左卡尼汀在动脉粥样硬化及冠心病中的抗炎抗氧化作用

发布时间：2018-04-22 08:19

本文选题：动脉粥样硬化 + 炎症标志物　；参考：《山东大学》2016年博士论文

【摘要】：第一部分左卡尼汀对动脉粥样硬化大鼠抗炎抗氧化的实验研究目的：动脉粥样硬化AS是心血管疾病的发生及发展最主要的原因,寻找能降低动脉粥样硬化的调节物非常重要。本研究的目的在于评价左卡尼汀(左旋肉碱、乙酰-L-肉碱、ALCAR)的在Wistar大鼠中调节改善动脉粥样硬化的能力及其在动脉粥样硬化大鼠中抗炎、抗氧化的作用,同时对左卡尼汀发挥抗动脉粥样硬化作用的机制进行更深层次的研究。方法：32只Wistar大鼠随机分为正常饮食组(对照组)、正常饮食+ALCAR组(ALCAR组)、动脉粥样硬化组(AS组)、动脉粥样硬化+ALCAR组(AS+ALCAR组),动脉粥样硬化模型采用高脂饲料喂养+肌肉注射维生素D3+主动脉球囊损伤建立。ALCAR组及AS+ALCAR组在常规饮食或高脂饮食的基础上给予进行左卡尼汀200mg/kg/d口服。16周后,大鼠麻醉处死,取血液样本,主动脉和心脏组织。检测血清脂质分布、氧化应激物质水平、炎症物质水平和脂联素水平；HE染色观察主动脉组织结构；检测主动脉组织和心脏组织氧化应激物质水平,检测主动脉组织血管紧张素Ⅱ含量；Real-timeQPCR法检测主动脉组织及心脏组织CRP、IL-1β、TNF-α和iNOS的mRNA表达；Western blot法检测主动脉组织及心脏组织CRP、IL-1β、TNF-α和iNOS蛋白表达。数据统计使用SPSS 17.0软件包进行,多组样本数据间比较,采用one-way ANOVA分析,多组间两两组间比较,如果方差齐性者,采用LSD检验,如果方差不齐者,采用Dunnett's T3检验,如果为非正态分布者,采用Mann-Whitney检验,P0.05表示具有显著性统计学差异。结果：1.大鼠血清血脂分布情况：与对照组相比,AS组大鼠血清TC、TG、LDL-C和VLDL-C含量显著上升,HDL-C含量显著下降(P0.01)。与AS组相比,AS+ALCAR组大鼠血清TC、TG、LDL-C和VLDL-C含量显著下降,HDL-C含量显著上升(P0.01)。正常饮食给予ALCAR后,与对照组相比,大鼠血清TC、 TG、LDL-C和VLDL-C含量有一定下降趋势,但无显著性统计学差异(P0.05),HDL-C含量显著升高(P0.05)。2.大鼠血清氧化应激物质水平：与对照组比较,AS组大鼠血清MDA浓度显著升高,SOD和GSH-Px活力显著降低(P0.01)；AS+ALCAR组大鼠与AS组大鼠相比,ALCAR可显著降低AS大鼠血清MDA浓度,提高SOD和GSH-Px活力(P0.05,P0.01)。3.大鼠血清炎症物质水平：与对照组比较,AS组大鼠血清TNF-α、IL-1β和CRP含量显著升高(P0.01)。与AS组比较,AS+ALCAR组大鼠血TNF-α、 IL-1β和CRP含量均显著降低(P0.01)。与对照组相比,AS组大鼠血清APN浓度显著降低(P0.05),AS+ALCAR组大鼠血清ADPN浓度较AS组无显著性变化(P0.05)。4.大鼠主动脉组织学检查：HE染色结果显示,对照组、ALCAR组大鼠的血管管腔较大,管壁较薄,血管内膜结构完整且平滑,中膜平滑肌细胞排列规整,平行于内弹力膜。AS组大鼠管腔较窄,管壁明显增厚,内皮细胞脱落,平滑肌细胞大多排列紊乱,粥样斑块形成,斑块内可见大量泡沫细胞和脂质沉积。AS+ALCAR组较AS组明显管腔面积大,可见斑块的严重程度远远低于AS组。5.大鼠主动脉组织氧化应激物质水平：与对照组比较,AS组大鼠主动脉组织MDA浓度显著升高,SOD和GSH-Px活力显著降低(P0.01)。与AS组大鼠相比,ALCAR可显著降低AS大鼠主动脉组织MDA浓度,提高SOD和GSH-Px活力(P0.01)。6.大鼠主动脉组织AngⅡ含量：与对照组组相比,AS大鼠主动脉AngⅡ含量显著上升(P0.01),ALCAR组大鼠主动脉AngⅡ含量无明显变化。与AS组相比,ALCAR可显著降低AS大鼠主动脉AngⅡ含量(P0.05)。7.主动脉组织炎症物质Real-time QPCR检测：AS可导致大鼠主动脉组织TNF-α、IL-1β和iNOS的mRNA相对表达量显著增加(P0.01)。ALCAR可显著降低由AS大鼠的主动脉组织CRP、TNF-α、IL-1β和iNOS mRNA过度表达(P0.01)。对照组和ALCAR组主动脉组织CRP、TNF-α、IL-1β和iNOS mRNA表达无明显差异(P0.05)。8.主动脉组织炎症物质Western blot检测：AS可显著诱导大鼠主动脉组织CRP、TNF-α、IL-1β和iNOS蛋白高表达(P0.01)。ALCAR对AS大鼠主动脉组织CRP、TNF-α、IL-1β和iNOS蛋白表达均有显著的下调作用(P0.01)。对照组和ALCAR组主动脉组织CRP、TNF-α、IL-1β和iNOS蛋白表达无明显差异(P0.05)。9.心脏组织氧化应激物质：与对照组比较,AS组大鼠心脏组织MDA浓度显著升高,SOD和GSH-Px活力显著降低(P0.01)；与AS组大鼠相比,ALCAR可显著降低AS大鼠心脏组织MDA浓度,提高SOD和GSH-Px活力(P0.05或P0.01)。10.心脏组织炎症物质Real-time QPCR检测：AS可导致大鼠心脏组织CRP、 TNF-α、IL-1β和iNOS的nRNA相对表达量显著增加。ALCAR可显著降低AS大鼠的心脏组织CRP、TNF-α、IL-1β和iNOS mRNA过度表达。ALCAR组较对照组CRP、TNF-α、IL-1β和iNOS mRNA表达低,有统计学意义(P0.01或P0.05)。11.心脏组织炎症物质Western blot检测：AS可显著诱导大鼠心脏组织CRP、TNF-α、IL-1β和iNOS蛋白高表达(P0.01)。ALCAR对正常和AS大鼠心脏组织CRP、TNF-α、IL-1β和iNOS蛋白表达均有显著的下调作用(P0.05或P0.01)。结论：1.在AS大鼠中,ALCAR可以通过调节血脂、抑制炎症因子基因及蛋白表达、抗氧化应激、调节动脉肾素血管紧张素系统来对抗动脉粥样硬化。2. ALCAR可以降低AS大鼠心脏组织的炎症因子的基因及蛋白的表达,增加其抗氧化能力,可能与其抗冠状动脉粥样硬化与心肌保护有关,后续需开展更深入的机制研究。第二部分左卡尼汀对冠心病不稳定性心绞痛的临床疗效及其抗氧化抗炎的作用目的：观察左卡尼汀对冠心病不稳定性心绞痛患者的临床疗效及其抗氧化、抗炎的影响,进一步探讨左卡尼汀在治疗冠心病中的机制。方法：以潍坊市人民医院作为样本来源,样本选取时间段为2012年8月～2013年12月,选取该院心血管内科在此时间段内因胸痛或胸闷入院,冠状动脉造影结果显示至少有一支主要冠状动脉的管腔狭窄≥50%,并符合2011年ACC/AHA制定的《2007年不稳定性心绞痛、非段抬高心肌梗死诊疗指南的更新》中不稳定性心绞痛诊断标准,确诊为不稳定性心绞痛,并符合排除标准的患者115例,随机分为两组,对照组(n=55)给予常规冠心病药物治疗,治疗组(n=60)在常规治疗基础上,给予左卡尼汀治疗(2G/d)静脉滴注,治疗7天,改为口服每日2g到56天。观察两组治疗前后的临床症状的改善情况、血脂分布情况、一般的血生化指标、监测药物的副反应,并于治疗前后测定GSH-Px、MDA、hs-CRP、IL-6、 TNF-a水平。以此来评价左卡尼汀的对冠心病不稳定性心绞痛的临床疗效及其对冠心病患者抗炎、抗氧化的作用。