【作者】 栾家杰；

【导师】 李卫平；

【作者基本信息】 安徽医科大学 ， 药理学， 2012， 博士

【摘要】 糖尿病（Diabetes Mellitus, DM）是由于胰岛素分泌缺乏及（或）机体产生胰岛素抵抗所引起的以血糖升高为主要特征的常见内分泌代谢性疾病，现已成为继肿瘤、心脑血管疾病之后第三大危害人类健康的慢性疾病。糖尿病肾病（Diabeticnephropathy, DN）是DM最常见和最严重的并发症之一，约20%～30%的1型糖尿病（Type1diabetes mellitus, T1DM）或2型糖尿病（Type2diabetes mellitus, T2DM）患者最终并发DN，其中相当一部分可发展为终末期肾病（End-stage renal disease,ESRD）。DN是糖尿病患者死亡的重要原因。DN发病机制尚未完全阐明，但其发生和发展是以高糖状态为始动因子的多因素综合作用的结果，已有研究表明，高糖状态可引起机体的氧化应激反应增强，氧化应激可能是导致DN等并发症的主要原因，在DN的发生、发展中起着重要的作用。对DN的防治，目前亦尚缺乏理想的方法与药物。因此探讨DN发病机制，寻找有效防治DN的方法与药物具有重要的理论与实践意义。祖国医学认为，糖尿病属于“消渴”、“水肿”病范畴，为“本虚标实、虚实夹杂”之证。治疗当以扶正与祛邪并举。临床以益气养阴、化瘀通络治疗为主。黄芪为临床常用中药，具有补气升阳、益气固表、利水消肿、扶正固本和延缓衰老等多种药理作用，可用于气虚水肿，内热消渴等多种病症的防治。黄芪作为君药是防治心、肝、肾脏等疾病的常用药物。单味黄芪对肾病综合征患者即可产生明显的降低尿蛋白的作用，是临床常用防治肾病综合征的中药之一。黄芪总苷（Astragalosides，AST）是黄芪的主要有效部位群。本课题组多年的研究发现，AST有抗应激、抗氧化和免疫调节等多种药理作用。关于以黄芪组方或黄芪提取物治疗糖尿病的临床研究以及基础研究报道较多，但尚未见系统研究AST防治DM及其并发症DN的作用与机制的研究报道。本课题组对黄芪的有效组份AST及单体黄芪甲苷的药理学进行了多年的研究，在此基础上，我们拟在体内外，对AST治疗DM的作用及可能的作用机制进行探讨，寻找有效的防治DM与DN的药物，为提高临床治疗DM与DN并发症的疗效提供理论基础。目的：研究AST对链脲佐菌素（Streptozotocin, STZ）诱导的大鼠及小鼠糖尿病模型治疗作用，探讨AST对糖尿病动物肾脏保护作用及可能机制；采用高糖培养人肾小球系膜细胞（Human Mesangial Cell, HMC）作为目标细胞，进一步研究AST防治DM及DN的分子机制。方法：1. STZ(100mg·kg^-1)单次腹腔注射建立糖尿病小鼠模型。取造模成功的小鼠随机分为模型组（Model）、Tempol组(90mg·kg^-1)、AST组(30,60,120mg·kg^-1)。每组20只动物。各组动物连续灌胃给药4-6W，正常组（Control）和模型组给予等容量的蒸馏水。分别在给药后4W、6W，各组随机取半数（10只）动物，取血检测空腹血糖（Fasting blood glucose, FBG）、甘油三酯（Triglyceride, TG）、总胆固醇（TotalCholesterol, TC）、糖化血清蛋白（Glycosylated serum protein, GSP）含量；取血后颈椎脱臼处死动物后迅速摘取肝脏、脾脏、肾脏，称重，计算脏器指数；制作10%肝脏组织匀浆，分离上清液检测T-SOD、MDA活性和GSH-Px含量；一侧肾脏制作病理切片，进行组织形态学检查，并用免疫组织化学方法检测肾脏TRPC6蛋白表达水平，TUNEL法检测STZ糖尿病小鼠肾小球细胞凋亡；另侧肾脏用于检测TGF-β₁mRNA、Col Ⅳ mRNA表达。2. STZ(60mg·kg^-1)单次腹腔注射建立糖尿病大鼠模型。取造模成功大鼠随机分为模型组（Model group）、Tempol组(60mg·kg^-1)与AST组(40mg·kg^-1)。每组20只动物。各组动物每日灌胃给药1次，连续4-6W，正常组（Control）和模型组给予等容量蒸馏水。实验过程中每日观察大鼠生存状态与活动情况，并采用代谢笼法，在造模前（Before）、造模后（0W）、给药后1W、2W，4W、6W分别记录大鼠体重，日摄食量、日饮水量、24h尿量（urine output，UO）等生理指标。尿液静置后，采用渗透压摩尔浓度测定仪测定尿液渗透压。取适量尿液离心，用于检测尿液生化指标：尿液肌酐（urine creatinine，Ucr）、24h尿蛋白（UAE）、β2-微球蛋白（β2-MG）、肿瘤坏死因子α（TNF-α）、转化生长因子-β₁（TGF-β₁）含量。分别在给药后4W及6W，各组随机取半数（10只）动物麻醉后，腹主动脉取血，检测空腹血糖（FBG），血清糖化血清蛋白（GSP）、空腹血清胰岛素（Fasting insulin, FINS）、胰岛素敏感指数（Insulin sensitivity index, ISI）、总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（High density lipoprotein cholesterol, HDL-C）、低密度脂蛋白胆固醇（Lowdensity lipoprotein cholesterol, LDL-C）含量、T-SOD、GSH-Px活性和MDA含量、血清尿素氮（Blood urea nitrogen, BUN）、血肌酐（Serum creatinine, Scr）含量。取血后颈椎脱臼处死动物，迅速摘取肝脏、脾脏、肾脏，称重，计算脏器指数。一侧肾脏制作病理切片，进行组织形态学检查，并采用免疫组织化学方法检测肾脏TRPC6蛋白表达水平；另侧肾脏用于检测TRPC6mRNA、TGF-β₁mRNA、Col ⅣmRNA表达。3.采用人肾小球系膜细胞（Human Mesangial Cell, HMC）作为目标细胞，分别设立正常对照组（NG）、甘露醇渗透压对照组（MA）、高糖组（HG）、Tempol组(100μmol·L^-1)、AST(50,100,200mg L^-1)组。NG组细胞予低糖DMEM培养液(含5.5mmol L^-1D-葡萄糖)培养，MA组予含24.5mmol L^-1甘露醇的低糖培养液培养，HG组予高糖DMEM培养液(含30mmol L^-1D-葡萄糖)培养，Tempol组予含100μmol L^-1Tempol的高糖培养液培养干预，AST各剂量组分别予含50,100,200mg L^-1AST高糖培养液培养。