本文選題：糖尿病腎�。―N）　切入點：紅景天苷（SAL）　出處：《河北醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：目前，糖尿病腎病(Diabetic Nephropathy, DN)已成為終末期腎臟病的主要原因之一，對DN發(fā)生發(fā)展機制的研究就顯得尤為重要，但至今尚未完全闡明,其機制可能與遺傳、糖代謝、血流動力學(xué)、炎癥及細(xì)胞因子等因素有關(guān)。探討其相關(guān)機制可以為糖尿病腎病防治提供新的理論依據(jù)。已有研究證實，氧化應(yīng)激與糖尿病腎病的發(fā)病密切相連。高糖狀態(tài)所致的氧化應(yīng)激可促使活性氧簇（Reactive oxygen species，ROS）產(chǎn)生過多,誘導(dǎo)炎性介質(zhì)和細(xì)胞因子生成，使細(xì)胞外基質(zhì)（Extracellular matrix，ECM）合成增加,加速腎小球硬化。長期的高血糖使紅細(xì)胞的攜氧能力下降，組織細(xì)胞H2O2、O2-等生成增多,谷胱甘肽(GSH)減少,影響腎臟的結(jié)構(gòu)和功能。Nrf2/ARE信號通路是機體重要的內(nèi)源性保護(hù)通路，該通路被認(rèn)為是調(diào)控體內(nèi)眾多抗氧化成分的關(guān)鍵通路，具有平衡內(nèi)環(huán)境的氧化-還原狀態(tài)、抗炎等生物作用。Nrf2/ARE信號通路激活使下游Ⅱ相解毒酶及抗氧化酶基因轉(zhuǎn)錄增加,如谷氨酰半胱氨酸合成酶（γ-GCS），增加機體對氧化應(yīng)激的抵抗。過氧化物酶體增殖物激活受體(PPARs)是核轉(zhuǎn)錄因子超家族的成員，其中PPAR-γ與腎臟關(guān)系十分密切。PPAR-γ的激活與抑制對維持機體功能狀態(tài)具有重要意義。目前已知的拮抗劑較局限，如BADGE、GW9662等藥物；PPAR-γ的激動劑主要包含兩類，即天然配體與合成配體，前者如15d-PGJ2等，后者主要包括噻唑烷二酮類藥物等。并且已有研究顯示Nrf2/ARE信號通路與PPAR-γ具有某些內(nèi)在聯(lián)系。紅景天苷作為一種中藥提取物，有著廣泛的藥理作用，如抗氧化、抗炎、抗腫瘤等。在動物實驗研究中，紅景天苷是否可以通過激活Nrf2/ARE信號通路與PPAR-γ因子減輕糖尿病腎臟的氧化應(yīng)激反應(yīng)，減少蛋白尿，文獻(xiàn)報道十分少見。本實驗通過建立糖尿病大鼠模型，給予紅景天苷干預(yù)，探討Nrf2/ARE信號通路與PPAR-γ因子之間的關(guān)系，揭示紅景天苷在糖尿病腎病發(fā)生發(fā)展中的作用，從而為治療糖尿病腎病提供新思路。 材料與方法：選擇SPF級健康雄性SD大鼠30只，體重200±20g，隨機選擇10只，即正常組（NC組），其余20只制造糖尿病大鼠模型。購買大鼠后先行適應(yīng)性喂養(yǎng)，在腹腔注射鏈尿佐菌素(STZ)之前需空腹12小時，造糖尿病大鼠模型即給予STZ65mg/kg。72小時后測血糖≥16.7mmol/L，，尿糖≥3+即為造模成功。NC組給予等量檸檬酸-檸檬酸鈉緩沖液腹腔注射。造模成功隨機分為兩組，即DM組、SAL組。次日開始進(jìn)行治療，即給予紅景天苷30mg/kg·d灌胃。各組大鼠分籠喂養(yǎng)，予普通飼料、自由進(jìn)食水，光照溫度濕度一定，共觀察12周。