代谢性内毒素血症在肝胰岛素抵抗中对肝细胞线粒体功能的影响
发布时间：2018-09-16 20:19
【摘要】：目的:1、通过建立大鼠代谢性内毒素血症及肝胰岛素抵抗模型,探讨代谢性内毒素血症对肝线粒体结构与能量代谢的影响;2、通过体外实验,探讨内毒素(LPS)对大鼠正常肝细胞线粒体结构及能量代谢的直接影响。方法:在体实验:30只雄性SD大鼠随机分三组(每组10只):正常对照组(NC组)、高果糖组(HFD组,10%果糖水喂养)、LPS组(皮下注射LPS 300μg·kg-1·d-1)。每周检测并记录大鼠体重,8周末,禁食12 h以上,腹腔注射糖耐量实验结束后,经乙醚麻醉腹主动脉采血,4℃3500 r/min离心10 min,取血浆分装后置于-40℃保存。分离肝脏组织,部分置于4%多聚甲醛溶液中固定12~24 h,冲洗修整,经常规石蜡包埋切片,行苏木素伊红染色(HE染色);剩余样本置液氮速冻后转-80℃保存。1、腹腔注射糖耐量实验(IPGTT)每周检测大鼠体重变化,8周末,空腹腹腔注射50%葡萄糖溶液(2 g/kg)。尾静脉采血,采用快速血糖仪(罗氏活力型)分别检测注射前(0 min)与注射后(15min、30 min、120 min)血糖水平。2、血浆肝酶、LPS检测及肝胰岛素抵抗评估采用酶法测定血糖、血浆AST及ALT;鲎试剂法检测血浆LPS水平;ELISA法检测胰岛素变化,计算胰岛素抵抗指数(Homeostasis model assessment-insulin resistance,HOMA-IR),公式为:HOMA-IR=空腹血糖(FPG,mmol/L)×空腹胰岛素(FINS,EU/ml)/22.5。3、氧化损伤产物及能量代谢指标检测采用酶法检测并计算血浆中GSH-PX的水平;ELISA法检测血浆中氧化损伤产物(8-Ohd G、MDA、4-HNE)及能量代谢指标(ADP、ATP)的表达。4、肝脏组织病理学检测于4%多聚甲醛固定液中取出肝脏组织,冲洗修整后,梯度酒精脱水,二甲苯透明,常规石蜡包埋切片,厚度5μm,行HE染色,光镜下观察肝组织病理学变化。5、Western blot分析采用Western blot检测肝组织胰岛素信号转导关键蛋白(p-IRS1Tyr632、IRS1、p-PI3KTyr458、PI3K)及线粒体内膜受体蛋白(UCP2)表达。离体实验:采用改良胶原酶二步灌流法,体外分离培养大鼠肝细胞并随机分为四组:正常对照组(NC组,DMEM培养液培养)、高果糖组(HFD组,培养液+4.5 g/L果糖水)、LPS组(培养液+10 mg/L LPS)、果糖并LPS干预组(H+L组,培养液+4.5g/L果糖水+10 mg/L LPS)。20 h后,吸取并分装细胞上清液,胰酶(含EDTA)消化,2000 r/min离心3 min后弃上清,收取肝细胞于-40℃冷冻保存。1、氧化损伤产物及能量代谢指标检测采用ELISA法检测细胞上清液中氧化损伤产物(8-Ohd G、MDA、4-HNE)及能量代谢指标(ADP、ATP)的表达。2、Western blot分析采用Western blot检测肝细胞中胰岛素信号转导关键蛋白(p-IRS1Tyr632、IRS1、p-PI3KTyr458、PI3K)、线粒体内膜受体蛋白(UCP2)表达。结果:在体实验:1、体重变化及糖耐量结果实验期间,各组大鼠总体状态良好。与NC组相比,HFD组与LPS组大鼠体重在2~8周显著增高(P0.01,P0.05)。HFD组与LPS组各时间点血糖均显著高于NC组(P0.01,P0.05),表明HFD组与LPS组大鼠发生糖耐量异常。2、血浆肝酶、LPS检测及肝胰岛素抵抗评估结果HFD组与LPS组的肝酶、FINS、FPG、LPS水平及HOMA-IR显著高于NC组(P0.01),HFD组与LPS组无统计学差异(P0.05)。3、氧化损伤产物及能量代谢指标变化HFD组与LPS组8-OhdG、MDA、4-HNE及GSH-PX显著高于NC组(P0.01),HFD组与LPS组差异无统计学意义。能量代谢指标,HFD组与LPS组ADP、ATP与NC组相比显著降低(P0.01),HFD组与LPS组差异无统计学意义(P0.05)。4、肝组织病理学变化NC组肝脏呈暗红色,无油腻感,HE染色后光镜下可见肝索呈放射状排列。HFD组与LPS组肝脏有明显油腻感;镜下可见肝细胞包浆内出现脂滴空泡并伴有气球样变。5、Western blot结果与NC组相比,HFD组与LPS组肝组织IRS1、PI3K表达、p-IRS1Tyr632/IRS1和p-PI3KTyr458/PI3K比值则显著降低(P0.01),UCP2表达显著升高(P0.01),HFD组与LPS组相比无统计学意义(P0.05)。离体实验:1、氧化损伤产物及能量代谢指标变化HFD组、LPS组及H+L组8-Ohd G、MDA及4-HNE显著高于NC组(P0.01,P0.05),能量代谢指标与NC组比较,HFD组、LPS组及H+L组ADP、ATP显著降低(P0.05)。HFD组、LPS组及HFD+LPS组差异无统计学意义(P0.05)。2、Western blot结果与NC组相比,HFD组、LPS组及H+L组肝细胞IRS1、PI3K表达、p-IRS1Tyr632/IRS1和p-PI3KTyr458/PI3K比值则显著降低(P0.01,P0.05),UCP2表达显著升高(P0.01),HFD组、LPS组及H+L组相比无统计学意义(P0.05)。结论:1、高果糖饮食及皮下注射LPS可诱发大鼠胰岛素抵抗和代谢性内毒素血症,并伴有氧化应激和肝细胞线粒体功能障碍。2、LPS可诱导氧化中间产物生成增多,清除减少,致使肝脏处于氧化应激状态,进而破坏肝细胞线粒体结构,影响其能量代谢,加速胰岛素抵抗及代谢性疾病的发生发展。
[Abstract]:Objective: 1. To investigate the effects of metabolic endotoxemia on hepatic mitochondrial structure and energy metabolism by establishing rat models of metabolic endotoxemia and hepatic insulin resistance; 2. To investigate the direct effects of lipopolysaccharide (LPS) on mitochondrial structure and energy metabolism of rat normal hepatocytes in vitro. Rats were randomly divided into three groups (10 rats in each group): normal control group (NC group), high fructose group (HFD group, fed with 10% fructose water) and LPS group (subcutaneous injection of LPS 300 UG kg 1 D 1). Liver tissues were separated and fixed in 4% paraformaldehyde solution for 12-24 hours, washed and trimmed, paraffin embedded sections were stained with hematoxylin-eosin (HE) staining, and the remaining samples were frozen and stored at - 80 C. 1. Intraperitoneal glucose tolerance test (IPGTT) was performed weekly for 8 weeks. At the end of the study, 