本研究采用统计软件SPSS 17.0对数据进行分析,如为计量资料,采用均数±标准差(x±s)的形式来表示,对照组、治疗组组内治疗前后对比应用配对t检验进行数据分析,治疗后对照组与治疗组比较使用独立样本t检验进行数据分析,如为计数资料,进行x2检验。P0.05表示具有显著性统计学差异。结果：1.随访情况,临床疗效比较：对照组有2例,治疗组有3例患者因失去随访,退出试验,随访过程中没有药物副反应的发生。治疗后治疗组的总有效率为91.22%和对照组84.91%无统计学意义(x 2=0.53,P=0.41),但显效率治疗组47.37%明显高于对照组24.53%(x 2=5.23,P=0.02),差异有统计学意义(P0.05)。2.两组血脂情况的比较：两组治疗前TC, TG, LDL-C, HDL-C水平无差异(P0.05),治疗后对照组TG, LDL-C水平较治疗前明显降低(P0.01),HDL-C水平无明显改变(P0.05),与治疗前相比治疗组的TG和LDL-C水平有明显的下降趋势(P0.01),而HDL-C水平有上升趋势(P0.05),治疗后治疗组TG水平较对照组降低更明显(1.65±0.21 mmol/L vs 1.79±0.10 mmol/L P0.01),HDL-C水平较对照组明显升高(1.02±0.26 mmol／L vs 0.89±0.41 mmol/L P=0.01)。TC,LDL-C和对照组无明显差异(4.65±1.31mmol/L vs 5.05 ±1.10mmol/L P=0.08;2.55±1.06mmol/L vs 2.85±0.90 mmol/L P=0.11)。3.两组抗氧化能力的比较：两组治疗前GSH-Px.MDA无差异(P0.05),两组治疗前后GSH-Px,MDA的比较,两组治疗前后,治疗后较治疗前GSH-Px均明显升高,MDA明显下降(P0.01),经过治疗后治疗组较对照组GSH-Px升高,MDA下降更明显(149.80±28.40 U/mL vs 125.45±23.32 U/mL P0.01; 5.99±0.52 mmol/L vs 6.25±0.60 mmol/L P=0.02)。4.两组炎症因子的比较：两组治疗前hs-CRP、IL-6.TNF-α无明显差异(P0.05),两组治疗前后hs-CRP,IL-6,TNF-α明显下降(P0.01),治疗组较对照组治疗后下降更为明显(1.65±0.62mmol/L vs 2.25±0.70mmol/L P0.01; 90.93±18.13 ng/L vs 115.82±17.50 ng/L P0.01；11.85±2.85 pmol/L vs 13.25± 3.17 pmol/L P=0.02),有统计学意义。结论：左卡尼汀在改善冠心病不稳定性心绞痛症状方面有确切疗效,在常规药物治疗的基础上,进一步可以缓解临床症状,并能改善血脂分布,提高抗氧化酶的活性,提高抗氧化的能力,降低脂质过氧化反应,降低炎症反应,治疗动脉粥样硬化,从而治疗冠心病。
[Abstract]:The aim of this study is to evaluate levocarnitine (L-carnitine, acetyl -L- carnitine, ALCA, ALCA), and the aim of the experimental study of anti inflammatory and antioxidant activities of Levocarnitine in atherosclerotic rats. R) to regulate the ability to improve atherosclerosis in Wistar rats and its anti-inflammatory and anti oxidative effect in atherosclerotic rats. At the same time, the mechanism of anti atherosclerotic effect of levocarnitine was further studied. Methods: 32 Wistar rats were randomly divided into normal diet group (control group) and normal diet +ALCAR Group (group ALCAR), atherosclerotic group (group AS), atherosclerotic +ALCAR group (group AS+ALCAR), atherosclerotic models were fed with high fat feed and intramuscular injection of vitamin D3+ aortic balloon injury to establish.ALCAR group and AS+ALCAR group on the basis of conventional diet or high fat diet on the basis of levocarnitine 200mg/kg/d oral.16 weeks after oral administration. Rats were killed, blood samples, aorta and cardiac tissue were taken. Serum lipid distribution, oxidative stress level, inflammatory substance level and adiponectin level were detected; HE staining was used to observe the structure of aorta; the levels of oxidative stress in aortic and cardiac tissues were detected, and the content of angiotensin II in aorta tissue was detected; Rea The l-timeQPCR method was used to detect the expression of CRP, IL-1 beta, TNF- alpha and iNOS in the aortic tissue and cardiac tissue, and the Western blot method was used to detect the expression of CRP, IL-1 beta, TNF- alpha and iNOS protein in the aorta and heart tissues. The data statistics were carried out by the 17 software package. The ratio of multiple groups of sample data was compared with that of the 22 groups. If the homogeneity of variance, if the LSD test, if the variance is not homogeneous, Dunnett's T3 test, if the non normal distribution, Mann-Whitney test, P0.05 showed significant statistical difference. Results: 