DEHP对青春期大鼠糖代谢的影响及其机制

发布时间：2018-05-21 19:23

本文选题：DEHP + 糖代谢　；参考：《吉林大学》2017年硕士论文

【摘要】：随着社会经济的发展和人民生活水平的提高,糖尿病患病率逐年呈上升趋势,已成为影响人类生命健康的主要慢性非传染性疾病之一。其中儿童青少年患病比例也在逐步扩大,特别是2型糖尿病。而2型糖尿病的发生又与代谢综合征联系紧密,已有研究提示环境中存在的内分泌干扰物等外源性化学物质可以干扰机体的正常代谢,特别是以DEHP为主要代表的邻苯二甲酸酯类物质,由于DEHP作为增塑剂使用广泛,而其又容易从材料中挥发和溶解出来,因此不但对生态环境造成污染,还可通过空气、水、食物等多种媒介进入机体,进而危害机体健康。目的:观察DEHP暴露对大鼠糖代谢的影响,了解大鼠肝脏和胰腺组织胰岛素受体、瘦素受体以及JAK2-STAT3-SOCS3通路相关蛋白的表达情况,探讨DEHP致大鼠糖代谢紊乱的可能机制,为深入了解环境内分泌干扰物DEHP的毒性,保护人群健康提供科学依据,为Met S和T2D等代谢性疾病的研究以及预防提供新思路。方法:选择健康清洁级Wistar大鼠80只,雌雄各半,21日龄,重量为(50±5)g,将80只青春期大鼠按照性别分别随机分为四组:(1)对照组(玉米油);(2)低剂量组(5mg/kg/d DEHP,1/6000 LD50);(3)中剂量组(50 mg/kg/d DEHP,1/600 LD50);(4)高剂量组(500 mg/kg/d DEHP,1/60 LD50)。适应性饲养一周后,用玉米油配制相应浓度的DEHP后,每天上午9:00进行灌胃染毒,称量饮水量、摄食量和体重,并对实验动物进行大体观察,持续染毒4周。染毒结束后,禁食12h,将大鼠麻醉,心脏取血,离心后收集血清,并剪取肝脏和胰腺;用生化分析仪检测空腹血糖,用ELISA法测定血清胰岛素和瘦素水平,并计算各组青春期大鼠的HOMA-IR值;计算肝脏和胰腺的脏器系数;HE染色法观察青春期大鼠肝脏和胰腺的病理组织变化;实时荧光定量PCR法测定肝脏和胰腺中JAK2、STAT3、SOCS3和Ob-R基因m RNA的表达情况;免疫组织化学法对JAK2、STAT3、SOCS3和Ob-R蛋白在肝脏和胰腺中进行定位,并应用Image-Pro Plus 6.0软件根据不同组间的IOD值进行半定量蛋白分析;Western Blot法测定肝脏和胰腺中JAK2、STAT3、SOCS3、Ob-R和IR蛋白表达水平,并进行分析。结果:1.DEHP对青春期大鼠的一般毒性(1)50mg/kg/d和500mg/kg/d DEHP暴露组的青春期大鼠出现行动迟缓、精神萎靡不振、被毛疏松无光泽并且伴有脱毛现象,而对照组和5mg/kg/d DEHP暴露组的青春期大鼠精神状态良好、活动正常、皮毛光亮。(2)50mg/kg/d和500mg/kg/d DEHP暴露组的青春期大鼠摄食、饮水和体重均显著低于5mg/kg/d DEHP暴露组(P0.05),而5mg/kg/d DEHP暴露组青春期大鼠摄食量、饮水量和体重显著高于对照组(P0.05)。(3)500mg/kg/d DEHP暴露组的青春期大鼠肝脏脏器系数显著高于其他各组(P0.05),而各组大鼠胰腺脏器系数均无显著差异(P0.05)。2.DEHP对青春期大鼠的血糖稳态和相关激素的影响50mg/kg/d和500mg/kg/d DEHP暴露组的青春期大鼠空腹血糖、血清胰岛素,血清瘦素和HOMA-IR值均显著高于对照组(P0.05),并且500mg/kg/d DEHP暴露组显著高于5mg/kg/d DEHP暴露组(P0.05)。3.DEHP对青春期大鼠肝脏和胰腺组织形态的影响(1)肝脏:50mg/kg/d和500mg/kg/d DEHP暴露组青春期大鼠肝细胞染色较浅、排列疏松、部分水肿、胞浆比较清亮,有炎性细胞浸润和空泡样变,并且500mg/kg/d染毒组青春期大鼠肝细胞出现轻度坏死现象。(2)胰腺:50mg/kg/d和500mg/kg/d染毒组青春期大鼠胰岛数量相对减少,分布零散,边界不清,并且细胞体积稍有增大4.DEHP对青春期大鼠肝脏和胰腺相关基因m RNA表达的影响(1)肝脏:500mg/kg/d DEHP暴露组大鼠肝脏Ob-R基因m RNA表达显著低于5mg/kg/d DEHP暴露组(P0.05);500mg/kg/d DEHP暴露组大鼠肝脏SOCS3 m RNA表达显著高于对照组和5mg/kg/d DEHP暴露组(P0.05);JAK2和STAT3 m RNA表达各组间均无显著差异(P0.05)。(2)胰腺:500mg/kg/d DEHP暴露组大鼠胰腺Ob-R基因m RNA表达显著低于其他各组(P0.05);500mg/kg/d DEHP暴露组大鼠胰腺SOCS3基因m RNA表达显著高于对照组(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露组大鼠胰腺STAT3基因m RNA表达均显著高于对照组(P0.05);JAK2 m RNA表达各组均无显著差异(P0.05)。5.DEHP对青春期大鼠肝脏和胰腺相关蛋白表达的影响(1)肝脏:免疫组织结果显示,500mg/kg/d DEHP暴露组大鼠肝脏Ob-R蛋白表达显著低于对照组(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露组大鼠肝脏SOCS3蛋白表达均显著高于对照组(P0.05);500mg/kg/d DEHP暴露组大鼠肝脏JAK2和STAT3蛋白表达均显著高于对照组(P0.05)。Western Blot结果显示,500mg/kg/d DEHP暴露组大鼠肝脏IR蛋白表达显著低于对照组(P0.05);5mg/kg/d、50mg/kg/d和500mg/kg/d DEHP暴露组大鼠肝脏Ob-R蛋白表达均显著低于对照组(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露组大鼠肝脏SOCS3蛋白表达均显著高于对照组和5mg/kg/d DEHP暴露组(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露组大鼠肝脏JAK2和STAT3蛋白表达均显著高于对照组(P0.05)。(2)胰腺:免疫组织结果显示,500mg/kg/d DEHP暴露组大鼠胰腺Ob-R蛋白表达显著低于其他各组(P0.05);500mg/kg/d和50mg/kg/d DEHP暴露组大鼠胰腺SOCS3蛋白表达均显著高于对照组(P0.05);JAK2和STAT3蛋白表达各组间均无显著差异(P0.05)。Western Blot结果显示,500mg/kg/d DEHP暴露组大鼠胰腺IR蛋白表达显著低于对照组和5mg/kg/d DEHP暴露组(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露组大鼠胰腺Ob-R蛋白表达均显著低于对照组(P0.05);500mg/kg/d DEHP暴露组大鼠胰腺SOCS3蛋白表达显著高于其他各组(P0.05);500mg/kg/d DEHP暴露组大鼠胰腺JAK2和STAT3蛋白表达均显著高于对照组(P0.05)。结论:(1)低剂量DEHP暴露可能会促进青春期大鼠生长发育,体重增速加快,而高剂量的DEHP暴露可能会减缓青春期大鼠体重增加;高剂量DEHP暴露可使青春期大鼠肝脏的脏器系数增加。(2)高剂量DEHP暴露可能会影响青春期大鼠正常糖代谢,扰乱血糖稳态,引起胰岛素和瘦素水平升高,出现以胰岛素抵抗为典型症状的代谢综合征。(3)高剂量DEHP暴露可降低青春期大鼠肝脏和胰腺胰岛素受体和瘦素受体的表达,受体信号传导受阻可能是高剂量DEHP暴露所导致青春期大鼠瘦素水平升高的机制之一(4)高剂量DEHP暴露可能会增加肝脏和胰腺SOCS3 m RNA表达,并且使青春期大鼠肝脏和胰腺JAK2、STAT3和SOCS3蛋白表达增加。由于SOCS3的高表达而引起的受体后信号转导抑制可能是青春期大鼠产生胰岛素抵抗和瘦素水平升高的机制之一。
