二甲双胍对糖尿病大鼠血清CTRP9水平的影响及其治疗效应的评估
发布时间：2018-09-03 06:16
【摘要】：1研究背景糖尿病作为一种常见的慢性疾病,其发病人数逐年增加。截止到目前,全球已经有大约2.5亿人口罹患该疾病。而到2025年,这一人数可能将达到4亿左右。世界卫生组织(World Health Organisation,简称WHO)预测,相比发达国家,发展中国家人群患糖尿病的人数将会上升更快,这与其不健康饮食、老龄化、肥胖等诸多因素有关。世界卫生组织做出统计显示,2015年,发展中国家患糖尿病的人群年龄大多数集中在35-65岁左右,这正是一个人工作生产的黄金时段;而发达国家患糖尿病年龄则集中在65岁左右。2016年,世界卫生日的主题是“应对糖尿病,我们需要更多的行动”。从以上数据我们可以看出,全人类必须开始更加重视糖尿病。一方面,我们需要重视糖尿病带来的各种机体损害,比如各种并发症的产生,如周围神经病变,自主神经病变,视网膜病变,心血管危害等等,另一方面我们要加强糖尿病患者的健康教育,宣扬糖尿病的相关知识,在糖尿病的高危人群中深入开展相关知识的讲解。总之,糖尿病的防治刻不容缓。糖尿病发病因素多,可由遗传、免疫、微生物及其毒素、自由基、精神因素等等各种原因作用于机体,进而导致胰岛功能减退、胰岛素抵抗,由于胰岛素分泌相对或绝对不足、或胰岛素作用缺陷,而引发机体糖、脂肪和电解质等一系列代谢紊乱综合征,临床上以高血糖为主要特点,而持久升高的血糖、血脂一方面会对机体造成损伤,还会诱发β细胞的功能损坏和诱发胰岛素抵抗,这些都与氧化应激和慢性炎症密切相关,可以说,氧化应激贯穿糖尿病的发生、发展。CTRP9作为近些年发现的一个脂肪细胞因子,它的家族一共有十五个成员,结构大致相似,具有正性作用,它在预防氧化应激、胰岛素抵抗、心血管疾病的发生等方面发挥着不可忽视的作用。已有证据显示,CTRPs家族中,特别是CTRP9和APN具有非常相似的结构,它们的生物学特点也有相同点。有学者发现,对小鼠进行APN基因消除,小鼠并未出现代谢紊乱的表现,这说明,可能体内有其他因素或者机制来代偿性的弥补APN的缺乏。这种作用,有可能是通过CTRPs来代偿性实现的。由于CTRPs家族与APN结构和生物学特点的相似性,有学者推测,CTRP9参与了上述的代偿机制。进一步阐明CTRP9的生理学效应,探索其与心血管疾病、肥胖、糖尿病之间的联系,可以为慢性代谢性疾病的治疗提供新的思路和方法。二甲双胍作为治疗糖尿病的一线用药之一,通常认为它通过以下三个途径达到降糖目的:1、延缓胃肠道对葡萄糖的吸收;2、增加组织对胰岛素的敏感性;3、抑制肝糖原异生作用,降低肝糖输出。近些年来,二甲双胍的一些其他作用也被越来越被人们所熟知,比如抗炎、抗肿瘤、抗氧化应激等,但是对于其抗氧化应激的作用机制,尚缺乏临床实验和基础实验的支持。2材料和方法2.1分组及饲喂方法SPF级SD雄性大鼠40只,体重为89.62±7.42g。适应性喂养7天后,随机分为2组,一组10只,一组30只,分别饲喂普通饲料、高脂高糖饮食,在第8周时腹腔注射STZ,使用STZ+高脂高糖联合造模,以30mg/kg腹腔注射造模,3天后测空腹血糖,将空腹血糖大于15mmol/L视作为造模成功。将造模组随机分为3组,每组10只,一组继续饲喂高脂高糖(M组),一组饲喂高脂高糖+二甲双胍(M+MET组),一组饲喂高脂高糖+胰岛素(M+INS);普通饲料组喂普通饲养(C组,10只)。MET按200mg/Kg剂量灌胃,不给予MET的大鼠使用生理盐水灌胃。共喂养12周。2.2观察指标(1)血标本用于检测血糖浓度(葡萄糖氧化酶法)及胰岛素含量(放射免疫法),以此计算胰岛素抵抗指数(2)ELISA法检测血清中CTRP9、TNF-α的表达水平(3)Western blot法检测IKK-β及ser181蛋白表达的表达水平(4)RT-PCR法检测CTRP9、TNF-α、IL-6基因的表达水平3结果3.1 HOMA-IR各组大鼠血清HOMA-IR水平之间的差异有统计学意义(F=169.923,P=0.0000.05)。M组、M+Ins组和M+Met组的HOMA-IR均比Control组高,差异有统计学意义(P0.05)。M+Ins组、M+Met组的HOMA-IR均比M组低,差异有统计学意义(P0.05)。见表3.1、3.2。3.2血清CTRP9的表达水平各组大鼠骨骼肌组织平均CTRP9水平之间的差别有统计学意义(F=98.773,P=0.0000.05)。进一步两两比较,M+Ins组、M+Met组的CTRP9水平均高于M组,差异有统计学意义(P0.05)。M+Ins组、M+Met组的CTRP9水平均低于C组,差异有统计学意义(P0.05)。见表3.3、3.4。3.3大鼠HOMA-IR与CTPR9水平的相关性分析M组,HOMA-IR与血清CTRP9水平呈负相关,r=-0.418,P=0.0190.05;M+MET组,HOMA-IR与血清CTRP9水平呈负相关,r=-0.306,P=0.0220.05。见图3.2。3.4各组大鼠骨骼肌组织CTRP9、TNF-α、IL-6基因的表达水平与C组比较,M组大鼠脂肪细胞CTRP9 mRNA表达水平显著降低,有统计学意义(P0.05);与M组比较,M+Ins组、M+Met组CTRP9 mRNA表达水平显著升高,有统计学意义(P0.05);与C组比较,M组大鼠骨骼肌TNF-αmRNA表达水平显著下降,有统计学意义;与M组比较,M+Ins组、M+Met组TNF-αmRNA表达水平显著升高,有统计学意义(P0.05)。与C组比较,M组大鼠骨骼肌IL-6 mRNA表达水平显著下降,有统计学意义;与M组比较,M+Ins组、M+Met组IL-6 mRNA表达水平显著升高,有统计学意义。3.5各组大鼠骨骼肌组织IKK-β、ser181蛋白的表达4结论(1)2型糖尿病大鼠血清中CTRP9表达水平降低。(2)2型糖尿病大鼠胰岛素抵抗与CTRP9的表达呈负相关。(3)二甲双胍降低糖尿病大鼠骨骼肌组织ser181蛋白的表达,对IKK-β影响不大。(4)二甲双胍的上述作用有可能是通过抑制IKK-β的磷酸化实现的。
[Abstract]:Background Diabetes mellitus, a common chronic disease, has increased year by year. Up to now, about 250 million people worldwide have suffered from the disease. By 2025, this number may reach about 400 million. The World Health Organization (WHO) predicts that compared with developed countries, developing countries have suffered from the disease. The number of people with diabetes will rise more quickly, due to unhealthy diet, aging, obesity and many other factors. According to the World Health Organization, the majority of people with diabetes in developing countries will be around 35-65 years old in 2015, which is the golden time for one person to work and produce diabetes in developed countries. In 2016, the theme of World Health Day is "We need more action to deal with diabetes." From the above data, we can see that the whole human race must begin to pay more attention to diabetes. On the one hand, we need to pay attention to the damage caused by diabetes, such as the occurrence of complications, such as week. Peripheral neuropathy, autonomic neuropathy, retinopathy, cardiovascular hazards, and so on. On the other hand, we should strengthen the health education of diabetic patients, promote the knowledge of diabetes, in-depth interpretation of the relevant knowledge in high-risk groups of diabetes. In short, the prevention and treatment of diabetes is urgent. There are many factors of diabetes, can be left behind. Transmission, immunity, microorganisms and their toxins, free radicals, mental factors and