【作者】 陈云；

【导师】 李赛美；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2011， 博士

【摘要】 第一部分文献研究糖尿病肾病是糖尿病最常见的微血管并发症之一,是导致终末期肾功能衰竭的重要原因,其特征性病理变化是早期肾小球高灌注、肾小球肥大、系膜区扩张、基底膜增厚及细胞外基质堆积,以致肾小球硬化、肾间质纤维化。DN发病机制非常复杂,其确切的发病机制尚未完全阐明,现代医学认为多个信号转导系统在糖尿病肾病发病过程中具有重要的作用,治疗主要通过控制血糖、控制血压、纠正脂质代谢紊乱以及血管紧张素转化酶抑制剂和血管紧张素受体阻断剂类药物的应用,干预这些系统中的相关环节在不同程度上推迟、控制DN的发展。因此,更好地了解和采取积极的预防措施,减缓疾病的进程是治疗的关键。糖尿病肾病乃现代医学的病名,在古代医籍中并没有相对应的病名记载。然而,综观糖尿病肾病的发病机理和临床表现应属于祖国医学中的消渴病及其并发症包括“水肿”、“虚劳”、“肾劳”、“水病”、“胀满”、“尿浊”、“关格”等范畴。中医学认为DN发生是多种因素引起,本虚标实为其基本病机。本虚指阴阳、气血、五脏之虚,标实指痰浊、水湿、瘀血等病理之实。肺、胃脾、肾三脏之阴亏虚,而导致肺燥、胃热、肾虚,其中肾阴不足是决定因素。临床表现多为虚实夹杂。治疗上以中医辨证论治思想为基础,根据病程发展的不同阶段,正邪双方的强弱、进退；机体气血阴阳及脏腑的功能状态,进行整体调节,早期以益气养阴逐瘀,佐以滋补肝肾；中期以温肾健脾为重,辅以益气养血祛瘀；晚期以温补脾肾、佐以利水消肿化瘀。达到减少蛋白尿、改善肾功能、延缓疾病的发展有一定的作用。中医药在治疗DN方面有着独特的优势,中药及其配伍的药理效应具有多层次、多靶点、多环节的功能特点,而且远期疗效亦较稳定,具有比西药更大的优势。在临床上,对于症状的改善,肾功能的保护以及延缓和阻止肾脏损害的病程进展,提高DN患者生存质量都有良好的作用。本学科在80年代已经开展对Ⅱ型糖尿病的中医防治研究,围绕气阴不足、瘀血阻络的病机,采用经方桃核承气汤为基础加味研制成降糖三黄片,在临床上治疗Ⅱ型糖尿病取得显著疗效。运用降糖三黄片治疗糖尿病肾病其理论依据是胃、肾之间无论在生理或病理上都相互作用、相互影响。因此,进一步深入探讨降糖三黄片对于糖尿病肾病的疗效及作用机制对于其在临床的广泛应用具有深远的意义。第二部分实验研究目的：以长期高脂饲养(与目前人类饮食生活方式更为接近)的方式,观察中药降糖三黄片对早期糖尿病肾病大鼠的血糖、血脂、肾功能、24小时尿蛋白定量、肾组织组织形态学及肾组织ACE活性、血管紧张素Ⅱ、转化生长因子-β1以及蛋白激酶C的影响,初步探讨其对糖尿病肾病的治疗及其作用机理,为临床应用泻热逐瘀,益气养阴方药治疗早期糖尿病肾病提供实验依据。方法：清洁级Wistar大鼠普通饲料适应性喂养2W后,随机分为2组,正常组(10只)和造模组,正常组继续以普通饲料喂养,造模组改用高脂高热量饲料喂养一个月。造模前禁食12h,造模组按30mg/kg剂量腹腔内注射STZ,正常组腹腔内注射等量柠檬酸缓冲液。1W后,测定空腹血糖和空腹胰岛素,计算胰岛素敏感指数。连续两次空腹血糖≥11.1mmol/L,查尿糖++以上,胰岛素敏感指数降低,且有多饮、多尿、多食现象确认为2型糖尿病模型。2型糖尿病大鼠成模后,继续以高脂饲料连续喂养8W,即出现糖尿病大鼠的肾脏损害(以电镜结果为证)。糖尿病造模成功后,造模组进一步分层,随机分为模型组、中药组(降糖三黄片干预),西药组(卡托普利干预)。共44只大鼠完成实验,正常组10只,模型组10只,降糖三黄片干预组12只,卡托普利干预组12只(余5只或死亡或不符合条件)。中药组药量按787.5mg/kg/d的剂量给予,西药组药量按照4mg/kg/d的剂量给予,正常组及模型组灌服等量蒸馏水。①STZ注射后1周后大鼠禁食12h后空腹眼眶静脉窦采血,糖尿病大鼠造模成功后8周,处死前腹主动脉采血检测FBG、胆固醇和甘油三酯以及胰岛素。②于造模后四周、八周分别检测大鼠24h尿量、尿蛋白。③无菌操作取双肾,剥离被膜称重,计算肾重/体重指数(KW/BW);取右肾组织4%多聚甲醛初步固定,用于观察肾脏病理形态学变化；血、尿生化指标均用全自动生化分析仪测定。④放免法检测各组肾肌组织中AngⅡ含量及紫外分光亮度法检测ACE活性；⑤免疫组化检测肾皮质TGF-β1蛋白水平和PKC活性；同时以光学显微镜和透视电子显微镜观查肾小球、肾小管-间质的病理形态学改变。结果：1.实验第8周末,观察大鼠各项指标。结果表明,降糖三黄片明显改善DN大鼠的一般状态,三多一少的症状,体重明显增加,与模型组和西药组大鼠相比,差异显著(P<0.01)；降糖三黄片组血糖、血脂水平明显下降,胰岛素敏感指数上升,与模型组和西药组比较有显著性差异(P<0.01；P<0.05)；提示中药组对糖代谢有改善作用,并且作用优于西药组；显著改善实验大鼠多尿、多饮的症状,降低24h尿蛋白排泄量,与模型组比较差异显著(P<0.01)。2.利用放射免疫法和紫外分光亮度法检测各组实验动物肾脏标本中的肾组织血管紧张素Ⅱ(AngⅡ)含量和血管紧张素转换酶(ACE)活性。结果表明：模型组肾组织ACE活性明显增强,与正常组、中药组、西药组比较有显著的差异(P<0.01)；说明降糖三黄片具有一定的抑制DN大鼠肾组织中ACE活性,但其作用强度则低于血管紧张素转换酶抑制剂(ACEI)的卡托普利。模型组大鼠肾组织AngⅡ的含量比正常组、西药组和中药组显著升高(P<0.01)；两治疗组之间比较无统计学意义(P>0.05)。实验结果说明,降糖三黄片和西药卡托普利均有一定降低DN大鼠肾组织中AngⅡ含量的作用,表明中西药都能通过调节AngⅡ的分泌,起到保护肾功能的作用。3.本实验通过光镜、电镜观察各组8周末肾组织形态学变化。结果表明：模型组肾脏局灶肾小管内见蛋白沉积,肾小球增大,毛细血管基质增生,肾小球基底膜增厚,部分肾小管上皮细胞水肿变性。透射电镜观察结果显示肾小球基底膜呈阶段性增厚,有的模糊不清,上皮足突广泛融合或部分融合,滤过膜间隙增大,系膜区扩大,系膜细胞增多。降糖三黄片组经治疗8周后上述病理改变比模型组显著减轻；提示造模组大鼠肾脏组织损害可能从模型建立初期已经发生,并且呈进行性加重。模型组及西药组的肾小球硬化指数、肾小球毛细血管基质增生程度均高于正常组和中药组,其中肾小球毛细血管基质增生程度与正常组和中药组比较差异明显(P<0.05)；肾小球平均横截面积(MGA)较正常组和中药组显著增大(P<0.01),提示模型组及西药组的肾小球硬化明显。降糖三黄片对于延缓DN的肾脏病理损害疗效确切。4.采用免疫组化方法观察降糖三黄片对DN大鼠肾组织中TGF-β1的表达和PKC活性。免疫组化结果显示,TGF-β1阳性表达部位主要在肾小管胞浆,模型组TGF-β1明显高于正常组和中、西药组(P<0.01),西药组略低于中药组但无显著性差异。