辛伐他汀激活棕色脂肪活性改善奥氮平引起的大鼠体重与代谢异常的初步研究

发布时间：2018-06-24 03:34

本文选题：辛伐他汀 + 奥氮平　；参考：《西南大学》2017年硕士论文

【摘要】：背景:精神分裂症是一类多发于青少年的重性精神病。青少年一旦患有此类疾病,须长期或终生服用抗精神病药物。而奥氮平作为常用的二代抗精神病药物之一,是现下被广泛应用于治疗精神分裂症的药物。与第一代抗精神病药物相比,它对脑中的5-HT与DA受体均有影响,对精神分裂症及各种性状的精神病都有一定的治疗效果。但值得关注的是,长期服用奥氮平会导致严重的与代谢相关的副作用疾病的发生例如肥胖,高血脂,胰岛素抵抗与高血糖等。这些副作用的发生会严重影响患者的生活质量,也可能使患者特别是青少年患者对奥氮平的治疗效果失去信心。但奥氮平引起的代谢方面的副作用机制尚未完全弄清。研究发现奥氮平可以使体内重要的产热器官——棕色脂肪的活性降低,产热水平下降,使棕色脂肪的形态趋近于白色脂肪。棕色脂肪(Brown adipose tissue,BAT)是体内一种极其重要的能量代谢的器官,可以通过非颤栗而产生热量。哺乳动物体内均有棕色脂肪,且婴幼儿时期的棕色脂肪远多于成年时期。因此,幼年机体的棕色脂肪也是常用的实验对象之一。提高棕色脂肪的产热活性,可以促进体内能量代谢,使体重下降,并改善血脂血糖异常。其细胞中线粒体内膜上的解偶联蛋白-1(Uncoupling protein-1,UCP1)是棕色脂肪特异性产热蛋白,且在棕色脂肪中高表达,并且UCP1蛋白可将化学能转化为热能释放。除了UCP1,棕色脂肪细胞中的过氧化物酶体增殖体激活受体γ共激活子α(Peroxisome-proliferator-activated receptor-γco-activator-1α,PGC-1α),过氧化物酶体增殖体激活受体γ(Peroxisome-proliferator-activated receptorγ,PPARγ)以及含PR结构域的蛋白16(PR domain-containing 16,PRDM16)等基因的表达水平也与产热活性相关。而下丘脑的阿片促黑色素原(Proopiomelanocortin,POMC)基因与脑干的酪氨酸羟化酶(Tyrosine hydroxylase,TH)基因的表达水平的变化也会影响棕色脂肪的产热活性的变化。以辛伐他汀(Simvastatin)为代表的他汀类药物,作为一线降脂药物,增加血清胆固醇的消除,改善血脂异常症状,控制心血管疾病与高血脂患者体重。虽然辛伐他汀在糖脂代谢方面的研究已日渐清晰,但在能量代谢方面特别是与棕色脂肪的作用方面却鲜有报道。因此,辛伐他汀是否可以提高棕色脂肪的活性用于改善代谢异常是值得研究的。目的:辛伐他汀可以控制高脂摄食肥胖模型的体重增长,降低血脂血糖水平并改善胰岛素的敏感性等。但辛伐他汀是否能激活棕色脂肪产热活性来干预奥氮平引起的体重增长与代谢失衡尚未有报道。本研究选择断奶幼鼠模拟幼年机体的代谢功能,通过检测辛伐他汀对奥氮平给药后大鼠的体重、摄食、血脂血糖、自发活动与棕色脂肪活性等方面变化的影响来研究辛伐他汀对于奥氮平引起的体重增长及代谢异常的干预功能。方法:1.选择36只断奶雌性幼鼠(体重:45-55 g),并将它们随机分配为两个组(n=18),并分别给予3.0 mg/kg/day奥氮平(t.i.d.)与空白糖丸连续14天,期间每两天检测大鼠体重、摄食、肛温变化。药物处理14天后,检测血浆TG、TC与空腹血糖值。2.将上述大鼠分为四组:空白组随机平均分为空白对照组与辛伐他汀组(10.0 mg/kg/day),奥氮平组随机平均分为奥氮平组(3.0 mg/kg/day,t.i.d.)与联合给药组(奥氮平:3.0 mg/kg/day,t.i.d.,辛伐他汀:10.0 mg/kg/day),继续给药35天,每两天检测大鼠体重、摄食、肛温变化;3.在联合给药第30天时,进行大鼠旷场实验。将大鼠放入黑色的开口盒中(50×50×50 cm3),普通摄影机跟踪拍摄大鼠活动,每只大鼠拍摄25分钟,并用Noldus Ethovision分析拍摄的大鼠活动录像分析大鼠的平均速率与总距离来反映大鼠的自发活动的变化;4.辛伐他汀与奥氮平联合给药中期(第20天)与动物实验结束时,测血浆TG、TC与空腹血糖值;在动物实验结束后检测棕色脂肪与白色脂肪的重量;将棕色脂肪进行冷冻切片并进行油红染色;5.采用RT-qPCR及Western blotting检测棕色脂肪产热相关基因与下丘脑和脑干能量代谢相关基因的mRNA与蛋白表达水平。结果:1.奥氮平/辛伐他汀对大鼠体重与摄食的影响奥氮平单独给药两周后,奥氮平给药大鼠的体重明显高于空白对照组(99.5±5.7 g vs.79.5±4.5 g,p0.05);与空白对照组相比,奥氮平增加大鼠的摄食量(40.2±3.6 g vs.33.5g±2.5 g,p0.05);联合给药5周后,辛伐他汀与奥氮平联合用药的大鼠体重与单独喂食奥氮平组相比(205.0±11.1 g vs.233.5±10.6 g,p0.05)明显下降;与空白对照组相比,辛伐他汀不影响大鼠摄食量(p0.05)。2.辛伐他汀降低奥氮平给药大鼠的血脂血糖值根据前两周奥氮平单独给药后大鼠空腹血浆甘油三酯、胆固醇与血糖值的检测可知,奥氮平组的甘油三酯(1.02±0.06 mmol/L vs.0.73±0.02 mmol/L,p0.01)、总胆固醇值(2.40±0.04 mmol/L vs.2.28±0.03 mmol/L,p0.05)与血糖值(7.74±0.13mmol/L vs.7.39±0.11 mmol/L,p0.05)明显高于空白对照组。联合给药中期(第20天)检测表明,联合给药的血脂血糖值与奥氮平单独给药组相比均有明显下降(p0.05)。联合给药35天后,奥氮平与辛伐他汀联合给药组的甘油三酯(0.84±0.03mmol/L vs.1.27±0.04 mmol/L,p0.01)、总胆固醇值(2.43±0.05 mmol/L vs.2.74±0.06mmol/L,p0.01)与血糖值(7.47±0.03 mmol/L vs.7.95±0.18 mmol/L,p0.01)明显低于奥氮平组。辛伐他汀组的甘油三酯(0.59±0.06 mmol/L vs.0.85±0.06 mmol/L,p0.05)与总胆固醇值(2.22±0.10 mmol/L vs.2.45±0.03 mmol/L,p0.05)低于空白对照,但血糖值与空白对照相比无明显差别(p0.05)。大鼠体重增长值与总胆固醇值呈显著性正相关(r=0.458,p0.01)。3.奥氮平/辛伐他汀对大鼠自发活动的影响奥氮平给药大鼠的总距离低于空白对照组(4091.3±200.6 cm vs.4998.3±157.6 cm,p0.05)且平均速率也低于空白对照组(2.7±0.1 cm/s vs.3.3±0.1 cm/s,p0.05)。但辛伐他汀对于奥大鼠的总距离与平均速率的影响并不明显(both p0.05)。4.奥氮平/辛伐他汀对大鼠产热活动的影响奥氮平单独给药组的大鼠体温与空白对照组相比下降明显(37.41±0.03℃vs.37.64±0.02℃,p0.01)。辛伐他汀与奥氮平联合给药5周后,与空白对照组相比,辛伐他汀对大鼠体温的调节不明显,但在联合给药后期(第26天后),体温相比于奥氮平单独给药组有上升趋势,但没有出现显著性差异(p0.05)。5.