【作者】 梁炜；

【导师】 魏连波；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 糖尿病肾病（diabetic nephropathy,DN）是糖尿病最常见的微血管并发症之一,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。DN的发生是多种因素综合作用的结果。其中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在糖尿病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的细胞外基质（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SIP1,导致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est-1原癌基因结合位点,而TGF-β₁也诱导调控est-1表达,est-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est-1调控miR-192。西医在治疗DN上主要是控制高血糖、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。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,本课题通过动物实验,从miR-192及其TGF-β₁介导途径出发,探讨肾康丸治疗DN的分子基因水平的作用机制,为其临床应用提供实验依据。具体如下:第1部分肾康丸对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:首先用链脲佐菌素（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4组:模型对照组、肾康丸组、开博通组和胰岛素组,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。开博通片组大鼠实验用量设定为4.5mg·kg-1大鼠体重,开博通片研磨后用蒸馏水配成0.9mg·mL-1溶液。肾康丸组和开博通组大鼠按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。胰岛素组以血糖11.1mmol/L为基准每日皮下注射胰岛素8～40u控制血糖。各组大鼠自由进食饮水,实验中除胰岛素组外,其他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、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）;放免法检测血清胰岛素和血清胰高血糖素含量与血栓素（TXB₂）、6-酮-前列环素(6-keto-PGF_la)、内皮素（ET）;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:1.55只大鼠用于造模,成模率80%（44/55）,成模后病死率4.55%（2/44）。2.8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、开博通及胰岛素治疗组大鼠一般情况均较模型对照有所改善;3.正常对照组及各治疗组大鼠体重均较治疗前增加（P＜0.01）,模型对照组大鼠体重未增,且较各治疗组降低（P＜0.01）。4.与正常对照组比较,各组大鼠血糖、GHb、24hUpro、Scr、BUN、TC、TG、LDL-c、胰高血糖素均升高,24h尿量及饮水量增加;与模型对照组比较,肾康丸、开博通及胰岛素治疗均可以降低24hUpro、BUN及24h尿量及饮水量;肾康丸与胰岛素治疗可以降低血糖、GHb和胰高血糖素（P＜0.01）。仅肾康丸组TC、TG、LDL-c明显下降（P＜0.01）。5.与正常对照组比较,各组大鼠HDL-c、胰岛素水平显著降低（P＜0.01）。仅肾康丸治疗组与模型对照组相比,对HDL-c具有显著升高作用（P＜0.01）;肾康丸与胰岛素治疗均可提高胰岛素水平（P＜0.01）。6.与正常对照组比较,DM各组大鼠TXB₂、ET水平均显著升高,6-keto-PGF_la均显著降低（P＜0.01）。而较之模型组,肾康丸组TXB₂、ET水平明显下降（P＜0.01）,对6-keto-PGF_la肾康丸组则有显著的恢复作用（P＜0.01）。开博通、胰岛素治疗对上述指标无改善。（P＞0.05）7.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、开博通和胰岛素组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:1.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。2.肾康丸能够促进胰岛素分泌,降低胰高血糖素。3.肾康丸能够降低实验性DN大鼠尿蛋白,抑制血清BUN及Scr,具有一定的保护肾功能作用。4.肾康丸能够降低TXB₂水平,提高6-keto-PGF_la水平,恢复TXB₂与6-keto-PGF_la的平衡。5.肾康丸能够降低血浆ET水平,缓解血管内皮损伤。6.肾康丸能够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。第2部分肾康丸对DN大鼠miR-192信号通路的影响目的:探讨肾康丸对DN大鼠肾脏miR-192及其TGF-β₁介导通路的影响。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SP（过氧化物酶标记的链霉卵白素Streptavidin/Peroxidase）法进行免疫组织化学染色,分别以兔抗大鼠TGF-β₁、Collagen1和SIP1多克隆抗体为一抗,检测肾脏组织TGF-β₁、Collagen1和SIP1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300μL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 PremixEx Taq^TM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、Collagen1、SIP1和miR-192 mRNA表达。结果:1.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、开博通和胰岛素治疗可以减少TGF-β₁蛋白表达,其中肾康丸组效果更明显。2.Collagen1蛋白在正常组肾小球内无表达,在肾小囊、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、开博通组和胰岛素组肾小球内无表达,肾小囊、肾小管表达量较模型组减少。3.SIP1蛋白在正常组肾小囊内无表达,在肾小球、肾小管、肾髓质表达明显;模型组表达量减低;肾康丸、开博通和胰岛素治疗后可以增强SIP1表达。4.与正常组比较,模型组大鼠肾脏TGF-β₁、Collagen1、miR-192 mRNA表达量增加,而SIP1 mRNA表达降低;肾康丸组与开博通组可以降低TGF-β₁、Collagen1、miR-192 mRNA表达,提高SIP1 mRNA表达。结论:1.肾康丸可以降低造模后升高的TGF-β₁ mRNA及蛋白表达;2.肾康丸可以下调Collagen1mRNA和蛋白表达;3.肾康丸可以上调DN降低的SIP1mRNA和蛋白表达;4.肾康丸可以降低造模后升高的miR-192的表达。全文结论:本研究通过DN动物模型实验研究,再次证明肾康丸具有治疗DN和保护肾脏的作用;其作用机理与药物抑制miR-192及其TGF-β₁介导通路的活性,减少ECM的分泌与基因合成有关。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 an important and refractory micro vascular complication of diabetes mellitus（DM）,as well as the common reason resulting in chronic renal failure.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.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.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（Ⅱstages） 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 on animal 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:ChartⅠLaboratory 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 4 groups:model control group, capoten group,Shenkangwan group and insulin 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）,content of insulin and glucagons, endothelin（ET）,thromboxane B₂（TXB₂）,6-keto-taglandine I₂(6-keto-PGF_1a).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:55 rats were used to established the DM models and the rate of successful models was 80%（44/55）.And the death rate of DM model rats was 4.55% （2/44）.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,LDL-c,glucagons content and TXB₂,ET were increased distinctly（P＜0.01）;and HDL-c,6-keto-PGF_1a and insulin 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,Collagen1,SIP1 with the method of immune histochemistry,and observed the expression of TGF-β₁,Collagen1,SIP1 and miR-192 mRNA in the renal tissue of the rats in every group with the method of the quantitative real-time PCR.Results:The renal tissue expression of TGF-β₁,Collagen1 and miR-192 mRNA in the rats of DN model group were increased markedly than those in the rats of normal control group.And the expressions of TGF-β₁,Collagenl protein were increased distinctly in these rats’ kidneys too.While the expressions of SIP1 protein and mRNA were decreased markedly in the rats of the model group.Compare with those in the rats of the DN model group,the expression of TGF-β₁,Collagenl and miR-192 in the rats of Shenkangwan and capoten group were decreased markedly.Conclusions: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.Conclusions of the whole paper:We carried out the experiment on animal 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