[Abstract]:With the development of social economy and the improvement of people's living standard, the prevalence of diabetes has been increasing year by year, and has become one of the main chronic non infectious diseases affecting human life and health. Among them, the proportion of children and adolescents is gradually expanding, especially type 2 diabetes. And the incidence of type 2 diabetes is associated with metabolic syndrome. Closely, it has been suggested that exogenous chemicals such as endocrine disruptors, such as endocrine disruptors, can interfere the normal metabolism of the body, especially the DEHP as the main representative of phthalic acid esters. Because DEHP is widely used as plasticizer, it is easily volatilized and dissolved from the material. Therefore, it is not only made to the ecological environment. In order to investigate the effect of DEHP exposure on the metabolism of sugar in rats, the effects of exposure on glucose metabolism in rats, the expression of insulin receptor, leptin receptor and JAK2-STAT3-SOCS3 pathway related egg white in rat liver and pancreas were observed, and the disorder of glucose metabolism caused by DEHP in rats was investigated. The possible mechanism provides a scientific basis for understanding the toxicity of environmental endocrine disrupting interferon DEHP, protecting the health of the population, and providing new ideas for the study and prevention of metabolic diseases such as Met S and T2D. Methods: 80 healthy and clean Wistar rats were selected, the male and the male were half, 21 days old, and the weight was (50 + 5) g, and 80 puberty rats were divided by sex respectively. Randomly divided into four groups: (1) the control group (corn oil); (2) low dose group (5mg/kg/d DEHP, 1/6000 LD50); (3) medium dose group (50 mg/kg/d DEHP, 1/600 LD50); (4) high dose group (500 mg/kg/d DEHP, 1/60 LD50). After adaptation to a week, using jade rice oil to prepare the corresponding concentration, at 9:00 every morning, gavage, weighing water quantity, feeding, feeding The quantity and weight were observed, and the experimental animals were observed for 4 weeks. After the end of the infection, the rats were fasted 12h, the rats were anaesthetized, the heart was collected, the serum was collected, the liver and the pancreas were collected, the liver and the pancreas were cut, the fasting blood glucose was detected by the biochemical analyzer, the serum islet and leptin levels were measured by the ELISA method, and the HOMA-IR value of the rats in each group was calculated. The organ coefficients of the liver and pancreas were calculated. The pathological changes of the liver and pancreas in the puberty rats were observed by HE staining. The expression of M RNA, JAK2, STAT3, SOCS3 and Ob-R in the liver and pancreas was measured