奥氮平/辛伐他汀对大鼠脂肪堆积与棕色脂肪形态的影响奥氮平作用后,大鼠的产热活动下降,辛伐他汀促进产热活动的增加,进而我们对大鼠体内重要调节产热的器官——棕色脂肪和白色脂肪进行研究。相较空白对照组,奥氮平组的白色脂肪积累明显增加(7.30±0.58 g vs.6.14±0.18 g,p0.05)。联合给药组比奥氮平组的白色脂肪积累明显减少(5.67±0.39 g vs.7.30±0.58 g,p0.05)。联合给药组的棕色脂肪比于奥氮平组有明显增加(0.69±0.04g vs.0.59±0.06 g,p0.05)。另外,根据棕色脂肪切片油红染色可知,奥氮平使棕色脂肪的脂滴变大,但辛伐他汀与奥氮平联合给药组的脂滴大小与空白对照组无明显变化。6.奥氮平/辛伐他汀对大鼠棕色脂肪产热相关基因mRNA与蛋白水平的影响奥氮平组相比于空白对照组,UCP1的转录水平显著下调了~44%(p0.05),PGC-1α下调了~40%(p0.05)而PPARγ下调了~55%(p0.05)。相比于奥氮平组,奥氮平与辛伐他汀联合给药组可以上调UCP1的基因转录水平(144%±14%vs.56%±11%,p0.01)。联合给药组在PGC-1α的转录水平上相比于奥氮平组也有明显提升(103%±13%vs.56%±11%,p0.05)。辛伐他汀联合给药组与奥氮平单独给药组相比,PPARγ的转录水平明显上调(75%±14%vs.45%±6%,p0.05)。而辛伐他汀组PRDM16的转录水平相比于空白对照组相比有上调(141%±11%vs.100%±22%,p0.05)。与空白对照组相比,UCP1与PGC-1α在奥氮平组中的蛋白表达量分别显著下调了~29%(p0.01)与~25%(p0.05)。奥氮平与辛伐他汀联合给药组的UCP1蛋白表达水平相比于奥氮平组显著上升(86%±6%vs.71%±5%,p0.05)。UCP1蛋白表达水平与大鼠总胆固醇呈显著性负相关(r=-0.484,p0.01)。7.奥氮平/辛伐他汀对大鼠POMC与TH基因的m RNA水平的影响与空白对照组相比,下丘脑的POMC与脑干TH在奥氮平的作用下分别降低了~36%(p0.05)与~45%(p0.05),辛伐他汀使POMC的表达水平显著上升了~99%(p0.01)。联合给药组与奥氮平组相比,TH转录水平有明显上升(108%±9%vs.55%±14%,p0.05)。结论:奥氮平使大鼠体重增长明显,糖脂代谢与能量代谢失衡。辛伐他汀不仅可以调节血脂血糖水平,还能通过提高棕色脂肪的活性改善奥氮平引起的体重增长与代谢异常。相关性分析显示,辛伐他汀的降血脂作用可能与激活棕色脂肪相关。这些结果为临床上治疗二代抗精分药物所引起的副作用疾病提供一个潜在思路与方案。
[Abstract]:Background: schizophrenia is a class of severe psychosis that occurs frequently in adolescents. Once a teenager is suffering from this disease, antipsychotics must be taken for a long time or a lifetime. Olanzapine, one of the two generation antipsychotic drugs commonly used, is widely used in the treatment of schizophrenia. Compared with the first generation of antipsychotic drugs, It has an effect on both 5-HT and DA receptors in the brain, and has a certain therapeutic effect on schizophrenia and all kinds of psychosis. But it is worth noting that olanzapine may lead to severe metabolic associated side effects such as obesity, hyperlipidemia, Isle resistance and hyperglycemia. It can seriously affect the quality of life of the patient and may also lose confidence in the treatment effect of olanzapine, especially in young patients. However, the metabolic side effects of olanzapine have not been fully understood. The brown fat (Brown adipose tissue, BAT) is an extremely important energy metabolic organ in the body that produces heat through non tremor. There are brown fat in mammals, and the brown fat of infants is far more than in adulthood. It is also one of the commonly used experiments. To improve the thermal activity of brown fat, it can promote energy metabolism in the body, reduce body weight, and improve blood glucose and blood glucose abnormality. The uncoupling protein -1 (Uncoupling protein-1, UCP1) on the mitochondrial inner membrane of the cells is a brown fat specific thermoprotein, and is highly expressed in brown fat, and UCP1 Protein can convert chemical energy into heat energy release. In addition to UCP1, peroxisome proliferators in brown adipocytes activate receptor gamma co activator alpha (Peroxisome-proliferator-activated receptor- gamma co-activator-1 alpha, PGC-1 alpha), peroxisome proliferator activated receptor gamma (Peroxisome-proliferator-activated receptor gamma, PPAR gamma). The expression level of protein 16 (PR domain-containing 16, PRDM16), including the PR domain, is also related