【作者】 王明；

【导师】 魏连波；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2011， 博士

【摘要】 糖尿病肾病(DN)是糖尿病(DM)最常见的微血管并发症之一,是导致终末期肾病(ESRD)和DM患者死亡的主要原因。约40%的1型DM和5-20%的2型DM患者可以发生DN。目前DN的发病机制尚不清楚,亦缺乏有效的治疗手段。因此,有效地预防、治疗DN已成为医学领域中极为重要的问题。DM可由多种途径损害肾脏,并累及肾脏的所有结构,导致肾小球细胞增殖、肥大,基底膜增厚和进行性细胞外基质(ECM)堆积,最终导致弥漫性或结节性肾小球硬化。转化生长因子-β1 (TGF-β1)是一种多功能的细胞因子,在DN发病中起着非常重要的作用。包括：促使肾脏固有细胞发生增殖、肥大,增加系膜细胞(MC)合成和分泌ECM,并通过增加基质降解酶抑制物活性而减少ECM的降解等方面作用。在DM肾损害中,几乎所有已证实的分子介质和细胞内传导途径都可刺激、介导肾内TGF-β1的活化。因此,探寻通过拮抗TGF-β1治疗DN的新方法,寻找其作用更特异、有效的下游因子或环节,具有重要的临床意义。microRNAs (miRNAs)作为一类小分子非编码RNA,由于其存在的普遍性以及所参与的调控过程的复杂性,成为近年来研究的热点。已有研究显示miRNAs在DM微血管并发症发病中起重要作用。miR-192作为在肾脏特异性表达的miRNA之一,可以经由其TGF-β1介导通路参与DN的ECM蛋白蓄积进程,可能机制为：TGF-β1正调节miR-192,miR-192减量调节smad相互作用蛋白1(SIP1),导致Ⅰ型胶原(CollgenⅠ)过度表达,蛋白蓄积。SIP1的3’UTR为miR-192调控靶点,miR-192通过mRNA降解途径减少SIP1表达。TGF-β1诱导miR-192表达的机制尚待进一步探讨,有证据显示miR-192启动子存在高度保守的上游区Ets-1原癌基因结合位点,而TGF-β1也诱导调控Ets-1表达,Ets-1在肾脏发育、保持肾小球完整性及基质金属蛋白酶表达中都是必需的,由此推断,TGF-β1可能是通过Ets-1调控miR-192。大多数DN患者,一旦进入DN临床蛋白尿病期,肾脏已发生不可逆的损害,病情将呈进行性发展,因此及早发现和治疗DN具有极大的临床价值。西医在治疗DN上主要是饮食治疗、控制高血糖、调节血脂、减少蛋白尿、控制高血压等对症治疗,但迄今尚未有能完全阻断DN进展的药物。因此,发挥中医药治疗特色和优势,积极探求中医药治疗DN的有效方法,防止或延缓其发展具有重要的意义。DN属于中医的“肾消”、“水肿”、“肾劳”、“关格”等范畴。脾肾亏虚是DN的发病基础,痰湿、浊毒、瘀血等是其发展过程中的促进因素,本虚标实是DN的病机特点。DN早期(Ⅲ期)多表现阴虚或气阴两虚；DN临床期(Ⅳ期)多表现脾肾亏虚、水湿内停或夹瘀血阻络；DN终末期(V期)多表现脾肾阴阳衰败、浊毒中阻。因此,对Ⅲ～Ⅳ期DN的治疗以益气健脾、滋肾养阴兼活血通络为法。中药复方制剂肾康丸由黄芪、芡实、金樱子、水蛭、益母草等药物组成,功能益气健脾、补肾固涩,利尿消肿,活血通络,符合Ⅲ～Ⅳ期DN的病因病机,具有标本兼治之功。肾康丸作为医院制剂,在临床治疗DN患者多年,疗效确切,并获得国家自然科学基金、南方医科大学开发基金及珠江医院医疗特色院长基金资助,其组方及工艺研究已获得国家发明专利。前期临床观察和动物实验研究都表明,该药在一定程度上可以改善DN一般症状,保护肾脏,延缓DN的进展。但其治疗DN的分子水平的作用机制还不清楚,有待进一步研究。因此,本课题在前期已完成的部分大鼠实验研究基础上,通过建立链脲佐菌素(STZ)致DN大鼠模型和体外高糖刺激下培养大鼠MC,观察DN大鼠肾脏TGF-β1、Ets-1、miR-192表达情况及MC中TGF-β1、Ets-1、SIP1、CollagenⅠ、miR-192的表达情况,探讨miR-192及其TGF-β1介导通路在DN发生发展中的作用机制,同时研究肾康丸对上述途径和指标的影响,进一步探讨肾康丸改善DN的细胞分子水平的作用机制,为其临床应用提供实验依据。具体如下：第1章肾康丸对DN大鼠的肾脏保护作用目的：观察肾康丸对DN大鼠模型的治疗及肾脏保护作用,为进一步研究药物作用机制提供实验依据。方法：首先用STZ按55mg·kg-1大鼠体重一次性腹腔内注射法建立大鼠DM模型,连续喂养2周,测血糖、尿蛋白、尿量。若血糖值>16.6mmol/L、尿量>原尿量150%、尿蛋白排泄>30mg/kg/24h,则造模成功,计算成模率。大鼠造模成功后,称重,随机分为3组：模型对照组、肾康丸组、美卡素组,另设正常对照组。肾康丸组大鼠实验用量设定为1.1g·kg-1大鼠体重,肾康丸研磨后用蒸馏水配成0.22g·mL-1混悬液。美卡素片组大鼠实验用量设定为7.2mg·kg-1大鼠体重,美卡素片研磨后用蒸馏水配成2.9mg·mL-1溶液。肾康丸组和美卡素组大鼠按5mL·kg-1体重,灌服相应药液；模型对照和正常对照组大鼠灌服等容积生理盐水。每日1次,连续8w。各组大鼠自由进食饮水,实验中各组不予任何降血糖药物。治疗8周,治疗期间及治疗后观察大鼠一般状况,每2w检测一次血糖、尿糖。末次给药后计24h尿量和饮水量,然后用10%水合氯醛腹腔麻醉大鼠,腹主动脉穿刺采血,分离血清,全自动生化分析仪检测大鼠血尿素氮(BUN)、血肌酐(Scr)、血总胆固醇(TC)、血甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-c)、低密度脂蛋白胆固醇(LDL-c)、用紫外分光光度法检测糖化血红蛋白(GHb)；考马斯亮兰法检测24h尿蛋白(Upro)；取大鼠肾脏,观察肾脏大小,计算肾重、相对肾重,并通过HE染色法观察大鼠肾脏组织病理变化。结果：46只大鼠用于造模,成模率78.9%(30/38),成模后病死率4.67%(2/30)。8周治疗期间,模型组大鼠血糖均维持在16.7mmol·L-1以上,尿糖+++以上；表现出多饮、多食、多尿、消瘦的DM症状,肾康丸、美卡素治疗组大鼠一般情况均较模型对照有所改善；正常对照组及各治疗组大鼠体重均较治疗前增加(P<0.05或P<0.01),与模型对照组相比较,肾康丸组与美卡素组增长量显著增加(P<0.01,P<0.05)。