丹参茎叶对慢性肾功能损伤的改善作用及机制研究

发布时间：2018-04-28 15:38

本文选题：丹参茎叶 + 丹参　；参考：《南京中医药大学》2017年硕士论文

【摘要】：一、文献研究较为系统地总结和归纳了国内外关于慢性肾功能损伤在中西医上的发病机制、诊断标准及防治手段等研究方面取得的进展,以期为慢性肾功能损伤的深入认识及临床治疗提供一定的线索。同时归纳和总结了丹参中主要活性成分,并分析了国内外学者近年来在丹参用于慢性肾功能损伤的药理学作用与机制及其在慢性肾功能损伤的临床应用等方面取得的研究进展,以期为丹参治疗慢性肾功能损伤作用机制的深入研究提供参考,为丹参资源的综合利用与开发提供依据。二、丹参茎叶及丹参提取制备与成分分析对回流、冷浸、超声的提取方法及提取溶剂进行考察,发现采用回流提取法时,丹参提取液中总丹酚酸、总丹参酮及丹参茎叶提取液中总丹酚酸、总黄酮含量最高。提取溶剂为80%乙醇时,丹参醇提取液中总丹参酮含量及丹参茎叶提取液中总黄酮含量最高。采用水提醇沉法制备的丹参水提物中总酚酸含量为16.74%;水提醇沉法制备的丹参茎叶水提物中总酚酸含量为15.26%,芦丁与异槲皮苷总含量为0.41%。丹参80%乙醇提取物中总酚酸含量为11.62%,总丹参酮含量为3.79%;丹参茎叶80%乙醇提取物中总酚酸含量为11.21%,芦丁与异槲皮苷总含量为1.21%。三、丹参茎叶对糖尿病肾病大鼠的肾功能改善作用及机制研究1、采用高糖高脂饲料伴随5%葡萄糖水喂养,加小剂量STZ腹腔注射建立糖尿病大鼠模型,以丹参茎叶及丹参提取物进行干预,待模型组大鼠出现蛋白尿时,收集血清、尿液、肾组织等生物样品,通过测定生化指标、分析病理切片及测定肾组织中相关蛋白的表达等评价丹参茎叶及丹参对糖尿病肾病模型大鼠的保护作用。结果发现,模型组大鼠空腹血糖可达29 mmol·L-1,且出现明显蛋白尿、肾小管损伤及肾小球基底膜略微增厚,给药组大鼠空腹血糖明显下降,肾组织病理结构趋向正常组,与模型组存在显著性差异。说明丹参茎叶与丹参能改善糖尿病症状,且可有效预防其继发性肾损伤,可能是通过抑制肾组织中wnt4、β-catenin、TGF-β等相关蛋白的表达来实现。2、采用代谢组学分析方法,借助UPLC-QTOF/MS技术并结合多变量统计学分析,探讨糖尿病肾病大鼠血清、尿液、肾组织的代谢轮廓特征,并对丹参茎叶及丹参提取物干预后的生物效应与作用机制进行研究。结果表明,在血清、尿液、肾组织中分别发现并鉴定了 21个、16个、22个潜在生物标志物,涉及磷脂代谢、花生四烯酸代谢、嘧啶代谢紊乱;正常组、模型组、丹参茎叶及丹参的提取物给药组在PLS-DA图上区分明显,给药组均向正常状态转归。3、采用16SrRNA技术研究正常大鼠、糖尿病肾病大鼠、丹参水提物与醇提物、丹参茎叶水提物与醇提物给药组大鼠粪便中菌群的多样性,并采用LEfSe分析寻找差异菌群,并利用PICRUSt软件对肠道菌群的生物学功能进行预测。结果发现,糖尿病肾病大鼠粪便中肠道菌群的丰富度及多样性较正常组低,各给药组大鼠肠道菌群生物多样性接近正常组。在科及属分类的菌群中寻找到的差异菌群包括双歧杆菌属、传代菌属、消化链球菌科、脱硫弧菌属、SMB53、乳酸杆菌属、梭菌属、理研菌科、瘤胃菌科。大鼠粪便中肠道菌群涉及的代谢通路有43条,主要包括膜转运、氨基酸代谢、核苷酸代谢、辅酶和维生素的代谢、碳水化合物代谢、能量代谢、基因复制与修复、基因转录与翻译等。4、用高糖建立肾小球系膜细胞损伤模型,以丹参茎叶及丹参水提物与醇提物、丹酚酸B、丹参酮IIA进行干预,利用Weatern blotting检测各细胞中Wnt-4、β-catenin、TGF-β的表达量,发现丹参茎叶及丹参均能显著抑制细胞中该蛋白的表达。四、丹参茎叶对慢性肾衰竭大鼠的肾功能改善作用及机制研究1、以腺嘌呤建立慢性肾衰竭模型,不同剂量丹参茎叶及丹参提取物进行干预后,发现丹参茎叶及丹参提取物均能降低慢性肾衰竭大鼠体内血清肌苷、血清尿素氮、尿蛋白的含量,抑制肾组织中α-SMA、FN、NOX1、NOX2、NOX4、p-ERK、TGF-β的表达,改善肾功能。2、采用代谢组学分析方法,借助UPLC-QTOF/MS技术并结合多变量统计学分析,探讨慢性肾衰竭大鼠血清、尿液、肾组织的代谢轮廓特征,并对丹参提取物干预后的生物效应与作用机制进行研究。结果表明,在血清、尿液、肾组织中分别发现并鉴定了 13个、27个、19个潜在生物标志物,其涉及的代谢通路包括色氨酸代谢、嘌呤代谢、嘧啶代谢、苯丙氨酸代谢。丹参水提物及丹参醇提物能显著调控模型大鼠体内的代谢紊乱。