论文标题:肾康丸治疗糖尿病肾病的实验研究
Experimental Studies of Shenkangwan in Diabetic Nephropathy
论文作者
论文导师 魏连波,论文学位 博士,论文专业 中西医结合临床
论文单位 第一军医大学,点击次数 82,论文页数 128页File Size7130K
2007-03-01论文网 http://www.lw23.com/lunwen_950111377/
Shenkangwan ;; Diabetic nephropathy ;; Oxidative stress ;;Angiotensin;; Advanced glycation end products
随着生活水平的提高、生活方式的改变和人口老龄化，糖尿病(diabetes mellitus，DM)的患病率在全球迅速增加，已经成为发达国家中继心血管病和肿瘤之后的第三大非传染性疾病，是严重危害人类健康的世界性公共卫生问题。糖尿病肾病(diabetic nephropathy，DN)是糖尿病的主要并发症之一，总发生率为47.66％，是1型DM患者的主要死亡原因，在2型DM，其严重性仅次于冠状动脉和脑动脉粥样硬化病变。在西方国家，17％-33.8％接受肾脏替代治疗的肾衰竭病人是由于DN引起的，DN已经成为慢性肾衰竭的首要病因。而且在接受肾脏替代治疗过程中，DM患者的死亡率远远高于非DM患者，其2年生存率仅50％左右。因此，如何有效地预防、治疗DN已成为医学界共同关注的问题。 DN的发病机制十分复杂，迄今尚不清楚。目前认为氧化应激(oxidative stress，OS)是重要的共同机制，在DM的慢性并发症中可能起主要作用。同时，近年来人们认识到DM时存在。肾脏局部肾素．血管紧张素系统(renin-angiotensin system，RAS)的异常，可能与DN的发生发展关系密切，血管紧张素Ⅱ(angiotensinⅡ，AngⅡ)是RAS中的主要活性中心，通过与其特异性1型受体AT_1R结合而发挥一系列致病作用，可导致肾小球硬化及肾间质纤维化。大量研究证实，应用血管紧张素转换酶抑制剂(angiotensin-converting enzyme inhibition，ACEI)或血管紧张素Ⅱ受体拮抗剂(angiotensinⅡreceptor antagonist，ARB)阻断RAS均可抑制肾脏肥大，降低尿白蛋白排泄，延缓DN的进展。另外，晚期糖基化终产物(advanced glycation end products，AGEs)也是DN发病机制中的重要因素之一，主要通过与其受体(receptor for advanced glycation end products，RAGEs)结合引发生物学效应，在DN的发生、发展过程中与RAGEs相互作用组成调控网络，因而AGEs-RAGEs系统对于研究DN的发病机制具有重要的意义。 目前DN的西医治疗措施除饮食治疗、控制血糖等之外，以ACEI、ARB的应用最为广泛和重要，但这些方法临床疗效均相当有限，迄今尚无针对DN的特效西药。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(Ⅰ～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的治疗上以益气健脾、滋肾养阴兼活血通络为法。虽然中医药在DN的治疗方面具有比较明显的优势，但绝大多数尚处于疗效观察和经验积累阶段，目前国内外未见对DN具有确切疗效的中药新药问世。 我们在对DN的长期临床治疗和研究过程中，精心筛选提炼药物组方，命名为肾康丸，经反复临床验证，疗效十分确切，并已申请国家发明专利(肾康丸的组方及工艺研究，专利号200610036515．2)。肾康丸由黄芪、金樱子、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，暗合Ⅲ～Ⅳ期DN的病因病机，具有标本兼治之功。但其治疗DN的细胞分子水平的作用机制还不清楚，有待进一步研究。本研究通过DN大鼠模型从多个方面探讨了肾康丸治疗DN的细胞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： 第一部分肾康丸对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，为进一步研究其作用机制提供实验依据。 方法：首先用链脲佐菌素(STZ)按55 mg╱kg大鼠体重一次性腹腔内注射法建立大鼠DM模型，连续2周测血糖、尿糖、尿量，以连续三次空腹血糖＞16.6mmol／L、尿糖强阳性、尿量＞原尿量150％、尿微量白蛋白定性测量阳性者为DN成模标准。造模过程中大鼠死亡率为17.65％。4周后将模型大鼠随机分为4组(n=7)：肾康丸组、厄贝沙坦组、肾康丸和厄贝沙坦合用组、DN模型组。所有大鼠均在同一动物房内用代谢笼分笼饲养，标准饮食，自由进水。另设正常对照组(n=6)。灌胃给药治疗8周，观察大鼠一般状况、血糖、尿糖。末次给药后将大鼠放入代谢笼中，计24 h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，检测大鼠血糖(Glu)、24 h尿蛋白定量、血肌酐(Cr)、尿素氮(UN)、血总胆固醇(TC)、血甘油三酯(TG)；取大鼠肾脏，观察肾脏大小，计算肾重、相对肾重，并通过HE染色法和电镜观察大鼠肾脏组织病理变化。 统计学方法：数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件，治疗前后两组间比较采用配对t检验、多组间比较采用重复测量方差分析，治疗后多组间比较采用One Way ANOVE、SNK检验；方差不齐者首先进行数据变换，若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验，检验水平a=0.05。 结果： 1．34只大鼠用于造模，成模率82.35％(28／34)，成模后病死率14.29％(4／28)。 2．在8周治疗期间内，DN模型组大鼠血糖均维持在16.6 mmol／L以上，尿糖(+++)以上；表现出多饮、多食、多尿、消瘦的DM症状。 3．与正常组大鼠比较，DN模型组大鼠UN、cr、TC、TG、24 h尿蛋白定量等指标均升高，具有显著性意义(P＜0.01)；肾脏重量及相对肾重增加，具有显著性意义(P＜0.01)，且出现了相当于Mogensen分期3期DN的肾脏病理损害。 4．与DN模型组大鼠比较，除厄贝沙坦组大鼠Glu、TC、TG无显著性意义(P＞0.05)外，三个治疗组均可见大鼠一般状况改善，上述指标水平降低，具有显著性意义(P＜0.01)，同时可见肾脏组织病理损害减轻。 5．肾康丸与厄贝沙坦对DN大鼠具有协同肾脏保护作用。 结论： 1．肾康丸对DN大鼠具有肾脏保护作用。 2．肾康丸与厄贝沙坦对DN大鼠肾脏具有协同保护作用。 第二部分肾康丸对DN大鼠肾脏氧化应激的影响 目的：探讨肾康丸对DN大鼠肾脏氧化应激的影响。 方法：取第一部分各组大鼠肾脏，检测肾组织丙二醛(MDA)含量以及谷胱甘肽过氧化物酶(GSH-Px)、铜锌超氧化物歧化酶(Cu，Zn-SOD)、过氧化氢酶(CAT)活性；采用逆转录聚合酶链反应(RT-PCR)法观察各组大鼠肾组织GSH-Px、Cu，Zn-SOD、CAT mRNA表达情况。 统计学方法：数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件，治疗后多组间比较采用One Way ANOVE、SNK检验；方差不齐者首先进行数据变换，若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验，检验水平a=0.05。 结果： 1．DN模型组大鼠与正常组大鼠比较，肾组织中MDA含量升亩，抗氧化酶GSH-Px、Cu，Zn-SOD、CAT活性下降，GSH-Px、Cu，Zn-SOD mRNA表达水平增高，均具有统计学意义(P＜0.01)；但肾组织中CAT mRNA表达水平无显著性差异(P＞0.05)。 2．治疗后五组肾组织MDA含量存在显著性差异(穿=26．