【作者】 武军；

【导师】 李延青；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 研究背景糖尿病是因为胰岛素分泌缺陷和／或胰岛素作用缺陷所引起的以慢性高血糖为特征的代谢性疾病。糖尿病的患病率在逐年增加,由于糖尿病的高患病率和高致残率,不仅影响患者的生产能力和生活质量,加上高昂的医疗费用,造成严重的社会问题和经济问题,成为威胁人类健康的全球性卫生问题。因此,加强对糖尿病的研究具有重要的意义。近年来,糖尿病与胃肠道特别是胃排空之间的关系越来越受到重视,一方面胃排空的速度会影响到餐后血糖的高低;另一方面,胃肠道可以分泌多种激素,参与血糖调节。众所周知,糖尿病病人存在胃排空减慢即胃轻瘫。但胃轻瘫是一种相对少见的糖尿病并发症,多发生在糖尿病病程的后期,其发生机制认为主要与糖尿病自主神经病变有关,然而越来越多的研究发现,在糖尿病的早期,包括无自主神经病变的1型糖尿病病人和2型糖尿病病人,也包括STZ诱导的糖尿病大鼠早期和自发性糖尿病BB大鼠,存在胃排空加快。虽然人们对糖尿病胃轻瘫给予许多关注,并对其机制进行了比较深入的研究,但对糖尿病早期出现的胃排空加快的机制研究很少。Ghrelin是1999年由Kojima等在大鼠胃组织中分离出的一种由28个氨基酸残基组成的活性多肽,是生长激素促分泌剂受体(GHSR)的内源性配体。Ghrelin主要由胃底部黏膜泌酸腺X／A样细胞合成并分泌入血,血液中的ghrelin主要来自胃。除了胃以外,在肠道、胰腺、垂体以及下丘脑等器官中也发现ghrelin内存在。Ghrelin可以促进生长激素(GH)分泌,增强食欲,减少脂肪利用、增加胃酸分泌、促进胃肠动力,增加体重等。研究证实,ghrelin可以促进大鼠和人的液体和固体排空。在正常大鼠和人中,空腹时血ghrelin水平明显升高,进食后血ghrelin水平明显下降并持续,在肥胖和初发1型糖尿病病人,进食后血ghrelin水平无明显下降,在STZ诱导的糖尿病大鼠中,空腹和进食后血ghrelin水平均明显高于正常大鼠。有研究发现,STZ诱导的糖尿病大鼠早期,胃组织合成ghrelin增加,释放入血增加导致高ghrelin血症,在基于以上研究,我们推测ghrelin是否在糖尿病早期的胃排空加快中起作用值得进一步研究,在这方面的研究很少。Obestatin是由Zhang等在2005年从大鼠胃组织中分离出来的一种由23个氨基酸残基组成的Ghrelin相关肽。它与ghrelin来源于同一前体蛋白,Obestatin来源于proghrelin的C末端,而ghrelin来源于proghrelin的N末端。Obestatin被认为是GPR39的内源性配体。尽管来源于同一前体蛋白,但研究发现obestatin的一些生物学功能与ghrelin相反,如obestatin可以减少食物摄入,降低体重的增加,抑制胃排空,抑制空肠肌肉的收缩等,但研究结果也存在争议。因此,Obestatin的确切生物学功能需要进一步研究。Obestatin在STZ诱导的糖尿病大鼠早期的胃排空中所起作用的研究尚未见报道。目的：本研究通过利用STZ腹腔注射建立胰岛素缺乏糖尿病大鼠模型,并同时给予外源性补充胰岛素治疗,观察治疗前后STZ诱导的糖尿病大鼠早期液体胃排空的变化,同时观察治疗前后血糖、胰岛素、血浆ghrelin和obestatin水平、胃底粘膜中ghrelin和obestatin的表达以及ghrelin mRNA在胃底表达情况、下丘脑ghrelin、obestatin水平及受体mRNA表达的变化,研究STZ诱导的糖尿病大鼠早期液体胃排空与ghrelin和obestatin的关系,并探讨其中的机制。方法：40只Wistar大鼠随机分为正常对照组(NC组)、糖尿病组(DM组)、低剂量胰岛素干预组(LD组)、高剂量胰岛素干预组(HD组)。DM组、LD组和HD组大鼠一次性腹腔注射链脲佐菌素(STZ) (50mg/kg体重)制备糖尿病大鼠模型,LD组和HD组分别给以中性胰岛素4单位和8单位皮下注射,于注射STZ后2周后用甲基纤维素-酚红溶液灌胃法测定液体胃排空,腹主动脉取血并留取胃底组织和下丘脑组织,用酶免法测定血浆和下丘脑ghrelin和obestatin水平,用免疫组化检测大鼠胃底Ghrelin和obestatin的积分吸光度、半定量RT-PCR技术检测大鼠胃底Ghrelin mRNA的表达和下丘脑GHSR mRNA和GPR-39 mRNA的表达。应用SPSS11.0统计软件进行统计,P<0.05为有显著性差异。结果：1、注射STZ3天至2周后,DM组大鼠体重明显低于NC组(P<0.01),DM组大鼠血糖明显高于NC组(P<0.01)；注射STZ2周后,DM组大鼠血insulin水平明显低于NC组(P<0.01),DM组大鼠胃液体排空率明显快于NC组(P<0.01)。2、注射STZ2周后,DM组大鼠血浆ghrelin水平明显高于NC组(P<0.01),DM组大鼠胃底ghrelin积分吸光度明显低于NC组(P<0.01),DM组大鼠胃底ghrelin mRNA的表达水平明显高于NC组(P<0.01),DM组大鼠下丘脑ghrelin水平明显高于NC组(P<0.01),DM组大鼠下丘脑GHSR mRNA表达水平明显高于NC组(P<0.01)。3、注射STZ2周后,DM组大鼠血浆obestatin水平均明显高于NC组(P<0.01),DM组大鼠胃底obestatin积分吸光度明显低于NC组(P<0.01),DM组大鼠下丘脑obestatin水平与NC组没有明显差别(p>0.05),DM组和NC组大鼠下丘脑中未检测到GPR-39 mRNA的表达。4、外源性补充胰岛素治疗后,LD组和HD组大鼠体重明显高于DM组(P<0.01),HD组大鼠体重明显高于LD组(P<0.05)；LD组和HD组大鼠血糖明显低于DM组(P<0.01),但仍高于NC组(P<0.01),HD组大鼠血糖明显低于LD组(P<0.01)；LD组和HD组大鼠insulin水平明显高于DM组(P<0.05,P<0.01),HD组大鼠insulin水平明显高于LD组(P<0.01)；LD组和HD组大鼠胃液体排空率明显低于DM组(P<0.01),但仍明显高于NC组(P<0.01),HD组大鼠胃液体排空率明显低于LD组(P<0.01)。5、外源性补充胰岛素治疗后,LD组和HD组大鼠血浆ghrelin水平明显低于DM组(P<0.01),LD组大鼠血浆ghrelin水平仍明显高于NC组(P<0.01),HD组和NC组大鼠血浆ghrelin水平没有明显差别(p>0.05)；LD组和HD组大鼠胃底ghrelin积分吸光度明显高于DM组(P<0.01),但仍明显低于NC组(P<0.01,P<0.05),HD组大鼠胃底ghrelin积分吸光度明显高于LD组(P<0.05)；LD组和HD组大鼠胃底ghrelin mRNA的表达水平明显低于DM组(P<0.05,P<0.01),LD组大鼠胃底ghrelin mRNA的表达水平仍明显高于NC组(P<0.01),HD组和NC组大鼠胃底ghrelin mRNA的表达水平没有明显差别(p>0.05)；LD组和HD组大鼠下丘脑ghrelin水平明显低于DM组(P<0.05,P<0.01),LD组大鼠下丘脑ghrelin水平仍明显高于NC组(P<0.05),HD组和NC组大鼠下丘脑ghrelin水平没有明显差别(p>0.05)；LD组和HD组大鼠下丘脑GHSR mRNA表达水平与DM组没有明显差别(p>0.05),均明显高于NC组(P＜0.01)。6、外源性补充胰岛素治疗后,LD组和HD组大鼠血浆obestatin水平与DM组之间没有明显差别(p>0.05),均明显高于NC组(P<0.01)；LD组和HD组大鼠胃底obestatin积分吸光度与DM组之间没有明显差别(p>0.05),均明显低于NC组(P<0.01)；LD组和HD组以及DM组和NC组四组之间大鼠下丘脑obestatin水平之间没有明显差别(p>0.05)；LD组和HD组大鼠下丘脑中未检测到GPR-39 mRNA的表达。结论：1、STZ诱导的糖尿病大鼠早期液体胃排空明显加快。