红景天苷对糖尿病大鼠肾脏Nrf2、γ-GCS、PPAR-γ表达的影响

发布时间：2018-03-18 17:57

  本文选题：糖尿病肾病（DN）　切入点：红景天苷（SAL）　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：目前，糖尿病肾病(Diabetic Nephropathy, DN)已成为终末期肾脏病的主要原因之一，对DN发生发展机制的研究就显得尤为重要，但至今尚未完全阐明,其机制可能与遗传、糖代谢、血流动力学、炎症及细胞因子等因素有关。探讨其相关机制可以为糖尿病肾病防治提供新的理论依据。已有研究证实，氧化应激与糖尿病肾病的发病密切相连。高糖状态所致的氧化应激可促使活性氧簇（Reactive oxygen species，ROS）产生过多,诱导炎性介质和细胞因子生成，使细胞外基质（Extracellular matrix，ECM）合成增加,加速肾小球硬化。长期的高血糖使红细胞的携氧能力下降，组织细胞H2O2、O2-等生成增多,谷胱甘肽(GSH)减少,影响肾脏的结构和功能。Nrf2/ARE信号通路是机体重要的内源性保护通路，该通路被认为是调控体内众多抗氧化成分的关键通路，具有平衡内环境的氧化-还原状态、抗炎等生物作用。Nrf2/ARE信号通路激活使下游Ⅱ相解毒酶及抗氧化酶基因转录增加,如谷氨酰半胱氨酸合成酶（γ-GCS），增加机体对氧化应激的抵抗。过氧化物酶体增殖物激活受体(PPARs)是核转录因子超家族的成员，其中PPAR-γ与肾脏关系十分密切。PPAR-γ的激活与抑制对维持机体功能状态具有重要意义。目前已知的拮抗剂较局限，如BADGE、GW9662等药物；PPAR-γ的激动剂主要包含两类，即天然配体与合成配体，前者如15d-PGJ2等，后者主要包括噻唑烷二酮类药物等。并且已有研究显示Nrf2/ARE信号通路与PPAR-γ具有某些内在联系。红景天苷作为一种中药提取物，有着广泛的药理作用，如抗氧化、抗炎、抗肿瘤等。在动物实验研究中，红景天苷是否可以通过激活Nrf2/ARE信号通路与PPAR-γ因子减轻糖尿病肾脏的氧化应激反应，减少蛋白尿，文献报道十分少见。本实验通过建立糖尿病大鼠模型，给予红景天苷干预，探讨Nrf2/ARE信号通路与PPAR-γ因子之间的关系，揭示红景天苷在糖尿病肾病发生发展中的作用，从而为治疗糖尿病肾病提供新思路。 材料与方法：选择SPF级健康雄性SD大鼠30只，体重200±20g，随机选择10只，即正常组（NC组），其余20只制造糖尿病大鼠模型。购买大鼠后先行适应性喂养，在腹腔注射链尿佐菌素(STZ)之前需空腹12小时，造糖尿病大鼠模型即给予STZ65mg/kg。72小时后测血糖≥16.7mmol/L，，尿糖≥3+即为造模成功。NC组给予等量柠檬酸-柠檬酸钠缓冲液腹腔注射。造模成功随机分为两组，即DM组、SAL组。次日开始进行治疗，即给予红景天苷30mg/kg·d灌胃。各组大鼠分笼喂养，予普通饲料、自由进食水，光照温度湿度一定，共观察12周。分别于6周、12周，随机选取5只大鼠，留取24小时尿液，检测蛋白定量，称取体重后腹腔注射10%水合氯醛麻醉，心脏取血，检测血糖、肌酐、尿素氮。双肾去包膜称重后，留取右肾，检测CAT、GSH指标及应用RT-PCR方法检测Nrf2、γ-GCS、PPAR-γ的表达。留取左肾，制作病理切片，分别采用HE和PAS染色方法，在光镜下观察大鼠肾脏组织病理变化；应用免疫组织化学方法检测肾脏中Nrf2、γ-GCS、PPAR-γ的表达，结果应用图像分析系统进行半定量分析。实验数据若计量资料用均数±标准差（X±S）表示，每组间比较采用单因素方差分析，两组间比较采用两独立样本t检验，若数据不满足正态性和方差齐则采用非参数检验，应用SPSS19.0统计分析软件处理实验数据，以P0.05有统计学意义。 结果：统计三组大鼠体重与肾指数（肾重/体重）、24小时尿蛋白定量及血生化指标，以P0.05具有统计学意义。其中，糖尿病大鼠随着病程进展逐渐出现多饮、多食、多尿、消瘦、精神萎靡、反应迟钝等症状，24小时尿蛋白增加，SAL组大鼠尿蛋白定量较DM组减少，血糖、血肌酐及尿素氮亦较DM组下降（P0.05），具有统计学意义。光镜下观察各组肾脏组织HE及PAS染色，NC组大鼠肾脏未见病变，随着高血糖的持续存在，其余两组在第6周时肾组织可有细胞及基质轻度增生，病变随时间推移逐渐加重，于第12周系膜基质增生更为明显。SAL组肾脏病变程度较DM组有所减轻。应用图像分析系统分析免疫组织化学染色切片，结果显示：Nrf2、γ-GCS主要表达于肾小球、肾小管上皮细胞胞浆中,表达强度为：SAL组DM组NC组（P0.05）；PPAR-γ主要在集合管、近端肾小管、远端肾小管的上皮细胞胞浆中表达，肾小球少见表达，该因子表达强度为： NC组DM组SAL组（P0.05）。氧化应激指标方面，肾脏中CAT与GSH在造模成功后6、12周后显著减低，SAL组水平明显高于DM组（P0.05）。RT-PCR结果显示Nrf2、γ-GCS、PPAR-γ的表达趋势与免疫组织化学结果一致。 结论：糖尿病大鼠造模成功后，氧化应激增强，Nrf2/ARE信号通路激活，其下游抗氧化蛋白γ-GCS表达增强，PPAR-γ的表达降低；SAL可以增强Nrf2/ARE信号通路的激活状态，诱导γ-GCS表达，并可抑制PPAR-γ表达，达到降低血糖、减少蛋白尿、减轻肾脏氧化应激损伤的作用。