干预时间为24h。用MTT法检测AST(0-400mg L^-1)对高糖培养HMC细胞增殖与细胞活力影响；流式细胞仪检测各组HMC细胞中ROS含量；荧光实时定量PCR法检测各组HMC细胞内TRPC6mRNA、TGF-β₁mRNA、NOX4mRNA表达情况；Western blot法检测各组HMC细胞内TRPC6、TGF-β₁、NOX4蛋白表达情况。采用生物素双抗体夹心酶联免疫吸附法（Enzyme-linked immunosorbentassaym, ELISA）测定各组HMC细胞上清液内SOD、MDA、GSH-Px、TGF-β₁、FN、Col Ⅳ含量。结果：1黄芪总苷对实验性糖尿病小鼠肾脏保护作用1.1AST可显著改善STZ糖尿病小鼠“三多一少”的症状，提高肝脏抗氧化能力，表现为升高T-SOD、GSH-Px活性，降低MDA含量；血清FBG、GSP、TC及TG水平亦有下降的趋势，但尚未能恢复至正常水平。1.2AST可在一定程度上降低肾脏指数，对减轻肾小球损伤与凋亡呈剂量依赖性。1.3AST可抑制肾脏Col Ⅳ mRNA、TGF-β₁mRNA高表达，显著提高糖尿病小鼠肾小球TRPC6蛋白表达水平。2黄芪总苷对实验性糖尿病大鼠肾脏保护作用2.1AST可显著改善STZ糖尿病大鼠“三多一少”症状，表现为体重降低减缓，日摄食量、日饮水量及尿量逐渐减少。2.2AST可降低STZ糖尿病大鼠FBG、GSP，升高ISI；但大鼠FBG仍处于较高水平。2.3AST可降低STZ糖尿病大鼠血清TC、TG及LDL-C水平，升高HDL-C含量，提示AST可在一定程度上纠正糖尿病大鼠的血脂代谢紊乱。2.4AST可提高STZ糖尿病大鼠抗氧化能力，提高血清T-SOD、GSH-Px活性，降低MDA含量。2.5AST可使STZ糖尿病大鼠UAE明显下降，显著降低糖尿病大鼠24h尿β2-MG、TNF-α、TGF-β₁排出量。改善STZ糖尿病大鼠肾功能，降低Scr、BUN含量，提高Ccr，降低肾脏指数，减轻肾小球损伤；降低肾脏Col Ⅳ mRNA、TGF-β₁mRNA表达，提高TRPC6mRNA表达量，同时也显著提高糖尿病大鼠肾小球TRPC6蛋白表达水平。这表明AST对STZ糖尿病大鼠具有较好的防治其肾脏功能损伤的作用。3黄芪总苷对高糖培养人肾小球系膜细胞保护作用3.1在0--400mg L^-1浓度范围内，AST可浓度依赖性的抑制高糖培养HMC细胞增殖。3.2高糖培养HMC上清液内T-SOD、GSH-Px活性显著降低，MDA含量明显升高；AST可在一定程度上提高HMC T-SOD、GSH-Px活性，降低MDA含量。3.3高糖培养HMC上清液TGF-β₁、FN、Col Ⅳ含量明显上升；各剂量AST均可明显降低TGF-β₁、FN、Col Ⅳ含量，除AST(50mg L^-1)组外，其他各剂量组已恢复至正常水平。3.4高糖培养HMC内ROS含量明显上升；各剂量AST均可明显降低HMC细胞内ROS含量，其中AST(200mg L^-1)组HMC内ROS含量已降至正常水平。3.5高糖培养HMC内TRPC6mRNA表达水平显著降低，TGF-β₁mRNA及NOX4mRNA表达水平明显上升； AST(100,200mg L^-1)可显著提高HMC细胞内TRPC6mRNA表达水平，降低TGF-β₁mRNA及NOX4mRNA表达水平。3.6高糖培养HMC内TRPC6蛋白表达水平显著降低，TGF-β₁及NOX4蛋白表达水平明显升高；各剂量AST均可显著提高HMC细胞内TRPC6蛋白表达水平，降低TGF-β₁蛋白表达水平；除AST组(50mg L^-1)外，其他各剂量组NOX4蛋白表达水平也出现明显下降。结论：1AST对实验性糖尿病动物肾脏具有一定的保护作用，其机制可能与AST提高糖尿病动物的抗氧化能力、调节血脂代谢、抑制高糖状态所致的肾组织Col Ⅳ mRNA、TGF-β₁mRNA高表达及显著提高糖尿病动物肾组织TRPC6mRNA与TRPC6蛋白表达水平抑制肾小球细胞凋亡有关。2AST对高糖培养HMC具有保护作用，其机制可能与AST提高HMC抗氧化能力、降低TGF-β₁、FN、Col Ⅳ含量、抑制TGF-β₁、NOX4高表达及提高TRPC6表达水平有关。更多还原

【Abstract】 BackgroundDiabetes Mellitus （DM）, a common endocrine-metabolic disease caused by defects ofinsulin secretion and/or insulin resistance, is chacterized by increased blood glucose. Ithas been the third major disease following tumor and cardio-cerebrovascular disease.Diabetic nephropathy （DN） is the most frequent and serious complications of DM andbecomes the main cause of deaths in DM. Approximately20to30persents of patientswith Type1diabetes mellitus （T1DM） or Type2diabetes mellitus （T2DM） have DN,most of which are further developed to end-stage renal disease （ESRD）. Pathogenesy ofDN has not been clarified so far. It is believed to be influenced by multiple determintswith the high glucose level as the initiation factor. Strong oxidative stress is observed inDM and plays an important role in occurance and development of DM. Therefore,oxidative stress is regarded as the key reason of DM complications. As to the treatmentand prevention of DN, it is lack of ideal strategies and drugs. Thus, it would be of of greatsignificant