2、STZ诱导的糖尿病大鼠早期存在高ghrelin血症,胃底粘膜ghrelin的表达明显减少,ghrelin mRNA在胃底组织表达明显增加,下丘脑ghrelin水平明显增加,下丘脑GHSR mRNA表达明显增加。3、STZ诱导的糖尿病大鼠早期存在高obestatin血症,胃底粘膜obestatin的表达明显减少,下丘脑中obestatin水平无明显变化,下丘脑中未见GPR-39 mRNA的表达。4、外源性补充胰岛素治疗后,在使STZ诱导的早期糖尿病大鼠高血糖和低胰岛素血症明显改善的同时,可以使其加快的液体胃排空明显下降。5、外源性补充胰岛素治疗后,可以使STZ诱导的糖尿病大鼠早期的血浆ghrelin水平明显下降,胃底粘膜ghrelin的表达明显增加,ghrelin mRNA在胃底组织表达明显减少,下丘脑ghrelin水平明显下降,但下丘脑GHSR mRNA表达无明显变化。6、外源性补充胰岛素治疗后,对STZ诱导的糖尿病大鼠早期的高obestatin血症和胃底粘膜obestatin的低表达无明显影响,对下丘脑中obestatin水平无明显影响,下丘脑中未见GPR-39mRNA的表达。7、STZ诱导的糖尿病大鼠早期胃液体排空明显加快,内源性ghrelin的增加可能在STZ诱导的糖尿病大鼠早期胃排空加快中起重要作用,内源性obestatin在其中的作用有待进一步探讨。更多还原

【Abstract】 BACKGROUNDDiabetes mellitus is a metabolic disease characterized by chronic hyperglycemia because of defects in insulin secretion and/or defects in insulin action. The prevalence rate of diabetes mellitus increased year by year. As the high prevalence and high disability of diabetes mellitus, it not only affects the quality of life but also high medical costs. Diabetes mellitus results in serious social problems and economic problems and become global health issues that a threat to human health. Therefore, to enhance the study of diabetes mellitus has important significance.In recent years, the relationship between diabetes and gastrointestinal tract, especially gastric emptying was received more and more attention. On the one hand the speed of gastric emptying may affect the postprandial blood glucose level, on the other hand, gastrointestinal tract can secrete multiple glucoregulatory hormones involved in regulating blood glucose.It is well recognized that certain populations of diabetic patients have delayed gastric emptying called gastroparesis. Gastroparesis is a relatively rare complication that occurs late in diabetes. Diabetic gastroparesis is explained by the autonomic neuropathy associated with diabetes. However, rapid gastric emptying can be seen in subgroups of patients in the early stages of Type 2 diabetes, non-insulin-dependent diabetes mellitus (Type 2 diabetes), and neuropathy-free, insulin-dependent diabetes mellitus (Type 1 diabetes) It has also been reported that gastric emptying is accelerated in the early stages of streptozotocin-induced diabetes in rats and in spontaneously diabetic BB rats. Although a great deal of attention has been paid to diabetic gastroparesis, the mechanism of accelerated gastric emptying in the early stages of diabetes has not been fully studied.Ghrelin is a 28 amino acid peptide that originally was isolated from the rat stomach in 1999 by.Kojima et al. Ghrelin, an orexigenic peptide, is the endogenous ligand for the growth hormone secretagogue receptor(GHS-R). Ghrelin is produced by A-like cells in the in the oxyntic glands of the gastric mucosa and is released into circulation. The stomach, especially the fundus part, is the major source of circulating ghrelin. Besides the stomach, the peptide is also expressed at lower levels in many other tissues such as intestine, pancreas, pituitary, hypothalamus, liver and so on. Ghrelin has potent stimulatory effects on GH secretion and food intake. In addition, it was shown that ghrelin promotes gastric emptying and a positive energy balance, thus increasing adiposity and body weightIt is well established that ghrelin stimulates gastrointestinal motility. Ghrelin accelerates solid gastric emptying in humans, rats, and mice. Gastric emptying of nonnutrient liquid is also accelerated by ghrelin in rats and mice. Circulating levels of ghrelin rise