50% glucose solution (2 g / kg) was injected intraperitoneally on an empty stomach. Blood samples were collected from the tail vein. Blood glucose levels were measured before injection (0 min) and after injection (15 min, 30 min, 120 min) by rapid glucose meter (Roche's activity type). Blood glucose levels were measured by enzymatic method, plasma AST and ALT by LPS detection and liver insulin resistance assessment. The levels of insulin were measured by ELISA, and the Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated. The formula was: HOMA-IR = fasting blood glucose (FPG, mmol/L) * fasting insulin (FINS, EU/ml) / 22.5.3. The levels of GSH-PX in plasma were measured by enzyme method. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in plasma were detected by LISA. The liver tissues were removed from 4% paraformaldehyde fixed solution by histopathological examination. After washing and dressing, the liver tissues were dehydrated by graded alcohol, dimethylbenzene was transparent, paraffin embedded and sliced, and the thickness was 5 microns. The liver tissues were observed by HE staining and light microscopy. Pathological changes. 5. Western blot analysis was used to detect the expression of key insulin signal transduction proteins (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) and mitochondrial endometrial receptor protein (UCP2) in liver tissue. In vitro experiment: rat hepatocytes were isolated and cultured by modified two-step perfusion of collagenase and randomly divided into four groups: normal control group Group C (NC group, DMEM culture medium), high fructose group (HFD group, culture medium + 4.5 g/L fructose water), LPS group (culture medium + 10 mg/L LPS), fructose and LPS intervention group (H + L group, culture medium + 4.5 g/L fructose water + 10 mg/L LPS). 20 hours later, the supernatant of cells was absorbed and packed, digested with trypsin (containing EDTA), and centrifuged for 3 minutes after 2000 r/min, the supernatant was discarded and collected. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in cell supernatant were detected by ELISA. 2. The key proteins of insulin signal transduction (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) were detected by Western blot. Mitochondrial endometrial receptor protein (UCP2) expression. Results: In vivo experiment: 1. Body weight changes and glucose tolerance results during the experiment, the overall state of rats in each group was good. Compared with NC group, the weight of HFD group and LPS group increased significantly in 2-8 weeks (P 0.01, P 0.05). The levels of liver enzymes, FINS, FPG, LPS and HOMA-IR in HFD group and LPS group were significantly higher than those in NC group (P 0.01). There was no significant difference between HFD group and LPS group (P 0.05). The changes of oxidative damage products and energy metabolism indexes in HFD group and LPS group were 8-OhdG, MDA, 4-HNE and GSH-PX. The ADP and ATP of HFD group and LPS group were significantly lower than those of NC group (P Compared with NC group, the expression of IRS1 and PI3K, the ratio of p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in HFD group and LPS group were significantly decreased (P 0.01), and the expression of UCP2 was significantly increased (P 0.01) in HFD group and LPS group. In vitro experiment: 1. The changes of oxidative damage products and