模型组肾小管PKC活性明显高于正常组,差异具有统计学意义(P<0.01)。中药组和西药组差异不大,但仍高于正常组(P<0.01),与模型组相比则明显降低,有统计学意义(P<0.01)。结论：1.中药降糖三黄片不仅能从客观指标上降低糖尿病大鼠的血糖、血脂水平,改善胰岛素抵抗,还能够明显改善糖尿病大鼠“三多一少”的症状,体现了中医学整体观念的思想。2.中药降糖三黄片能够降低DN大鼠尿蛋白排泄,抑制早期肾脏肥大,减轻肾脏病理损害和延缓肾脏病理进展,其作用机制与纠正脂代谢紊乱及血液流变学异常有关。3.降糖三黄片对DN大鼠肾组织AngⅡ有一定的抑制作用,这可能是其具有改善肾功能、减轻肾脏病理损害和延缓肾脏病理进展的作用机制之一4.降糖三黄片能抑制DN大鼠PKC和TGF-β1的过度表达,减少细胞外基质积聚,是减轻肾脏病理损害的重要环节。5.降糖三黄片对DN大鼠肾组织的保护作用可能是通过抑制肾组织AngⅡ活性、调控大鼠肾组织PKC活性的升高及转位,下调TGF-β1的过度表达,进而减少肾小球细胞外基质的积聚,使肾脏病理得到改善甚至逆转,从而达到保护肾脏的作用。更多还原

【Abstract】 Part One:Literature ReviewDiabetic nephropathy (DN) is one of the micro-vascular complication caused by diabetic mellitus (DM) and it has been the leading cause of end-stage renal disease with diabetes-related patients. The concept in understanding the pathogenesis of DN is that the results from a series of specific progressive renal pathological changes that have occurred incidence early in the course of diabetes. The early renal pathological changes is characterized by accumulation of extracellular matrix, leading to expansion of glomerular mesangial regions at the expense of filtration surface area, thickening of the glomerular and tubular basement membranes. Important changes in arterioles and in the renal interstitium also occur as well. These changes cause glomerular hypertension and progressive decline in GFR. Systemic hypertension may accelerate progression. The determinants of these early structural abnormalities are, however, largely remain unknown.Medical research has demonstrated that a number of signal transduction pathways may have played the important roles in onset of DN. Nowadays, the key element of treatments are patient’s education, dietary and exercise counseling, monitoring of glucose control, improving of lipid metabolism disorder and taking ACE inhibitors or angiotensin II receptor blockers accordingly. The treatments may in certain extent to improve and control the symptom of DN and prevent ESRD developing. Therefore, it is crucial to better understand and take the necessary aggressive-preventive measures to slow this progressive disease process.Diabetic nephropathy is known as modern medical term and there is no corresponding name has mentioned in ancient Chinese medicine literature. According to pathogenesis and clinical manifestations of Diabetic Nephropathy, it should be categorized in TCM as "wasting-thirsting" or "xiao ke" accompany with its complications, such as "edema", "consumptive disease", "kidney consumption", "dropsy disease", "swollen disease", "turbid urine", "Vomiting and Dysuria Diseases". TCM believes that DN is caused by many factors, its basic pathogenesis is deficiency in origin and excess in superficiality. Deficiency stands for insufficiency in zang-fu organs of qi, blood, yin and yang. Excess represents those pathological products, such as phlegm accumulation, fluid retention, and blood stasis. Yin