本文選題：黃芪甲苷 + 糖尿病腎病��； 參考：《山東大學(xué)》2017年博士論文

【摘要】：研究背景糖尿病(diabetes mellitus,DM)是嚴(yán)重影響人類健康的慢性非傳染性疾病,隨著飲食結(jié)構(gòu)和生活方式的改變,其患病率、發(fā)病率和患者數(shù)量急劇上升,給家庭及社會(huì)帶來(lái)沉重的經(jīng)濟(jì)負(fù)擔(dān)。糖尿病腎病(diabetic kidney disease,DKD)是糖尿病微血管病變的主要并發(fā)癥之一,即使血壓及血糖控制達(dá)標(biāo),糖尿病患者最終有30%—40%發(fā)展至糖尿病腎病。在全球范圍內(nèi),糖尿病腎病是引起終末期腎病(end-stage renal disease,ESRD)的主要原因。既往認(rèn)為糖尿病腎病在進(jìn)展過(guò)程中,最重要的病理生理變化為不可逆的腎小球纖維化及瘢痕形成,腎小管損傷只是繼發(fā)現(xiàn)象。但隨著研究的深入人們發(fā)現(xiàn),腎小管病變,尤其是腎小管間質(zhì)的纖維化(tubulointerstitial fibrosis, TIF)在推動(dòng)糖尿病腎病進(jìn)展中起了非常重要的作用,腎小管病變可不依賴于腎小球的病變。因此,探討延緩腎小管間質(zhì)纖維化的治療策略,對(duì)于改善患者預(yù)后具有十分重要的意義。盡管TIF的發(fā)病機(jī)理尚未明了,但越來(lái)越多的證據(jù)指向腎小管上皮細(xì)胞凋亡及腎小管上皮細(xì)胞-成纖維細(xì)胞(間充質(zhì)細(xì)胞)轉(zhuǎn)分化(epithelial-to-mesenchymal transition,EMT)。EMT是糖尿病腎臟疾病小管損傷的經(jīng)典病理改變,且與腎間質(zhì)纖維化的程度相平行。越來(lái)越多的研究強(qiáng)有力地證實(shí)了EMT在不同類型的腎臟疾病中發(fā)揮著重要作用,而逆轉(zhuǎn)EMT則能有效改善腎臟纖維化。雖然大量研究揭示了各種EMT的始動(dòng)因素、調(diào)節(jié)因子及存在的信號(hào)通路,但在所有因素中,TGF-β1是多種組織和器官纖維化的關(guān)鍵調(diào)控因子,是糖尿病腎病進(jìn)展過(guò)程中的主要炎性因子,其啟動(dòng)并調(diào)節(jié)EMT的全過(guò)程,其主要通過(guò)①誘導(dǎo)細(xì)胞凋亡導(dǎo)致細(xì)胞丟失②直接作用于成纖維細(xì)胞、系膜細(xì)胞等上調(diào)細(xì)胞外基質(zhì)(extracellular matrix,ECM)合成,抑制ECM降解③促進(jìn)腎小管上皮細(xì)胞EMT參與腎臟纖維化過(guò)程。已有研究表明,TGF-β1可誘導(dǎo)腎小管上皮細(xì)胞呈現(xiàn)肌成纖維細(xì)胞的形態(tài),即極性改變、出現(xiàn)肌動(dòng)蛋白微絲及致密小體,且膠原蛋白產(chǎn)生增加。盡管TGF-β1可通過(guò)RhoA,ERK,P38-MAPK, JNK和Wnt/β-catenin途徑介導(dǎo)上述改變,但目前TGF-β/Smad被認(rèn)為是介導(dǎo)這一過(guò)程最主要的信號(hào)通路�；罨腡GF-β1使得Smad2和Smad3磷酸化,隨后,磷酸化的Smad2和Smad3同Smad4結(jié)合后形成Smad低聚復(fù)合物,并轉(zhuǎn)入細(xì)胞核調(diào)節(jié)靶基因轉(zhuǎn)錄。糖尿病時(shí),許多促炎因子能激活Smad信號(hào)通路,在糖尿病腎病患者腎小球及間質(zhì)纖維化區(qū)域可見(jiàn)到磷酸化的Smad2及Smad3沉積,證明有TGF-β/Smad信號(hào)通路的活化。目前臨床上尚無(wú)治療糖尿病腎病的特效藥物,只能通過(guò)控制飲食、降糖、降壓等措施進(jìn)行治療,效果有限。中醫(yī)藥治療歷史悠久,中藥因其多成分、治療多靶點(diǎn)的特點(diǎn),具有良好的應(yīng)用前景。黃芪為豆科植物蒙古黃芪或膜莢黃芪的干燥根,具有補(bǔ)氣固表、利尿托毒、排膿、斂瘡生肌的功效。黃芪甲苷(AstragalosideⅣ,ASI)是黃芪皂苷的單體成分,具有抗氧化、減輕缺血再灌注損傷等作用,在防治急性腎損傷、膜性腎病、糖尿病腎病等腎臟疾病中發(fā)揮重要作用。目前,對(duì)ASI防治糖尿病腎病的報(bào)道大多局限于足細(xì)胞及系膜細(xì)胞,對(duì)腎小管間質(zhì)纖維化研究較少,因此本研究擬通過(guò)體內(nèi)及體外實(shí)驗(yàn)探討ASI抑制糖尿病腎病腎間質(zhì)纖維化的作用和機(jī)制。研究目的觀察ASI對(duì)2型糖尿病模型KKAy小鼠腎組織TGF-β/Smad信號(hào)通路及其對(duì)高糖誘導(dǎo)下腎小管上皮細(xì)胞NRK-52E的影響,探討ASI對(duì)腎間質(zhì)纖維化的保護(hù)作用及機(jī)制,為臨床應(yīng)用ASI提供實(shí)驗(yàn)依據(jù)。研究方法1.動(dòng)物分組:購(gòu)入小鼠適應(yīng)性喂養(yǎng)2周,KKAy小鼠予以KK飼料(高脂)喂養(yǎng)至14周,隨機(jī)血糖超過(guò)13.9mmol/L提示造模成功;血糖水平相近的KKAy小鼠隨機(jī)分為模型組(10只)和ASI組(10只),正常飲食的同齡10只雄性C57BL/6J小鼠作為對(duì)照組。其中對(duì)照組和模型組每日予以生理鹽水予以40mg/kg灌胃,ASI組予以ASI 40mg/kg灌胃;2.血糖、血肌酐、尿ACR檢測(cè):每周測(cè)量小鼠體重,于第16周、20周、24周采取提尾反射法收集隨機(jī)尿液,ELISA試劑盒檢測(cè)尿微量白蛋白和尿肌酐,計(jì)算尿ACR,尾靜脈取血測(cè)血糖,內(nèi)眥取血測(cè)血肌酐;3. 24周時(shí)斷頸處死小鼠,分離腎臟,行HE染色、Masson染色觀察各組腎臟病理變化;4.