糖尿病肾病炎症因子的表达及黄芪甲苷的肾脏保护作用

发布时间：2018-06-08 06:20

本文选题：文本挖掘 + 气阴两虚　；参考：《广州中医药大学》2016年博士论文

【摘要】：目的：1、探讨中医药治疗糖尿病肾病的用药特点；2、观察气阴两虚挟瘀证糖尿病肾病患者血清CXCL12等炎症因子的表达情况；3、观察黄芪甲苷对2型糖尿病db/db小鼠的肾脏保护作用。方法：1、文献研究采用文本挖掘的方法,探索中医治疗糖尿病肾病的的用药特点。通过参考《中药大辞典》(第二版)、《中华人民共和国药典》(增补版)及全国高等中医药院校规划教材《中药学》(第二版)建立词表库。登录中国知网(CNKI),对中医治疗糖尿病肾病理论研究、经验总结、临床试验相关文献进行搜索。将搜索到的文献按程序进行筛选并转换格式后建立数据库,以该词表库作为关键词苦对相关文献中的关键词进行提取并保存。采用Cytoscape软件对提取到的关键词进行网络分析,总结其用药特点。2、临床观察参考相关文献建立诊断及纳排标准,对2015年3月至2016年1月在广州中医药大学附属深圳市中医院门诊及住院就诊的糖尿病肾病患者进行筛选,纳入符合标准的气阴两虚挟瘀型糖尿病肾病患者进行观察。观察内容包括一般资料、生化指标水平及相关炎症因子的表达。3、动物实验基于以上两项研究的结果,采用中药黄芪提取物黄芪甲苷对2型糖尿病db/db小鼠进行治疗。用药期间,应用代谢笼、血糖仪和动物无创血压计测量小鼠的代谢指标、血糖和血压。治疗12周后杀检小鼠,留取血液和尿液。应用全自动生化分析仪检测小鼠肾功能、血白蛋白等生化指标；应用ELISA检测小鼠尿白蛋白排泄率和肾小管损伤指标；应用PAS染色剂透射电镜检测肾小球和肾小管病理和超微病理损伤；采用免疫印迹法(Western Blot)检测肾脏内P-Akt和P-NF-k B蛋白表达。结果：1、文献研究通过制定检索式,检索CNKI糖尿病肾病相关文献,共获得中医治疗糖尿病肾病理论研究、经验总结及临床试验相关文献共3778篇。根据筛选标准,经筛选后得到目标文献理论研究文献58篇,经验总结文献264篇,临床试验文献618篇。将这些文献转换格式后进行关键词提取,得到关键的一维频次及二维频次结果,并利用Cytoscape软件对提取结果进行网络关系分析。在理论研究文献中,使用频次处于前三位的中药方剂为肾气丸、生脉散、金匮肾气丸,使用频次处于前三位的中药为黄芪、丹参、茯苓。网络分析结果发现理论研究中,黄芪、丹参处于整体用药的核心位置,与其他药物连系最多。经验总结文献中方剂应用居于前三位的是真武汤、六味地黄丸、五苓散,网络分析结果发现真武汤与前十位中其他方剂几乎均存在连系,而济生肾气丸、金匮肾气丸、六味地黄丸、生脉散关系密切,而其他方剂相对独立。单味中药挖掘结果前三位是黄芪、丹参、地黄,网络分析结果发现黄芪、地黄位于药物应用的中心位置。临床试验文献中方剂挖掘结果前三位分别为六味地黄丸、补阳还五汤、真武汤,网络分析结果金匮肾气丸、六味地黄丸处于核心位置。单位中药挖掘结果前三位为黄芪、丹参、地黄,网络分析结果发现黄芪仍然处于用药的核心位置。2、临床观察研究共纳入2015年3月至2016年1月在广州中医药大学附属深圳市中医院门诊及住院病例共80例,并纳入在体检门诊的21例健康人群作为对照组。所有纳入病例分为正常组(N组)21例、糖尿病组(DM组)19例、糖尿病肾病1-2期组(DKD1-2组)19例、糖尿病肾病3期组(DKD3组)20例、糖尿病肾病4期组(DKD4组)21例、糖尿病肾病5期组(DKD5组)21例。各组病例在年龄、性别、体重等基础资料上差异无统计学意义。但BMI、血压、血糖及糖化血红蛋白指数方面N组明显低于DN各组。肾功方面,SCr由N组至DN5组患者逐渐升高,BUN含量DN4、5组含量最高,GFR水平在DKD早期升高,后期逐渐下降,末期时将至最低。脂代谢方面除HDL外,各组结果较为一致,N组的HDL水平明显高于其他各组。CXCL12检测结果,N组与DKD5组含量最高,DM组含量最低,且与N组差异具有统计学意义。MCP-1在DKD4、5组含量最高,且与N组比较差异具有统计学意义。TGF-β1检测结果显示DKD各组均高于N组。CRP记过显示,N组与DKD5组含量最低,而其他DKD各组与N组差异均具有统计学意义。3、动物实验动物实验将db/db小鼠随机分为模型组和治疗组,每组8只,治疗组采用黄芪甲苷给药,持续12周。8只db/m小鼠作为对照组。模型组与治疗组体重明显高于正常组,且模型组体重增加快于治疗组。实验结束时,模型组血压低于正常组,而治疗组血压明显升高,且明显高于模型组,其差异具有统计学意义。治疗组心率降低,与模型组相比其差异具有统计学意义。模型组、治疗组小鼠左、右肾重高于正常值,模型组与正常组差异具有统计学意义,而与治疗组相比差异无统计学意义。在血糖及糖化血红蛋白方面,模型组与治疗组都远高于正常组,但治疗组与模型组差异无统计学意义。小鼠UAE方面,模型组、治疗组均高于正常组。治疗组尿白蛋白含量一直低于模型组,且在第8、12周时与模型组相比,两者差异具有统计学意义。生化指标方面,除SCr、TG、LDL外,其余各项指标模型组均高于正常组,且差异具有统计学意义。而治疗组除TC、HDL外,其余各项结果与模型组相比,差异均具有统计学意义。病理改变方面,模型组小鼠肾小球、肾小管肥大,FPW增加、GBM和TBM增厚,与正常组相比其差异均具有统计学意义。治疗组可减低肾小球、肾小管面积,抑制GBM、 TBM增厚。足突宽度方面,模型组最高,与正常组比较,远高于正常组。治疗组低于模型组,与模型组差异有统计学意义。通路蛋白方面,p-Akt结果显示模型组与正常组、治疗组比较其差异具有统计学意义。p-NF-k B p65的表达结果与p-Akt一致。ELISA方面,db/db模型组尿中TGF-β1表达远高于正常组,其与治疗组差异具有统计学意义。小鼠肾脏TGF-β1治疗组远低于正常组和模型组,且差异具有统计学意义。NGAL结果显示,模型组小鼠中含量最高,治疗组低于模型组,其差异具有统计学意义。KIM-1结果模型组高于治疗组,差异具有统计学意义。结论：1、中药黄芪是中医治疗DKD的核心用药之一；2、DKD患者的发病与代谢紊乱、血流动力学异常等关系密切。气阴两虚挟瘀型DKD患者血清中炎症因子表达增加且增加程度与疾病发展进程有关；3、黄芪的单体提取物黄芪甲苷对2型糖尿病db/db小鼠肾脏具有保护作用。黄芪甲苷可能通过抑制db/db小鼠肾内Akt/NF-k B活化,减轻肾脏损伤,降低db/db小鼠的尿白蛋白,起到肾脏保护作用。
[Abstract]:Objective: 1, to explore the characteristics of traditional Chinese medicine in the treatment of diabetic nephropathy; (2) to observe the expression of serum CXCL12 and other inflammatory factors in diabetic nephropathy patients with Qi Yin deficiency and stasis syndrome; 3, observe the renal protective effect of Astragaloside on db/db mice of type 2 diabetes. Method: 1, the literature study used the method of text mining to explore the treatment of traditional Chinese medicine. The characteristics of the drug use of diabetic nephropathy. Through the reference to the Chinese Dictionary of Chinese medicine (Second Edition), the People's Republic of China Pharmacopoeia (supplement Edition) and the national medicine colleges and universities, the Second Edition (Chinese pharmacy > (Second Edition), set up a thesaurus library. Logon to the Chinese CNKI, the theoretical study on the treatment of diabetic nephropathy by Chinese medicine, the summary of experience and the related literature of clinical trials. The search is searched. After screening and converting the searched documents according to the program, the database is set up. The key words in the related literature are extracted and preserved by the word list as the key words. Cytoscape software is used to analyze the extracted key words and summarize the characteristics of the medicine.2. The clinical observations refer to the related literature. Screening of diabetic nephropathy patients from March 2015 to January 2016 at the Shenzhen Chinese Traditional Medical Hospital affiliated to Guangzhou University of Chinese Medicine and hospitalized in the affiliated Shenzhen Chinese Traditional Medical Hospital of Guangzhou University of Chinese Medicine. The observation was carried out in the patients with deficiency of Qi Yin deficiency and stasis type diabetic nephropathy. The contents included general data, biochemical index level and related inflammation. The expression of factor.3, based on the results of the above two studies, using Astragalus membranaceus glycoside of Astragalus membranaceus to treat type 2 diabetic db/db mice. During the period of drug use, the metabolic index, blood glucose and blood pressure of mice were measured by the metabolic cage, blood glucose meter and animal noninvasive blood pressure meter. After 12 weeks, the mice were killed and the blood and urine were left. A full automatic biochemical analyzer was used to detect the renal function of mice, serum albumin and other biochemical indexes. The urinary albumin excretion rate and renal tubule injury index were detected by ELISA. The pathological and ultrastructural pathological damage of glomeruli and renal tubules were detected by transmission electron microscopy with PAS stain, and P-Akt and P in kidney were detected by Western Blot The expression of -NF-k B protein. Results: 1. The literature study was made by formulating the retrieval formula and retrieving the related literature of CNKI diabetic nephropathy. A total of 3778 articles related to the theoretical study of diabetic nephropathy, experience summary and clinical trials were obtained. According to the screening criteria, 58 articles were obtained from the theoretical study of target literature and 264 articles were summarized by experience. 618 articles in clinical trial. The key key one-dimensional frequency and two dimensional frequency results were obtained after the conversion of these documents. The results were analyzed by Cytoscape software. In the theoretical study literature, the traditional Chinese medicine prescription in the first three places was used as kidney qi pill, Shengmai Powder and Jingui kidney qi pill. The results of the network analysis showed that Astragalus membranaceus and Salvia miltiorrhiza were in the core position of the whole drug use in the theoretical study, and the best use of Chinese medicine in the first three was Zhenwu soup, six flavour rehmannia pill, five Ling powder, and network analysis results found Zhenwu