与正常对照组比较,各组大鼠血糖、GHb、24hUpro、Scr、BUN、TC、TG、LDL-c均升高(P<0.01),24h尿量及饮水量增加(P<0.01)；与模型对照组比较,肾康丸、美卡素治疗均可以降低24hUpro、BUN及24h尿量及饮水量(P<0.01或P<0.05)；肾康丸治疗可以降低血糖、GHb (P<0.05)。各组中,仅肾康丸组TC、TG、LDL-c明显下降(P<0.01)。与正常对照组比较,各组大鼠HDL-c显著降低(P<0.01)。与模型对照组相比,肾康丸治疗组对HDL-c具有显著升高作用(P<0.01)。与正常对照组比较,模型组大鼠肾重及相对肾重增加(P<0.01),且出现了相当于Mogensen分期的3期左右DN病理损害。肾康丸、美卡素病理改变较模型组均有不同程度的减轻,其中肾康丸组效果更为显著。结论：肾康丸能够改善实验性DN大鼠一般状态,具有一定的调节血糖、血脂代谢的作用,能够降低实验性DN大鼠尿蛋白,抑制血清BUN及Scr,改善肾脏组织病理学改变,在一定程度上缓解肾脏病理损伤,具有治疗DN大鼠,保护大鼠肾脏的作用。第2章肾康丸对DN大鼠miR-192信号通路的影响目的：探讨DN大鼠肾脏miR-192及其TGF-β1介导通路的作用机制及肾康丸的影响。方法：取第一部分各组大鼠左肾组织,沿正中矢状面剖开,用PBS冲洗干净,除去包膜,投入10%甲醛中性缓冲液中固定,应用过氧化物酶标记的链霉卵白素(SP)法进行免疫组织化学染色,分别以兔抗大鼠TGF-β1Ets-1多克隆抗体为一抗,检测肾脏组织TGF-β1、Ets-1蛋白的表达。取第一部分各组大鼠右肾组织,PBS冲洗干净后,-70℃冻藏,20mg左右肾组织冰浴中匀浆后溶解于300-μL裂解液RL中,按试剂盒说明书提取总RNA。取1.5μg总RNA采用M-MLV转录成cDNA,采用SYBR Premix Ex TaqTM在FTC-2000型实时荧光定量PCR仪上进行荧光定量PCR,内参照采用GAPDH,检测肾脏组织TGF-β1、Ets-1和miR-192 mRNA表达。结果：正常组大鼠肾脏在肾小管上皮细胞中有少量TGF-β1蛋白表达；与正常组大鼠相比,模型组在肾小管上皮细胞和髓质的TGF-β1蛋白表达量明显增加；与模型组相比较,肾康丸、美卡素治疗可以减少TGF-β1蛋白表达,具有统计学意义(P<0.01),其中肾康丸组效果更明显。Ets-1蛋白在正常组肾小球、肾小囊、肾小管散在表达；模型组则在肾小球内、肾小囊、肾小管表达增加；肾康丸组、美卡素组肾小球、肾小囊、肾小管表达量较模型组减少(P<0.01)。与正常组比较,模型组大鼠肾脏TGF-β1、Ets-1、miR-192 mRNA表达量增加；与模型组相比较,肾康丸组与美卡素组可以降低TGF-β1、Ets-1、miR-192mRNA表达。结论：DN大鼠中TGF-β1高表达,可上调Ets-1表达,从而增加miR-192表达；’肾康丸可有效抑制miR-192及其TGF-β1介导通路的表达,从而延缓DN进展。第3章肾康丸对高糖培养的MC miR-192信号通路的影响目的：探讨肾康丸对高糖培养MC miR-192信号通路的影响方法：16只大鼠随机分为4组,正常大鼠血清组、美卡素组、肾康丸高低剂量组。大鼠灌服相应的药物7天后,分离药物血清。体外培养大鼠MC。将体外高糖培养的MC分为5组：高糖对照组、正常大鼠血清组、美卡素组及肾康丸高、低剂量组。另设正常葡萄糖组。培养24h后,酶联免疫吸附试验法(ELISA)检测各组MC培养上清TGF-β1含量；采用Western blot法检测各组MC细胞中Ets-1、SIP1和CollagenⅠ蛋白表达的情况；采用实时荧光定量聚合酶链反应(RT-PCR)法观察各组MC细胞中TGF-β1、Ets-1、miR-192、SIP1、CollagenⅠmRNA表达情况。结果：与正常葡萄糖组比较,高糖培养组MC细胞上清中TGF-β1含量增加(P<0.01),Ets-1和CollagenⅠ蛋白表达增加(P<0.01),SIPl蛋白表达降低(P<0.01)。与高糖组比较,含药血清干预后,TGF-β1含量降低(P<0.01),Ets-1和CollagenⅠ蛋白表达减少(P<0.01),SIP1蛋白表达增加(P<0.01)。其中肾康丸高剂量组效果更明显。与正常葡萄糖组比较,高糖培养组MC细胞中TGF-β1、Ets-1、miR-192和CollagenⅠmRNA基因表达上调,SIP1 mRNA基因表达下调,肾康丸和美卡素干预后可以降低高糖诱导的TGF-β1、Ets-1、miR-192、CollagenⅠmRNA基因表达,上调高糖MC中SIP1 mRNA表达。其中肾康丸高剂量组效果显著。结论：.MC中miR-192及其TGF-β1介导通路的活性增加,提示高糖可以激活该通路,进而诱导ECM蛋白的蓄积；肾康丸可以抑制高糖MC细胞中激活的miR-192及其TGF-β1介导通路,减少ECM的蓄积,延缓DN进程。全文结论：本研究通过DN大鼠模型实验研究,再次证明肾康丸具有治疗DN和保护肾脏的作用；通过观察DN大鼠及体外高糖培养MC中miR-192及其TGF-β1介导通路的表达情况及肾康丸的干预效果,提示肾康丸的作用机理可能与抑制该通路活性,减少ECM的分泌与合成,延缓肾脏纤维化有关。为肾康丸应用于DN的临床治疗,提供了较为全面、深入的分子水平作用机制的理论和实验依据。更多还原

【Abstract】 Diabetic nephropathy (DN) is one of the most familiar and refractory vascular complications of diabetes mellitus (DM) in clinic. And it is a leading cause resulting in death among the end-stage renal disease and DM patients. About 40% type 1 and 5～20% type 2 DM patients might run to DN. DN whose pathogenesis has not been fully understood, doesn’t been well treated in the days. So how to treat and prevent DN effectively is becoming a common concerned problem among the international medical researchers.DM might damage the patients’ kidney