1. rats serum lipid distribution: compared with the control group, the serum TC, TG, LDL-C and VLDL-C content in the AS group was significantly higher than that of the control group. The content of HDL-C decreased significantly (P0.01). Compared with group AS, the content of TC, TG, LDL-C and VLDL-C in the AS+ALCAR group decreased significantly, and the HDL-C content increased significantly (P0.01). The level of oxidative stress substance in serum of (P0.05).2. rats was significantly increased: compared with the control group, the concentration of MDA in the serum of AS rats increased significantly, and the activity of SOD and GSH-Px decreased significantly (P0.01). Compared with the AS rats, AS+ALCAR group rats significantly reduced the serum MDA concentration in AS rats and increased the serum inflammation in the rats. Compared with the control group, the content of serum TNF- a, IL-1 beta and CRP in the AS group increased significantly (P0.01). Compared with the AS group, the content of TNF- a, IL-1 beta and CRP decreased significantly in group AS+ALCAR (P0.01). Compared with the control group, the concentration of serum albumin in the rats of the group of AS groups was significantly lower than that in the control group. Change (P0.05).4. rat aorta histological examination: HE staining results showed that in the control group, the vascular lumen of the group ALCAR rats was larger, the wall of the tube was thinner, the intima structure of the vessel was complete and smooth, the smooth smooth muscle cells of the middle membrane were arranged, the lumen in the.AS group of the internal elastic membrane was narrower, the wall of the tube thickened obviously, the endothelial cells fell off, the smooth muscle cells were large. There was a lot of disorder and atheromatous plaque formation. There was a large number of foam cells and lipid deposition in.AS+ALCAR group than that of AS group. The severity of plaque was much lower than that of AS group.5. rats. Compared with the control group, the concentration of MDA in the aorta tissue of the AS group was significantly higher, SOD and GSH-Px. Compared with the AS group, ALCAR significantly reduced the MDA concentration in the aorta of the AS rats and increased the content of Ang II in the aorta of SOD and GSH-Px (P0.01).6. rats: compared with the control group, the content of the aorta Ang II in the AS rats increased significantly. Compared with the group, ALCAR could significantly reduce the Ang II content of aorta (P0.05) in the aorta of AS rats and the Real-time QPCR detection of the inflammatory substances in the aorta of.7.: AS can lead to a significant increase in the relative expression of TNF- alpha, IL-1 beta and iNOS mRNA. Expression (P0.01). The expression of CRP, TNF- alpha, IL-1 beta and iNOS mRNA in the aorta of the control group and the ALCAR group had no significant difference (P0.05) Western blot detection in the.8. aorta tissue: AS could significantly induce the aorta tissue of the rat aorta. The expression of protein had a significant downregulation effect (P0.01). There was no significant difference in the expression of CRP, TNF- a, IL-1 beta and iNOS protein in the control group and the ALCAR group (P0.05).9. cardiac tissue