二甲雙胍對糖尿病大鼠血清CTRP9水平的影響及其治療效應(yīng)的評估
發(fā)布時間：2018-09-03 06:16
【摘要】：1研究背景糖尿病作為一種常見的慢性疾病,其發(fā)病人數(shù)逐年增加。截止到目前,全球已經(jīng)有大約2.5億人口罹患該疾病。而到2025年,這一人數(shù)可能將達(dá)到4億左右。世界衛(wèi)生組織(World Health Organisation,簡稱WHO)預(yù)測,相比發(fā)達(dá)國家,發(fā)展中國家人群患糖尿病的人數(shù)將會上升更快,這與其不健康飲食、老齡化、肥胖等諸多因素有關(guān)。世界衛(wèi)生組織做出統(tǒng)計顯示,2015年,發(fā)展中國家患糖尿病的人群年齡大多數(shù)集中在35-65歲左右,這正是一個人工作生產(chǎn)的黃金時段;而發(fā)達(dá)國家患糖尿病年齡則集中在65歲左右。2016年,世界衛(wèi)生日的主題是“應(yīng)對糖尿病,我們需要更多的行動”。從以上數(shù)據(jù)我們可以看出,全人類必須開始更加重視糖尿病。一方面,我們需要重視糖尿病帶來的各種機(jī)體損害,比如各種并發(fā)癥的產(chǎn)生,如周圍神經(jīng)病變,自主神經(jīng)病變,視網(wǎng)膜病變,心血管危害等等,另一方面我們要加強(qiáng)糖尿病患者的健康教育,宣揚(yáng)糖尿病的相關(guān)知識,在糖尿病的高危人群中深入開展相關(guān)知識的講解�？傊�,糖尿病的防治刻不容緩。糖尿病發(fā)病因素多,可由遺傳、免疫、微生物及其毒素、自由基、精神因素等等各種原因作用于機(jī)體,進(jìn)而導(dǎo)致胰島功能減退、胰島素抵抗,由于胰島素分泌相對或絕對不足、或胰島素作用缺陷,而引發(fā)機(jī)體糖、脂肪和電解質(zhì)等一系列代謝紊亂綜合征,臨床上以高血糖為主要特點,而持久升高的血糖、血脂一方面會對機(jī)體造成損傷,還會誘發(fā)β細(xì)胞的功能損壞和誘發(fā)胰島素抵抗,這些都與氧化應(yīng)激和慢性炎癥密切相關(guān),可以說,氧化應(yīng)激貫穿糖尿病的發(fā)生、發(fā)展。CTRP9作為近些年發(fā)現(xiàn)的一個脂肪細(xì)胞因子,它的家族一共有十五個成員,結(jié)構(gòu)大致相似,具有正性作用,它在預(yù)防氧化應(yīng)激、胰島素抵抗、心血管疾病的發(fā)生等方面發(fā)揮著不可忽視的作用。已有證據(jù)顯示,CTRPs家族中,特別是CTRP9和APN具有非常相似的結(jié)構(gòu),它們的生物學(xué)特點也有相同點。有學(xué)者發(fā)現(xiàn),對小鼠進(jìn)行APN基因消除,小鼠并未出現(xiàn)代謝紊亂的表現(xiàn),這說明,可能體內(nèi)有其他因素或者機(jī)制來代償性的彌補(bǔ)APN的缺乏。這種作用,有可能是通過CTRPs來代償性實現(xiàn)的。由于CTRPs家族與APN結(jié)構(gòu)和生物學(xué)特點的相似性,有學(xué)者推測,CTRP9參與了上述的代償機(jī)制。進(jìn)一步闡明CTRP9的生理學(xué)效應(yīng),探索其與心血管疾病、肥胖、糖尿病之間的聯(lián)系,可以為慢性代謝性疾病的治療提供新的思路和方法。二甲雙胍作為治療糖尿病的一線用藥之一,通常認(rèn)為它通過以下三個途徑達(dá)到降糖目的:1、延緩胃腸道對葡萄糖的吸收;2、增加組織對胰島素的敏感性;3、抑制肝糖原異生作用,降低肝糖輸出。近些年來,二甲雙胍的一些其他作用也被越來越被人們所熟知,比如抗炎、抗腫瘤、抗氧化應(yīng)激等,但是對于其抗氧化應(yīng)激的作用機(jī)制,尚缺乏臨床實驗和基礎(chǔ)實驗的支持。2材料和方法2.1分組及飼喂方法SPF級SD雄性大鼠40只,體重為89.62±7.42g。適應(yīng)性喂養(yǎng)7天后,隨機(jī)分為2組,一組10只,一組30只,分別飼喂普通飼料、高脂高糖飲食,在第8周時腹腔注射STZ,使用STZ+高脂高糖聯(lián)合造模,以30mg/kg腹腔注射造模,3天后測空腹血糖,將空腹血糖大于15mmol/L視作為造模成功。