from varies of ways and including all structure of the kidney, which could cause the proliferation and hypertrophy in glomerular cells, thickening of the glomerular basement membrance, ceaseless accumulation of extracellular matrix (ECM) and lead to glomerulosclerosis in the end. Transforming growth factor beta 1 (TGF-β1)is a multiple functional cytokine and has a assured effect in the pathogenesis of DN. These include inducing the proliferation and hypertrophy of the cells in the kidney, increasing the synthesizing and secretion of the ECM, decreasing the decomposition of the ECM via increasing the active effect of inhibitors of metalloproteinases and so on.. The activation of TGF-β1 can be stimulated and mediated by almost all confirmed media and the molecular pathway in Kidney damage of DM. Therefore, It has important clinical significance to find a new method for treatment of DN by antagonizing TGF-β1, and to find its more specific and efficient downstream factors or links.MicroRNAs (miRs) are short noncoding RNAs that recently have been shown to play important roles in mammalian gene expression. Recent evidence suggests that miR-192 involves in TGF-β1 mediated collagen regulation. Thus, TGF-β1 induced down-regulation of SIP1 via miR-192 and can enhance collagenⅠexpression via derepression at E-box elements. The observations provide the first functional role for a miR expressed in the kidney. Small noncoding RNAs and miRs such as miR-192 and their inhibitors may be targets for diseases such as DN and other diabetic complications.The majority of DN patients, once enter the clinical proteinuria, irreversible kidney damage has occurred, the disease will show a progressive development, so early detection and treatment is of great clinical value in DN. Now the treatment of the DN in western medicine mainly aims at its symptom which include controlling the intensive blood glucose, reducing the albuminuria, control the hypertension, and so on. But so far neither one medicine can prevent the progression of the DN. So it is important to prevent and treat DN by exerting the superiority and characteristic of traditional chinese medicine (TCM) in treating DN and explore actively effective treatment from TCM.DN belongs to the domain of the "Shenxiao", "Dropsy", "Shenlao", "Guange", and so on in TCM. "Splenic and renal weaken" is the basal pathogenesis of DN and the "Phlegm and damp", the "Grime and toxin", the "Gore" are the stimulative factors. So the "fundamentality weaken and sign mighty" is the TCM characteristic pathogenesis of DN. Early DN (Ⅲstages) always appears the symptoms of "Yin weaken" or "both Qi and Yin weaken". Clinic DN (Ⅳstages)always comes forth the symptoms of "splenic and renal weaken", "liquid and