energy metabolism indexes in HFD group, LPS group and H + L group were significantly higher than those in NC group (P 0.01, P 0.05). Compared with NC group, ADP and ATP in HFD group, LPS group and H + L group were significantly lower (P 0.05). There was no significant difference between HFD group, LPS group and HFD + LPS group (P 0.05). (2) Compared with NC group, the expression of IRS1, PI3K, p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in hepatocytes of HFD group, LPS group and H+L group were significantly decreased (P 0.01, P 0.05), and the expression of UCP2 was significantly increased (P 0.01). There was no significant difference between HFD group, LPS group and H+L group (P 0.05). Insulin resistance and metabolic endotoxemia, accompanied by oxidative stress and hepatocyte mitochondrial dysfunction. 2, LPS can induce the production of oxidative intermediates increased, clearance decreased, resulting in oxidative stress in the liver, thereby destroying the structure of hepatocyte mitochondria, affecting its energy metabolism, accelerating insulin resistance and metabolic diseases. The development of life.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R58
本文编号：2244679
[Abstract]:Objective: 1. To investigate the effects of metabolic endotoxemia on hepatic mitochondrial structure and energy metabolism by establishing rat models of metabolic endotoxemia and hepatic insulin resistance; 2. To investigate the direct effects of lipopolysaccharide (LPS) on mitochondrial structure and energy metabolism of rat normal hepatocytes in vitro. Rats were randomly divided into three groups (10 rats in each group): normal control group (NC group), high fructose group (HFD group, fed with 10% fructose water) and LPS group (subcutaneous injection of LPS 300 UG kg 1 D 1). Liver tissues were separated and fixed in 4% paraformaldehyde solution for 12-24 hours, washed and trimmed, paraffin embedded sections were stained with hematoxylin-eosin (HE) staining, and the remaining samples were frozen and stored at - 80 C. 1. Intraperitoneal glucose tolerance test (IPGTT) was performed weekly for 8 weeks. At the end of the study, 50% glucose solution (2 g / kg) was injected intraperitoneally on an empty stomach. Blood samples were collected from the tail vein. Blood glucose levels were measured before injection (0 min) and after injection (15 min, 30 min, 120 min) by rapid glucose meter (Roche's activity type). Blood glucose levels were measured by enzymatic method, plasma AST and ALT by LPS detection and liver insulin resistance assessment. The levels of insulin were measured by ELISA, and the Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated. The formula was: HOMA-IR = fasting blood glucose (FPG, mmol/L) * fasting insulin (FINS, EU/ml) / 22.5.3. The levels of GSH-PX in plasma were measured by enzyme method. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in plasma were detected by LISA. The liver tissues were removed from 4% paraformaldehyde fixed solution by histopathological examination. After washing and dressing, the liver tissues were dehydrated by graded alcohol, dimethylbenzene was transparent, paraffin embedded and sliced, and the thickness was 5 microns. The liver tissues were observed by HE staining and light microscopy. Pathological changes. 