將造模組隨機(jī)分為3組,每組10只,一組繼續(xù)飼喂高脂高糖(M組),一組飼喂高脂高糖+二甲雙胍(M+MET組),一組飼喂高脂高糖+胰島素(M+INS);普通飼料組喂普通飼養(yǎng)(C組,10只)。MET按200mg/Kg劑量灌胃,不給予MET的大鼠使用生理鹽水灌胃。共喂養(yǎng)12周。2.2觀察指標(biāo)(1)血標(biāo)本用于檢測血糖濃度(葡萄糖氧化酶法)及胰島素含量(放射免疫法),以此計算胰島素抵抗指數(shù)(2)ELISA法檢測血清中CTRP9、TNF-α的表達(dá)水平(3)Western blot法檢測IKK-β及ser181蛋白表達(dá)的表達(dá)水平(4)RT-PCR法檢測CTRP9、TNF-α、IL-6基因的表達(dá)水平3結(jié)果3.1 HOMA-IR各組大鼠血清HOMA-IR水平之間的差異有統(tǒng)計學(xué)意義(F=169.923,P=0.0000.05)。M組、M+Ins組和M+Met組的HOMA-IR均比Control組高,差異有統(tǒng)計學(xué)意義(P0.05)。M+Ins組、M+Met組的HOMA-IR均比M組低,差異有統(tǒng)計學(xué)意義(P0.05)。見表3.1、3.2。3.2血清CTRP9的表達(dá)水平各組大鼠骨骼肌組織平均CTRP9水平之間的差別有統(tǒng)計學(xué)意義(F=98.773,P=0.0000.05)。進(jìn)一步兩兩比較,M+Ins組、M+Met組的CTRP9水平均高于M組,差異有統(tǒng)計學(xué)意義(P0.05)。M+Ins組、M+Met組的CTRP9水平均低于C組,差異有統(tǒng)計學(xué)意義(P0.05)。見表3.3、3.4。3.3大鼠HOMA-IR與CTPR9水平的相關(guān)性分析M組,HOMA-IR與血清CTRP9水平呈負(fù)相關(guān),r=-0.418,P=0.0190.05;M+MET組,HOMA-IR與血清CTRP9水平呈負(fù)相關(guān),r=-0.306,P=0.0220.05。見圖3.2。3.4各組大鼠骨骼肌組織CTRP9、TNF-α、IL-6基因的表達(dá)水平與C組比較,M組大鼠脂肪細(xì)胞CTRP9 mRNA表達(dá)水平顯著降低,有統(tǒng)計學(xué)意義(P0.05);與M組比較,M+Ins組、M+Met組CTRP9 mRNA表達(dá)水平顯著升高,有統(tǒng)計學(xué)意義(P0.05);與C組比較,M組大鼠骨骼肌TNF-αmRNA表達(dá)水平顯著下降,有統(tǒng)計學(xué)意義;與M組比較,M+Ins組、M+Met組TNF-αmRNA表達(dá)水平顯著升高,有統(tǒng)計學(xué)意義(P0.05)。與C組比較,M組大鼠骨骼肌IL-6 mRNA表達(dá)水平顯著下降,有統(tǒng)計學(xué)意義;與M組比較,M+Ins組、M+Met組IL-6 mRNA表達(dá)水平顯著升高,有統(tǒng)計學(xué)意義。3.5各組大鼠骨骼肌組織IKK-β、ser181蛋白的表達(dá)4結(jié)論(1)2型糖尿病大鼠血清中CTRP9表達(dá)水平降低。(2)2型糖尿病大鼠胰島素抵抗與CTRP9的表達(dá)呈負(fù)相關(guān)。(3)二甲雙胍降低糖尿病大鼠骨骼肌組織ser181蛋白的表達(dá),對IKK-β影響不大。(4)二甲雙胍的上述作用有可能是通過抑制IKK-β的磷酸化實現(xiàn)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2
本文編號：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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2
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