Rg1、Rb1对糖尿病肾病大鼠肾脏保护作用及其对MCP-1蛋白及其mRNA表达调控的研究

【作者】 张学凯；

【导师】 赵宗江；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2008， 硕士

【摘要】 糖尿病肾病（Diabetic Nephropathy，DN）是指糖尿病（Diabetes mellitus，DM）微血管病变所引起的肾脏结构和功能的改变，是糖尿病常见和严重的远期并发症之一。世界范围内，随着DM发病率的增加DN日益增多，在西方国家DN已成为终末期肾脏病（End sta ge rena l disease，ESRD）的主要原因。在我国DM患病率也不断增高，据估计我国糖尿病患者已超过4000万。我国因DN而发生慢性肾衰竭的人数也逐年上升。DN的病理特征包括：早期的肾脏肥大、肾小球和肾小管基底膜增厚及肾小球内高灌注、高跨膜压；病程进展可有肾小球细胞外基质积聚、肾小管间质纤维化。这些特征性病理变化是细胞外基质（Extracellula r Matrix，ECM）在肾小球系膜区以及肾小管间质堆积所致，而ECM的聚集与肾组织内单核/巨噬细胞（Monocytes/macropha ges）的广泛浸润有关。单核细胞趋化蛋白-1（Monocyte Chemoa ttracta nt Protein-1，MCP-1）是单核巨噬细胞特异性的趋化因子，对单核巨噬细胞有很强的趋化激活作用。从Ⅰ型或Ⅱ型DN患者肾组织活检以及DN大鼠肾脏病理学检查中均确认MCP-1是单核巨噬细胞聚集的主要因素。在DM过程中，代谢和血流动力学的变化相互作用，促进DN的发生。其中多重因素都可刺激肾脏固有细胞、间质浸润细胞产生MCP-1。而MCP-1能趋化单核/巨噬细胞，激活粘附分子，诱导溶酶体的释放，活化TGF-βl，促进超氧阴离子和胶原的产生，对DN的进展起了重要作用。本实验所用药物是从云南文山三七中提取出的人参皂甙单体Rb1、Rg1，是三七总皂甙（Pana x notoginseng saponins，PNS）的主要活性单体。中医认为，三七性温、味甘、微苦，归肝、胃经，具有化瘀止血、活血定痛、补虚强壮的效果，兼活血补血之功效于一身。其主要活性成分是PNS，其中含20多种达玛烷型皂甙，与人参主要成分一致，即人参二醇型皂甙Rb1（Panaxad iolof saponin Rb1，Rb1）、人参三醇型皂甙Rg1（Panaxad iol of saponin Rg1，Rg1）。Rb1、Rg1具有多种生物学功能，现在的研究主要集中在抗肿瘤、抗氧化、诱导细胞凋亡以及扩张血管、抑制血小板聚集、延长凝血时间、抗炎等方面。中医理论认为DN属于中医学的“水肿”、“肾消”和“尿浊”等病症范畴，其病机特点是本虚标实。《内经》云“虚则补之、实则泻之”。虚实夹杂、正邪交争之病在治疗时最应谨慎。正所谓补虚防助邪，祛邪防伤正。三七活血化瘀、补虚强壮，兼活血补血之功效于一身，很切合DN的中医病机，一方面活血化瘀不伤正，一方面补血益气不助邪。另外研究还发现：不管是PNS还是其主要成分单体Rg1、Rb1，都具有明显的肾脏保护作用，对多种肾脏疾病都有减轻作用。本文通过整体动物实验和体外细胞实验，应用免疫组织化学法（Immunohistochemistry，IHC）、原位杂交法（In situ hybrid iza tion，ISH）、MTT法以及Western blot等方法从组织、细胞、蛋白质、基因水平进行研究，旨在探讨Rg1、Rb1防治DN的作用机制。目的：通过建立DN大鼠模型，从整体动物实验研究Rg1、Rb1是否能够有效的改善DN大鼠肾功能、延缓肾脏病理损伤，进而从组织、细胞、蛋白质、基因水平研究Rg1、Rb1对MCP-1表达的影响，以期阐明Rg1、Rb1防治DN的作用机制。方法：整体实验采用腹腔内一次性注射STZ复制DM大鼠模型。清洁级雄性SD大鼠75只，正常组10只，其余造模成功后随机分组，各组分别给予相应药物或蒸馏水灌胃治疗；1．每周测量大鼠体重，于第4、8、12周末分别留尿测24h尿蛋白。12周后处死大鼠，取血清检测生化指标。取肾脏称重，计算肾重/体重比；染色后观察大鼠肾脏病理学变化。同时采用病理评分标准评价各组大鼠肾组织病理改变。2．IHC：检测大鼠肾组织中MCP-1蛋白表达情况；3．ISH：检测肾组织中MCP-1mRNA的表达情况。细胞实验，采用肾小球系膜细胞（Glomerular mesa ngia l cells，GMCs）体外培养。除空白组外，其余各组均用高糖刺激，并应用相应的纯药，检测下列指标。1．MTT：检测Rg1、Rb1对高糖刺激12h，24h，48h和60h时GMCs增殖的影响情况。2．Western blot：检测Rg1、Rb1对高糖刺激48h时GMCs表达的MCP-1蛋白含量的影响情况。结果：1．各治疗组大鼠一般情况及肾脏病理损害与模型组相比均有所改善。其中，①体重：与模型组比较各治疗组大鼠体重各周均有所增加，但没有统计学差异。②肾重/体重比：与模型组比，Rb1组大鼠肾重/体重比减轻明显，有极显著性差异（P<0.01）；Rg1大、Rg1小也有减轻，有显著性差异（P<0.05）。③24h尿蛋白定量：与模型组比较，第4周、第8周Rg1大、Rg1小及Rb1组明显减轻，有极显著性差异（P<0.01）；第12周，Rg1大、Rg及Rb1组有显著性差异（P<0.05）。④血生化：Rg1、Rb1可以降低BUN、Scr与TG，与模型组比较，各治疗组BUN均有极显著性差异（P<0.01），Scr方面Rg1大及Rb1组有极显著性差异（P<0.01），Rg1小组有显著性差异（P<0.05）；TG治疗组均有降低趋势，但无统计学差异。Rg1、Rb1均可以减轻DN大鼠肾脏病理损害损伤指数。2．Rg1、Rb1可以减少肾组织MCP-1蛋白及其mRNA的表达，与模型组比较，Rg1小、大及Rb1肾组织MCP-1蛋白的表达均显著减少，具有极显著性统计学意义（P<0.01）；Rg1小、Rg1大及Rb1组大鼠肾组织MCP-1mRNA表达明显减少，其中Rg1大剂量组与模型组相比有显著差异（P<0.05）。3．Rg1、Rb1能够抑制高糖刺激的GMCs增殖，各时相点以Rg1大剂量抑制效果最好。4．Rg1、Rb1能够明显抑制高糖刺激的GMCs分泌MCP-1蛋白，其中Rg1大剂量、Rb1大剂量与高糖组相比有极显著性差异（P<0.01）。Rg1小剂量、Rb1小剂量与高糖组相比有降低，但无统计学意义。结论：1．初步的药效学试验发现：Rg1、Rb1对DN大鼠的肾脏损伤有肯定的改善作用。⑴Rg1、Rb1能显著改善DN大鼠的一般状况；能降低DN大鼠肾重/体重比；调整血脂，减轻24h尿蛋白排泄量，改善肾功能。⑵Rg1、Rb1能减轻DN大鼠肾脏病理学改变，减轻肾小球系膜区增宽、系膜细胞增生，肾小管管腔扩张、小管上皮细胞胞质减少、细胞萎缩或空泡变性以及减轻肾小球和肾间质纤维化等。2．进一步的机理研究发现：Rg1、Rb1防治DN大鼠肾脏损伤的机理可能主要在于以下两个方面：⑴Rg1、Rb1能够下调肾组织MCP-1蛋白及mRNA的表达。⑵Rg1、Rb1能够不同程度的抑制高糖刺激下的GMCs的增殖和MCP-1蛋白的表达。总之，Rg1、Rb1可以改善DN大鼠肾功能，减轻DN肾脏病理损伤。抑制GMCs的增殖和MCP-1蛋白的表达；下调肾组织MCP-1蛋白及mRNA的表达可能是Rg1、Rb1防治DN的机理之一。更多还原