753，JF，<0．001)，DN模型组秩次最高；三个治疗组比较，厄贝沙坦组秩次最高。 3．治疗后五组间肾组织CAT、GSH-Px与Cu,Zn-SOD存在显著性差异(F值分别为100.231、91.88、133.868，P=0.000)，肾康丸组、厄贝沙坦组、两药合用组与DN模型组比较CAT、GSH-Px与Cu，Zn-SOD活性明显升高(P＜0.001)。三个治、疗组比较，肾康丸组、厄贝沙坦组之间无显著性差异(P分别为0.097，0.452，0.086)，但均与两药合用组存在显著性差异(P＜0.001)。 4．治疗后五组间肾组织CAT mRNA表达水平无显著性差异(P=0.216)，GSH-Px、Cu，Zn-SOD mRNA表达水平存在显著性差异(X~2=23.287、F=29.099，P＜0.001)。五组间比较，DN模型组GSH-Px mRNA相对表达量秩次最高；三个治疗组比较，肾康丸组GSH-Px mRNA相对表达量秩次最高。三个治疗组Cu，Zn-SOD mRNA相对表达量与DN模型组比较均显著升高(P＜0.001)，其中两药合用组与肾康丸组、厄贝沙坦组比较存在显著性差异(P＜0.01)，但肾康丸组、厄贝沙坦组之间比较均无统计学差异(P=0.350) 结论： 1．肾康丸可以抑制DN大鼠肾脏的氧化应激。 2．肾康丸与厄贝沙坦对DN大鼠肾脏的氧化应激具有协同抑制作用。 第三部分肾康丸对DN大鼠肾脏AngⅡ及其受体AT_1R表达的影响 目的：探讨肾康丸对DN大鼠AngⅡ及其1型受体AT_1R表达的影响。 方法：取第一部分各组大鼠血浆、肾脏，采用放射免疫法检测血浆和肾组织AngⅡ含量，免疫组化法检测肾组织AngⅡ、AngⅡ1型受体AT_1R的表达，RT-PCR法检测肾组织AT_1R mRNA的表达。 统计学方法：数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件，治疗后多组间比较采用One Way ANOVE、SNK检验；方差不齐者首先进行数据变换，若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验，检验水平a=0.05。 结果： 1．DN模型组大鼠与正常对照组比较，血浆及肾组织AngⅡ含量均明显升高(P＜0.001)。治疗后五组间血浆及肾组织AngⅡ均存在显著差异(F值分别为50.152、126.110，P=0.000)。与DN模型组比较，肾康丸组血浆及肾组织AngⅡ含量明显降低(P＜0.001)，厄贝沙坦组、两药合用组血浆及肾组织AngⅡ含量均较肾康丸组明显升高(P＜0.001)。 2．治疗后五组间肾组织AngⅡ免疫组化相对表达量也存在显著性差异(X~2=23.837，P=0.000)，厄贝沙坦组肾组织AngⅡ免疫组化相对表达量秩次最高。 3．治疗后五组间肾组织AT_1R含量及其mRNA相对表达量均存在显著性差异(X~2分别为24.366、22.748，P=0.000)，DN模型组肾组织AT_1R含量及其mRNA相对表达量秩次均最高。三个治疗组比较，两药合用组肾组织AT_1R含量及其mRNA相对表达量秩次最低，厄贝沙坦组次之。 结论： 1．肾康丸可以降低DN大鼠血浆、肾组织AngⅡ水平以及肾组织AT_1R含量，抑制肾组织AT_1R mRNA表达。 2．肾康丸可以部分拮抗厄贝沙坦引起的DN大鼠血浆及肾组织AngⅡ水平增高。 第四部分肾康丸对DN大鼠肾脏AGEs及其受体RAGEs表达的影响 目的：探讨肾康丸对DN大鼠AGEs及其受体RAGEs表达的影响。 方法：取第一部分各组大鼠血清、肾脏，检测血清和肾组织AGEs含量，免疫组化法检测肾组织RAGEs的表达，RT-PCR法检测肾组织RAGEs mRNA的表达。 统计学方法：数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件，治疗后多组间比较采用One Way ANOVE、SNK检验；方差不齐者首先进行数据变换，若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验，检验水平a=0.05。结果： 1．DN模型组大鼠与正常对照组比较，血清及肾组织AGEs含量均明显升高(P＜0.01)。与DN模型组比较，三个治疗组血清及肾组织AGEs含量均明显降低(P＜0.01)；与肾康丸组、厄贝沙坦组比较，两药合用组血清(P＜0.05)及肾组织(P＜0.01)AGEs含量均明显降低；肾康丸组、厄贝沙坦组之间比较均无显著性差异(P＞0.05)。 2．DN模型组大鼠与正常对照组比较，肾组织RAGEs含量及其mRNA表达水平均明显升高(P＜0.01)。与DN模型组比较，三个治疗组肾组织RAGEs含量及其mRNA表达水平均明显降低(肾康丸组、厄贝沙坦组P＜0.05，两药合用组P＜0.01)；与肾康丸组、厄贝沙坦组比较，两药合用组肾组织RAGEs含量及其mRNA表达水平均明显降低(P＜0.05)；肾康丸组、厄贝沙坦组之间比较均无显著性差异(P＞0.05)。 结论： 1．肾康丸可以降低DN大鼠血清、肾组织AGEs水平以及肾组织RAGEs含量，抑制肾组织RAGEs mRNA表达。 2．肾康丸与厄贝沙坦对DN大鼠AGEs-RAGEs系统具有协同抑制作用。 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，其作用机理与抑制氧化应激，减少AngⅡ的生成及其受体AT_1R的表达，降低AGEs的含量及其受体RAGEs的表达有关，为其临床推广应用于治疗DN提供了较为全面、深入的分子水平的理论依据。
With the enhance of living standard, the changing of life style and the aging of the population, the incidence of diabetic mellitus (DM) is rising rapidly in world widely, which has become the third non-infectious disease listed behind cardiovascular diseases and tumour in developed country. So, it has become a cosmopolitan public health problem threatened human health. Because of a total incidence of 47.66% in DM, diabetic nephropathy(DN)is one of the primary complications of DM in clinic. And it is a leading cause resulting in death among type 1 DM patients, and only atherosclerosis of coronary and cerebral arteries preponderate over it in severity in type 2 DM patients. According to some surveys, about 17～33.8% renal function failure patients receiving renal replacement treatment(RRT) is resulting from DN, and it is a leading cause resulting in end-stage renal disease(ESRD) in DM patients too. And it was observed that the death rate in DM patients which received RRT, with a survival rate of two years accounting for 50% or so, were far more than that in non-DM patients. So how to treat