to the thermal activity, and the changes in the expression level of the Proopiomelanocortin, POMC and Tyrosine hydroxylase, TH in the hypothalamus also affect the heat production of brown fat. Changes in activity. The statins, represented by simvastatin (Simvastatin), serve as a first-line lipid-lowering drug, increase the elimination of serum cholesterol, improve dyslipidemia and control the body weight of patients with cardiovascular disease and hyperlipidemia. Although the study of simvastatin in glycolipid metabolism is increasingly clear, it is especially in energy metabolism. There are few reports on the effect of brown fat. Therefore, it is worth studying whether simvastatin can improve the activity of brown fat to improve metabolic abnormalities. The effect of activating the thermal activity of brown fat to interfere with the body weight growth and metabolic imbalance caused by olanzapine has not been reported. This study selected the young rats of weaning to simulate the metabolic function of the young body, and the effects of simvastatin on the body weight, feeding, blood lipid, blood glucose, self activity and brown fat activity after olanzapine administration. To study the intervention function of simvastatin on the body weight growth and metabolic abnormalities caused by olanzapine. Methods: 1. selected 36 weanling female young rats (weight: 45-55 g), and randomly assigned them to two groups (n=18), and were given 3 mg/kg/day olanzapine (t.i.d.) and blank pills for 14 days, and the weight of rats was detected every two days, feeding, 14 days after the treatment, the plasma TG, TC and fasting blood glucose.2. were divided into four groups. The blank group was randomly divided into blank control group and simvastatin group (10 mg/kg/day). The olanzapine group was randomly divided into olanzapine group (3 mg/kg/day, t.i.d.) and combined administration group (olanzapine: 3 mg/kg/day, t.i.d., octyl. Statins: 10 mg/kg/day), continue to give medicine for 35 days, test rats weight, feeding, and anus temperature changes every two days. 3. in thirtieth days of combined administration, the rat field experiment was carried out. Rats were put into the black open box (50 x 50 * 50 cm3), the ordinary camera tracked the rats' activity, each rat was photographed for 25 minutes, and used Noldus Ethovision to analyze and shoot. The rat activity video was used to analyze the average rate and the total distance of rats to reflect the changes in the spontaneous activity of rats; 4. in the mid-term (Twentieth days) administration of the combined administration of simvastatin and olanzapine, the plasma TG, TC, and fasting blood glucose were measured, and the weight of brown fat and white fat was detected after the end of the animal experiment; and the brown fat was added into the rat. Frozen section and oil red staining; 5. RT-qPCR and Western blotting were used to detect the mRNA and protein expression level of the genes related to the heat production related to the brown fat and the energy metabolism of the hypothalamus and brain stem. Results: 1. olanzapine / simvastatin on the influence of oranzapine on the weight and feeding of