so on all kinds of reasons affect the body, and then lead to islet dysfunction, insulin resistance, due to the relative or absolute insufficiency of insulin secretion, or insulin deficiency, resulting in the body sugar, fat and electrolyte and a series of metabolic disorders syndrome, clinical hyperglycemia as The main characteristics, and persistently elevated blood glucose, lipids on the one hand will cause damage to the body, but also induce beta cell dysfunction and insulin resistance, which are closely related to oxidative stress and chronic inflammation, it can be said that oxidative stress throughout the occurrence and development of diabetes mellitus. There are fifteen members in the CTRPs family with similar structure and positive effect. It plays an important role in the prevention of oxidative stress, insulin resistance and cardiovascular disease. Some scholars have found that there is no metabolic disorder in mice after APN gene deletion, which indicates that there may be other factors or mechanisms in the body to compensate for the lack of APN. This effect may be compensated by CTRPs. Because of the similarity of the CTRPs family and APN structure and biological characteristics, it is learned. It is speculated that CTRP9 is involved in the above mechanisms. Further clarification of the physiological effects of CTRP9 and its association with cardiovascular diseases, obesity and diabetes mellitus may provide new ideas and methods for the treatment of chronic metabolic diseases. In recent years, some other effects of metformin have been known by more and more people, such as anti-inflammatory, anti-tumor, anti-oxidative stress and so on, but for its antioxidant effect The mechanism of chemical stress is still lack of clinical and basic experimental support. 2 Materials and Methods 2.1 Grouping and feeding SPF SD male rats with a body weight of 89.62+7.42 g were randomly divided into two groups after 7 days of adaptive feeding. One group of 10 rats and the other 30 rats were fed with normal diet, high fat and high sugar diet respectively, and STZ was injected intraperitoneally at the 8th week. The model was established by STZ+high-fat and high-glucose combined with intraperitoneal injection of 30mg/kg. The fasting blood glucose was measured 3 days later. The fasting blood glucose was more than 15mmol/L. The model group was randomly divided into three groups, 10 rats in each group. One group was fed with high-fat and high-sugar (M+MET), the other with high-fat and high-glucose + insulin (M+I). Normal diet group was fed with normal feeding (group C, 10 rats). MET was given 200 mg/kg by gastric lavage, rats without MET were given normal saline by gastric lavage. Blood samples were fed for 12 weeks. 2.2 observation indexes (1) Blood glucose concentration (glucose oxidase method) and insulin content (radioimmunoassay) were used to calculate insulin resistance index (2) ELISA method to detect blood glucose concentration (glucose oxidase method). The expression levels of CTRP9, TNF-alpha in serum (3) Western blot were used to detect the expression levels of IKK-beta and ser181 protein (4) RT-PCR was used to detect the expression levels of CTRP9, TNF-alpha and IL-6 gene. Results 3.1 The difference of HOMA-IR levels in serum of rats in each HOMA-IR group was statistically significant (F=169.923, P=0.0000.05). The HOMA-IR levels in M group, M+Ins group and M+Met group were all significantly different. Compared with the control group, the difference was statistically significant (P 0.05). The HOMA-IR of M+Ins group and M+Met group was lower than that of M group, and the difference was statistically significant (P 0.05). See Table 3.1, 3.2.3.2, the expression of CTRP9 in serum of rats skeletal muscle tissue in each group was statistically significant (F = 98.773, P = 0.0000.05). The levels of CTRP9 in group E were higher than those in group M (P 0.05). The levels of CTRP9 in group M + Ins and group M + M ET were lower than those in group C (P 0.05). The expression levels of CTRP9, TNF-a and IL-6 in skeletal muscle tissue of rats in each group were significantly lower than