and prevent the DN effectively is becoming a common concerned problem among the international medical researchers. The pathogenesis of DN is complicated and some of them remain poorly understood. Nowadays, it is widely accepted that oxidative stress(OS) is a primary common mechanism in the occurring of chronic complications of DM. Simultaneously, many researchers have found there was a abnormity of renin-angiotensin system(RAS) in local diabetic kidney tissue, and believed it might play a pivotal-role in the occurrence and progression ofDN. AngiotensinⅡ(AngⅡ) is the principal effector of RAS, and has multiple direct pathopoiesia actions via the combination with type 1 angiotensin receptor(ATIR), including glomerular sclerosis and interstitial fibrosis. Many studies have confirmed that the inhibition of the RAS by the use of angiotensin converting enzyme inhibitors (ACEI) or angiotensinⅡreceptor blockers(ARB) offer renoprotection, reduce the albuminuria excretory rate and delay the progression in DN. In addition to OS and RAS, advanced glycation end products(AGEs), which development its biological effect via combining with receptor for AGEs (RAGEs), also play an important role in the pathogenesis of DN. By interacting with each other AGEs and RAGEs compose a regulation network. Therefore, to illuminate AGEs and RAGEs system possess an important meaning to elucidating the pathogenesis of DN. The use of ACEI and ARB is the most extensive and important therapy in the treatment of the DN in western medicine at present, in addition to the regulation of diets and controlling of the intensive blood glucose. However, the effects of these therapies are proved to be limited, and so far neither one specificness medicine can treat DN effectively. DN belongs to the domain of the "Shenxiao", "Dropsy", "Shennao", "Guange", and so on in traditional chinese medicine(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(Ⅰ～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 "impelling blood stream and dredging vas". Although TCM is superior to western medicine in treatment of DN, there are no effective Chinese materia medica to treat the patients with DN, because most of them are still in the period of therapeutic effect observation or experience accumulating. On the basis of our long-term clinic experience on DN treatment and research on it, we created a new compound preparation with well selected and essential traditional Chinese drug, and named it "Shenkangwan". Long-term use of Shenkangwan in clinic have proved it could treat DN effectively, and has attained national invention patent ("the study on the prescription and craft of Shenkangwan"; patent number: 200610036515.2). Shenkangwan was mainly constituted of huangqi, gordon guryale seed, chrokee rose fruit, leech, herba leonuri, 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 ofⅢ～V stages DN fitly and can treat the DN from the fundamentality to the sign. But its eytomolecular mechanism in treating DN is not clear and need farther research. So we carried out the experiment on animal to explore the cytomolecular and genie mechanism of Shenkangwan in treating DN and provide the laboratorial evidence for its clinic application. The details were as follows: ChartⅠRenoprotective effect of Shenkangwan in diabetic nephropathy rats Objective: 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 uric glucose and the urine volume. The rats, which value of blood glucose exceeding 16.7mmol·L~(-1), uric