soup. Almost all the other prescriptions in the first ten places are connected, while Jisheng Shenqi Pill, Jinkui kidney qi pill, six flavored rehmannia pill, and the other prescription are relatively independent. The first three of the results of single Chinese medicine mining are Astragalus membranaceus, Salvia miltiorrhiza, and Rehmannia glutinosa, and Rehmannia glutinosa is located at the center of the drug application. The first three places were six flavors of rehmannia pill, Buyang return five soup, Zhenwu Decoction, zhenkui Shenqi Pill and six flavour Rehmannia Pill in the core position. The three place of the results of traditional Chinese medicine excavation were Astragalus membranaceus, Salvia miltiorrhiza and Rehmannia glutinosa. The results of network analysis found that Astragalus was still at the core of drug use,.2, and clinical observation and research included 201 A total of 80 cases of outpatient and hospitalized cases in Shenzhen Chinese Traditional Medical Hospital of Guangzhou University of Chinese Medicine from March to January 2016 were included, and 21 healthy people were included in the medical check-up clinic as control group. All the cases were divided into normal group (group N) 21 cases, diabetes group (Group DM) 19 cases, diabetic nephropathy 1-2 group (group DKD1-2) 19 cases, diabetic nephropathy 3 phase group (DKD3 There were 20 cases, 21 cases of diabetic nephropathy (DKD4) group (Group 4), 5 stage diabetic nephropathy group (group DKD5) 21 cases. There was no significant difference in age, sex, weight and other basic data in each group. But BMI, blood pressure, blood sugar and glycosylated hemoglobin index were significantly lower in group N than in DN. SCr from group N to DN5 group gradually increased, BUN contained The content of DN4,5 group was the highest, and the level of GFR increased in the early stage of DKD, gradually decreased at the later stage and was the lowest at the end of the stage. The result of lipid metabolism except HDL was the same. The HDL level in N group was significantly higher than that of other.CXCL12 tests. The content of N and DKD5 groups was the highest, and the DM group was the lowest, and the difference between the N group and the N group was statistically significant. The 5 groups had the highest content, and compared with the N group, the difference of.TGF- beta 1 showed that all the DKD groups were higher than the N group.CRP, and the N group and the DKD5 group were the lowest, while the other DKD groups and the N group had statistical significance.3. The animal experimental animal experiment randomly divided the db/db rats into the model group and the treatment group, with 8 rats in each group. The group was administered with astragaloside, and only db/m mice were used as control group for 12 weeks. The weight of the model group and the treatment group was significantly higher than that of the normal group, and the weight of the model group was increased faster than the treatment group. The blood pressure of the model group was lower than the normal group at the end of the experiment. The blood pressure of the model group was significantly higher than that in the treatment group, and the treatment group was significantly higher than the model group, and the difference was statistically significant. The treatment group was significantly higher than the model group. Compared