[Abstract]:Objective: at present, diabetic nephropathy (Diabetic Nephropathy DN) has become one of the major cause of end-stage renal disease, to study the occurrence and development mechanism of DN is particularly important, but has not yet been fully clarified, glucose metabolism and its mechanism may be related to genetic, hemodynamics, inflammation and cytokines to explore the factors. The related mechanism can provide a new theoretical basis for the prevention and treatment of diabetic nephropathy. Studies have confirmed that the pathogenesis of oxidative stress and diabetic nephropathy is closely linked to oxidative stress induced by high glucose. The state promotes reactive oxygen species (Reactive oxygen, species, ROS) to produce too much, inducing the production of inflammatory mediators and cytokines, extracellular matrix (Extracellular. Matrix, ECM) increased synthesis, accelerated glomerulosclerosis. High blood glucose for a long time the oxygen carrying capacity of red blood cells decreased, cell H2O2, increase of O2- formation, glutathione (GSH) decreased Influence of the structure and function of the kidney,.Nrf2/ARE signaling pathway is an important endogenous protection pathway, this pathway is considered a key pathway in many antioxidants, oxidation with balanced environment in the reduction state, anti-inflammatory and other biological role of.Nrf2/ARE signaling pathway activation of the downstream phase II detoxification enzymes and antioxidant enzyme gene transcription increased. Such as glutamylcysteine synthetase (gamma -GCS), increase the body's resistance to oxidative stress. Peroxisome proliferator activated receptor (PPARs) is a member of the nuclear transcription factor superfamily, which is very close relationship between PPAR- and renal.PPAR- gamma gamma activation and inhibition to maintain body function has important significance. At present known antagonists a limitation, such as BADGE, GW9662 and other drugs; PPAR- gamma agonist consists mainly of two types, namely, natural and synthetic ligands such as 15d-PGJ2 ligand, the former, the latter Including the two thiazolidinediones. And studies have shown that Nrf2/ARE signaling pathway and PPAR- gamma has some internal relations. As a traditional Chinese medicine extract salidroside, has extensive pharmacological effects such as antioxidant, anti-inflammatory, anti-tumor and so on. In animal studies, Rhodiola glycosides could day by activating Nrf2/ARE signaling pathway PPAR- and gamma factor attenuates oxidative stress in diabetic kidney, reduce proteinuria, reported in the literature are very rare. By establishing the model of diabetic rats, given salidroside intervention, to investigate the relationship between Nrf2/ARE signaling pathway and PPAR- gamma factor, reveal the Salidroside in role in the development of diabetic nephropathy, so as to provide new ideas the treatment of diabetic nephropathy.
鏉愭枡涓庢柟娉曪細閫夋嫨SPF绾у仴搴烽泟鎬�SD澶ч紶30鍙

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