【Abstract】 Diabetic nephropathy (DN) is an important microvascular complication of diabetic mellitus (DM) which caused the injury of the renal. As DM is growing quickly in the world wide rage, DN has been the first reason result in end-stage renal disease (ESRD) in the western country. The prevalence of DM is also growing fast in our country, according to one estimate there are more than 40,000,000 DM patients, so the ESRD induced by DN is growing continuously.There are characteristic pathological changes in the early stages of DN, such as the hypertrophy of kidney, the thickening of glomerular and tubular basement, the high perfusion and cross membrane pressure in the glomerulus. As DN progresses, there will be extracellular matrix accumulation , such as glomerulosclerosis and interstitial fibrosis.A central concept in understanding the pathogenesis of DN is that it results from a series of specific progressive renal pathological changes that have their onset early in the course of diabetes. These changes develop during along silent period, before features of clinical renal disease-including proteinuria, hypertension, and declining glomerular filtration rate (GFR)-are evident. Central to the nephropathological changes is the accumulation of extracellular matrix,leading to expansion of glomerular mesangial regions at the expense of filtration surface area, and thickening of the glomerular and tubular basement membranes.Evidence is accumulating that macrophages and their products play a significant role in those pathological changes. Increased glomerular macrophages have been observed in rats with experimental diabetes, as well as in biopsy samples of kidney from DM patients. There are associations prove that macrophages accumulate in kidney glomerulus and interstitium of patients with diabetic nephropathy is due to monocyte chemoattractant protein-1 (MCP-1), which is a chemokine produced by both tubular epithelial and glomerular mesangial cells (GMCs). The prolonged hyperglycemia, excessive production of Advanced Glycation End Products, overactivation of Protein kinase C, vasoactive substances like Angiotensin II and increased kidney chemokine production all can induce the production of MCP-1, and then the MCP-1 will recruiting and activating monocytes/macrophages from the circulation to kidney, activating the adhesion molecules and induceing the release of lysosomes, inducing TGF- P 1 and promoting the generation of superoxide anion and Collagens, thus play a key role in the progress of DN.There are clinical findings that urinary MCP-1 levels were significantly elevated in patients with diabetic nephrotic syndrome. And urinary MCP-1 excretion is proportional to the number of MCP-1 positive cells in the renal biopsy specimens. And patients used HMG-coA reducing enzyme inhibitor, angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor (ARB), and euglycemic agent like rosiglitazone all can reduce the levels of urinary MCP-1 and albuminuria