by real time fluorescence quantitative PCR, and the immunohistochemical method was used to locate JAK2, STAT3, SOCS3 and Ob-R protein in the liver and pancreas, and should be located in the liver and pancreas. Image-Pro Plus 6 software was used for semi quantitative protein analysis based on IOD values between different groups. Western Blot was used to determine the protein expression levels of JAK2, STAT3, SOCS3, Ob-R and IR in the liver and pancreas. Results: 1.DEHP toxicity of 1.DEHP on adolescent rats (1) action in adolescent rats Retardation, depressed spirit, glossy hair loosening and hair removal, while the adolescent rats in the control group and the 5mg/kg/d DEHP exposed group had good mental state, normal activity and bright fur. (2) the feeding of puberty rats in 50mg/kg/d and 500mg/kg/d DEHP exposure groups were significantly lower than those of 5mg/kg/d DEHP exposure group (P0.05). In 5mg/kg/d DEHP exposure group, the intake of puberty rats was significantly higher than that of the control group (P0.05). (3) the liver organ coefficient of the puberty rats in the 500mg/kg/d DEHP exposure group was significantly higher than that of the other groups (P0.05), but the pancreas organ coefficient of the rats in each group had no significant difference (P0.05).2.DEHP to the blood glucose homeostasis and related excitation in the puberty rats. The effect of 50mg/kg/d and 500mg/kg/d DEHP exposure group on fasting blood glucose, serum insulin, serum leptin and HOMA-IR were significantly higher than that of the control group (P0.05), and the 500mg/kg/d DEHP exposure group was significantly higher than 5mg/kg/d DEHP exposure group (P0.05).3.DEHP on the liver and pancreatic tissue morphology of the puberty rats (1) livers: 50mg/ The liver cells of kg/d and 500mg/kg/d DEHP exposed rats were dyed shallow, loosely arranged, partial edema, cytoplasm relatively clear, inflammatory cell infiltration and vacuolating, and there was a slight necrosis in the liver cells of the puberty rats of 500mg/kg/d. (2) pancreas: the number of pancreatic islets in 50mg/ kg/d and 500mg/kg/d exposed rats was relative to the number of islets of the rats. The effect of 4.DEHP on the expression of M RNA in liver and pancreas related genes in puberty rats (1) liver: Ob-R gene m RNA expression in liver of 500mg/kg/d DEHP exposure group was significantly lower than 5mg/kg/d DEHP exposure group (P0.05). There was no significant difference between the control group and the 5mg/kg/d DEHP exposure group (P0.05), and there was no significant difference in the expression of JAK2 and STAT3 m RNA (P0.05). (2) the pancreas Ob-R gene was significantly lower in the pancreas of the 500mg/kg/d DEHP exposure group than in the other groups. The expression of STAT3 gene m RNA in the pancreas of g/kg/d and 500mg/kg/d DEHP exposed rats was significantly higher than that in the control group (P0.05), and there was no significant difference in the expression of JAK2 m RNA in each group (P0.05).5.DEHP on the expression of liver and pancreas related proteins in puberty rats (1) liver: immune tissue results showed the liver