降糖三黄片对糖尿病肾病microRNA-21信号通路的影响
发布时间：2018-08-12 15:53
【摘要】：文献研究:糖尿病肾病(diabetic nephropathy,DN)是糖尿病较为常见的微血管并发症之一,也是导致患者出现终末期肾脏疾病以及进行肾替代治疗的首要原因,严重地威胁着人们的健康。据研究报道,大约15%～40%的糖尿病患者会发展为糖尿病肾病。在我国,DN导致的终末期肾病的比例正在逐年上升,预计在不久的将来DN将成为终末期肾病最主要的原因。然而,DN的发病机制极其复杂,其具体机制至今尚未完全阐明清楚,可能是多种因素综合作用的结果,由于众多生物活性物质参与了 DN的发病过程,使其病理生理机制具有多途径、多环节相互作用的特点,故对其进行有效的防治一直是临床的难题。因此,探究DN的发病机制,寻求延缓DN进展的治疗方法具有重要的社会意义及经济价值。在整体观念、辨证论治等思想指导下,中医药在防治糖尿病及其并发症方面有独特的优势。中药复方在干预防治疾病方面具有多途径、多靶点的特点,因此,运用中药复方防治DN具有较大的潜力。中医学对糖尿病及其并发症的认识源远流长,早在《黄帝内经》就有消渴病的相关记载,历代医家、后世学者对消渴病及其并发症的病因病机认识不断发展,诊治水平不断提高。总结古今中医药辨治DN的文献,DN早期以气阴两虚、瘀热互结为基本病机,治疗宜益气养阴、泻热逐瘀。降糖三黄片乃我校熊曼琪教授及其研究团队在经方桃核承气汤的基础上加减化裁而成,在临床上治疗糖尿病肾病已二十余年,取得了良好的临床疗效。为进一步证实该方的治疗作用,更深入探讨其可能的作用机制,我们进行了本项研究,即借助糖尿病肾病的动物模型,观察其对早期糖尿病肾病大鼠干预作用及对microRNA-21信号通路的影响。实验研究:目的:利用糖尿病肾病模型大鼠,观察降糖三黄片对模型大鼠糖脂代谢水平、肾功能、肾组织病理改变以及microRNA-21信号通路的影响,以探明降糖三黄片防治DN的作用机制,为降糖三黄片的临床应用提供现代生物学方面的实验依据与支持。方法:选取80只雄性SD大鼠,适应性喂养1周后,依据体重随机将其分成正常组10只和造模组70只。造模前禁食不禁水12 h,将配制好的STZ溶液按45 mg· kg-1的剂量一次性腹腔注射,正常组腹腔注射等量的柠檬酸缓冲液。全部大鼠在同一动物房内用代谢笼分笼饲养,标准饮食,不使用胰岛素及其他降糖药物。连续喂养3周,然后测血糖、尿量,若血糖16.7 mmol ·L-1、尿量原尿量150%、24小时尿蛋白定量30mg以上,则可确定大鼠DN模型造模成功。造模期间大鼠死亡3只,血糖自行恢复3只,血糖达标但尿蛋白低于标准的大鼠8只,因此成模的DN大鼠共56只。将成模大鼠随机分为模型组12只,降糖三黄片低剂量组11只,降糖三黄片中剂量组11只,降糖三黄片高剂量组11只,厄贝沙坦组11只。造模前后均以清洁级普通饲料喂养。降糖三黄片低剂量组按0.675g·kg-1给药,降糖三黄片中剂量组1.350 g·kg-1给药,降糖三黄片高剂量组2.700g·kg-1给药,厄贝沙坦组0.0135g·kg-1给药,灌胃前药物均用纯净水配成2ml混悬液,每日1次灌胃,连续8w。正常组及模型组予以灌服等量蒸馏水。实验期间大鼠自由进食、饮水。实验期间观察大鼠的一般情况,每隔4周测大鼠体重及尾尖FBG,末次给药后计24 h尿量及饮水量。实验结束时测大鼠肾功能(BUN、Scr),血脂(TC、TG、LDL-C、HDL-C),糖化血红蛋白(HbA1c),24h尿蛋白(Upro);取大鼠肾脏,计算肾重、相对肾重;光镜及电镜观察大鼠肾脏组织病理形态;免疫组化法观察大鼠肾脏TGF-β1、Smad3、Smad7的染色情况;Western blotting法检测各组大鼠肾脏TGF-β1、Smad3、Smad7蛋白的表达;RTQ-PCR法检测各组大鼠肾脏TGF-β1、Smad3、Smad7、microRNA-21 mRNA 的表达。结果:1.体重、饮水量及尿量方面:体重方面,药物干预4周及8周后,较模型组,药物干预组体重均有明显增加(P0.01),其中以降糖三黄片高剂量组效果最好,降糖三黄片中剂量组次之,而降糖三黄片低剂量组与厄贝沙坦组无差异(P0.05)。饮水方面,较模型组,降糖三黄片中剂量组、降糖三黄片高剂量组、厄贝沙坦组有所减少(P0.01),而降糖三黄片低剂量组则无明显差异(P0.05);与厄贝沙坦组比较,降糖三黄片低剂量组、降糖三黄片中剂量组无明显差异(P0.05),而降糖三黄片高剂量组明显少于厄贝沙坦组(P0.01)。尿量方面,降糖三黄片中剂量组、降糖三黄片高剂量组、厄贝沙坦组有所减少(P0.01),而降糖三黄片低剂量组则无明显差异(P0.05);与厄贝沙坦组比较,降糖三黄片高剂量组尿量减少(P0.05),而降糖三黄片中剂量组无差异(P0.05),降糖三黄片低剂量组则高于厄贝沙坦组(P0.05)。2.糖脂代谢方面:空腹血糖方面,治疗4周后及8周后,降糖三黄片低剂量组、降糖三黄片中剂量组、降糖三黄片高剂量组均明显低于模型组及厄贝沙坦组(P0.01),模型组与厄贝沙坦组则无差异(P0.05)。糖化血红蛋白方面,治疗8周后,降糖三黄片低剂量组、降糖三黄片中剂量组、降糖三黄片高剂量组均明显低于模型组及厄贝沙坦组(P0.01),模型组与厄贝沙坦组则无差异(P0.05)。TC、TG、LDL-C方面,治疗8周后,降糖三黄片低剂量组、降糖三黄片中剂量组、降糖三黄片高剂量组均明显低于模型组及厄贝沙坦组(P0.01),模型组与厄贝沙坦组则无差异(P0.05)。HDL-C方面,与模型组比较,降糖三黄片中剂量组、降糖三黄片高剂量组明显升高(P0.01),而降糖三黄片低剂量组、厄贝沙坦组与模型组无明显差异(P0.05)。3.肾重、相对肾重、肾功能及尿蛋白方面:肾重方面,降糖三黄片中剂量组、降糖三黄片高剂量组、厄贝沙坦组明显低于模型组(P0.01),而降糖三黄片低剂量组与模型组无明显差异(P0.05),降糖三黄片中剂量组、降糖三黄片高剂量组与厄贝沙坦组无明显差异(P0.05),而而降糖三黄片低剂量组则明显高于厄贝沙坦组(P0.01)。相对肾重方面,药物干预组明显低于模型组(P0.01),降糖三黄片中剂量组、降糖三黄片高剂量组与厄贝沙坦组无明显差异(P0.05),而降糖三黄片低剂量组则明显高于厄贝沙坦组(P0.01)。肾功能方面(BUN、Scr),药物干预组明显低于模型组(P0.01),降糖三黄片高剂量组相较厄贝沙坦组无明显差异(P0.05),而降糖三黄片低剂量、降糖三黄片中剂量组则明显高于厄贝沙坦组(P0.01)。24 h尿蛋白方面,治疗前模型组与药物干预组,组间无明显差异(P0.05),治疗8周后,与模型组比较,降糖三黄片低剂量组降低(P0.05),降糖三黄片中剂量组、降糖三黄片高剂量组、厄贝沙坦组降低更为明显(P0.01);与厄贝沙坦组比较,降糖三黄片高剂量组无明显差异(P0.05),降糖三黄片低剂量组、降糖三黄片中剂量组则明显高于厄贝沙坦组(P0.01)。4.光镜及电镜下观察肾脏病理形态:正常组大鼠肾脏未见病理改变,模型组大鼠肾脏病变严重,可见肾小球体积增大明显,肾小球肿胀、充血,肾小囊变窄,肾小球系膜细胞增生,部分肾小管细胞肥大,部分可见空泡变性,毛细血管管腔狭窄,足突扁平,结构不清,线粒体空泡样变,系膜基质增多,肾小管上皮细胞脱落、减少或萎缩。降糖三黄片低剂量组、降糖三黄片中剂量组、降糖三黄片高剂量组、厄贝沙坦组均能改善DN肾脏组织病变,其中以降糖三黄片高剂量组效果最好。5.肾脏TGF-β 1、Smad3、Smad7蛋白表达:免疫组化染色结果显示,较正常组,模型组TGF-β1、Smad3蛋白表达明显增多(P0.01),呈棕黄色颗粒,着色极深,药物干预组介于正常组与模型组之间,明显低于模型组(P0.01);与厄贝沙坦组比较,降糖三黄片高剂量组无明显差异(P0.05),降糖三黄片低剂量组、降糖三黄片中剂量组高于厄贝沙坦组(P0.05)。