Apelin多肽对胰脏和肾脏损伤的保护机制研究

发布时间：2018-04-02 07:47

本文选题：apelin　切入点：1型糖尿病　出处：《华中科技大学》2014年博士论文

【摘要】：糖尿病是由胰岛素的分泌障碍和／或胰岛素的作用障碍所引起的,以长期高血糖为特征的代谢性疾病。糖尿病的长期慢性高血糖会引起器官的功能障碍和衰竭,造成各种糖尿病并发症的发生。糖尿病肾病是一种常见糖尿病微血管并发症,以肾小球硬化和肾病综合征为特征,也被认为是导致终末期肾衰竭的主要原因。肥胖型糖尿病肾病的病发过程中常伴随着肾脏缺血再灌注损伤(ischemia/reperfusion injury, I/R injury)的发生,但对于糖尿病及糖尿病肾病和肾脏缺血再灌注损伤的分子机制及其防治手段尚需进一步研究。Apelin是一类新型脂肪因子,具有调节血压、胰岛素分泌和能量代谢等多种生物功能,其中apelin-13是apelin家族中最丰富且活性最高的成员。本论文研究了apelin-13是否能有效地抑制糖尿病所致的胰脏和肾脏病变,是否能抑制缺血/再灌注引起的肾脏损伤。本论文以1型糖尿病模型Akita小鼠为实验对象,研究1型糖尿病导致的胰脏和肾脏病变；并以高糖刺激的HBZY-1和MES13细胞作为模拟糖尿病肾病的体外细胞模型。我们还以单侧肾结扎的wistar大鼠作为肾脏缺血再灌注损伤的动物模型；以在低氧(1%02)条件下进行无糖无血清培养基培养的HBZY-1和NRK-52E细胞作为模拟肾脏缺血再灌注损伤的体外模型。本论文通过蛋白质免疫印迹、免疫组织化学染色、免疫荧光染色、聚合酶链式反应和染色质免疫共沉淀等手段,从分子水平、细胞水平和病理水平研究了apelin对胰脏和肾脏损伤的治疗作用的分子机理。我们首先对Akita小鼠进行为期两个月的apelin-13的尾静脉注射,结果显示apelin-13治疗明显改善糖尿病引起的胰脏中胰岛面积和胰岛素含量减少。进一步的研究表明,apelin-13能抑制糖尿病诱导内质网应激反应(endoplasmic reticulum stress, ER stress)中PERK和IREla通路的激活,降低分子伴侣(GRP78, calnexin和HSP70)在Akita小鼠胰脏中的表达水平,并激活糖尿病所抑制的AKT、AMPK和ERK通路。其次,在Akita小鼠中, apelin-13不仅降低了糖尿病引起的总尿量、尿总蛋白和肾小球滤过率等肾功能相关生理指标的上升,还抑制了糖尿病导致的肾小球肥大、系膜扩张和肾脏炎症。本论文首次发现apelin-13明显的抑制了由糖尿病或高糖或丁酸钠引起的NF-κB和组蛋白的乙酰化通路激活导致的炎症因子的表达。并且apelin-13可以上调糖尿病或高糖或丁酸钠抑制的HDAC1表达。另外,在缺血再灌注引起肾损伤的大鼠中,损伤前腹腔注射apelin-13能显著降低肾脏缺血再灌注损伤引起的肾小管坏死,细胞凋亡和组蛋白的高甲基化水平。在HBZY-1和NRK-52E细胞进行的缺氧实验显示：apelin-13通过抑制Tgf-β和组蛋白的高甲基化水平引起的凋亡反应,达到对细胞的保护作用。此外,在HBZY-1细胞中过表达apelin也能抑制I/R损伤引起的Tgf-P和组蛋白的高甲基化水平,有效抑制I/R损伤引起的细胞凋亡。本论文研究结果显示,在Akita小鼠中,apelin-13通过抑制糖尿病引起的内质网应激反应并促进胰脏中胰岛素的表达。Apelin-13能降低糖尿病引起的肾脏中组蛋白的高乙酰化水平,抑制NF-κB和MAPK通路的激活,进而抑制肾脏的炎症反应。此外,apelin-13还可抑制缺血再灌注损伤所致的Tgf-β通路和组蛋白的高甲基化水平,最终抑制细胞凋亡。Apelin-13不但对1型糖尿病及糖尿病肾病有良好治疗效果,对急性肾损伤也有显著疗效。本研究不仅为探索1型糖尿病及糖尿病肾病、急性肾损伤的分子机制提供指导意见,并且为1型糖尿病及糖尿病肾病、急性肾损伤的药物治疗提供了新的研究方向。
[Abstract]:Diabetes is caused by insulin secretion function disorder disorder and / or insulin, metabolic disease characterized by long-term high glucose. Diabetes chronic hyperglycemia can cause organ dysfunction and failure, caused by various complications of diabetes. Diabetic nephropathy is a common microvascular complication of diabetes, glomerular sclerosis and nephrotic syndrome characteristics, is also considered to be the leading cause of end-stage renal failure. Renal obese diabetic disease process often accompanied by renal ischemia reperfusion injury (ischemia/ reperfusion injury, I/R injury) occurred, but the molecular mechanism for diabetes and diabetic nephropathy and renal ischemia reperfusion injury and the treatment needs further study.Apelin is a kind of new fat factor, can regulate the blood pressure, insulin secretion and energy metabolism etc. Apelin-13 is the most abundant and highly active member of Apelin family. In this paper, we studied whether apelin-13 can effectively inhibit pancreatic and renal lesions caused by diabetes and whether it can inhibit renal injury induced by ischemia / reperfusion.
In this paper, a model of type 1 diabetes Akita mice as experimental subjects, type 1 diabetes mellitus in pancreas and kidney disease; and glucose stimulated HBZY-1 and MES13 cells as in vitro cell model of diabetic nephropathy. We also used Wistar rats as the animal model of unilateral renal ligation of renal ischemia reperfusion injury; to in hypoxia (1%02) in vitro model of sugar free cultured HBZY-1 and NRK-52E cells as a model of renal ischemia reperfusion injury conditions. This paper through Western blot, immunohistochemistry, immunofluorescence staining, polymerase chain reaction and chromatin immunoprecipitation method, from the molecular level, molecular study on the mechanism of the therapeutic effect of apelin on the pancreas and kidney damage in the cell level and pathological level.