before and decrease after a meal in normal-weight subjects. In contrast, the postprandial reduction of ghrelin is not obvious in obese subjects and patients with new onset childhood Type 1 diabetes In STZ-induced diabetic rats, postprandial ghrelin concentrations are higher than in control rats and return to fasted levels rapidly after feeding. Gastric ghrelin-immunoreactive cells are slightly decreased in STZ-induced diabetic rats, whereas preproghrelin mRNA levels are extremely higher in STZ rats than in control rats. The increased plasma ghrelin levels and the decreased gastric ghrelin cells in the diabetic rats may be due to an increase in ghrelin release from the stomach into the bloodstream.Based on these evidences, we hypothesize that ghrelin contributes to rapid gastric emptying in the early stages of STZ-induced diabetes.Obestatin is a newly discovered peptide of 23 amino acids isolated from rat stomach in 2005 by Zhang et al. It derived from the same precursor protein as ghrelin. Obestatin is derived from the carboxy-terminal part of proghrelin, whereas ghrelin is derived from the N-terminal part of the same precursor. The distribution of obestatin-and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats was characterized. Obestatin is proposed to be the endogenous ligand for GPR39. Notwithstanding the same precursor, the biological activity of obestatin is reportedly opposite to that of ghrelin. It has been reported that obestatin has inhibitory effects on feeding and digestive motility and thus antagonizes the stimulatory effect of ghrelin. Zhang et al first reported that obestatin can suppresse cumulative food intake, decrease body weight gain, and inhibited gastric emptying and jejunal muscle contraction in mice. Since then, however, the inhibitory effects of obestatin on food intake and gastrointestinal motility have remained controversial, and further studies are needed to determine the physiological function of obestatin. The role of obestatin in the gastric emptying in the early stages of streptozotocin-induced diabetic rats has not been studied.Objective:This study was conducted in streptozotocin-induced diabetic rats featuring with insulin deficiency and treated with insulin at the same time. The aim of our study was to investigate the gastric emptying, plasma levels of ghrelin and obestatin, the expression levels of ghrelin, obestatin and ghrelin mRNA in the fundus of stomach, levels of ghrelin and obestatin in the hypothalamus, the expression levels of GHSR mRNA and GPR-39 mRNA in the hypothalamus in the early stages of streptozotocin-induced diabetic rats, and explore the relation between them.Methods:Forty Wistar rats were randomly divided into four groups:normal control group(NC group), diabetic mellitus group(DM group), low-dose insulin-treated group(LD group) and high-dose insulin-treated group(HD group). Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ). Rats in LD group and HD group were received 4U/animal/day and 8U/animal/day recombinant human insulin (Humulin N) by subcutaneous injection beginning the 4th day after the STZ injection. Two weeks after the STZ injection, Gastric emptying was measured by intragastric administration of methylcelluose-phenol red solution. The levels of ghrelin and obestatin in the plasma and hypothalamus were measured by EISIA method. The expression levels of ghrelin and obestatin in the fundus of stomach were detected by immunohistochemistry