较模型组,药物干预组Smad7表达明显增多(P0.01);厄贝沙坦组Smad7表达明显高于降糖三黄片低剂量组、降糖三黄片中剂量组、降糖三黄片高剂量组(P0.01)。Western blotting法测量TGF-β1、Smad3、Smad7蛋白表达显示:较正常组,模型组TGF-β1、Smad3蛋白表达明显增加(P0.01),降糖三黄片低剂量组低于模型组(P0.05),降糖三黄片中剂量组、降糖三黄片高剂量组、厄贝沙坦组明显低于模型组(P0.01);降糖三黄片高剂量组TGF-β1、Smad3蛋白表达与厄贝沙坦组相比无明显差异(P0.05),且厄贝沙坦组优于降糖三黄片低剂量组及降糖三黄片中剂量组(P0.01或P0.05)。较正常组,模型组Smad7蛋白表达明显减少(P0.01),药物干预组明显高于模型组(P0.01),厄贝沙坦组Smad7蛋白表达明显高于降糖三黄片低剂量组、降糖三黄片中剂量组(P0.01),而与降糖三黄片高剂量组无明显差异(P0.05)。6.RTQ-PCR 法检测大鼠肾脏 TGF-β1、Smad3、Smad7、microRNA-21 mRNA 表达显示:以正常组基因表达为参照,模型组TGF-β1、Smad3、microRNA-21mRNA基因表达显著增多(P0.01),药物干预组较模型组明显降低(P0.01),降糖三黄片高剂量组与厄贝沙坦组无明显差异(P0.05),而降糖三黄片低剂量组、降糖三黄片中剂量组明显高于厄贝沙坦组(P0.01)。以正常组基因表达为参照,模型组Smad7 mmRNA基因表达显著减少(P0.01),药物干预组较模型组明显升高(P0.01),降糖三黄片高剂量组与厄贝沙坦组无明显差异(P0.05),而降糖三黄片低剂量组、降糖三黄片中剂量组明显低于厄贝沙坦组(P0.01)。结论:1.在总结前人各医家关于消渴病因病机及证治的基础上,总结糖尿病肾病病机属虚实夹杂,正虚为本,邪实为标,治以扶正祛邪为法,集益气养阴、泻热逐瘀、通阳活血、通腑泄浊于一体,可有效防治本病。2.降糖三黄片可以改善DN大鼠的一般状态,具降低血糖、调节血脂的作用。3.降糖三黄片可以降低DN大鼠的尿蛋白,降低血清BUN及Scr,具有一定保护DN大鼠肾功能的作用。4.降糖三黄片可以改善DN大鼠肾脏组织病理损害,具一定的延缓肾脏病理损伤的作用。5.降糖三黄片可以下调DN大鼠肾脏中TGF-β 1、Smad3蛋白的表达,上调Smad7蛋白的表达;降糖三黄片可以下调DN大鼠肾脏中TGF-β1、Smad3、microRNA-21mRNA的表达,上调Smad7 mRNA的表达,从而调控了 DN的microRNA-21及其TGF-β 1/Smad的信号通路转导,延缓了 DN的病程进展。
[Abstract]:Literature review: Diabetic nephropathy (DN) is one of the most common microvascular complications in diabetes mellitus, and is also the leading cause of end-stage renal disease and renal replacement therapy, which seriously threatens people's health. According to research reports, about 15%-40% of diabetic patients will develop diabetes mellitus. Nephropathy. In China, the proportion of end-stage kidney disease caused by DN is increasing year by year. It is expected that DN will be the main cause of end-stage kidney disease in the near future. However, the pathogenesis of DN is extremely complex, and its specific mechanism has not been fully clarified yet. It may be the result of a combination of many factors, due to many bioactive substances. Therefore, it is of great social and economic significance to explore the pathogenesis of DN and seek treatment methods to delay the progress of DN. Under the guidance of traditional Chinese medicine, it has unique advantages in the prevention and treatment of diabetes mellitus and its complications. Traditional Chinese medicine compound prescriptions have many ways and targets in the prevention and treatment of diseases. Therefore, the use of traditional Chinese medicine compound prescriptions for the prevention and treatment of DN has great potential. Summarize the ancient and modern Chinese medicine treatment of DN literature, DN early to Qi and Yin deficiency, blood stasis and heat interlinked as the basic pathogenesis, treatment appropriate Qi Yang Yin, Xie Fei Zhuyu. In order to further confirm the therapeutic effect of this prescription and further explore its possible mechanism of action, we carried out this study, that is, with the help of animal model of diabetic nephropathy. Objective: To observe the effects of Jiangtang Sanhuang Tablets on glucose and lipid metabolism, renal function, pathological changes of kidney tissue and microRNA-21 signaling pathway in early diabetic nephropathy rats. Methods: Eighty male SD rats were randomly divided into normal group (10 rats) and model group (70 rats) according to their body weight after one week of adaptive feeding. All rats were fed in a metabolic cage in the same animal room without insulin or other hypoglycemic drugs. After feeding for 3 weeks, blood glucose and urine volume were measured. If blood glucose was 16.7 mmol L-1, urine prouria was 150% and urine protein was measured for more than 30 mg in 24 hours. During the modeling period, 3 rats died, 3 blood glucose recovered, 8 rats with blood glucose up to the standard but urinary protein down to the standard, so 56 DN rats were established. 