excretion. The present study demonstrated that up-regulation of locally produced MCP-1 involved in advanced renal lesions in DN patients possibly through macrophages recruitment.The ginsenoside Rbl and Rgl used in this experiment are extracted from the yunna wenshan notoginseng radix, which are the principal active monomers in Panaxnotoginoside (PNS). Notoginseng is the radix of Araliaceae perennial herb Panax notoginseng ( Burk) F. H. Chen (PNG). The characteristic of PNG according to Chinese medicine is warm, sweet flavor, mini-bitter and entering liver, stomach meridians, with the effect of stop bleeding by removing stasis and arrest pain by promoting blood circulation, reinforcing deficiency and invigorating. With the two effects of promoting blood flow and nourish blood. The ginsenoside Rbl and Rgl are the major active components of PNS and ginseng, interestingly the content of the former is higher than the latter. Rbl, Rgl have many biological functions, the study of their pharmacologic action is range from expanding blood vessel, inhibiting platelet agglutination, prolonging coag time, anti-inflammatory, antioxygen, and to Inducing apoptosis. According to Chinese medicine theory, the change occur in DN is due to blood stasis and zheng qi deficiency, thus medicine with the effects of invigorating blood circulation and reinforcing deficiency have great effect. Notoginseng, radix with the advantage of nourishing the blood without aggravating haemostasis and promoting blood flow without injuring blood is very suitable for DN. The animal experiment and clinical research also indicate that either PNS or its key components Rgl and Rbl all have renal protective effect.In this thesis we investigate the mechanism of Rbl and Rgl in relief DN from levels of tissue, cell, protein and gene.Objective:In order to investigate the mechanism of Rbl and Rgl in relief DN from levels of organ, tissue, cell, protein and gene, we established DN rat model and high glucose cell model then observed the efficiency of Rbl and Rgl in influence MCP-1 gene and protein’s expression.Methods:Diabetes induced in SD rats by intraperitoneal injection of streptozotocin (STZ) was used in vivo experiments. 75 SD rats were used in the study, 10 of the rats in normal group except that all the other were injected by STZ, after that the mice whose blood glucose were over 16.7mmol/l and urinary glucose were always over ++++ were recognized as DM animal model. After randomly divided into 5 groups, model group, irbesartan group, Rgl high dose group, Rgl low dose group and Rbl middle dose group, each group were treated accordingly.1.The urine were collected respectively at the end of the 4, 8, 12th week, and24-hour urinary protein were determined by coomassie briliant blue method.12 weeks after intraperitoneal injection of streptozotocin (STZ), all the ratswere sacrificed, the serum was collected for serum biochemical indexesdetermination, such as blood urea nitrogen (BUN), serum creatinine (Scr)and Triglyceride(TG). Kidneys were taken for pathological examination(HE, HE-PASM, and Mallory).2.Immunohistochemistry was used to examine the kidney’s expression of MCP-1 protein. 