| BackgroundWith the improvement of people’s living standard and the evolution of disease patterns,chronic non-infectious diseases have become one of the primary threats to human health and life, among which, cardiovascular disease, with its high rate of morbidity and mortality, has ranked forefront among the death-related diseases. Atherosclerosis(AS) is the pathophysiological basis of multiple cardiovascular diseases.With respect to the etiology and pathogenesis of atherosclerosis, there are many different views. In recent years, a large number of epidemiological evidences have brought increasing attention to hyperhomocysteinemia(HHcy)--- an independent risk factor involved in the development and progress of atherosclerosis. The underlying mechanisms by which homocysteine triggers atherosclerosis at the level of biochemistry and molecular biology are still obscure, which makes the effective intervention and prevention of atherosclerosis and other cardiovascular diseases not possible. Therefore, the clarification of the precise mechanism in the carcinogenesis of atherosclerosis at the biochemical and molecular level will be of great theoretical and practice significance to find effective ways of preventing and treatment of the disease.Lutein, a kind of natural phytochemical, is structurally classified as thecarotenoid containing oxygen. Its molecular formula is C40H56O2,and molecular weight is 568.85. Compared with other hydroxyl group containing carotenoids, lutein structurally possesses a β-ionone ring and a ε-inonoe ring on both ends, which endows it with strong antioxidant capacity and multiple biological functions. A large number of Epidemiological studies and animal experiments have demonstrated that lutein can exerts an effective protective effect during the initiation and progression of atherosclerosis, but the exact mechanism is not clear. We focus our study on homocysteine and its relationship with AS as no articles have been published on this aspect yet.Objectives1. To unveil the relationships between elevated plasma levels of homocysteine and oxidative stress status in patients suffering from coronary disease.2. To explore the possible HHcy-mediated mechanism of atherosclerosis from a biochemical and molecular biology point of view by examining the oxidative stress status, in vivo levels of nitric oxide(NO) and endothelin-1(ET-1) and the expression of other related inflammatory molecules with or without the intervention of lutein, the known anti-oxidant, in experimental model rat with HHcy, whereby to seek the effective new ways to prevent or intervene the development of atherosclerosis.Methods1. To explore the interrelationship between plasma Hcy level and in vivo oxidative stress status in coronary heart disease patients, the plasma Hcy level, the activities of superoxide dismutase(SOD), glutathione peroxidase(GPx), catalase(CAT) and the MDA content were determined.2. The experimental model of HHcy was established in wistar rats. Rats were randomly divided into four groups: control, model group, folic acid group and lutein group. The control group rats(10 rats) were administered with 2ml 1% Carboxy Methyl Cellulose(CMC) solution by intragastric pathway once per day, following chow feed for eight weeks. The HHcy animal model(10 rats) was established by intragastric administration with with 3% methionine suspension(1.5g/Kg body weight) once per day, following chow feed for eight weeks. The folic acid intervebtion group rats(10 rats) were intragastric administered with 3% methionine suspension plus folic acid suspension(60mg/kg body weight) once per day, following chow feed for eight weeks. Lutein intervebtion group rats(10 rats) were intragastric administered with 3% methionine suspension plus lutein suspension(20mg/kg body weight) once per day, following normal chow feed for eight weeks.3. At the end of the experiments, rats were sacrificed under anesthesia by infusing 10% hydrated chloraldehyde intraperitonealy(0.36g/Kg). 10 ml blood was collected from heart immediately and blood samples were stored at-80 °C. Segments of descending aorta were collected for RT-PCR and Western blot analysis. Serum content of Hcy and other parameters like the activities of SOD, GPx, as well as the levels of MDA, T-AOC, NO and ET-1 were measured.4. Both the m RNA expression and the protein expression levels of SOD2、GPx1、NF-κBp65 and ICAM-1 in abdominal aorta were determined by RT-PCR and Western blot respectively.Results1. Our results demonstrated that the plasma Hcy levels in patients with coronary heart diseases were significantly higher than those of control subjects. These research findings furtherly testified that elevated plasma Hcy level is a dependent risk factor in the initiation and progression of coronary heart disease. And the MDA levels were significantly higher than those of control subjects and the activities