11 rats in the dosage group and 11 rats in the Irbesartan group were fed with clean general diet before and after modeling. The rats were fed and drinked freely during the experiment. The general condition of the rats was observed during the experiment. The body weight and FBG of the tail tip were measured every four weeks. The urine volume and water consumption were measured 24 hours after the last administration. The renal function (BUN, Scr), blood lipid (TC, TG, L) were measured at the end of the experiment. DL-C, HDL-C, HbA1c, 24-hour urinary protein (Upro); kidney of rats was taken out to calculate kidney weight, relative kidney weight; pathological morphology of rat kidney was observed by light and electron microscopy; TGF-beta 1, Smad3, Smad7 staining was observed by immunohistochemistry; TGF-beta 1, Smad3, Smad7 protein expression was detected by Western blotting; Results: 1. Body weight, water intake and urine volume: Body weight. After 4 and 8 weeks of drug intervention, the body weight of the drug intervention group increased significantly compared with the model group (P Compared with the model group, the middle dose group of Jiangtang Sanhuang Tablets and the high dose group of Jiangtang Sanhuang Tablets, the Irbesartan group decreased (P 0.01), while the low dose group of Jiangtang Sanhuang Tablets had no significant difference (P 0.05). There was no significant difference in the middle dose group of Tangsanhuang Tablets (P 0.05), but the high dose group of Jiangtangsanhuang Tablets was significantly less than that of Irbesartan Tablets (P 0.01). The urine volume of the high dose group of Jiangtang Sanhuang Tablets decreased (P 0.05), but the middle dose group of Jiangtang Sanhuang Tablets had no difference (P 0.05). The low dose group of Jiangtang Sanhuang Tablets was higher than the Irbesartan group (P 0.05). 2. Glucose and lipid metabolism: fasting blood glucose, after 4 weeks and 8 weeks of treatment, the low dose group of Jiangtang Sanhuang Tablets, the middle dose group of Jiangtang Sanhuang Tablets, the high dose group of Jiangtang Sanhuang Tablets Compared with the model group and irbesartan group (P 0.01), the glycosylated hemoglobin of the model group and irbesartan group was not significantly different (P 0.05). In the aspect of glycosylated hemoglobin, after 8 weeks of treatment, the low dose group of Jiangtang Sanhuang tablets, the middle dose group of Jiangtang Sanhuang tablets, the high dose group of Jiangtang Sanhuang tablets were significantly lower than the model group and irbesartan group (P 0.01). TC, TG, LDL-C were no difference (P 0.05). After 8 weeks of treatment, the low-dose group, middle-dose group and high-dose group of Jiangtang Sanhuang tablets were significantly lower than the model group and Irbesartan group (P 0.01). There was no difference between the model group and Irbesartan group (P 0.05). HDL-C, compared with the model group, the middle-dose group of Jiangtang Sanhuang tablets