dysfunction in lung, kidneys, spleen and stomach, caused lung dryness, heat of stomach and deficiency in kidneys Yin. Deficiency in kidney Yin is the main determinants of all. Clinical manifestations shows that intermingled deficiency and excess for many cases. The therapeutic principles are formulated according to the basic theory of TCM and syndrome differentiation. According to the different course of the disease, the conflict between the anti-pathogenic qi and pathogenic depend on strong or weak, advance and retreat, of either side. The principle of treatment is based on syndrome differentiation, development of disease, the strength of human body against the disease, and the function of Zang-fu with its qi and blood, yin and yang to have overall regulation. Treatment is concentrated on replenishing Qi and nourishing Yin as well as removing blood stasis in assistant with tonifying the kidney and liver at early stage. Treatment for invigorate the spleen, warm the kidney supported by benefiting Qi and nourishing blood and removing blood stasis is suggested during metaphase stage; to warm and tonify the spleen and kidney accompany with inducing diuresis and alleviating edema at the advanced stage as a result to reduce proteinuria and improve renal function, controlling the development of the disease. TCM hold unique advantages in treating DN. Pharmacological effects of Chinese medicine and its formulation have multi-level, multi-target and multi-connection features with long term stability of curative effects, which are superior to Western medicine. On clinical practice, it proves that TCM has remarkable curative effects in improving symptoms, protecting renal function, preventing or postpone the progression of renal damage and improving the living quality of patients.The research base on TCM classical prescription "Taohe Chengqi Decoction" has already carried out since 80’s regarding to the treatment on type 2 diabetes against the pathogenesis of deficiency in Qi and Yin, blood stasis obstruction of collaterals has gained the notable effects. According to the former studies, Jiangtangsanhuang (JTSH) tablet has been developed for the treatment of diabetic nephropathy under the theoretical basis of stomach and kidney in both physiology and pathology are interaction and interfere. Thus, further study on the mechanism of JTSH tablet for DN in order to be broadly adopted by clinical practice has become profound significance.Part two:Experimental researchObjective:The study had used high cholesterol diet pattern which is closer to the human lifestyle to observe the effects of JTSH tablet on blood glucose, urinary protein in 24hours, renal function, rennin activity, the content of angiotensinⅡ, the expressions of transforming growth factor-β1, the function of PKC and the pathological changes of renal tissue for the early stage of diabetic nephropathy rats, to reveal