with the model group, the left and right kidney weight of the model group was higher than that of the normal group. The difference between the model group and the normal group was statistically significant, but there was no significant difference between the model group and the normal group. In the blood sugar and glycated hemoglobin, the model group and the treatment group were far higher than the normal group, but the model group and the treatment group were far higher than the normal group. There was no significant difference between the treatment group and the model group. The UAE, the model group and the treatment group were all higher than the normal group. The urine albumin content in the treatment group was lower than the model group, and the difference was statistically significant compared with the model group at week 8,12. The biochemical indexes, except SCr, TG and LDL, were all higher than the normal group. The difference was statistically significant in the treatment group except TC and HDL, and the other results were statistically significant compared with the model group. In the pathological changes, the glomerulus, renal tubule hypertrophy, FPW increased, GBM and TBM thickening in the model group, and the difference was statistically significant compared with the normal group. The treatment group could reduce the glomerular and kidney. The area of canaliculus, inhibition of GBM, TBM thickening. The width of the foot process was the highest in the model group. Compared with the normal group, it was far higher than the normal group. The treatment group was lower than the model group. The difference between the model group and the model group was statistically significant. The p-Akt results showed that the model group was compared with the normal group, and the treatment group had a statistically significant difference in.P-NF-k B p65 expression. The TGF- beta 1 expression in db/db model group was much higher than that of the normal group, and the difference of TGF- beta 1 in the model group was significantly higher than that in the treatment group. The TGF- beta 1 group in the mice kidney was far lower than the normal group and the model group, and the difference was statistically significant.NGAL results showed that the content of the model group was the highest, the treatment group was lower than the model group, and the difference between the model group and the model group was lower. The statistical significance.KIM-1 result model group is higher than the treatment group, the difference has statistical significance. Conclusion: 1, Astragalus membranaceus is one of the core drugs for the treatment of DKD in traditional Chinese medicine; 2, the incidence of DKD patients is closely related to metabolic disorders and hemodynamic abnormalities. The expression of inflammatory factors in serum of patients with Qi Yin deficiency and blood stasis type DKD increases and increases. 3, Astragalus membranaceus, the single extract of Astragalus membranaceus, has protective effect on the kidney of type 2 diabetic db/db mice. Astragaloside may inhibit the activation of Akt/NF-k B in the kidney of db/db mice, reduce renal damage and reduce the urinary albumin in db/db mice, and play the role of renal protection.
【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】：2016
【分类号】：R259

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