and the high-dose group of Jiangtang Sanhuang tablets were significantly lower than the model group. The kidney weight, relative kidney weight, renal function and urinary protein were significantly higher in the high dose group (P 0.01), but significantly lower in the low dose group (P 0.01) than in the model group (P 0.05). There was no significant difference between the low dose group and the model group (P 0.05), the middle dose group, the high dose group and the Irbesartan group (P 0.05), but the low dose group was significantly higher than the Irbesartan group (P 0.01). There was no significant difference between the high dose group and irbesartan group (P 0.05), but the low dose group was significantly higher than irbesartan group (P 0.01). The renal function (BUN, Scr), the drug intervention group was significantly lower than the model group (P 0.01), the high dose group of Jiangtang Sanhuang tablets was no significant difference compared with irbesartan group (P 0.05), and the low dose group was significantly higher than irbesartan group (P 0.05). There was no significant difference in urinary protein between the model group and the drug intervention group before treatment (P 0.05). After 8 weeks of treatment, compared with the model group, the low dose group of Jiangtang Sanhuang tablets decreased (P 0.05), the middle dose group of Jiangtang Sanhuang tablets and the high dose group of Jiangtang Sanhuang tablets decreased (P 0.05). Compared with irbesartan group, there was no significant difference (P 0.05) in the high-dose group of Jiangtang Sanhuang Tablets, the low-dose group of Jiangtang Sanhuang Tablets, and the middle-dose group of Jiangtang Sanhuang Tablets were significantly higher than that of irbesartan group (P 0.01). In type I group, the kidney lesions were serious. The glomerulus enlarged obviously, the glomerulus swelled and congested, the renal vesicle narrowed, the glomerular mesangial cells proliferated, some renal tubular cells hypertrophy, some vacuoles degenerated, the capillary lumen narrowed, the foot process flattened, the structure unclear, mitochondrial vacuole-like change, the increase of mesangial matrix, the renal tubular epithelial cells. Low-dose Jiangtang Sanhuang Tablets group, middle-dose Jiangtang Sanhuang Tablets group, high-dose Jiangtang Sanhuang Tablets group, irbesartan group can improve the renal pathological changes of DN, of which high-dose Jiangtang Sanhuang Tablets group has the best effect. 5. Expression of TGF-beta 1, Smad3, Smad7 protein in kidney: Immunohistochemical staining results showed that compared with normal group, model group T GF-beta 1, Smad3 protein expression increased significantly (P 0.01), showing brown-yellow granules, very deep coloring, drug intervention group between the normal group and the model group, significantly lower than the model group (P 0.01); compared with Irbesartan group, high-dose Jiangtang Sanhuang tablets group had no significant difference (P 0.05), low-dose Jiangtang Sanhuang tablets group, middle-dose Jiangtang Sanhuang tablets group was higher than Irbesartan group. Compared with the model group, the expression of Smad7 in the drug intervention group was significantly increased (P 0.01); the expression of Smad7 in the Irbesartan group was significantly higher than that in the low-dose group, the middle-dose group and the