rats for two weeks, olanzapine was administered to rats The weight of the rats was significantly higher than that in the blank control group (99.5 + 5.7 g vs.79.5 + 4.5 g, P0.05), and olanzapine increased the intake of rats (40.2 + 3.6 g vs.33.5g + 2.5 g, P0.05), compared with the blank control group; the body weight of the rats combined with the combined administration of simvastatin and olanzapine was compared with the olanzapine group (205 + 11.1 g vs.233.5 + 10.6) after 5 weeks of combined administration G, P0.05) decreased significantly. Compared with the blank control group, simvastatin did not affect the diet of rats (P0.05).2. Simvastatin to reduce the blood lipid of olanzapine in rats. The triglycerides (1.02 + 0.06 mmol) of the olanzapine group were detected in the fasting plasma triglycerides of the rats after olanzapine alone. /L vs.0.73 + 0.02 mmol/L, P0.01), the total cholesterol (2.40 + 0.04 mmol/L vs.2.28 + 0.03 mmol/L, P0.05) and blood glucose (7.74 + 0.13mmol/L vs.7.39 + 0.11 mmol/L, P0.05) were significantly higher than that in the blank control group. The combined administration of the medium-term (Twentieth days) test showed that the blood lipids and blood glucose levels of the combined administration were significantly lower than that of olanzapine alone. (P0.05). After 35 days of combined administration, the triglyceride (0.84 + 0.03mmol/L vs.1.27 + 0.04 mmol/L, P0.01) of olanzapine and simvastatin, total cholesterol (2.43 + 0.05 mmol/L vs.2.74 + 0.06mmol/L, P0.01) and blood glucose (7.47 + 0.03 mmol/L vs.7.95 + 0.18 mmol/L) were significantly lower than that of olanzapine group. The glycerol of simvastatin group was three. The value of the ester (0.59 + 0.06 mmol/L vs.0.85 + 0.06 mmol/L, P0.05) and total cholesterol (2.22 + 0.10 mmol/L vs.2.45 + 0.03 mmol/L, P0.05) was lower than that of the blank control, but there was no significant difference between the blood sugar and the blank (P0.05). The weight growth value of the rats was significantly positively correlated with the total cholesterol (r=0.458, P0.01).3. olanzapine / simvastatin to rats The total distance of olanzapine in rats was lower than that in the blank control group (4091.3 + 200.6 cm vs.4998.3 + 157.6 cm, P0.05) and the average rate was lower than that in the blank control group (2.7 + 0.1 cm/s vs.3.3 + 0.1 cm/s, P0.05). But the effect of simvastatin on the total distance and average rate of the rats was not obvious (both P0.05).4. olanzapine The effect of simvastatin on the heat production in rats was significantly lower than that in the blank control group (37.41 + 0.03 vs.37.64 + 0.02, P0.01). After 5 weeks of combined administration of simvastatin and olanzapine, the regulation of simvastatin on the body temperature of rats was not obvious, but in the later period of the combined Administration ( Twenty-sixth days later), the body temperature was higher than that of olanzapine alone, but there was no significant difference (P0.05) the effect of olanzapine / simvastatin on the fat accumulation and the shape of brown fat in rats. After the effect of olanzapine, the heat production of rats decreased, and simvastatin promoted the