those in group C (P 0.05), and the expression levels of CTRP9 mRNA in adipocytes of rats in group M were significantly higher than those in group M + Ins and group M + Met (P 0.05). Compared with group M, the expression level of TNF-alpha mRNA in skeletal muscle of rats in group M was significantly decreased (P 0.05); compared with group M, the expression level of TNF-alpha mRNA in group M + Ins and group M + Met was significantly increased (P 0.05). Compared with group C, the expression level of IL-6 mRNA in skeletal muscle of rats in group M was significantly decreased (P 0.05). The expression of IL-6 mRNA in M+Met group was significantly higher than that in M+Met group. 3.5 The expression of IKK-beta and ser181 protein in skeletal muscle tissue of rats in each group was statistically significant. Conclusion (1) The expression of CTRP9 in serum of type 2 diabetic rats was decreased. (2) Insulin resistance in type 2 diabetic rats was negatively correlated with the expression of CTRP9. (3) Metformin decreased the expression of CTRP9 in skeletal muscle tissue of diabetic rats The expression of ser181 protein had little effect on IKK-beta. (4) Metformin may inhibit the phosphorylation of IKK-beta.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2
本文编号：2219133
[Abstract]:Background Diabetes mellitus, a common chronic disease, has increased year by year. Up to now, about 250 million people worldwide have suffered from the disease. By 2025, this number may reach about 400 million. The World Health Organization (WHO) predicts that compared with developed countries, developing countries have suffered from the disease. The number of people with diabetes will rise more quickly, due to unhealthy diet, aging, obesity and many other factors. According to the World Health Organization, the majority of people with diabetes in developing countries will be around 35-65 years old in 2015, which is the golden time for one person to work and produce diabetes in developed countries. In 2016, the theme of World Health Day is "We need more action to deal with diabetes." From the above data, we can see that the whole human race must begin to pay more attention to diabetes. On the one hand, we need to pay attention to the damage caused by diabetes, such as the occurrence of complications, such as week. Peripheral neuropathy, autonomic neuropathy, retinopathy, cardiovascular hazards, and so on. On the other hand, we should strengthen the health education of diabetic patients, promote the knowledge of diabetes, in-depth interpretation of the relevant knowledge in high-risk groups of diabetes. In short, the prevention and treatment of diabetes is urgent. There are many factors of diabetes, can be left behind. Transmission, immunity, microorganisms and their toxins, free radicals, mental factors and so on all kinds of reasons affect the body, and then lead to islet dysfunction, insulin resistance, due to the relative or absolute insufficiency of insulin secretion, or insulin deficiency, resulting in the body sugar, fat and electrolyte and a series of metabolic disorders syndrome, clinical hyperglycemia as The main characteristics, and persistently elevated blood glucose, lipids on the one hand will cause damage to the body, but also induce beta cell dysfunction and insulin resistance, which are closely related to oxidative stress and chronic inflammation, it can be said that oxidative stress throughout the occurrence and development of diabetes mellitus. There are fifteen members in the CTRPs family with similar structure and positive effect. It plays an important role in the prevention of oxidative stress, insulin resistance and cardiovascular disease. Some scholars have found that there is no metabolic disorder in mice after APN gene deletion, which indicates that there may be other factors or mechanisms in the body to compensate for the lack of APN. This effect may be compensated by CTRPs. Because of the similarity of the CTRPs family and APN structure and biological characteristics, it is learned. It is speculated that CTRP9 is involved in the above mechanisms. Further clarification of the physiological effects of CTRP9 and its association with cardiovascular diseases, obesity and diabetes mellitus may provide new ideas and methods for the treatment of chronic metabolic diseases. In recent years, some other effects of metformin have been known by more and more people, such as anti-inflammatory, anti-tumor, anti-oxidative stress and so on, but for its antioxidant effect The mechanism of chemical stress is still lack of clinical and basic experimental support. 