high-dose group (P 0.01). The expression of TGF-beta 1, Smad3 and Smad7 protein in the model group was measured by Western blotting. The expression of TGF-beta 1 and Smad3 protein in the high-dose group was not significantly different from that in the Irbesartan group (P 0.05). Compared with the normal group, the expression of Smad7 protein in the model group was significantly decreased (P 0.01), that in the drug intervention group was significantly higher than that in the model group (P 0.01), that in the Irbesartan group was significantly higher than that in the low dose group, and that in the middle dose group (P 0.01). There was no significant difference in TGF-1, Smad3, Smad7, microRNA-21 mRNA expression between high-dose Jiangtang Sanhuang Tablets group and control group (P 0.05). RTQ-PCR assay showed that TGF-1, Smad3, microRNA-21 mRNA expression in the kidney of rats was significantly higher in model group than in model group (P 0.01). There was no significant difference between the high-dose group and irbesartan group (P 0.05), but the low-dose group of Jiangtang Sanhuang tablets and the middle-dose group of Jiangtang Sanhuang tablets were significantly higher than that of irbesartan group (P 0.01). There was no significant difference between the dosage group and irbesartan group (P 0.05), but the low dosage group of Jiangtang Sanhuang tablets and the middle dosage group of Jiangtang Sanhuang tablets were significantly lower than that of irbesartan group (P 0.01). Conclusion: 1. On the basis of summing up the etiology, pathogenesis and treatment of diabetic nephropathy, the pathogenesis of diabetic nephropathy is a mixture of deficiency and excess. It can effectively prevent and treat DN rats. 2. Jiangtang Sanhuang tablets can improve the general state of DN rats, reduce blood sugar and regulate blood lipids. 3. Jiangtang Sanhuang tablets can reduce urinary protein of DN rats, reduce serum BUN and Scr, and protect the kidney of DN rats to a certain extent. 4. Jiangtang Sanhuang Tablet can improve the pathological damage of kidney tissue in DN rats, and can delay the pathological damage of kidney to some extent. 5. Decrease
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5
[Abstract]:Literature review: Diabetic nephropathy (DN) is one of the most common microvascular complications in diabetes mellitus, and is also the leading cause of end-stage renal disease and renal replacement therapy, which seriously threatens people's health. According to research reports, about 15%-40% of diabetic patients will develop diabetes mellitus. Nephropathy. In China, the proportion of end-stage kidney disease caused by DN is increasing year by year. It is expected that DN will be the main cause of end-stage kidney disease in the near future. However, the pathogenesis of DN is extremely complex, and its specific mechanism has not been fully clarified yet. It may be the result of a combination of many factors, due to many bioactive substances. Therefore, it is of great social and economic significance to explore the pathogenesis of DN and seek treatment methods to delay the progress of DN. Under the guidance of traditional Chinese medicine, it has unique advantages in the prevention and treatment of diabetes mellitus and its complications. Traditional Chinese medicine compound prescriptions have many ways and targets in the