to seek for effective methods for DN control.In Traditional Chinese Medicine （TCM）, it is considered that DM belongs to consumptivethirst and oedema and characterised with asthenia in origin, asthenia in superficiality andasthenia and sthenia. It treatment must focus on strengthening body resistance andeliminating pathogen. In clinical medication of TCM, supplementing qi and nourishingyin and disperse blood stasis and dredge collateral are suggested to be the predominanttreatments.Astragalus membranaceus （AS） is one of the most commonly used herbal medicines in TCM, which possesses “Buqishengyang, Yiweigubiao, Tudushengji, Lishuituizhong”function. It can be used in deficiency of vital energy and calor internus. AS is a principaldrug to control diseases in heart, liver and kidney. Single AS can decrease urine protein innephritic syndrome and is one of the commonest traditional medicine to control nephriticsyndrome. Astragalosides （AST） is the effective fraction extracted from astragalus.Through many years of research by our team, AST is found to have many biologicaleffects, including anti-stress, anti-oxidation and immunological regulation. There havebeen lots of clinical and experimantal researches on DM treatment with astragalus orextract of astragalus, but the systematic study about the effect of AST on DM and itspotential mechanisms is still limited. In this thesis, we take the pharmacologic action ofastragalus effective component as the particular target and aim to understand the effect ofAST on DM and its potential mechanisms with both in vivo experiment and ex vivoexperiment. Findings generated from the exproments are expected to provide elementaryinformation on DM treatment and drug design for DN.ObjectivesTo understand effect of AST on DM and its potential mechanisms by treating diabetic ratsand diabetic mice induced by streptozotocin （STZ） and the molecular mechanismsunderline the effect is investigated using high-glucose-incubated human mesangial cell（HMC） as the targeted cell.Methods1The diabetic mouse model was established by intraperitoneal injection with STZ (100mg·kg^-1) and the model mice were randomly divided into Model group, Tempol group (90mg·kg^-1), AST-L group (30mg·kg^-1), AST-M group (60mg·kg^-1), and AST-H group (120mg·kg^-1). The animals were given the drugs with successive intragastric administration for4-6weeks, the same volume of distilled water were admistered to Control group and Model group, respectively. After4weeks and6weeks of administration, half of the mice（10） in each group were randomly selected and blood sampkes were drawn to assessfasting blood glucose （FBG）, triglyceride （TG）, total cholesterol （TC）, glycosylated serumprotein （GSP）. The mice were sacrificed with cervical dislocation after blood sampleswere taken. And then, livers, spleens