應(yīng)用免疫組化染色檢測(cè)各組腎臟TGF-β1、Smad2/3、α-SMA的表達(dá);5. ASI對(duì)NRK-52E細(xì)胞活力的影響:NRK-52E細(xì)胞生長(zhǎng)至融合狀態(tài)時(shí),同步化培養(yǎng)24小時(shí),以不同濃度的ASI (0、10、20、40、80、100μg/ml,依次簡(jiǎn)寫(xiě)為 0、ASI10、ASI20、ASI40、ASI80、ASI100)刺激 NRK-52E 細(xì)胞 24小時(shí),細(xì)胞計(jì)數(shù)試劑盒CCK-8檢測(cè)細(xì)胞活性;6. ASI對(duì)高糖誘導(dǎo)下NRK-52E細(xì)胞凋亡的影響:①細(xì)胞長(zhǎng)至融合狀態(tài)時(shí),同步化培養(yǎng)24小時(shí),設(shè)正常對(duì)照組、高糖組、ASI各組:高糖DMEM中加入ASI,濃度分別為 20、40、80、100μg/ml (依次簡(jiǎn)寫(xiě)為 HG+ASI20、HG+ASI40、HG+ASI 80、HG+ASI 100),分別刺激NRK-52E細(xì)胞24小時(shí),或②用高糖+ASI 100μg/ml 分別刺激細(xì)胞 0、4、8、12、24、48 小時(shí),用 AnnexinV-FITCPI 試劑盒檢測(cè)細(xì)胞凋亡;7. ASI 對(duì)高糖誘導(dǎo)的NRK-52E 細(xì)胞 Smad2、Smad3、α-SMA、TGF-β1 表達(dá)的影響:細(xì)胞生長(zhǎng)至融合狀態(tài)時(shí),同步化培養(yǎng)24小時(shí),設(shè)正常對(duì)照組、高糖組、ASI各組(同前述),分別刺激NRK-52E細(xì)胞24小時(shí),以Real-Time PCR檢測(cè) Smad2、Smad3、α-SMA、TGF-β1 核酸水平的表達(dá),Western Blot 檢測(cè) Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、TGF-β1 蛋白水平的表達(dá);8.統(tǒng)計(jì)學(xué)方法:計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)誤表示,根據(jù)方差齊性檢驗(yàn)結(jié)果,兩組間比較采用獨(dú)立樣本t檢驗(yàn),多組比較采用單因素方差分析,所有數(shù)據(jù)均由統(tǒng)計(jì)軟件SPSS 19.0完成,以P0.05為差異有統(tǒng)計(jì)學(xué)意義,P0.01為差異有顯著統(tǒng)計(jì)學(xué)意義。研究結(jié)果1. 一般狀態(tài):對(duì)照組小鼠精神狀態(tài)好,反應(yīng)靈敏,毛色順滑;模型組小鼠精神萎靡不振,步履遲緩,煩渴多尿,反應(yīng)遲鈍,毛發(fā)無(wú)光澤,且隨著周齡的增加,上述癥狀更加明顯。ASI組小鼠的狀態(tài)介于上述兩組之間。在16、20、24周齡時(shí),模型組及ASI組小鼠體重均比同齡對(duì)照組小鼠大(均P0.01);與模型組小鼠相比,ASI組體重增長(zhǎng)減緩(均P0.05);2.血糖、血肌酐、尿ACR檢測(cè):在各觀察時(shí)間點(diǎn),與同齡對(duì)照組相比,模型組及ASI組血糖、尿ACR明顯升高(均P0.01);與同齡模型組相比,ASI組血糖、尿ACR較低(P0.05, P0.01)。三組小鼠血清肌酐檢測(cè)結(jié)果無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);3.各組小鼠腎組織病理改變:光鏡下可觀察到對(duì)照組腎小球及腎小管結(jié)構(gòu)清晰,系膜細(xì)胞數(shù)量正常,間質(zhì)中未見(jiàn)炎細(xì)胞浸潤(rùn)及纖維化;模型組腎小球肥大,系膜基質(zhì)增寬,系膜細(xì)胞增多,腎小管上皮細(xì)胞胞漿出現(xiàn)空泡、腎小管腔可見(jiàn)透明管型,腎間質(zhì)炎癥細(xì)胞增多,充血水腫;ASI組腎小球及系膜區(qū)改變介入對(duì)照組及模型組之間,腎小管上皮細(xì)胞輕度腫脹,胞漿較少,未間明顯的間質(zhì)纖維化;4.免疫組化染色檢測(cè)各組腎臟TGF-β1、Smad2/3及α-SMA的表達(dá)情況:α-SMA、TGF-β1及Smad2/3主要在腎小管-間質(zhì)表達(dá);對(duì)照組少見(jiàn)TGF-β1表達(dá),而模型組及ASI組TGF-β1表達(dá)增強(qiáng)(P0.01,P0.05),與模型組相比,ASI組TGF-β1表達(dá)減弱(P0.01);對(duì)照組α-SMA多數(shù)表達(dá)于腎血管平滑肌,在腎小管間質(zhì)偶有表達(dá),但在模型組可見(jiàn)α-SMA在腎小管上皮細(xì)胞、腎小管周?chē)兴磉_(dá)(P0.01),與模型組相比,ASI組α-SMA表達(dá)明顯下調(diào)(P0.01);對(duì)照組腎小管與腎小球細(xì)胞核有少量Smad2/3表達(dá),模型組及ASI組表達(dá)增加(P0.01,P0.05),但同模型組比較,ASI組Smad2/3表達(dá)減少(P0.01);5. ASI對(duì)NRK-52E細(xì)胞活力的影響:不同濃度ASI分別作用于NRK-52E細(xì)胞24小時(shí),各組之間細(xì)胞活性差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);6. ASI對(duì)高糖誘導(dǎo)下NRK-52E細(xì)胞凋亡的影響:與對(duì)照組相比,高糖組細(xì)胞凋亡增加(P0.01),加入 ASI 后,HG+ASI 40、HG+ASI 80、HG+ASI 100組的細(xì)胞凋亡均較高糖組減輕(均P0.05 ): HG+ASI 100μg/ml作用于細(xì)胞8h后,細(xì)胞凋亡顯著受抑,且隨著時(shí)間的延長(zhǎng),此作用有逐漸增強(qiáng)的趨勢(shì)(與Oh相比,均P0.05);7. ASI 對(duì)高糖誘導(dǎo)的 NRK-52E 細(xì)胞 Smad2、Smad3、α-SMA、TGF-β1 mRNA的影響:與對(duì)照組相比,高糖組(HG)、HG+ASI20組Smad2、TGF-β1mRNA表達(dá)上調(diào)(均P0.01),Smad3表達(dá)上調(diào)(均P0.05) , HG、HG+ASI20、HG+ ASI 40 α-SMA 表達(dá)上調(diào)(均P0.01);與 HG 相比,HG+ASI 80、HG+ASI 100 組 α-SMA、Smad3 mRNA 表達(dá)下調(diào)(均P0.05),HG+ASI 40、HG+ASI 80、HG+ASI 100 TGF-β1 表達(dá)下調(diào)(均P0.01),HG+ASI 20、HG+ASI 40、HG+ASI 80、HG+ASI100Smad2 表達(dá)下調(diào)(P0.05);8. ASI 對(duì)高糖誘導(dǎo)的 NRK-52E 細(xì)胞 Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、TGF-β1蛋白水平的影響:與對(duì)照組相比,高糖組(HG)、HG+ASI20、HG+ASI40組α-SMA、Smad2、Smad3表達(dá)上調(diào),差異有統(tǒng)計(jì)學(xué)意義(均P0.01),p-Smad2、p-Smad3 在 HG、HG+ASI20、HG+ASI40、HG+ASI80 組表達(dá)均上調(diào)(均P0.01 ),TGF-β1在HG、HG+ ASI 20組表達(dá)上調(diào)(均P0.01);與 HG 相比,HG+ASI80、HG+ASI 100 組 α-SMA、Smad2、Smad3、p-Smad2、p-Smad3表達(dá)下調(diào),差異有統(tǒng)計(jì)學(xué)意義(均P0.05),TGF-β1在HG+ASI40、HG+ASI80、HG+ASI100 組表達(dá)下調(diào)(均P0.05)。研究結(jié)論1.黃芪甲苷可減輕糖尿病KKAy小鼠的高血糖、肥胖及尿ACR,但不影響血肌酐;2.黃芪甲苷可改善糖尿病KKAy小鼠的腎小管間質(zhì)纖維化,其作用機(jī)制可能與ASI下調(diào)TGF-β/Smad信號(hào)通路有關(guān);3.黃芪甲苷可呈劑量及時(shí)間依賴性抑制高糖誘導(dǎo)的NRK-52E細(xì)胞凋亡;4.黃芪甲苷可抑制高糖誘導(dǎo)的NRK-52E細(xì)胞α-SMA表達(dá)及TGF-β1/Smad信號(hào)通路活性,從而減輕EMT,延緩腎間質(zhì)纖維化。
[Abstract]:Diabetes mellitus (DM) is a chronic noncommunicable disease which seriously affects human health. With the change of diet structure and lifestyle, the prevalence, incidence and number of patients rise sharply and bring heavy economic burden to family and society. Diabetic nephropathy (diabetic kidney disease, DKD) is diabetic Microblood. One of the major complications of vascular disease, even if the blood pressure and blood glucose control standard, diabetes patients eventually have 30% - 40% to develop to diabetic nephropathy. Diabetic nephropathy is the main cause of end-stage renal disease (ESRD) worldwide. The changes are irreversible glomerular fibrosis and scar formation, and renal tubular injury is only secondary. But with the study, renal tubular lesions, especially tubulointerstitial fibrosis (TIF), play a very important role in promoting diabetic nephropathy, and renal tubular lesions can not be found. It is dependent on the glomerular lesion. Therefore, it is of great significance to explore the treatment strategy of postponing renal tubule interstitial fibrosis. Although the pathogenesis of TIF is not clear, more and more evidence points to the apoptosis of renal tubular epithelial cells and the transdifferentiation of renal tubular fibroblasts (mesenchymal cells). (epithelial-to-mesenchymal transition, EMT).EMT is a classic pathological change of tubular injury in diabetic nephropathy and is parallel to the degree of renal interstitial fibrosis. More and more studies have strongly confirmed that EMT plays an important role in different types of renal diseases, while reversing EMT can effectively improve renal fibrosis. A large number of studies have revealed the initiating