prevention and treatment of diseases. Therefore, the use of traditional Chinese medicine compound prescriptions for the prevention and treatment of DN has great potential. Summarize the ancient and modern Chinese medicine treatment of DN literature, DN early to Qi and Yin deficiency, blood stasis and heat interlinked as the basic pathogenesis, treatment appropriate Qi Yang Yin, Xie Fei Zhuyu. In order to further confirm the therapeutic effect of this prescription and further explore its possible mechanism of action, we carried out this study, that is, with the help of animal model of diabetic nephropathy. Objective: To observe the effects of Jiangtang Sanhuang Tablets on glucose and lipid metabolism, renal function, pathological changes of kidney tissue and microRNA-21 signaling pathway in early diabetic nephropathy rats. Methods: Eighty male SD rats were randomly divided into normal group (10 rats) and model group (70 rats) according to their body weight after one week of adaptive feeding. All rats were fed in a metabolic cage in the same animal room without insulin or other hypoglycemic drugs. After feeding for 3 weeks, blood glucose and urine volume were measured. If blood glucose was 16.7 mmol L-1, urine prouria was 150% and urine protein was measured for more than 30 mg in 24 hours. During the modeling period, 3 rats died, 3 blood glucose recovered, 8 rats with blood glucose up to the standard but urinary protein down to the standard, so 56 DN rats were established. 11 rats in the dosage group and 11 rats in the Irbesartan group were fed with clean general diet before and after modeling. The rats were fed and drinked freely during the experiment. The general condition of the rats was observed during the experiment. The body weight and FBG of the tail tip were measured every four weeks. The urine volume and water consumption were measured 24 hours after the last administration. The renal function (BUN, Scr), blood lipid (TC, TG, L) were measured at the end of the experiment. DL-C, HDL-C, HbA1c, 24-hour urinary protein (Upro); kidney of rats was taken out to calculate kidney weight, relative kidney weight; pathological morphology of rat kidney was observed by light and electron microscopy; TGF-beta 1, Smad3, Smad7 staining was observed by immunohistochemistry; TGF-beta 1, Smad3, Smad7 protein expression was detected by Western blotting; Results: 1. Body weight, water intake and urine volume: Body weight. After 4 and 8 weeks of drug intervention, the body weight of the drug intervention group increased significantly compared with the model group (P Compared with the model group, the middle dose group of Jiangtang Sanhuang Tablets and the high dose group of Jiangtang Sanhuang Tablets, the Irbesartan group decreased (P 0.01), while the low dose group of Jiangtang Sanhuang Tablets had no significant difference (P 0.05). There was no significant difference in the middle dose group of Tangsanhuang Tablets (P 0.05), but the high dose group of Jiangtangsanhuang Tablets was significantly less than that of Irbesartan Tablets (P 0.01). The urine volume of the high dose group of Jiangtang Sanhuang Tablets decreased (P 0.05), but the middle dose group of Jiangtang Sanhuang Tablets had no difference (P 0.05). The low dose group of Jiangtang Sanhuang Tablets was higher than the Irbesartan group (P 0.05). 