the mechanism of reno-protective effect of JTSH Tablet against the experimental DN rats. So as to provide experimental basis of clinical service for treating early stage of Diabetic Nephropathy by using purge Heat and blood stasis with nourish qi and yin therapy. Methods:Healthy Wistar rats were randomly divided into normal control group (n=10) with regular feed and diabetic model groups with high cholesterol diet (HCD) throughout the study period. The model groups induced lower-dosage streptozocin (STZ) (30mg/kg) after 28 days of HCD and were randomly divided into three group with fasting blood glucose≥11.1mmol/L:early DN group(n=10), JTSH group(787.5mg·kg-1·d-1,n=12) and Captopril group(4mg·kg-1·d-1,n=12). The course of treatment lasted for 8 weeks. The general status (psychosis, diet, hair color, weight, etc.), blood glucose, glycosuria, renal weight/body weight ratio, urine protein in 24 hours were monitored.①One and eight weeks after STZ rejection, fasting blood glucose, cholesterol and insulin levels were detected at first and eighth week after STZ-induced;②Renal tissue were detected for the expression of TGF-β1 and activity of PKC by the method of immune histo-chemistry.③Renal Angiotensin converting enzyme(ACE)activity and angiotensinⅡ(AngⅡ) expression were measured by radio immunity.④The renal pathological changes were observed by optics microscope and electronic microscope.Result:1. The various items were observed at the end of 8weeks of experiment. The general conditions and the symptoms of polyphagia, polydipsia, polyuria were improved in JTSH group, the body weight had boosted faster than DN and Captopril groups with obvious significant (P<0.01). JTSH group had significantly reduced FBG, cholesterol, glycerin trimyristate level and increase ISI level after 8 weeks treatment comparing with DN and Captopril groups (P<0.01; P<0.05). It indicated that JTSH tablet have better effect in improving glucose metabolism than Captopril. Besides, decrease obviously in the amount of urine and urine protein of 24 hours of treated groups compared with DN group (P<0.05). Kidney coefficient of JTSH group compared with DN and Captopril groups were decreased (P<0.05 and P<0.01) respectively.2. The radiative immune method and biochemical method were adopt for detecting angiotensin converting enzyme(ACE)activity and the content of angiotensinⅡ(AngⅡ) in renal tissue. The results indicated that ACE activity increased obviously in DN group compared with the others (P<0.01); ACE activity was low in Captopril group compared with the Normal, JTSH and DN groups respectively (P<0.05, P<0.01). It proved that JTSH can suppress ACE activity in certain extend, while its contribution is not as strong as Captopril as angiotensin converting enzyme inhibitor. The content of AngⅡin renal tissue was raised in DN group compared with others (P<0.01). The comparison between two treated