肌肉注射sRAGE基因对高脂餐大鼠脂质代谢、慢性炎症状态及重要器官的作用
发布时间：2018-08-26 07:19
【摘要】： 背景及目的:高脂餐饮食促使动脉粥样硬化、血脂异常、胰岛素抵抗等发病,现代生活习惯使之呈流行趋势,近年研究表明慢性炎症和胰岛素抵抗相辅相成。现代饮食以高脂、高热量为特征,高温烹制的食物富含晚期糖基化终末产物(AGE),AGE与与其受体(RAGE)结合,通过NADPH及NF-κB通路,激活巨噬细胞,可导致氧化应激,引起多种促炎因子产生,一次摄入虽可使循环中AGE升高,使机体短时间内产生氧化应激状态,炎症因子水平升高,但停止摄入后机体AGE水平逐渐下降,氧化应激逐渐消退,但如果长期大剂量摄入,则会超出机体的代偿能力,表现为慢性炎症状态。减少食物中的AGE、阻断AGE与RAGE结合可降低机体慢性炎症状态,使db/db大鼠胰岛素抵抗减轻,NOD小鼠和高脂餐C57/B6小鼠糖尿病发生减少,糖尿病慢性并发症(动脉粥样硬化、微血管并发症)减轻。新近研究发现体内存在一种缺失细胞跨膜段及细胞内段的RAGE(又称为sRAGE)具有与AGE结合的能力,限制了AGE与细胞膜上完整的RAGE结合的能力,阻断AGE所导致的细胞氧化应激,减轻机体慢性免疫反应状态。本研究的目的是通过肌肉注射sRAGE基因,观察sRAGE对高脂餐大鼠机体氧化应激状态、脂质代谢、胰岛素抵抗及主动脉内膜、肝脏、肾脏的改变的影响。探讨肌肉注射sRAGE基因防治高脂餐大鼠代谢紊乱及重要器官损伤的作用。 方法: 1.动物实验:取SD大鼠28只(体重150～160g,6周龄),随机分为3组:高脂餐治疗组、高脂餐对照组和普通餐对照组。高脂餐大鼠先以高脂餐喂养8周,第9周10只在双侧后腿肌内各注射pLNCX_2-sRAGE 300μg(高脂餐治疗组),10只注射pLNCX_2质粒300μg/侧(高脂餐对照组):分别于第12周、第15周重复注射一次,第17周处死。8只同期普通饲料喂养的大鼠(普通餐对照组)在第17周处死。骨骼肌注射pLNCX_2-sRAGE和pLNCX_2质粒方法:4%水合氯醛腹腔麻醉大鼠,暴露双侧后腿肌肉,各注射质粒300μg,以电极相距20mm给予方波电脉冲(电压200V/cm,波宽40CM,脉冲次数9,频率1HZ)刺激,以增加基因转移效率。 2.三组大鼠分别于每次注射后的1、2、3周断尾取血测定SOD、MDA值。第17周处死前测空腹血糖和体重,处死后立即取心脏血、主动脉、肝脏和肾脏,进行血清胰岛素、甘油三脂、总胆固醇、TNF-α、超敏CRP、SOD、MDA测定,肾脏和主动脉病理HE染色,应用免疫组织化学染色观察经不同措施处理的大鼠的肾脏TGF-β1、肝脏NF-κB的表达。 结果: 1.高脂餐对照组体重、血糖、血清胰岛素、甘油三脂、总胆固醇、超敏CRP、TNF-α较普通餐对照组明显升高,与高脂餐对照组相比高脂餐治疗组除体重和超敏CRP轻度下降无统计学差异、甘油三脂显著升高外,其余各指标下降有统计学差异。 2.高脂餐治疗组大鼠注射基因后SOD水平逐渐上升,到第1次注射后3周与高脂餐对照组呈现统计学差异(P0.05),高脂餐治疗组大鼠注射基因后血MDA水平开始下降,1次注射后2周与高脂餐对照组有统计学差异(P0.05),在1次注射后3周有所升高;重复注射SOD继续升高,至接近普通餐对照组不再升高,MDA再次下降。 3. sRAGE对重要器官作用情况:普通餐对照组和高脂餐治疗组均未见动脉粥样硬化,但高脂餐对照组动脉内膜不完整,内皮坏死脱落,局部内膜略增厚,有动脉粥样硬化早期损伤的表现;高脂餐对照组肾小球系膜细胞和细胞外基质增多,TGF-β1呈现高表达,而高脂餐治疗组以上变化则较轻微,普通餐对照组皮质部肾小体和肾小管结构完好,TGF-β1低水平表达,TGF-β1三组间差异显著(P0.01);高脂餐对照组肝脏NF-κB表达增加,普通餐对照组呈低水平表达,高脂餐治疗组介于两者之间,三组间差异显著(P0.01)。 结论: 1.高脂饮食使大鼠体内氧化应激水平升高,抗氧化能力减弱,骨骼肌注射sRAGE基因可以降低高脂餐大鼠的氧化应激水平,抗氧化能力增强。 2.高脂餐可引起胰岛素抵抗,胆固醇水平升高;出现动脉粥样硬化、肾小球硬化的早期表现,骨骼肌注射sRAGE可一定程度地减轻此种改变。
[Abstract]:BACKGROUND & OBJECTIVE: High-fat diet promotes atherosclerosis, dyslipidemia, insulin resistance and other diseases. Modern living habits make it popular. Recent studies have shown that chronic inflammation and insulin resistance complement each other. Modern diet is characterized by high fat and high calorie content. High-temperature cooked food is rich in advanced glycation end products (AGE), AGE. Combining with its receptor (RAGE) and activating macrophages through NADPH and NF-kappa B pathway can lead to oxidative stress and induce the production of many pro-inflammatory factors. One intake can elevate the circulating AGE, which results in oxidative stress in a short period of time. The level of inflammatory factors increases, but the level of AGE decreases gradually after stopping intake and oxidative stress drives. Reducing AGE in food and blocking the combination of AGE and RAGE can reduce the chronic inflammatory state of the body, reduce insulin resistance in dB / DB rats, decrease the incidence of diabetes in NOD mice and C57 / B6 mice with high fat diet, and reduce the chronic complications of diabetes mellitus. Recent studies have found that a kind of RAGE (also known as sRAGE) lacking cell transmembrane and intracellular segments in vivo has the ability to bind to AGE, which limits the ability of AGE to bind to the complete RAGE on the cell membrane, blocks the oxidative stress induced by AGE and alleviates the chronic immune response symptoms. The aim of this study was to observe the effects of sRAGE on oxidative stress, lipid metabolism, insulin resistance and changes of aortic intima, liver and kidney in rats fed high-fat diet by intramuscular injection of sRAGE gene.