oxidative stress substance: compared with the control group, the concentration of MDA in the cardiac tissue of the group AS rats was significantly increased and the SOD and GSH-Px activity decreased significantly. ALCAR significantly reduced the concentration of MDA in the cardiac tissue of AS rats and increased the detection of Real-time QPCR in SOD and GSH-Px activity (P0.05 or P0.01).10. cardiac tissue inflammatory substances: AS could lead to rat cardiac tissue CRP. S mRNA overexpressed CRP, TNF- alpha, IL-1 beta and iNOS mRNA in.ALCAR group, and had statistical significance (P0.01 or P0.05).11. cardiac tissue inflammatory substances. The expression of iNOS protein has a significant downregulation effect (P0.05 or P0.01). Conclusion: 1. in AS rats, ALCAR can modulate blood lipid, inhibit gene and protein expression of inflammatory factors, antioxidant stress, and regulate arterial renin angiotensin system to counter atherosclerotic.2. ALCAR can reduce the basis of inflammatory factors in cardiac tissue of AS rats The expression of protein and protein, increasing its antioxidant capacity, may be related to its anti coronary atherosclerosis and myocardial protection, and further mechanism study should be carried out. Second the clinical efficacy and antioxidation and anti-inflammatory effects of levocarnitine on unstable angina pectoris of coronary heart disease: To observe the instability of CHD in coronary heart disease The clinical effect, antioxidation and anti-inflammatory effects of the patients with angina pectoris, and further explore the mechanism of Levocarnitine in the treatment of coronary heart disease. Methods: Taking the Weifang People's Hospital as a sample source, the selected time period was from August 2012 to December 2013, and the hospital was selected to be hospitalized for chest pain or chest tightness in this period of cardiovascular medicine. The results of arteriography showed that at least one of the main coronary artery stenosis was more than 50%, and was consistent with the <2007 years of unstable angina pectoris made in 2011 ACC/AHA, the updating of the non segment elevation myocardial infarction diagnosis guide, the diagnosis of unstable angina pectoris, the diagnosis of unstable angina pectoris, and the compliance with the exclusion criteria of 115 patients. The control group was divided into two groups, the control group (n=55) was given conventional coronary drug therapy, and the treatment group (n=60) was given levocarnitine treatment (2G/d) intravenous drip on the basis of routine treatment, and the treatment was treated for 7 days and changed into oral 2G to 56 days. The changes of clinical symptoms, blood lipid distribution, general blood biochemical indexes, and monitoring drugs were observed before and after the treatment of two groups. The effects of GSH-Px, MDA, hs-CRP, IL-6 and TNF-a were measured before and after treatment to evaluate the clinical efficacy of levocarnitine on coronary heart disease unstable angina and its anti-inflammatory and antioxidant effects on patients with coronary heart disease. This study was analyzed by the statistical software SPSS 17 logarithms, such as measurement data, using a mean number of standards. In the form