The expression of ghrelin mRNA in the fundus of stomach and the expression levels of GHSR mRNA and GPR-39 mRNA in the hypothalamus were measured by semi-quantitative RT-PCR. Statistical analysis was performed by using SPSS11.0 statistical software. P values<0.05 were considered as significant.Results1.From there days to two weeks after the STZ injection, the body weight decreased significantly and the blood glucose levels increased significantly in DM group compared to those in NC group(P<0.01). Two weeks after the STZ injection,the blood insulin levels decreased significantly in DM group compared to those in NC group(P<0.01). Two weeks after the STZ injection, the liquid gastric emptying accelerated significantly in DM group compared to those in NC group(P<0.01).2.Two weeks after the STZ injection, the plasma ghrelin levels increased significantly in DM group compared to those in NC group(P<0.01). The ghrelin integral optical density in the gastric fundus decreased significantly in DM group compared to those in NC group(P<0.01). The ghrelin mRNA expression levels in the gastric fundus increased significantly in DM group compared to those in NC group(P<0.01). The ghrelin levels in the hypothalamus increased significantly in DM group compared to those in NC group(P<0.01). The GHSR mRNA expression levels in the hypothalamus increased significantly in DM group compared to those in NC group(P<0.01).3.Two weeks after the STZ injection, the plasma obestatin levels increased significantly in DM group compared to those in NC group(P<0.01).The obestatin integral optical density in the gastric fundus decreased significantly in DM group compared to those in NC group(P<0.01).The obestatin levels in the hypothalamus were no statistical differnce between DM and NC group(P> 0.05). There was no GPR-39 mRNA expression detected in the hypothalamus in DM and NC group.4. After insulin treatments, the body weight increased significantly in LD and HD group compared to those in DM group(P<0.01) and the body weight increased significantly in HD group compared to those in LD group(P<0.05). The blood glucose levels decreased significantly in LD and HD group compared to those in DM group(P<0.01) and increased significantly in LD and HD group compared to those in NC group(P<0.01). The blood glucose levels decreased significantly in HD group compared to those in LD group (P<0.01). The blood insulin levels increased significantly in LD and HD group compared to those in DM group(P<0.01) and increased significantly in HD group compared to those in LD group(P<0.01). The liquid gastric emptying slowed significantly in LD and HD group compared to those in DM group(P<0.01) and accelerated significantly in LD and HD group compared to those in NC group(P<0.01). The liquid gastric emptying slowed significantly in HD group compared to those in LD group(P<0.01).5.After insulin treatments, the plasma ghrelin levels decreased significantly in LD and HD group compared to those in DM group(P<0.01). The plasma ghrelin levels increased significantly in LD group compared to those in NC group(P<0.01) and were no statistical differnce in HD group compared to those in NC group(P>0.05). The ghrelin integral optical density in the gastric fundus increased significantly in LD and HD group compared to those in DM group(P<0.01) and decreased significantly in LD and HD group compared to those in NC group(P<0.01,P<0.05). The ghrelin integral optical density in the