3. In situ Hybridization was used to examine the kidney’s expression of MCP-lmRNA.In the vitro study, we investigated the influence of Rbl and Rgl in GMCs’ proliferation and expression of MCP-1 protein stimulated by high glucose. The cells were divided into control group, high glucose group, irbesartan group, and Rgl high dose group, Rgl low dose group and Rbl high dose group, Rbl low dose group.1.The rat mesangial cells were cultured successfully in vitro. High glucosewas used to induce proliferation of mesangial cells. Changes of mesangial cells’proliferation were detected by MTT reduction assay at 12 hours, 24 hours, 48hours and 60 hours.2.Western-blot was used to detecting the expression of MCP-1 protein in theintracytoplasm of GMCs stimulated by high glucose.Result:1.Compared with model group, the body weight increased in all the othergroups, but there is no significant statistics difference; The kidney weight/body weight ratio of Rbl group were significantly decreased(P<0.01), Rgl high andlow dose group were also decreased (P<0.05); the Urine protein of Rgl high and low dose group and Rbl group were significantly decreased (P<0.01) at the end of the 4 and 8 week, and alsodecreased (P<0.05) at the end of the 12 week. And Rgl, Rbl also decreasethe biochemical parameters such as BUN, Scr and TG. Compared withmodel group, the BUN of all the other groups were significantly decreased(P<0.01); Scr of Rgl high dose and Rbl group were significantly decreased(P<0.01), and the Rgl low dose group were also decreased (P<0.05); the TGof all the other groups were decreased, but there is no significant statisticsdifference.2.The severe pathological changes of kidney structure in rats with DN wereobserved, while the Ginsenoside-Rgl, Rbl groups were obviously lessened. 3.The expression of MCP-imRNA and protein in the normal group is lowwhile markedly increased in the model group. In compare with model group,the expression of MCP-1 protein of Ginsenoside-Rgl, Rbl groups showedsignificant decrease (P<0.01); And the expression of MCP-lmRNA of Ginsenoside-Rgl, Rbl groups all showed decrease, but only the Rgl high dose group showed statistics difference (P<0.05),4.Rgl and Rbl both have the inhibiting effect on the proliferation of GMCsstimulated by high glucose, while the Rgl high dose group is the best.5.Rgl and Rbl can significantly decrease the over production of MCP-1protein in high glucose stimulated GMCs and with the dose dependenteffect.Conclusion:1.Pharmacodynamic Study found that Rg1, Rb1 may regulate the serum biochemical indexes, reduce the urine protein excretion, and alleviate severe pathological changes of kidney in rats with DN.2.Further study found that Rg1, Rb1 can reduce proliferation of GMCs, anddecrease the over expression of MCP-1 protein and mRNA in both renaltissue and the high glucose stimulated GMCs in vitro. This maybe one ofthe mechanisms in retarding DN renal injury by Rgl, Rbl.更多还原