Method:
1. Animal experiment: 28 SD rats (weighing 150-160g, 6 weeks old) were randomly divided into three groups: high-fat meal treatment group, high-fat meal control group and normal meal control group. Eight rats fed with normal diet were sacrificed at the seventeenth week. The skeletal muscles were injected with pLNCX_2-sRAGE and pLNCX_2 plasmids. The rats were anesthetized with 4% chloral hydrate intraperitoneally. The hind leg muscles were exposed. The plasmids were injected at 300 UG and the distance between the electrodes was 20 mm. The gene transfer efficiency was increased by stimulating with square wave electric pulse (voltage 200V/cm, wave width 40CM, pulse number 9, frequency 1HZ).
2. Blood samples were taken at the end of 1, 2, and 3 weeks after each injection to determine SOD and MDA. Fasting blood glucose and body weight were measured at the seventeenth week before execution. Cardiac blood, aorta, liver and kidney were taken immediately after execution. Serum insulin, triglyceride, total cholesterol, TNF-a, hypersensitive CRP, SOD, MDA were measured. Pathological HE staining of kidney and aorta was used. Histochemical staining was used to observe the expression of TGF- NF- 1 and liver NF- kappa B in rats treated with different measures.
Result:
1. The body weight, blood glucose, serum insulin, triglyceride, total cholesterol, hypersensitive CRP and TNF-alpha in the high-fat meal control group were significantly higher than those in the normal meal control group. Compared with the high-fat meal control group, there was no significant difference in weight and hypersensitive CRP, except triglyceride.
2. The level of SOD in the high-fat diet group increased gradually after gene injection, and there was significant difference between the high-fat diet group and the control group at 3 weeks after the first injection (P 0.05). Repeated injection of SOD continued to rise to nearly normal meal. The control group no longer increased, and MDA decreased again.
3. Effect of sRAGE on important organs: There was no atherosclerosis in the control group and the high-fat diet group, but in the high-fat diet control group, the intima was incomplete, endothelial necrosis and exfoliation, local intima was slightly thickened, showing early atherosclerotic injury; in the high-fat diet control group, mesangial cells and extracellular matrix increased, TGF-ECM increased. The expression of TGF-beta 1 was significantly higher in the control group than that in the control group (P There was a significant difference between the three groups (P0.01).
Conclusion:
1. High-fat diet can elevate the level of oxidative stress and weaken the antioxidant capacity of rats. Injecting sRAGE gene into skeletal muscle can reduce the level of oxidative stress and enhance the antioxidant capacity of rats with high-fat diet.