gastric fundus increased significantly in HD group compared to those in LD group(P＜0.01) The ghrelin mRNA expression levels in the gastric fundus decreased significantly in LD and HD group compared to those in DM group(P<0.05,P<0.01). The ghrelin mRNA expression levels in the gastric fundus increased significantly in LD group compared to those in NC group (P<0.01) and were no statistical differnce in HD group compared to those in NC group(P>0.05). The ghrelin levels in the hypothalamus decreased significantly in LD and HD group compared to those in DM group(P<0.01). The ghrelin levels in the hypothalamus increased significantly in LD group compared to those in NC group(P<0.01) and were no statistical differnce in HD group compared to those in NC group(P>0.05). The GHSR mRNA expression levels in the hypothalamus were no statistical differnce in LD and HD group compared to those in DM group(P>0.05) and increased significantly in LD and HD group compared to those in NC group (P<0.01).6.After insulin treatments, the plasma obestatin levels were no statistical difference in LD and HD group compared to those in DM group(P> 0.05) and increased significantly in LD and HD group compared to those in NC group(P＜0.01). The obestatin integral optical density in gastric fundus were no statistical difference in LD and HD group compared to those in DM group(P>0.05)and decreased significantly in LD and HD group compared to those in NC group(P<0.01). The obestatin levels in the hypothalamus were no statistical differnce among LD, HD, DM and NC group (P> 0.05). There was no GPR-39 mRNA expression detected in the hypothalamus in LD and HD group.Conclusionl.The liquid gastric emptying accelerated significantly in the early stages of STZ-induced diabetic rats.2.In the early stages of STZ-induced diabetic rats, the plasma ghrelin levels increased significantly, the ghrelin expression levels and the ghrelin mRNA expression levels in the gastric fundus increased significantly, the ghrelin levels and the GHSR mRNA expression levels in the hypothalamus increased significantly. 3.In the early stages of STZ-induced diabetic rats, the plasma obestatin levels increased significantly, the obestatin expression levels in the gastric fundus decreased significantly. The obestatin levels in the hypothalamus had no significant change and there was no GPR-39 mRNA expression detected in the hypothalamus.4.Treatments with insulin attenuated significantly increased blood glucose levels, decreased blood insulin levels and accelerated liquid gastric emptying in the early stages of STZ-induced diabetic rats.5.Treatments with insulin attenuated significantly increased plasma ghrelin levels, decreased ghrelin expression levels and increased ghrelin mRNA expression levels in the gastric fundus, increased ghrelin levels in the hypothalamus, but did not attenuate significantly increased GHSR mRNA expression levels in the hypothalamus in the early stages of STZ-induced diabetic rats.6.Treatments with insulin had no significant effect on increased plasma obestatin levels, decreased obestatin expression levels in the gastric fundus and the obestatin levels in the hypothalamus in the early stages of STZ-induced diabetic rats.7. Our study suggests that elevated endogenous ghrelin may play important role in the mechanism of accelerated liquid gastric emptying in the early stages of STZ-induced diabetic rats and the role of endogenous obestatin need further study.更多还原