5. Western blot analysis was used to detect the expression of key insulin signal transduction proteins (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) and mitochondrial endometrial receptor protein (UCP2) in liver tissue. In vitro experiment: rat hepatocytes were isolated and cultured by modified two-step perfusion of collagenase and randomly divided into four groups: normal control group Group C (NC group, DMEM culture medium), high fructose group (HFD group, culture medium + 4.5 g/L fructose water), LPS group (culture medium + 10 mg/L LPS), fructose and LPS intervention group (H + L group, culture medium + 4.5 g/L fructose water + 10 mg/L LPS). 20 hours later, the supernatant of cells was absorbed and packed, digested with trypsin (containing EDTA), and centrifuged for 3 minutes after 2000 r/min, the supernatant was discarded and collected. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in cell supernatant were detected by ELISA. 2. The key proteins of insulin signal transduction (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) were detected by Western blot. Mitochondrial endometrial receptor protein (UCP2) expression. Results: In vivo experiment: 1. Body weight changes and glucose tolerance results during the experiment, the overall state of rats in each group was good. Compared with NC group, the weight of HFD group and LPS group increased significantly in 2-8 weeks (P 0.01, P 0.05). The levels of liver enzymes, FINS, FPG, LPS and HOMA-IR in HFD group and LPS group were significantly higher than those in NC group (P 0.01). There was no significant difference between HFD group and LPS group (P 0.05). The changes of oxidative damage products and energy metabolism indexes in HFD group and LPS group were 8-OhdG, MDA, 4-HNE and GSH-PX. The ADP and ATP of HFD group and LPS group were significantly lower than those of NC group (P Compared with NC group, the expression of IRS1 and PI3K, the ratio of p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in HFD group and LPS group were significantly decreased (P 0.01), and the expression of UCP2 was significantly increased (P 0.01) in HFD group and LPS group. In vitro experiment: 1. The changes of oxidative damage products and energy metabolism indexes in HFD group, LPS group and H + L group were significantly higher than those in NC group (P 0.01, P 0.05). Compared with NC group, ADP and ATP in HFD group, LPS group and H + L group were significantly lower (P 0.05). There was no significant difference between HFD group, LPS group and HFD + LPS group (P 0.05). (2) Compared with NC group, the expression of IRS1, PI3K, p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in hepatocytes of HFD group, LPS group and H+L group were significantly decreased (P 0.01, P 0.05), and the expression of UCP2 was significantly increased (P 0.01). There was no significant difference between HFD group, LPS group and H+L group (P 0.05). Insulin resistance and metabolic endotoxemia, accompanied by oxidative stress and hepatocyte mitochondrial dysfunction. 2, LPS can induce the production of oxidative intermediates increased, clearance decreased, resulting in oxidative stress in the liver, thereby destroying the structure of hepatocyte mitochondria, affecting its energy metabolism, accelerating insulin resistance and metabolic diseases. The development of life.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R58
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