2. High-fat diet can induce insulin resistance and elevated cholesterol levels, and early manifestations of atherosclerosis and glomerulosclerosis can be alleviated by intramuscular injection of sRAGE.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
本文编号：2204134
[Abstract]:BACKGROUND & OBJECTIVE: High-fat diet promotes atherosclerosis, dyslipidemia, insulin resistance and other diseases. Modern living habits make it popular. Recent studies have shown that chronic inflammation and insulin resistance complement each other. Modern diet is characterized by high fat and high calorie content. High-temperature cooked food is rich in advanced glycation end products (AGE), AGE. Combining with its receptor (RAGE) and activating macrophages through NADPH and NF-kappa B pathway can lead to oxidative stress and induce the production of many pro-inflammatory factors. One intake can elevate the circulating AGE, which results in oxidative stress in a short period of time. The level of inflammatory factors increases, but the level of AGE decreases gradually after stopping intake and oxidative stress drives. Reducing AGE in food and blocking the combination of AGE and RAGE can reduce the chronic inflammatory state of the body, reduce insulin resistance in dB / DB rats, decrease the incidence of diabetes in NOD mice and C57 / B6 mice with high fat diet, and reduce the chronic complications of diabetes mellitus. Recent studies have found that a kind of RAGE (also known as sRAGE) lacking cell transmembrane and intracellular segments in vivo has the ability to bind to AGE, which limits the ability of AGE to bind to the complete RAGE on the cell membrane, blocks the oxidative stress induced by AGE and alleviates the chronic immune response symptoms. The aim of this study was to observe the effects of sRAGE on oxidative stress, lipid metabolism, insulin resistance and changes of aortic intima, liver and kidney in rats fed high-fat diet by intramuscular injection of sRAGE gene.
Method:
1. Animal experiment: 28 SD rats (weighing 150-160g, 6 weeks old) were randomly divided into three groups: high-fat meal treatment group, high-fat meal control group and normal meal control group. Eight rats fed with normal diet were sacrificed at the seventeenth week. The skeletal muscles were injected with pLNCX_2-sRAGE and pLNCX_2 plasmids. The rats were anesthetized with 4% chloral hydrate intraperitoneally. The hind leg muscles were exposed. The plasmids were injected at 300 UG and the distance between the electrodes was 20 mm. The gene transfer efficiency was increased by stimulating with square wave electric pulse (voltage 200V/cm, wave width 40CM, pulse number 9, frequency 1HZ).
2. Blood samples were taken at the end of 1, 2, and 3 weeks after each injection to determine SOD and MDA. Fasting blood glucose and body weight were measured at the seventeenth week before execution. Cardiac blood, aorta, liver and kidney were taken immediately after execution. Serum insulin, triglyceride, total cholesterol, TNF-a, hypersensitive CRP, SOD, MDA were measured. Pathological HE staining of kidney and aorta was used. Histochemical staining was used to observe the expression of TGF- NF- 1 and liver NF- kappa B in rats treated with different measures.
Result:
1. The body weight, blood glucose, serum insulin, triglyceride, total cholesterol, hypersensitive CRP and TNF-alpha in the high-fat meal control group were significantly higher than those in the normal meal control group. Compared with the high-fat meal control group, there was no significant difference in weight and hypersensitive CRP, except triglyceride.
2. The level of SOD in the high-fat diet group increased gradually after gene injection, and there was significant difference between the high-fat diet group and the control group at 3 weeks after the first injection (P 0.05). Repeated injection of SOD continued to rise to nearly normal meal. The control group no longer increased, and MDA decreased again.
3. Effect of sRAGE on important organs: There was no atherosclerosis in the control group and the high-fat diet group, but in the high-fat diet control group, the intima was incomplete, endothelial necrosis and exfoliation, local intima was slightly thickened, showing early atherosclerotic injury; in the high-fat diet control group, mesangial cells and extracellular matrix increased, TGF-ECM increased. The expression of TGF-beta 1 was significantly higher in the control group than that in the control group (P There was a significant difference between the three groups (P0.01).
Conclusion:
1. High-fat diet can elevate the level of oxidative stress and weaken the antioxidant capacity of rats. Injecting sRAGE gene into skeletal muscle can reduce the level of oxidative stress and enhance the antioxidant capacity of rats with high-fat diet.
2. High-fat diet can induce insulin resistance and elevated cholesterol levels, and early manifestations of atherosclerosis and glomerulosclerosis can be alleviated by intramuscular injection of sRAGE.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
【参考文献】
相关期刊论文 前3条
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2 蒋家华，，熊爱华，钟玲;糖尿病大鼠心肌MDA含量、SOD和Na￣+-K￣+-ATP酶活性的变化[J];中国病理生理杂志;1997年01期
3 徐成,王莉莉,曹颖林,李松;PPARs:脂代谢调节与胰岛素增敏治疗药物的作用靶标[J];中国药理学通报;2004年03期
本文编号：2204134
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