2 Materials and Methods 2.1 Grouping and feeding SPF SD male rats with a body weight of 89.62+7.42 g were randomly divided into two groups after 7 days of adaptive feeding. One group of 10 rats and the other 30 rats were fed with normal diet, high fat and high sugar diet respectively, and STZ was injected intraperitoneally at the 8th week. The model was established by STZ+high-fat and high-glucose combined with intraperitoneal injection of 30mg/kg. The fasting blood glucose was measured 3 days later. The fasting blood glucose was more than 15mmol/L. The model group was randomly divided into three groups, 10 rats in each group. One group was fed with high-fat and high-sugar (M+MET), the other with high-fat and high-glucose + insulin (M+I). Normal diet group was fed with normal feeding (group C, 10 rats). MET was given 200 mg/kg by gastric lavage, rats without MET were given normal saline by gastric lavage. Blood samples were fed for 12 weeks. 2.2 observation indexes (1) Blood glucose concentration (glucose oxidase method) and insulin content (radioimmunoassay) were used to calculate insulin resistance index (2) ELISA method to detect blood glucose concentration (glucose oxidase method). The expression levels of CTRP9, TNF-alpha in serum (3) Western blot were used to detect the expression levels of IKK-beta and ser181 protein (4) RT-PCR was used to detect the expression levels of CTRP9, TNF-alpha and IL-6 gene. Results 3.1 The difference of HOMA-IR levels in serum of rats in each HOMA-IR group was statistically significant (F=169.923, P=0.0000.05). The HOMA-IR levels in M group, M+Ins group and M+Met group were all significantly different. Compared with the control group, the difference was statistically significant (P 0.05). The HOMA-IR of M+Ins group and M+Met group was lower than that of M group, and the difference was statistically significant (P 0.05). See Table 3.1, 3.2.3.2, the expression of CTRP9 in serum of rats skeletal muscle tissue in each group was statistically significant (F = 98.773, P = 0.0000.05). The levels of CTRP9 in group E were higher than those in group M (P 0.05). The levels of CTRP9 in group M + Ins and group M + M ET were lower than those in group C (P 0.05). The expression levels of CTRP9, TNF-a and IL-6 in skeletal muscle tissue of rats in each group were significantly lower than those in group C (P 0.05), and the expression levels of CTRP9 mRNA in adipocytes of rats in group M were significantly higher than those in group M + Ins and group M + Met (P 0.05). Compared with group M, the expression level of TNF-alpha mRNA in skeletal muscle of rats in group M was significantly decreased (P 0.05); compared with group M, the expression level of TNF-alpha mRNA in group M + Ins and group M + Met was significantly increased (P 0.05). Compared with group C, the expression level of IL-6 mRNA in skeletal muscle of rats in group M was significantly decreased (P 0.05). The expression of IL-6 mRNA in M+Met group was significantly higher than that in M+Met group. 3.5 The expression of IKK-beta and ser181 protein in skeletal muscle tissue of rats in each group was statistically significant. Conclusion (1) The expression of CTRP9 in serum of type 2 diabetic rats was decreased. (2) Insulin resistance in type 2 diabetic rats was negatively correlated with the expression of CTRP9. (3) Metformin decreased the expression of CTRP9 in skeletal muscle tissue of diabetic rats The expression of ser181 protein had little effect on IKK-beta. (4) Metformin may inhibit the phosphorylation of IKK-beta.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2
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