groups was no statistical significance. The result showed that both JTSH tablet and Captopril can protect the renal function by reducing and adjusting the AngⅡin renal tissue of the DN rats.3. The rats’renal histopathology was observed by optical microscope and electric microscope at the end of the experiment. It was found that DN group glomerular basement membrane thick, mesangial matrix expansion and part of glomerulor vascular abnormally changed. The treated groups were found the above pathological changes had improved after 8 weeks treatment. It indicates that the damage of renal tissue might have occurred at the very beginning and increased gradually. Glomerular capillaries stromal hyperplasia significantly in DN group and Captopril group compared with JTSH (P<0.05) while index of glomerulosclerosis is increased significantly compared with normal and JTSH group (P<0.01). It looks that JTSH tablets were more effective in improving glomerular sclerosis and renal tubular edema degeneration of DN rats than Captopril, but difference is not outstanding. The result indicates that JTSH can slow down and relieve the kidney damage.4. The immunohisto-chemical Method is used for detecting the expression of TGF-β1 and the activity of PKC in kidney among groups. The expression of TGF-β1 among DN group was significantly higher than normal group (P<0.01), as well as JTSH and Captopril groups (P<0.01). The PKC activity in renal tubular was raised in DN group compared with JTSH and Captopril groups (P<0.01), whereas still higher than the normal group (P<0.01).Conclusion:1. JTSH tablets can significantly reduce FBG, cholesterol and glycerin trimyristate level, as well as ameliorate the insulin resistance. In addition, it can improve "three more and one less" symptoms which embody the holistic concept of TCM.2. JTSH tablets can reduce the excretion of urine protein of the DN rat and suppress the hypertrophy of kidney and reduce renal pathological damage at the same time, so as to relief the renal pathological progression. The mechanism of the improvement might have related to JTSH rectified the disorder of lipid metabolism and abnormal of blood rheology.3. JTSH can inhibit the expression of AngⅡin renal tissue of the DN rats which could be one of the mechanism of improving renal function and relief renal pathological damage.4. JTSH can restrain the PKC and TGF-β1 from over expression which is the important part to reduce the accumulation of extracellular matrix and prevent the renal being damaged.5. The mechanism of reno-protective effect of JTSH tablets may probably worked by inhibiting the Angll activities, controlling and transferring the up raising of PKC activities and reducing the expression of TGF-β1 as a result to reduce the accumulation of glomerular extracellular matrix and improved the renal pathology in order to reach the goal in protecting the renal function.更多还原