factors, regulatory factors and existing signaling pathways of various EMT, but in all factors, TGF- beta 1 is a key regulator of fibrosis in various tissues and organs. It is the main inflammatory factor in the progression of diabetic nephropathy. It initiates and regulates the whole process of EMT. It mainly induces apoptosis by inducing cell apoptosis. Cell loss (2) directly acts on fibroblasts, mesangial cells and so on, up the synthesis of extracellular matrix (extracellular matrix, ECM), inhibiting ECM degradation and promoting renal tubular epithelial cell EMT to participate in the process of renal fibrosis. It has been shown that TGF- beta 1 can induce the morphology of myofibroblast in renal tubular epithelial cells, that is, polarity change, Actin microfilaments and compact bodies appear and collagen production increases. Although TGF- beta 1 can mediate these changes through RhoA, ERK, P38-MAPK, JNK and Wnt/ beta -catenin pathways, TGF- beta /Smad is currently considered as the most important signaling pathway to mediate this process. Activated TGF- beta 1 makes Smad2 and Smad3 phosphorylation, followed by phosphorylation The Smad oligomer complex is formed with the combination of Smad3 with Smad4 and transferred into the nuclear regulation target gene transcription. In diabetes, many proinflammatory factors can activate the Smad signaling pathway. In the glomeruli and interstitial fibrosis areas of diabetic nephropathy patients, phosphorylated Smad2 and Smad3 deposition can be seen, proving the activation of TGF- beta /Smad signaling pathway. There is no special drug to treat diabetic nephropathy in bed. It can only be treated by controlling diet, reducing sugar and lowering blood pressure, and the effect is limited. Traditional Chinese medicine has a long history. The traditional Chinese medicine has a good application prospect because of its multi-component and multi target characteristics. The radix astragali is the dry root of Mongolia Astragalus or membranous membranous membranous Astragalus membranaceus. The effects of Astragalus glycoside (Astragaloside IV, ASI) are the monomers of Astragalus saponins, which have the effect of antioxidation and ischemia reperfusion injury, and play an important role in the prevention and treatment of renal diseases such as acute renal injury, membranous nephropathy, diabetic nephropathy and so on. At present, the prevention and treatment of diabetic nephropathy by ASI is reported. Most of the channels are limited to podocyte and mesangial cells, and there are few studies on renal tubulointerstitial fibrosis. Therefore, this study intends to explore the role and mechanism of ASI inhibition of renal interstitial fibrosis in diabetic nephropathy in vivo and in vitro. The purpose of