We first intravenous injection for two months apelin-13 to Akita mice, showed decreased islet area and insulin content of apelin-13 treatment significantly improved diabetic pancreas. Further studies showed that apelin-13 can inhibit the diabetes induced endoplasmic reticulum stress response (endoplasmic reticulum stress, ER stress and IREla PERK) in the activation pathway the lower molecular chaperones (GRP78, calnexin and HSP70) on the expression level of Akita mice in the pancreas, and activate diabetes inhibition of AKT, AMPK and ERK pathways. Secondly, in Akita mice, apelin-13 not only reduces the total amount of urine caused by diabetes, the rise of related physiological indexes of total urinary protein and glomerular filtration rate. Renal function, also inhibits diabetes induced glomerular hypertrophy, mesangial expansion and inflammation of the kidneys. This is the first time that apelin-13 significantly suppressed by diabetes. The expression of acetylation pathway disease or high glucose or sodium butyrate induced NF- kappa B and histone activation of inflammatory factors caused. And apelin-13 can up regulate the expression of diabetes or high glucose or sodium butyrate inhibited HDAC1. In addition, in ischemia reperfusion induced renal injury in rats, injury of anterior abdominal cavity injection can significantly reduce the apelin-13 renal ischemia reperfusion injury of renal tubular necrosis caused by high levels of methylation, apoptosis and histone. Hypoxia experiments were carried out in HBZY-1 and NRK-52E cells: the apoptosis reaction induced by apelin-13 through inhibition of Tgf- methylation level and protein beta group, achieve protective effect on cells. In addition, the high level of methylation Tgf-P and histone Apelin overexpression in HBZY-1 cells can inhibit I/R damage induced apoptosis, inhibit I/R damage caused.
The results of this study demonstrate that in Akita mice, apelin-13 via endoplasmic reticulum stress inhibition caused by diabetes and high levels of acetylation promotes the expression of.Apelin-13 in pancreatic insulin can reduce diabetes induced renal histone, inhibited the activation of NF- K B and MAPK pathway, and then inhibit the inflammatory reaction of the kidney. In addition, apelin-13 can inhibit the ischemia and reperfusion injury caused by high levels of methylation and histone Tgf- beta pathway, ultimately inhibit apoptosis of.Apelin-13 not only has a good effect in treating type 1 diabetes mellitus and diabetic nephropathy, also has significant curative effect on acute renal injury. This study not only for type 1 diabetes mellitus and diabetic nephropathy, provide guidance molecules the mechanism of acute renal injury, and type 1 diabetes mellitus and diabetic nephropathy, which provides a new research direction in the treatment of acute kidney injury.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R692

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