protein table of rats exposed group The expression of SOCS3 protein in liver of 50mg/kg/d and 500mg/kg/d DEHP exposed rats was significantly higher than that of control group (P0.05), and the expression of JAK2 and STAT3 protein in liver of 500mg/kg/d DEHP exposed rats was significantly higher than that of control group (P0.05) (P0.05). The expression of Ob-R protein in liver of rats exposed to 5mg/kg/d, 50mg/kg/d and 500mg/kg/d DEHP was significantly lower than that of the control group (P0.05), and the expression of SOCS3 protein in the liver of 50mg/kg/d and 500mg/kg/d DEHP exposed rats was significantly higher than that of the control group and the 5mg/kg/d exposed group. The expression of JAK2 and STAT3 protein was significantly higher than that of the control group (P0.05). (2) pancreas: the results of immuno tissue showed that the expression of Ob-R protein in the pancreas of 500mg/kg/d DEHP exposed rats was significantly lower than that of the other groups (P0.05), and the expression of SOCS3 protein in the pancreas of 500mg/kg/d and 50mg/kg/d DEHP exposed rats was significantly higher than that of the control group (P0.05). There was no significant difference between each group (P0.05).Western Blot results showed that the expression of IR protein in the pancreas of 500mg/kg/d DEHP exposed rats was significantly lower than that of the control group and 5mg/kg/d DEHP exposure group (P0.05), and the expression of pancreatic protein in the pancreas of 50mg/kg/d and 500mg/kg/d DEHP exposed rats was significantly lower than that of the control group. The expression of S3 protein was significantly higher than that in other groups (P0.05), and the expression of JAK2 and STAT3 protein in the pancreas of 500mg/kg/d DEHP exposed rats was significantly higher than that of the control group (P0.05). Conclusion: (1) low dose DEHP exposure may promote the growth and development of puberty rats and accelerate the growth of body weight, and high dose of DEHP exposure may slow the weight gain of adolescent rats. High dose DEHP exposure can increase the organ coefficient of liver in puberty rats. (2) high dose DEHP exposure may affect normal glucose metabolism in puberty rats, disturb blood glucose homeostasis, increase insulin and leptin levels, and develop metabolic syndrome with insulin resistance as typical symptoms. (3) high dose DEHP exposure can reduce the liver of puberty rats The expression of insulin receptor and leptin receptor in the pancreas and pancreas, and the obstruction of receptor signal conduction may be one of the mechanisms of high levels of leptin in puberty rats induced by high dose DEHP exposure (4) high dose DEHP exposure may increase the expression of SOCS3 m RNA in the liver and pancreas, and make the JAK2, STAT3 and SOCS3 protein tables of the liver and pancreas of the puberty rats The inhibition of post receptor signal transduction caused by the high expression of SOCS3 may be one of the mechanisms of insulin resistance and leptin levels in puberty rats.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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