humidity logjam" or "gore stagnateing vas". While end DN (Ⅴstages) always emergences the symptoms of "downfall of Yin and Yang in spleen and kidney" and "grime and toxin blocking". So the therapeutic principle of DN inⅢ-Ⅴstages includes "increasing the Qi and toughen the spleen", "nourishing the kidney and maintain Yin" and "impeling blood stream and dredging vas".The compound preparation Shenkangwan was mainly constituted of astragalus mongholicus, rhubarb, leech, chrokee rose fruit, and so on. It has the effect of increasing the Qi and toughen the spleen, nourishing the kidney and constringency, diuresis and detumescence, impeling blood stream and dredging vas. So it accords with the TCM characteristic of pathogenesis of DN fitly and can treat the DN from the fundamentality to the sign. As a hospital preparation, Shenkangwan has been used to treating DN patients in clinic for many years and has a assured curative effect. The study from clinic observation and animal experiment indicated that Shenkangwan could ameliorate the general symptom, protect the kidney and stay the development of the DN. But its molecular mechanism in treating DN is not clear and need farther research.So we carried out the experiment to investigate the expression of TGF-β1、Ets-1、miR-192 in the kidneys of the DN rats and the expression of TGF-β1、Ets-1、SIP1、Collagenl、miR-192 in the cultured mesangial cells (MC) induced by high glucose on the basis of experiment results before, to explore the molecular and genic mechanism of Shenkangwan in treating DN from TGF-β1 induced collagen expression via miR-192 signaling pathway and provide the laboratorial evidence for its clinic application. The details were as follows:Chart I Laboratory study on the protective effect of Shenkangwan on the kidney of the DN RatsObjective:To observe the effect of Shenkangwan in treating rats of DN model and protecting their kidneys and provide the laboratorial foundation for further studying its therapeutic mechanism.Methods:Firstly, we established the DM rat models by intraperitoneal injection of streptozotocin(STZ) according to the dose of 55 mg·kg-1 body weight. In the following two weeks, we detected the blood glucose, the 24 hour urine protein and the urine volume. The rats, which value of blood glucose exceeding 16.6 mmol·L-1 and 24 hour urine protein exceeding 30 mg/kg/24h, urine volume 1.5 times as baseline were taken for the successful DM rat models and calculated the rate of them. Then they were randomly divided into 3 groups:model control group, micardis group, and Shenkangwan group. Other eight normal rats were used as normal control group. All rats were treated with corresponding drugs for 8 weeks. During and after the treatment, the general state, blood and