7%and60.7%after18h and24h of reoxygenation respectively.Cells were pre-or post-treated with ALA (0,0.25,0.5,1.OmM, respectively) for4h hypoxia followed by12h reoxygenation treatment. We found that pre-or post-treatment with various concentrations of ALA could successfully attenuate the decrease of cell viability caused by H/R treatment.Exposure to4h hypoxia followed by12h reoxygenation significantly decreased the GSH, TAOC and SOD level, meanwhile, elevated LDH, MDA level in comparison to that of the control group. However, those effects could be reduced by pre-or post-treatment with ALA.Conclusion1. We selected treatment with4h hypoxia followed by12h of reoxygenation as a successful model for experiments. 2.1mM ALA pre-or post-treatment takes the most effective protection.3. ALA pre-or post-treatment can reduce HUVEC injury induced by hypoxia/reoxygenation. Part II Alpha lipoic acid protects against oxidative stress injury of HUVEC-cell apoptosis mechanismObjectiveTo explore whether apoptosis was happened in HUVEC cells under the oxidative stress and whether ALA pre-or post-treatment has an intervention effect on apoptosis.MethodCell morphology was observed under inverted phase contrast microscope. Cell apoptosis rate was detected by Annexin-VIPI flow cytometry instrument. Cell apoptosis was detected using Tunnel kit. Change of Mitochondria membrane potential was measured by JC-1staining under fluorescence microscope. The expression of apoptosis related proteins cleaved caspase-3was detected by western blot.ResultThe cell morphology was observed under inverted phase contrast microscope. The control group HUVEC showed vigorous growth, epithelioid adherent growth, the cells were polygonal or fusiform shape with the bottom. The gap between cells increases, cell shrinkage and became round, a few cells were suspended off in the hypoxia/reoxygenation group; While the gap between cells get smaller, the degree of cell shrinkage and became round was decreased, there are less cells to suspension off in the ALA pre-or post-treatment group compared with the model group. Cell apoptosis rate was detected by Annexin-VIPI flow cytometry instrument. Few cells were under the state of early apoptosis, late apoptosis and necrosis in the control group. Apoptotic cells were significantly increased in model group, and the most were the state of early apoptosis and late apoptosis. However, apoptotic cells were significantly decreased in the ALA pre-or post-treatment group.TUNNEL staining was used to evaluate apoptosis. There is almost no positive TUNNEL staining cells in the control group. There are more positive TUNNEL staining cells in the model group compared with the control group. However, in the ALA pre-or post-treatment group, the number of positive cells of TUNNEL staining was lower than that of model group.Change of Mitochondria membrane potential was measured by JC-1staining under fluorescence microscope. Control group cells have a higher membrane potential, the formation of JC-1is polymer (J-aggregates), and produced red fluorescence under fluorescence microscope. While the cells were exposed to hypoxia/reoxygenation treatment, most formation of the JC-1is monomer (monomer), and produced green fluorescence under fluorescence microscope. The fluorescence ratio of red to green is higher when cells were pre-or post-treated with1mmol/L ALA compared with the model group.The expression of apoptosis related protein cleaved caspase-3was detected by western blot. The expression level of cleaved caspase-3protein of model group was significantly increased compared with the control group. However, compared with model group, the expression of cleaved caspase-3protein of ALA pre-or post-treatment group decreased significantly. ConclusionALA pre-or post-treatment can significantly reduce the HUVEC apoptosis caused by hypoxia/reoxygenation. Part III Alpha lipoic acid protects against oxidative stress injury of HUVEC-cell autophagy mechanismObjectiveTo explore whether autophagy was happened in HUVEC cells under the oxidative stress and whether ALA pre-or post-treatment has an intervention effect on autophagy. MethodThe formation of autophagic vesicles was observed by transmission electron microscope and MDC staining. Expression of autophagy related protein LC3B and beclinl proteins were detected by western blot. ResultWe observed cells morphology under transmission electron microscope and found that cells were round approximately, intracellular organelles, nucleus, the morphology and distribution of chromosome were normal in the control group. Intracellular components form a large number of double and multilayer membrane autophagosome and the outline clearly visible in the model group. In addition, contents of some autophagic vacuoles were degradation like myelin sheath structure. Mitochondrial swell obviously. However, cytoplasmic components in different degradation stages were wrapped by single or double membrane of cell cytoplasm. The degree of organelle which was digested and formed the lamellar structure was significantly lower than those in the model group and the degree of mitochondria swelling reduced in the ALA pre-or post-treatment group.We found that fluorescent staining is weak in the control group by MDC staining.Green fluorescent was obviously increased in model group. We treated cells with ALA pre-or post-treatment, when cells were stained with MDC, we found that the expression of of intracellular fluorescence in these two groups was decreased significantly.Expression of autophagy related protein LC3B and beclinl protein was detected by western blot. The ratio of LC3-II/LC3-I increased significantly in the hypoxia/reoxygenation group, suggesting that part of the LC3-I to LC3-II conversion The ratio of LC3-II/LC3-I reduced significantly in the ALA pre-or post-treatment group. Level of beclinl is low in the control group. However, level of beclin1was significantly increased after hypoxia treatment. While cells were pre-or post-treated with ALA, Level of beclinl was reduced compared with the model group. ConclusionALA pre-or post-treatment can significantly reduce the HUVEC autophagy caused by hypoxia/reoxygenation. |
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