of poor (x + s), the control group was compared with the paired t test before and after treatment in the treatment group. After treatment, the control group was compared with the treatment group with independent sample t test for data analysis. For example, the x2 test.P0.05 showed significant statistical difference for the count data. Results: 1. follow up and clinical treatment. Comparison: there were 2 cases in the control group. There were 3 cases in the treatment group because of the loss of follow-up. There was no drug side reaction in the follow-up process. The total effective rate of the treatment group was 91.22% and the control group 84.91% had no statistical significance (x 2=0.53, P=0.41), but the significant efficiency treatment group was 47.37% significantly higher than the control group 24.53% (x 2=5.23, P=0.02), and the difference between the treatment group and the control group was 47.37%. The comparison of blood lipid in different groups of statistical significance (P0.05).2. two groups: there was no difference in the level of TC, TG, LDL-C and HDL-C before treatment (P0.05). The level of LDL-C in the control group was significantly lower than before the treatment (P0.01), and the HDL-C level was not significantly changed (P0.05), and the level of the treatment group was significantly lower than before treatment. The level of HDL-C had a rising trend (P0.05). The level of TG in the treatment group was more obvious than that of the control group (1.65 + 0.21 mmol/L vs 1.79 + 0.10 mmol/L P0.01), and the level of HDL-C was significantly higher than that of the control group (1.02 + 0.26 mmol / L vs 0.89 + 0.41 mmol/L). Comparison of antioxidant capacity in group 2.55 + 1.06mmol/L vs 2.85 + 0.90 mmol/L P=0.11).3. two: there was no difference between the two groups before treatment (P0.05), the comparison of GSH-Px, MDA in the two groups before and after treatment. The two groups before and after treatment were significantly higher than those before the treatment and MDA obviously descended (P0.01), and the treatment group was higher than the control group after treatment. The decrease was more obvious (149.80 + 28.40 U/mL vs 125.45 + 23.32 U/mL P0.01, 5.99 + 0.52 mmol/L vs 6.25 + 0.60 mmol/L P=0.02) in.4. two group, and there was no significant difference between the two groups before treatment and IL-6.TNF- alpha (P0.05). The treatment group decreased more after treatment than in the control group. It was significant (1.65 + 0.62mmol/L vs 2.25 + 0.70mmol/L P0.01; 90.93 + 18.13 ng/L vs 115.82 + 17.50 ng/L P0.01; 11.85 + 2.85 pmol/L vs 13.25 + 3.17 pmol/L P=0.02), with statistical significance. Conclusion: levocarnitine has a definite effect on improving the symptoms of coronary heart disease unstable angina, and further on the basis of conventional medicine treatment It can relieve the clinical symptoms, improve the distribution of blood lipid, improve the activity of antioxidant enzymes, improve the ability of antioxidation, reduce the reaction of lipid peroxidation, reduce the inflammatory reaction, and treat atherosclerosis, thus treating coronary heart disease.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5;R541.4

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