uric glucose levels of the rats in every group were observed. After treated in the last time, all rats were put into the metabolizing cage to be measured the volume of their 24 hours urine and drinking water. Then we hocused all the rats with 10% chloral hydrate, collected the blood via ventral artery, gathered the serum by centrifuge and detected the content of the blood glucose and the glucosylated hemoglobin (GHb), the excretion of the 24 hour urine protein (Upro), the levels of serum creatinine (SCr), blood urea nitrogen (BUN), total serum cholesterol (TC),triglyceride (TG),high density lipoprotein (HDL-c),low density lipoprotein (LDL-c),.And we took out the kidney of the rats, observed the size and volume of them and measured the kidney weight and relative kidney weight. Then we investigated their renal pathological changes by optical microscope with the coloration method of Hematoxylin and Eosin (HE)Results:46 rats were used to established the DM models and the rate of successful models was 78.9%(30/38). And the death rate of DM model rats was 4.67%(2/30). During the treating eight weeks, The model rats blood glucose were all over 16.7 mmol·L-1, the uric glucose positive experiment were exceeded+++. And they appeared the symptom of the DM such as the quantum of drinking, eating and urinating increased. Compare with the normal rats, their following indexes including GHb,24 h Upro, Scr, BUN, TC, TG, and LDL-c were increased distinctly (P<0.01); and HDL-c were decreased distinctly (P<0.01). Their kidney weight and relative kidney weight increased distinctly and came forth about the 3 stages renal pathological lesions according to the stages of Mogensen. Compare with the rats in model group, Shenkangwan could ameliorate the rat’ general state, amelioration the above indexes markedly and mitigate the renal pathological lesion.Conclusions:Shenkangwan could treat DN rats and had a certain protective effect on the kidney of DN rats.ChartⅡMicroRNA-192 in the kidneys of DN rats and its function in TGF-β1 induced collagen expressionObjective:To explore the effect of the Shenkangwan on TGF-β1 induced collagen expression via miR-192 signaling pathway in the kidneys of the DN rats.Methods:The kidneys used of this study were from the rats in chartⅠ. We detected the protein expression of TGF-β1; Ets-1 with the method of immune histochemistry, and observed the expression of TGF-β1; Ets-1 and miR-192 mRNA in the renal tissue of the rats in every group with the method of the quantitative real-time PCRResults:The renal tissue expression of TGF-β1, Ets-1 and miR-192 mRNA in the rats of DN model group were increased markedly than those in the rats of normal control group (P<0.01). And the expressions of TGF-β1, Ets-1 protein were