this study is to observe the TGF- beta /Smad signaling pathway and the high glucose induced by ASI in the renal tissue of type 2 diabetes model KKAy mice. The effect of NRK-52E on renal tubular epithelial cells was guided to explore the protective effect and mechanism of ASI on renal interstitial fibrosis, and to provide experimental basis for clinical application of ASI. Methods 1. animals were divided into groups: 2 weeks of adaptive feeding in mice, KKAy mice were fed with KK feed (high fat) for 14 weeks, and the blood glucose was more than 13.9mmol/L. The KKAy mice were randomly divided into model group (10 rats) and group ASI (10 rats), and 10 male C57BL/6J mice of the same age of normal diet were used as control group. The control group and model group were given 40mg/kg gavage with normal saline daily, ASI group was given ASI 40mg/kg, 2. blood glucose, blood creatinine, and urine ACR test: the weight of mice was measured every week, sixteenth weeks, 20 weeks, 24 weeks to take the tail reflection method to collect random urine, ELISA kit to detect urine microalbumin and urine creatinine, calculate urine ACR, take blood from the tail vein to measure blood sugar, and take blood to measure creatinine in the inner canthus; 3.24 weeks when the neck was killed, the kidneys were separated, HE staining was performed, and Masson staining was used to observe the pathological changes of kidney in each group; 4. the immunohistochemical staining was used to detect each group. The expression of TGF- beta 1, Smad2/3, and alpha -SMA, and the effect of 5. ASI on the vitality of NRK-52E cells: synchronization culture for 24 hours when NRK-52E cells grew to fusion state, and stimulated the cells for 24 hours at different concentrations of ASI (0,10,20,40,80100 mu g/ml, ASI10, ASI20, ASI40, etc.) for 24 hours, cell count kit inspection The effect of 6. ASI on the apoptosis of NRK-52E cells induced by high glucose: (1) when the cells grew to the fusion state, the cell culture was synchronized for 24 hours, and the normal control group, the high sugar group, and the ASI groups were added to the high sugar DMEM, and the concentration was 20,40,80100 g/ml (HG +ASI20, HG+ASI40, HG+ASI 80, HG+ASI 100) respectively. Cells were stimulated for 0,4,8,12,24,48 hours with high glucose +ASI 100 g/ml for 24 hours, and cell apoptosis was detected by AnnexinV-FITCPI kits, and 7. ASI on the expression of Smad2, Smad3, alpha -SMA, TGF- beta 1 induced by high glucose induced NRK-52E cells: cell growth to fusion state, synchronized culture for 24 hours, normal control group, high sugar Group (ASI), the NRK-52E cells were stimulated for 24 hours, respectively, and the expression of Smad2, Smad3, alpha -SMA, TGF- beta 1 nucleic acid level was detected by Real-Time