2. Glucose and lipid metabolism: fasting blood glucose, after 4 weeks and 8 weeks of treatment, the low dose group of Jiangtang Sanhuang Tablets, the middle dose group of Jiangtang Sanhuang Tablets, the high dose group of Jiangtang Sanhuang Tablets Compared with the model group and irbesartan group (P 0.01), the glycosylated hemoglobin of the model group and irbesartan group was not significantly different (P 0.05). In the aspect of glycosylated hemoglobin, after 8 weeks of treatment, the low dose group of Jiangtang Sanhuang tablets, the middle dose group of Jiangtang Sanhuang tablets, the high dose group of Jiangtang Sanhuang tablets were significantly lower than the model group and irbesartan group (P 0.01). TC, TG, LDL-C were no difference (P 0.05). After 8 weeks of treatment, the low-dose group, middle-dose group and high-dose group of Jiangtang Sanhuang tablets were significantly lower than the model group and Irbesartan group (P 0.01). There was no difference between the model group and Irbesartan group (P 0.05). HDL-C, compared with the model group, the middle-dose group of Jiangtang Sanhuang tablets and the high-dose group of Jiangtang Sanhuang tablets were significantly lower than the model group. The kidney weight, relative kidney weight, renal function and urinary protein were significantly higher in the high dose group (P 0.01), but significantly lower in the low dose group (P 0.01) than in the model group (P 0.05). There was no significant difference between the low dose group and the model group (P 0.05), the middle dose group, the high dose group and the Irbesartan group (P 0.05), but the low dose group was significantly higher than the Irbesartan group (P 0.01). There was no significant difference between the high dose group and irbesartan group (P 0.05), but the low dose group was significantly higher than irbesartan group (P 0.01). The renal function (BUN, Scr), the drug intervention group was significantly lower than the model group (P 0.01), the high dose group of Jiangtang Sanhuang tablets was no significant difference compared with irbesartan group (P 0.05), and the low dose group was significantly higher than irbesartan group (P 0.05). There was no significant difference in urinary protein between the model group and the drug intervention group before treatment (P 0.05). After 8 weeks of treatment, compared with the model group, the low dose group of Jiangtang Sanhuang tablets decreased (P 0.05), the middle dose group of Jiangtang Sanhuang tablets and the high dose group of Jiangtang Sanhuang tablets decreased (P 0.05). Compared with irbesartan group, there was no significant difference (P 0.05) in the high-dose group of Jiangtang Sanhuang Tablets, the low-dose group of Jiangtang Sanhuang Tablets, and the middle-dose group of Jiangtang Sanhuang Tablets were significantly higher than that of irbesartan group (P 0.01). In type I group, the kidney lesions were serious. The glomerulus enlarged obviously, the glomerulus swelled and congested, the renal vesicle narrowed, the glomerular mesangial cells proliferated, some renal tubular cells hypertrophy, some vacuoles degenerated, the capillary lumen narrowed, the foot process flattened, the structure unclear, mitochondrial vacuole-like change, the increase of mesangial matrix, the renal tubular epithelial cells. Low-dose Jiangtang Sanhuang Tablets group, middle-dose Jiangtang Sanhuang Tablets group, high-dose Jiangtang Sanhuang Tablets group, irbesartan group can improve the renal pathological changes of DN, of which high-dose Jiangtang Sanhuang Tablets group has the best effect. 