and kidneys were removed immediately, douchedwith ice physiological saline, dried with filter papers, weighed and organ index werecalculated. Liver tissue bomogenate （10%） was made by liver tissue of0.5g. Activities ofT-SOD and MDA and contents of GSH-Px were evaluated after isolating supernatants.One side of kidney was put into4%formaldehyde solution and made to paraffin sectionfor histomorphological examination. This side of kidney was also used to measure thelevel of TRPC6expression by immunohistochemistry as well as the apoptosis ofglomerular cells in glomeruli by TUNEL. The other side of kidney, however, was undercryopreservation at-80℃for the assessment of TGF-β₁mRNA、 Col Ⅳ mRNAexpression.2The diabetic rat model was established by intraperitoneal injection with STZ (60mg·kg^-1) and the model rats were randomly divided into Model group, Tempol group (60mg·kg^-1) and AST group (40mg·kg^-1). The animals were given the drugs with successiveintragastric administration for4-6weeks, the same volume of distilled water wererespectively admistered to Control group and Model group. Survival status and activitiesof the animals were observed every day. Via metabolic cage, body weight, daily foodintake, daily water intake and daily urine output （UO） were recorded respectivelypre-therapy,0W,1W,4W and6W after administration. After urion prepared in standing,uric osmotic pressure was assessed by osmotic pressure molar concentration determinator.Proper amount of urion was centrifugated and prepared for measurement of biochemicalindicators under cryopreservation at-80℃, such as urine creatinine （Ucr）,urinaryalbumin excretion （UAE） β2-microglobulin （β2-MG）, tumor necrosis factor （TNF-α）, transforming growth factor （TGF-β₁）. After4weeks and6weeks of administration, halfof the rats （10） in each group were randomly selected and blood samples were drawn fromabdominal aorta after anesthesia to assess FBG, GSP, fasting serum insulin （FINS）, insulinsensitivity index （ISI）, TC, TG, high density lipoprotein （HDL）, low density lipoprotein（LDL）, as well as activities of T-SOD and GSH-Px and contents of MDA, blood ureanitrogen （BUN）, serum creatinine （Sc）. The rats were sacrificed with cervical dislocationafter blood samples were taken. And then, livers, spleens and kidneys were removedimmediately, douched with ice physiological saline, dried with filter papers, weighed andorgan index were calculated. Liver tissue bomogenate （10%） was made by liver tissue of0.5g. One side of kidney was put into4%formaldehyde solution and made to paraffinsection for histomorphological examination. This side of kidney was also used to measurethe level of TRPC6expression by immunohistochemistry. The other side of kidney,however, was under cryopreservation at-80℃for the assessment of TRPC6mRNA,TGF-β₁mRNA and Col