分別于6周、12周，隨機選取5只大鼠，留取24小時尿液，檢測蛋白定量，稱取體重后腹腔注射10%水合氯醛麻醉，心臟取血，檢測血糖、肌酐、尿素氮。雙腎去包膜稱重后，留取右腎，檢測CAT、GSH指標(biāo)及應(yīng)用RT-PCR方法檢測Nrf2、γ-GCS、PPAR-γ的表達(dá)。留取左腎，制作病理切片，分別采用HE和PAS染色方法，在光鏡下觀察大鼠腎臟組織病理變化；應(yīng)用免疫組織化學(xué)方法檢測腎臟中Nrf2、γ-GCS、PPAR-γ的表達(dá)，結(jié)果應(yīng)用圖像分析系統(tǒng)進(jìn)行半定量分析。實驗數(shù)據(jù)若計量資料用均數(shù)±標(biāo)準(zhǔn)差（X±S）表示，每組間比較采用單因素方差分析，兩組間比較采用兩獨立樣本t檢驗，若數(shù)據(jù)不滿足正態(tài)性和方差齊則采用非參數(shù)檢驗，應(yīng)用SPSS19.0統(tǒng)計分析軟件處理實驗數(shù)據(jù)，以P0.05有統(tǒng)計學(xué)意義。 結(jié)果：統(tǒng)計三組大鼠體重與腎指數(shù)（腎重/體重）、24小時尿蛋白定量及血生化指標(biāo)，以P0.05具有統(tǒng)計學(xué)意義。其中，糖尿病大鼠隨著病程進(jìn)展逐漸出現(xiàn)多飲、多食、多尿、消瘦、精神萎靡、反應(yīng)遲鈍等癥狀，24小時尿蛋白增加，SAL組大鼠尿蛋白定量較DM組減少，血糖、血肌酐及尿素氮亦較DM組下降（P0.05），具有統(tǒng)計學(xué)意義。光鏡下觀察各組腎臟組織HE及PAS染色，NC組大鼠腎臟未見病變，隨著高血糖的持續(xù)存在，其余兩組在第6周時腎組織可有細(xì)胞及基質(zhì)輕度增生，病變隨時間推移逐漸加重，于第12周系膜基質(zhì)增生更為明顯。SAL組腎臟病變程度較DM組有所減輕。應(yīng)用圖像分析系統(tǒng)分析免疫組織化學(xué)染色切片，結(jié)果顯示：Nrf2、γ-GCS主要表達(dá)于腎小球、腎小管上皮細(xì)胞胞漿中,表達(dá)強度為：SAL組DM組NC組（P0.05）；PPAR-γ主要在集合管、近端腎小管、遠(yuǎn)端腎小管的上皮細(xì)胞胞漿中表達(dá)，腎小球少見表達(dá)，該因子表達(dá)強度為： NC組DM組SAL組（P0.05）。氧化應(yīng)激指標(biāo)方面，腎臟中CAT與GSH在造模成功后6、12周后顯著減低，SAL組水平明顯高于DM組（P0.05）。RT-PCR結(jié)果顯示Nrf2、γ-GCS、PPAR-γ的表達(dá)趨勢與免疫組織化學(xué)結(jié)果一致。 結(jié)論：糖尿病大鼠造模成功后，氧化應(yīng)激增強，Nrf2/ARE信號通路激活，其下游抗氧化蛋白γ-GCS表達(dá)增強，PPAR-γ的表達(dá)降低；SAL可以增強Nrf2/ARE信號通路的激活狀態(tài)，誘導(dǎo)γ-GCS表達(dá)，并可抑制PPAR-γ表達(dá)，達(dá)到降低血糖、減少蛋白尿、減輕腎臟氧化應(yīng)激損傷的作用。
[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(xì)閫夋嫨SPF綰у仴搴烽泟鎬D澶ч紶30鍙

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