PCR, Western Blot was detected in Smad2, Smad3, alpha, alpha, and beta 1 protein level; 8. statistical formula: measurement data were mistaken for mean number, and according to the homogeneity test of variance Results, the two groups were compared with the independent sample t test, and the multiple groups were compared with single factor analysis of variance. All the data were completed by the statistical software SPSS 19. The difference was statistically significant with the difference of P0.05. The difference of P0.01 was statistically significant. The results of the study were 1. general states: the control group had good mental state, sensitive reaction, smooth hair color slippery; The mice in the type group were depressed, slow, thirsty, polyuria, slow reaction and glossy hair, and with the increase of age, the above symptoms were more obvious between the two groups of.ASI mice. At the age of 16,20,24, the weight of the model group and the ASI group was larger than the same age group (P0.01); compared with the model mice, ASI group weight growth slowed (P0.05); 2. blood sugar, blood creatinine, urine ACR detection: compared with the same age control group, the blood glucose of the model group and the ASI group increased significantly (P0.01) at the observation time point. Compared with the same age model group, the blood glucose of the ASI group was lower (P0.05, P0.01). The serum creatinine detection results of the three groups were not statistically significant (P0.05); 3. Pathological changes of renal tissue in each group: the glomerular and tubular structure of the control group could be observed clearly, the number of mesangial cells was normal, the number of mesangial cells was normal, there was no inflammatory cell infiltration and fibrosis in the interstitium; the glomerular hypertrophy, the mesangial matrix increased, the mesangial cells increased, the cytosolic vacuoles appeared in the renal tubules, and the small tubule of the renal tubules could be seen in the renal tubule. The renal interstitial inflammatory cells increased and hyperemia and edema, and the glomerular and mesangial areas of the ASI group changed between the interventional control group and the model group. The renal tubular epithelial cells were slightly swollen, with less cytoplasm and no obvious interstitial fibrosis. 4. immunohistochemical staining was used to detect the expression of TGF- beta 1, Smad2/ 3 and alpha -SMA in each group: alpha -SMA, TGF- beta 1 and Smad2/3 were mainly in the group. In the control group, the expression of TGF- beta 1 was rare in the control group, while the expression of TGF- beta 1 in the model group and ASI group was enhanced (P0.01, P0.05). Compared with the model group, the expression of TGF- beta 1 