ⅣmRNA expression.3Human mesangial cells （HMC） was taken as target cells and divided into Normal group（NG）, Mannitol （MA）, high glucose group （HG）, Tempol (100μmol·L^-1), AST-L(50mg L^-1), AST-M (100mg L^-1) and AST-H (200mg L^-1). NG group were incubated withlow glucose DMEM culture solution (5.5mmol L^-1D-glocose), MA group wereincubated with low glucose culture solution (24.5mmol L^-1Mannitol) and HG groupwere with high glucose DMEM culture solution (30mmol L^-1D-glocose). Similarly,Tempol group were incubated with high glucose culture solution (100μmol L^-1Tempol),and high glucose culture solution (50mg L^-1AST) in AST-L group, high glucose culturesolution (100mg L^-1AST) in AST-M group and high glucose culture solution (200mg L^-1AST) in AST-H group. The duration of administration was all defined as24hours. Then,different methods were adopted to assess various indicators. For instance, MTT Assay forthe effect of AST on proliferation and viability of high-glucose-incubated HMC, flow cytometry for ROS contents in HMC, fluorescent quantitative real time PCR for TRPC6mRNA、TGF-β₁mRNA、NOX4mRNA expression in HMC, Western blot for TRPC6、TGF-β₁、NOX4expression in HMC. In addition, enzyme-linked immunosorbent assaym（ELISA） was introduced to assess contents of SOD、MDA、GSH-Px、TGF-β₁、FN、Col Ⅳ in HMC supernatant in each group.Results1Protective effects of AST on experimental diabetic mice1.1AST significantly improved the typical symptoms （polyuria, polydipsia, polyphagia,weight loss） and enhanced the anti-oxidative ability in liver of STZ diabetic mice. Indetail, it increased activities of T-SOD and GSH-Px and decreased MDA level. Althoughthe levels of serum FBG, GSP, TC and TG decreased, the reduction could not return tonormal level.1.2AST decreased kidney index to some extent. The effects of alleviating glomerulusdefects and apoptosis were shown dose-dependent and this effect was most significant atthe dosage of60mg·kg^-1.1.3AST inhibited the high expression of collagen Type Ⅳ mRNA and TGF-β₁mRNA inkidney. AST raised the TRPC6expression in glomerulus in STZ diabetic mice.2Protective effects of AST on experimental diabetic rats2.1AST significantly improved the typical symptoms （polyuria, polydipsia, polyphagia,weight loss） in STZ diabetic rats. The body weights of rats didn’t continuously decrease,while daily food intake, daily water intake and daily urine output decreased gradually.2.2AST decreased the levels of FBG and GSP and increase ISI. But the content of FBGstill kept at a relatively high level. 