胰岛素对脂多糖诱导脓毒症大鼠脑损伤的保护作用及机制

发布时间：2018-06-25 12:25

本文选题：脓毒症 + 脑　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景及目的解偶联蛋白2(UCP2)是位于线粒体内膜的阴离子转运蛋白,它可调节能量代谢并减少ROS的生成。UCP2的神经保护作用在帕金森病、脑缺血、癫痫等多种神经系统疾病中得到广泛证实。研究表明,脓毒症时,胰岛素不仅有降低血糖、调节代谢的作用,还具有减少ROS产生、对抗氧化应激、抗凋亡、保护线粒体等作用,但其具体机制并不十分明确。既往有体外研究表明胰岛素可能通过上调UCP2的表达,减少活性氧(ROS)的生成,进而发挥抗氧化的保护作用。本研究拟探讨胰岛素对脓毒症脑组织损伤的保护作用,探讨UCP2在脓毒症大鼠脑组织中的表达,及胰岛素对其表达的影响,探讨UCP2在胰岛素保护作用中的可能作用,进一步为脓毒症时胰岛素的临床应用提供实验及理论依据。方法行胰岛素浓度梯度预实验,根据大鼠血糖情况及生存率确定胰岛素浓度后,将50只SPF级雄性SD大鼠,按随机数字表法随机分为正常对照组(n=10)、脓毒症组(n=20)和胰岛素组(n=20)。通过腹腔注射革兰阴性菌脂多糖(LPS)15 mg/kg建立脓毒症大鼠模型。胰岛素组在造模前30分钟予皮下注射长效胰岛素1U/kg(预实验确定),脓毒症组皮下注射等量生理盐水。LPS造模后24h,每组各取8只处死,留取大脑皮质。提取大脑皮质线粒体测定线粒体活性氧(ROS)、丙二醛(MDA)水平及超氧化物歧化酶(SOD)活力;Western blot检测 Bcl-2、Bax、活化的 Caspase-9(Cleaved Caspase-9)的蛋白表达;TUNEL染色检测大脑皮质神经细胞凋亡;苏木精-伊红(HE)染色观察大脑皮质病理改变;RT-PCR检测大脑皮质UCP2的mRNA表达;Western blot和免疫组化检测皮质组织UCP2蛋白的表达。结果1.与正常对照组相比,脓毒症组及胰岛素组大鼠皮质组织线粒体ROS和MDA水平明显升高,而SOD活力明显下降,均P0.05;而与脓毒症组相比,胰岛素组大鼠皮质组织线粒体ROS和MDA水平下降,且SOD活力回升,均P0.05;2.与正常对照组相比,脓毒症组及胰岛素组大鼠大脑皮质组织神经细胞凋亡增加,脓毒症组抗凋亡蛋白Bcl-2表达下降,而促凋亡蛋白Bax和CleavedCaspase-9表达升高,均P0.05;而与脓毒症组相比,胰岛素组大鼠皮质组织神经细胞凋亡减少,抗凋亡蛋白Bcl-2表达升高,而促凋亡蛋白Bax和Cleaved Caspase-9 表达下降,均 P0.05;3.与正常对照组相比,脓毒症组和胰岛素组大鼠大脑皮质组织HE染色可见明显异常病理改变,而与脓毒症组相比,胰岛素组大鼠大脑皮质组织病理改变较脓毒症组减轻;4.与正常对照组相比,脓毒症组大鼠皮质组织UCP2 mRNA和蛋白表达均升高;而与脓毒症组相比,胰岛素组大鼠皮质组织UCP2 mRNA和蛋白表达较均脓毒症组升高。结论1.U/kg长效胰岛素皮下注射可有效改善脓毒症大鼠生存2.UCP2在脓毒症大鼠脑组织中表达上调;3.胰岛素可上调脓毒症大鼠脑组织中UCP2的表达;4.胰岛素通过减少ROS生成,减轻线粒体氧化应激,抑制线粒体凋亡通路的激活并减少神经细胞凋亡,在脓毒症大鼠脑组织损伤中发挥保护作用,其机制之一可能是通过上调UCP2的表达,进而减少ROS产生、减轻氧化应激并减少细胞凋亡,从而在脓毒症脑组织损伤中发挥保护作用。
[Abstract]:Background and objective uncoupling protein 2 (UCP2) is an anion transporter in the mitochondrial membrane. It regulates energy metabolism and reduces the neuroprotective effect of ROS producing.UCP2 in many kinds of nervous system diseases, such as Parkinson's disease, cerebral ischemia, and epilepsy. Sugar, which regulates metabolism, also reduces the effect of ROS production, anti oxidative stress, anti apoptosis, and mitochondria protection, but its specific mechanism is not very clear. In the past, in vitro studies have shown that insulin may reduce the production of active oxygen (ROS) by up regulation of UCP2 expression and then play a protective role in antioxidant. This study is to explore the pancreas. The protective effect of isisin on the brain tissue injury of sepsis, to explore the expression of UCP2 in the brain tissue of sepsis rats, and the effect of insulin on its expression, explore the possible role of UCP2 in the protective effect of insulin, and further provide experimental and theoretical basis for the clinical application of insulin in sepsis. After determining the insulin concentration in rats, 50 SPF grade male SD rats were randomly divided into normal control group (n=10), sepsis group (n=20) and insulin group (n=20). The rat model of sepsis was established by intraperitoneal injection of gram-negative lipopolysaccharide (LPS) 15 mg/kg by intraperitoneal injection. The insulin group was 30 before the model. Subcutaneous injection of long acting insulin 1U/kg (pre test), the sepsis group was subcutaneously injected with the same amount of normal