was weakened (P0.01), and the alpha -SMA majority of the control group was expressed in the renal vascular smooth muscle and the tubulointerstitium was occasionally expressed, but the alpha -SMA in the renal tubular epithelial cells and renal tubules was found in the model group. Compared with the model group, the expression of alpha -SMA in the ASI group was significantly down (P0.01), and in the control group, the renal tubules and the glomerular nuclei had a small amount of Smad2/3 expression, the expression of the model group and the ASI group increased (P0.01, P0.05), but the Smad2/3 expression of the ASI group decreased (P0.01) compared with the model group, and the effect of 5. ASI on the vitality of the 5. ASI cells: different concentrations The effect of I on NRK-52E cells was 24 hours respectively, and there was no significant difference in cell activity between each group (P0.05); the effect of 6. ASI on the apoptosis of NRK-52E cells induced by high glucose: compared with the control group, the apoptosis of the high glucose group increased (P0.01). After adding ASI, the apoptosis of HG+ASI 40, HG+ASI 80, HG+ASI 100 was lower than that of the high sugar group (P0.05): H After the action of G+ASI 100 mu g/ml on cell 8h, apoptosis was significantly inhibited, and with the prolongation of time, the effect was gradually enhanced (compared with Oh, P0.05); 7. ASI to high glucose induced NRK-52E cell Smad2, Smad3, alpha -SMA, TGF- beta 1 mRNA. All P0.01), Smad3 up regulation (P0.05), HG, HG+ASI20, HG+ ASI 40 alpha -SMA expression up-regulated (P0.01), HG+ASI 80 compared with HG, HG+ASI 100, 40, 80, 100, 1, 20, 40, 80, downregulation The effect of 8. ASI on the level of Smad2, Smad3, p-Smad2, p-Smad3, alpha -SMA, TGF- beta 1 protein in high glucose induced NRK-52E cells: compared with the control group, the high glucose group (HG), HG+ASI20, HG+ASI40 group was up regulated. .01), TGF- beta 1 was up-regulated in HG, HG+ ASI 20 groups (P0.01); compared with HG, HG+ASI80, HG+ASI 100 groups of -SMA, Smad2, Smad3, and decreased expression was statistically significant. Blood glucose, obesity and urine ACR, but do not affect the blood creatinine; 2. astragaloside can improve the renal tubulointerstitial fibrosis in diabetic KKAy mice, its mechanism may be related to the downregulation of TGF- beta /Smad signaling pathway in ASI; 3. astragaloside can inhibit high glucose induced apoptosis in a dose and time dependent manner; 4. astragaloside can inhibit high glucose induction The expression of NRK-52E -SMA and TGF- beta 1/Smad signaling pathway can alleviate EMT and delay renal interstitial fibrosis.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R692.9

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本文編號(hào)：1983644