increased distinctly in these rats’ kidneys too (P<0.01). Compare with those in the rats of the DN model group, the expression of TGF-β1, Ets-1 and miR-192 in the rats of Shenkangwan and micardis group were decreased markedly (P<0.01)Conclusions:The TGF-β1 could effect the miR-192 by control the expression of the Ets-1. Shenkangwan could restrain the activation of collagen expression via miR-192 signaling pathway in the renal tissue of DN rats and reduced the synthesizing and secretion of the ECM, which would conduce to staying the course of the DN.ChartⅢMicroRNA-192 in the MC cultured by high glucose and its function in TGF-β1 induced collagen expressionObjective:To explore the effect of Shenkangwan on TGF-β1 induced collagen expression via miR-192 signaling pathway in the the MC cultured by high glucose.Methods:MC cultured in high glucose in vivo was divided into five groups: high glucose control group, normal rats’serum control group, micardis group and Shenkangwan group (high dose and low dose).A normal glucose control group was used too. Cultured for 24 hours, the secretion of TGF-β1 of MC were detected by using enzyme linked immunosorbent assay (ELISA), the expression of TGF-β1、Ets-1、collagen 1、SIP1、miR-192mRNA were determined by using RT-PCR method and the expression of its protein were detected by Western blot method.Results:Compare with the normal glucose, high glucose could accelerate the secretion of TGF-β1 of MC, increase their expression of TGF-β1、Ets-1、collagen 1、miR-192 mRNA (P<0.01), enhance their expression of its protein (P<0.01), and depress the expression of SIP1 mRNA and protein in the cultured MC in vivo (P<0.01). While intervening and treating with Shenkangwan and micardis could reduce the secretion mRNA and the protein expression of TGF-β1、Ets-1、collagen 1、miR-192 in the cultured MC induced by high glucose (P<0.01), and increase the mRNA and the protein expression of SIP1 (P<0.01)Conclusions:High concentrations glucose could activate the miR-192 signaling pathway and stimulate the secretion of ECM in the cultured MC in vivo. Shenkangwan could restrain the activation of miR-192 signaling pathway in the cultured MC induced by high glucose and reduce their synthesizing and secretion of the ECM.Conclusions of the whole paper:We carried out the experiment on animal and the MC cultured by high glucose, and proved again that Shenkangwan could treat DN and had a certain protective effect on the kidney of DN. Its therapeutic mechanism was related to restraining the activation of collagen expression via miR-192 signaling pathway and reducing the synthesizing and secretion of the ECM. And those provided the molecular and genic therapeutic mechanism and laboratorial evidence for its clinic treatment on DN.更多还原