glucose positive experiment over +++, urinary volume surpassing 150% basal level and observation measurements on urine microalbumin showing positive were taken for the successful DM rat models and calculated the rate of them. The death rate of rats was 17.65% after model created. After 4 weeks, they were randomly divided into 4 groups(n=7): model control group; Shenkangwan group; Irbesartan group and conjunct therapy group. All rats were reared in metabolic cage in the same animal room, fed with standard diet and freely drunk. Other six 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, the excretion of the 24 hour urine protein(Upro)and the levels of serum creatinine(SCr), blood urea nitrogen(BUN), triglyceride(TG), total cholesterol(TC). 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) and electron microscope. Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results of two groups before and after treatment were analyzed using paired t-test for comparisons. Repeated measurement experiment was used to analyze the statistical significance among several groups. Results after treatment among multi-groups were analyzed using one-way ANOVE, Student-Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P＜0.05 was considered to be statistically significant. Results: 1.32 rats were used to established the DM models and the rate of successful models was 82.35%(28/34). And the death rate of DM model rats was 14.29%(4/28). 2.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. 3. Compare with the normal rats, their following indexes including UN、Cr、TC、TG、24h Upro were increased distinctly(P＜0.01). Their kidney weight and relative kidney weight increased distinctly(P＜0.01). And kidney tissue came forth about the 3 stages renal pathological lesions according to the stages of Mogensen. 4. Compare with the rats in model group, all three treatment could ameliorate the rat" general state, decrease the above indexes markedly(P＜0.01)and mitigate the renal pathological lesion. 5. Shenkangwan and Irbesartan have a synergetic renoprotective effect on DN rats. Conclusions: 1. Shenkangwan can play a certain renoprotective role in diabetic nephropathy rats. 2.Shenkangwan and Irbesartan perform a synergetic renoprotective effect on DN rats. ChartⅡEffect of the Shenkangwan on the renal oxidative stress in diabetic nephropathy rats Objective: To investigated the effect on the renal oxidative stress in early diabetic nephropathy rats in order to study the renalprotection mechanism of Shenkangwan. Methods: The kidneys used of this study were from the rats in chartⅠ. The renal intrinsic malondialdehyde(MDA) levels and antioxidatant enzyme activities such as glutathione peroxidase(GSH-Px), catalase(CAT) and Cu-Zn superoxide dismutase(SOD) were measured by corresponding methods. Meanwhile, the expression of GSH-Px, CAT, Cu-Zn SOD in the renal tissues of the rats in every group were detected with the method of the reverse transcription-polymerase chain reaction(RT-PCR). Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results after treatment among multi-groups were analyzed using one-way ANOVE、Student- Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups. P＜0.05 was considered to be statistically significant. Results: 1. Comparing with normal group, the content of MDA was increased significantly and the activity of GSH-Px, CAT and Cu-Zn SOD were down-regulated markedly in the renal tissue of DN model rats(P＜0.01). The renal tissue expression of GSH-Px, Cu-Zn SOD mRNA were increased distinctly(P＜0.01), but the expression of CAT mRNA was not significantly changed(P＞0.05). 