of SOD and GPx were significantly lower than those of control subjects. All these results revealed that there existed in the imbalance of free radical metabolism and the augment status of oxidative stress. Our results also showed that there existed a significant positive correlation between Hcy level and MDA level in patients with coronary heart disease. So the oxidative stress augment status might be one possible cause of Hcy- mediated As pathogenesis in patients with coronary heart disease.2. The HHcy rat model was successfully established to explore the interventioneffect and the possible mechanism of Hcy-mediated in the pathogenesis of As by using lutein, a known strong natural antioxidative agent.3. The SOD and GPx activities in the HHcy model group significantly were decreased compared with the control group, and the MDA content was greatly increased, whereas the T-AOC activities were also significantly decreased in HHcy model rats. All the above findings indicated that HHcy rats were in a status of over oxidative stress augment. However, above mentioned parameter results can be reversed by lutein intervention. These results showed that lutein could antagonize HHcy-induced oxidative stree status.4. The plasma NO levels in model group were significantly lower than those of the control group, and the ET-1 levels significantly were elevated compared with the control group. These findings indicated that HHcy can lead to the endothelial dysfunction. But this effect can be reverted effectively by lutein intervention as our results showed elevated NO level and decreased ET-1 level demonstrating its ameliorating effects in HHcy- induced endothelial dysfunction.5. RT-PCR and Western blot results also showed decreased expression of SOD2 and GPx1 but increased expression of inflammatory molecules NF-κBp65 and adhesion molecule ICAM-1 in aorta epithelial tissues of HHcy model rats. With lutein intervention, the m RNA and protein expression of SOD2 and GPx1 significantly were increased and the expression of NF-κB P65 and ICAM-1 were greatly down regulated.Conclusions1. Plasma Hcy levels in patients with coronary heart disease were apparently higher than those of the control subjects, and there existed oxidative stress augment status. The correlative analysis indicated that plasma Hcy levels in patients with coronary heart disease were postively correlated with the oxidative stress augment status. All these results further supported the idea that elevated plasma Hcy level is an important risk factor in the carcinogenesis of atherosclerosis and possibly mediated by oxidative stress mechanisms.2. There also existed oxidative stress augment status in experimental HHCy ratmodels and these were mirrored by decreased activities of SOD, GPx and increased lipid peroxidation. Also the Increased plasma ET-1 level and the decreased plasma NO content indicated HHcy can induce vascular endothelial dysfunction. RT-PCR and Western blot analysis results also showed that decreased gene expression of two important enzymes including both SOD2 and GPx1. However, the increased gene expression of inflammatory-related molecules including both NF-κBp65 and adhesion molecule ICAM-1 existed in aorta epithelial tissues of HHcy model rats.3. The lutein, a kind of pyotochemicals, was first utilized to intervene in HHcy rat models, and the results indicated that although lutein can not significantly lower the elevayted plasma Hcy levels in experimental HHcy rats, it can obviously interrupt and change the Hcy-mediated biochemical behavior including up-regulating the gene expression of SOD2 and GPx1 so as to enhance the ability of antioxidative stress and down-regulate the gene expression of NF-κBp65 and ICAM-1. All these findings indicated the intervening protective effect of lutein on the carcinogenesis of early atherosclerosis may due to the direct role of lutein acting as the strong antioxidant, and not the indirect role of lutein acting to lower the elevated plasma Hcy levels.4. Taken together, our results demonstracted that HHcy is one of the independent risk factors in the pthogenesis of aterosclerosis. Oxidative stress and inflammation are two important molecular mechanisms of HHcy-meditated aterosclerosis. Especically, the HHcy-induced oxidative stress augment may be the initial mechanism of HHcy-meditated aterosclerosis, and further induces the expression of NF-κB and its downstream inflammatory factor including ICAM-1, thereby correlating with the carcinogenesis and progression of aterosclerosis. Lutein can significantly attenuate Hcy-induced oxidative stress augment, down-regulate the expression of inflammation related molecules, which offers new clues and a pathway in the molecular level to use lutein to effectively intervene in the carcinogenesis and progression of aterosclerosis. |
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