5. Expression of TGF-beta 1, Smad3, Smad7 protein in kidney: Immunohistochemical staining results showed that compared with normal group, model group T GF-beta 1, Smad3 protein expression increased significantly (P 0.01), showing brown-yellow granules, very deep coloring, drug intervention group between the normal group and the model group, significantly lower than the model group (P 0.01); compared with Irbesartan group, high-dose Jiangtang Sanhuang tablets group had no significant difference (P 0.05), low-dose Jiangtang Sanhuang tablets group, middle-dose Jiangtang Sanhuang tablets group was higher than Irbesartan group. Compared with the model group, the expression of Smad7 in the drug intervention group was significantly increased (P 0.01); the expression of Smad7 in the Irbesartan group was significantly higher than that in the low-dose group, the middle-dose group and the high-dose group (P 0.01). The expression of TGF-beta 1, Smad3 and Smad7 protein in the model group was measured by Western blotting. The expression of TGF-beta 1 and Smad3 protein in the high-dose group was not significantly different from that in the Irbesartan group (P 0.05). Compared with the normal group, the expression of Smad7 protein in the model group was significantly decreased (P 0.01), that in the drug intervention group was significantly higher than that in the model group (P 0.01), that in the Irbesartan group was significantly higher than that in the low dose group, and that in the middle dose group (P 0.01). There was no significant difference in TGF-1, Smad3, Smad7, microRNA-21 mRNA expression between high-dose Jiangtang Sanhuang Tablets group and control group (P 0.05). RTQ-PCR assay showed that TGF-1, Smad3, microRNA-21 mRNA expression in the kidney of rats was significantly higher in model group than in model group (P 0.01). There was no significant difference between the high-dose group and irbesartan group (P 0.05), but the low-dose group of Jiangtang Sanhuang tablets and the middle-dose group of Jiangtang Sanhuang tablets were significantly higher than that of irbesartan group (P 0.01). There was no significant difference between the dosage group and irbesartan group (P 0.05), but the low dosage group of Jiangtang Sanhuang tablets and the middle dosage group of Jiangtang Sanhuang tablets were significantly lower than that of irbesartan group (P 0.01). Conclusion: 1. On the basis of summing up the etiology, pathogenesis and treatment of diabetic nephropathy, the pathogenesis of diabetic nephropathy is a mixture of deficiency and excess. It can effectively prevent and treat DN rats. 2. Jiangtang Sanhuang tablets can improve the general state of DN rats, reduce blood sugar and regulate blood lipids. 3. Jiangtang Sanhuang tablets can reduce urinary protein of DN rats, reduce serum BUN and Scr, and protect the kidney of DN rats to a certain extent. 4. Jiangtang Sanhuang Tablet can improve the pathological damage of kidney tissue in DN rats, and can delay the pathological damage of kidney to some extent. 5. Decrease
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5
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