saline.LPS to make 24h, each group took 8 dead and left the cerebral cortex. The mitochondria of the cerebral cortex were extracted and the mitochondrial reactive oxygen species (ROS), the malondialdehyde (MDA) water and the superoxide dismutase (SOD) activity were measured, and Western blot detected Bcl-2, B. Ax, the protein expression of activated Caspase-9 (Cleaved Caspase-9); TUNEL staining to detect the apoptosis of cerebral cortex nerve cells; hematoxylin eosin (HE) staining to observe the pathological changes in cerebral cortex; RT-PCR detection of mRNA expression in cerebral cortex UCP2; Western blot and immunohistochemical detection of the expression of cortical tissue UCP2 protein. Results 1. and normal control group phase In the sepsis group and the insulin group, the level of mitochondrial ROS and MDA increased significantly, while the activity of SOD decreased significantly, all P0.05, while the level of ROS and MDA in the cortical mitochondria of the insulin group decreased and the SOD activity increased, all P0.05, compared with the sepsis group, 2. compared with the normal control group, the sepsis group and the insulin group were larger than the normal control group. The apoptosis of neural cells in cerebral cortex was increased, the expression of anti apoptotic protein Bcl-2 in sepsis group decreased, while the expression of apoptotic protein Bax and CleavedCaspase-9 increased, all of which were P0.05. Compared with the sepsis group, the apoptosis of cortical neurons in the insulin group decreased and the expression of anti apoptotic protein Bcl-2 increased, while apoptotic protein Bax and Cleaved Caspase were promoted. The expression of -9 decreased in P0.05. 3. compared with the normal control group, the HE staining in the cerebral cortex of the sepsis group and the insulin group showed obvious abnormal pathological changes. Compared with the sepsis group, the pathological changes in the cerebral cortex of the insulin group were less than that of the sepsis group, and 4. compared with the normal control group, the cortical tissue of the sepsis group was UCP2. The expression of mRNA and protein increased, but compared with the sepsis group, the expression of UCP2 mRNA and protein in the cortical tissue of the insulin group was higher than that of the sepsis group. Conclusion the subcutaneous injection of 1.U/kg long acting insulin can effectively improve the expression of 2.UCP2 in the brain tissue of sepsis rats, and 3. insulin can up regulate the brain of sepsis rats. The expression of UCP2 in the weave; 4. insulin can reduce oxidative stress by reducing ROS, inhibit the activation of mitochondrial apoptosis pathway and reduce the apoptosis of neural cells. It may play a protective role in the brain tissue damage of sepsis rats. One of the mechanisms may be to reduce the expression of UCP2 and then reduce the production of ROS and reduce oxidative stress and decrease Apoptosis can play a protective role in sepsis brain tissue injury.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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