increase of heat production, and then we weighed the rats' body weight. The white fat accumulation in the olanzapine group was significantly increased (7.30 + 0.58 g vs.6.14 + 0.18 g, P0.05), compared with the blank control group. The white fat accumulation in the combined administration group was significantly reduced (5.67 + 0.39 g vs.7.30 + 0.58 g, P0.05). The brown fat accumulation in the combined administration group was brown. There was a significant increase in fat ratio in the olanzapine group (0.69 + 0.04g vs.0.59 + 0.06 g, P0.05). In addition, according to brown fat red staining, olanzapine made the fat droplets of brown fat larger, but the size of the lipid droplets of the combined administration of simvastatin and olanzapine had no significant changes in the brown fat of.6. olanzapine / simvastatin in the control group. Compared with the control group, the transcriptional level of UCP1 was significantly reduced by ~44% (P0.05), PGC-1 alpha was down regulated by ~40% (P0.05) and PPAR gamma lowered ~55% (P0.05). Compared to olanzapine group, the combined administration of olanzapine and simvastatin could increase the UCP1 gene transcriptional level (144% + 14%vs). .56% + 11%, P0.01). Compared with olanzapine group at the level of PGC-1 alpha, the combined administration group was also significantly increased (103% + 13%vs.56% + 11%, P0.05). The transcriptional level of PPAR gamma in the combined administration of simvastatin group was significantly up (75% + 14%vs.45% + 6%, P0.05) compared with the olanzapine group alone, while the transcriptional level of the simvastatin group was compared with that of the simvastatin group. Compared with the blank control group, the protein expression of UCP1 and PGC-1 alpha in olanzapine group was significantly reduced by ~29% (P0.01) and ~25% (P0.05), compared with the blank control group. The level of UCP1 protein expression in the combined administration of olanzapine and simvastatin group was significantly higher than that in olanzapine group (86% + 6%vs.71% + 5%, P). 0.05) the expression level of.UCP1 protein was significantly negatively correlated with rat total cholesterol (r=-0.484, P0.01). The effect of.7. olanzapine / simvastatin on the m RNA level of POMC and TH genes in rats was compared with that of the blank control group. The hypothalamic POMC and brainstem TH decreased respectively under the action of olanzapine. The expression level of TH was significantly increased by ~99% (P0.01). Compared with olanzapine group, the TH transcriptional level increased significantly (108% + 9%vs.55% + 14%, P0.05). Conclusion olanzapine could increase the weight growth of rats and the imbalance of glucose and lipid metabolism and energy metabolism. Improving the body weight growth and metabolic abnormalities caused by olanzapine. Correlation analysis shows that the action of simvastatin may be associated with the activation of brown fat. These results provide a potential idea and scheme for the clinical treatment of side effects caused by the two generation of antiseminal drugs.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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