2.3AST decreased the level of serum TC, TG and LDL while enhance the level of HDL.This kind of effect could rectify lipid metabolic disorders in STZ diabetic rats.2.4AST enhanced the anti-oxidative ability in STZ diabetic rats. It increased activities ofT-SOD and GSH-Px and decreased MDA level.2.5AST decreased daily amount of urine protein excretion as well as daily amount ofurine β2-MG, TNF-α and TGF-β₁excretion. AST improved kidney function of STZdiabetic rats. It decreased serum levels of creatinine, urea nitrogen and enhanced thecreatinine clearance rate. It decreased kidney index and alleviate the defects of glomerulus.It also decreased expression of collagen Type Ⅳ mRNA and TGF-β₁mRNA, enhancedexpression of TRPC6mRNA and increased expression of TRPC6in glomerulus in STZdiabetic rats.3Protective effect of AST on high glucose incubated glomerular mesangial cells3.1At the range of0-400mg L^-1, the cell proliferation inhibiting rate caused by ASTshowd dose-dependent in high glucose incubated incubated glomerular mesangial cells.3.2Activities of T-SOD and GSH-Px in HMC supernatant decreased and increasedcontent of MDA. AST increased activities of T-SOD and GSH-Px in HMC and decreasedthe level of MDA.3.3The level of TGF-β₁, FN and Col Ⅳ increased in high glucose incubated HMCsupernatant. AST with high, median and low dosage all decreased the contents of TGF-β₁,FN and Col Ⅳ. The three indicators returned to normal level in groups with high andmedian dosage.3.4The level of ROS significantly increased in high glucose incubated HMC. AST withhigh, median and low dosage all decreased the contents of ROS in HMC. And ROSdecreased to normal level in group with high AST dosage. 3.5The expression of TRPC6mRNA decreased in high glucose incubated HMC while theexpression of TGF-β₁mRNA and NOX4mRNA significantly increased. AST with highand median dosage both increased the expression of TRPC6mRNA and decreased theexpression of TGF-β₁mRNA and NOX4mRNA.3.6The expression of TRPC6protein decreased in high glucose incubated HMC while theexpression of TGF-β₁and NOX4protein significantly increased. AST with high, medianand low dosage all decreased the the expression of TRPC6protein in HMC and decreasedthe expression of TGF-β₁protein. The expression of NOX4protein significantlydecreased in group with high and median dosage.Conclusions1There is protective effect of AST on high glucose incubated glomerular mesangial cells.The potential mechanisms may include the enhancement of anti-oxidative ability of HMC,reduction of the levels of TGF-β₁, FN and Col Ⅳ, inhibitation in the high expression ofTGF-β₁and NOX4and promotion of the TRPC6expression by AST.2There are some protective effects of AST on kidney of experimental diabetic rats andmice. The potential mechanisms may include improvement of anti-oxidative ability inserum and liver, regulation of lipid metabolism, protection of kidney function, inhibitationin apoptosis in glomerulus, inhibitation in the high expression of Col ⅣmRNA,TGF-β₁mRNA in kidney and enhancement of the expression of TRPC6mRNA andTRPC6protein in glomerulus by AST.更多还原