2. There was a significant difference in the content of MDA among five groups after treatment(X~2=26.753, P＜0.001), and the mean rank of DN model was the highest. Compared among three treated groups, that of Irbesartan group was the highest. 3.There were significant difference in the activity of GSH-Px, CAT and Cu-Zn SOD(F=100.231、91.88、133.868 respectively; P=0.000). Compare with those in the rats of the DN model group, the activity of GSH-Px, CAT and Cu-Zn SOD in the rats of Shenkangwan, Irbesartan group and conjunct therapy group were up-regulated markedly(P＜0.001).Multiple comparison showed no significant difference in these between Shenkangwan and Irbesartan group(P=0.097, 0.452, 0.086 respectively), while compared with conjunct therapy group significant difference were found in these of both two groups(P＜0.001). 4.Significant difference were observed in the expression level of GSH-Px and Cu-Zn SOD mRNA in the kidney tissue of rats in all groups(X~2=23.287, F=29.099, P＜0.001), but not in CAT mRNA(P=0.216). The mean rank of the relative expression level of GSH-Px mRNA was the largest in all groups, but that in Skenkangwan was the largest in three treated groups. Compare with that in model group, the relative expression level of Cu-Zn SOD mRNA were markedly up-regulated in all treated groups(P＜0.001), while compare with that in conjunct therapy group, significant difference were found in Shenkangwan and Irbesartan groups, but no significant change was found in that between Shenkangwan and Irbesartan groups(P=0.350). Conclusions: 1.Shenkangwan can inhibit the renal oxidative stress in early diabetic nephropathy rats. 2.Shenkangwan can play a synergistic inhibiting role on the renal oxidative stress with Irbesartan in early diabetic nephropathy rats. ChartⅢEffect of Shenkangwan on the renal angiotensinⅡand its typeⅠreceptor expression in diabetic nephropathy rats Objective: To explore the effect of Shenkangwan on the expression of AngⅡand AT_1R in the kidney tissue of DN model rats. Methods: The blood plasm and kidneys used of this study were from the rats in chartⅠ. Radio-immunity was used to measure the content of AngⅡin the blood plasm and kidneys. The expression of AngⅡand AT_1R protein in the renal tissues were analyzed with the method of immunohistochemistry. RT-PCR was used to detect the expression of AT_1R mRNA in the tissues. Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results after treatment among multi-groups Were analyzed using one-way ANOVE、Student- Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups. P＜0.05 was considered to be statistically significant. Results: 1. Comparing with normal group, the content of AngⅡwere increased significantly in all samples of DN model rats(P＜0.001). Significant difference were observed in the content of AngⅡboth in the blood plasm and kidney tissues of rats in all five groups(F=50.152, 126.110, respectively, P=0.000). Compare with that in model group, the content of AngⅡboth in the blood plasm and kidney tissues of rats in Shenkangwan group were decreased distinctly(P＜0.001). And such did it when compared with Irbesartan and conjunct therapy groups(P＜0.001). 2. Immunohistochemistry analysis showed significant difference in the renal tissue relative expression of AngⅡin all five groups after treatment(X~2=23.837, P=0.000). The mean rank of that in Irbesartan group was the largest in all groups. 3.There were significant difference in the relative expression level of AT_1R protein and mRNA in the renal tissues in all groups after treatment(X~2=24.366, 22.748, respectively; P=0.000). The mean rank of those in model group was the largest in all groups. In three treated groups, that in conjunct therapy group was the smallest, then is that in Irbesartan group. Conclusions: 1. Shenkangwan can decrease the expression level of AngⅡboth in the blood plasm and kidney tissue, reduce the content of AT_1R in the kidney tissue and inhibit the renal expression of AT_1R mRNA in early diabetic nephropathy rats. 2. Shenkangwan can antagonize partly the increase of the expression level of AngⅡboth in the blood plasm and kidney tissue induced by Irbesartan. ChartⅣEffect of Shenkangwan on the renal advanced glycation end products and its receptor expression in diabetic nephropathy rats Objective: To investigated the effect of Shenkangwan on the AGEs and RAGEs in diabetic nephropathy rats. Methods: The blood plasm and kidneys used of this study were from the rats in chartⅠ. Radio-immunity was used to measure the content of AGEs in the blood plasm and kidneys. The expressions of RAGEs protein in the renal tissues were analyzed with the method of immunohistochemistry. RT-PCR was used to detect the expression of RAGEs mRNA in the kidney tissues. Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results after treatment among multi-groups were analyzed using one-way ANOVE、Student-Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups. P＜0.05 was considered to be statistically significant. Results: 1.Compare with those in normal group, the content of AGEs both in the blood plasm and renal tissue were increased significantly(P＜0.01). Compare with those in model group, those in all three treated groups were decreased matrkedly(P＜0.01). Significant decrease were found in the content of AGEs both in the blood plasm(P＜0.05) and renal tissue(P＜0.01) in conjunct therapy group compared with Shenkangwan and Irbesartan groups. 2. Compare with those in normal group, the expression level of RAGEs protein and mRNA were up-regulated distinctly(P＜0.01) in the renal tissue of rats in model group. Compare with those in model group, those in three treated groups were down-regulated significantly(P＜0.01 in Shenkangwan and Irbesartan groups, P＜0.05 in conjunct therapy group, respectively). Markedly down-regulations were observed in those in conjunct therapy group compared with those in Shenkangwan and Irbesartan groups(P＜0.05). And no significant difference was found in those between Shenkangwan and Irbesartan groups(P＞0.05). Conclusions: 1. Shenkangwan can decrease the expression level of AGEs both in the blood plasm and kidney tissues, reduce the content of RAGEs in the kidney tissue and inhibit the renal expression of RAGEs mRNA in early DN rats. 2. Shenkangwan and Irbesartan have a coordinate repression on AGEs-RAGEs system in DN rats model. Conclusions of the whole paper: We carried out the experiment on animal and cell 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 retarding OS, reducing the synthesizing AngⅡand down-regulating the expression of AT_1R, decreasing the content of AGEs and the expression of RAGEs. And those provided the cytomolecular and genic therapeutic mechanism and laboratorial evidence for its clinic treatment on DN.
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