3、采用16SrRNA技术研究腺嘌呤诱导的慢性肾衰竭大鼠粪便中菌群的多样性,采用LEfSe分析在科与属分类水平上发现了多个差异菌群,包括Mucispirillum、Kurthia、Clostridium、Blautia、Butyrivibrio、Shuttleworthia、Peptococcus、Ruminococcus、Bradyrhizobium、Methylobacterium、Azospirillum、Thalassospira、Methylophilus、Pseudomonas、Peptostreptococcaceae、S24-7等。丹参水提物与醇提物能在一定程度上改善腺嘌呤诱导的慢性肾衰竭大鼠粪便菌群多样性。其中,丹参醇提物可显著调节Clostridium、Pseudomonas及Peptostreptococcaceae的相对丰度,丹参水提物可显著调节Peptococcus 及 Peptostreptococcaceae。4、通过活化正常大鼠与慢性肾衰竭大鼠粪便中的菌群,将其与丹参中效应成分进行共培养,借助UPLC/Q-TOF/MS技术鉴定代谢产物,并分析代谢途径。发现正常与模型大鼠肠道菌群对丹参中丹参酮ⅡA、隐丹参酮、丹参酮Ⅰ、二氢丹参酮Ⅰ的代谢,以甲基化、脱甲基化、氧化、加氢化和羟基化等代谢途径为主,且4种丹参酮在肠道菌群的作用下可以相互转化;对丹酚酸B、丹酚酸C、迷迭香酸的代谢途径以葡糖醛酸化、甲基化及加氢化等代谢途径为主。5、建立硫酸吲哚酚诱导的肾小管上皮细胞(HK-2)损伤模型,以丹参茎叶及丹参水提物与醇提物进行干预,利用Weatern blotting检测各细胞中相关蛋白的表达量,发现丹参茎叶及丹参能显著抑制细胞中α-SMA、Fibronectin、p-ERK、NOX1、NOX2、NOX4、TGF-β、TGF-βRⅠ、TGF-βRⅡ、Smad2、Smad3 的相对表达,从而对 HK-2 细胞NADPH/ROS/ERK及TGF-β/smad信号通路起调控作用。五、丹参多组分对肾功能损伤的作用靶点-信号通路网络分子机制以丹参中10个化合物为研究对象,运用网络药理学研究方法,分析丹参多成分-多靶点-多途径之间的相互关系,为进一步明确丹参治疗慢性肾功能损伤的分子作用机制提供依据。发现结果表明,丹参中主要酚酸类成分与丹参酮类成分涉及的靶点有195个,信号通路有108条,如嘌呤代谢、嘧啶代谢、苯丙氨酸代谢、TGF-β信号通路、Wnt信号通路、色氨酸代谢、花生四烯酸代谢等。其中与肾功能损伤疾病直接相关的信号通路有15条,如TGF-β信号通路、Wnt信号通路、嘌呤代谢等,与实验研究结果相一致。
[Abstract]:First, the literature research systematically summarizes and summarizes the progress of the research on the pathogenesis, the diagnostic criteria and the means of prevention and treatment of chronic renal impairment in Chinese and Western medicine, in order to provide some clues for the deep understanding and clinical treatment of chronic renal function injury, and summarize and summarize the main activities in the salvia miltiorrhiza. The pharmacological action and mechanism of Salvia miltiorrhiza in chronic renal function injury and its clinical application in chronic renal function injury have been analyzed in recent years, in order to provide reference for the in-depth study of the mechanism of Salvia miltiorrhiza in the treatment of chronic renal function injury, and the comprehensive utilization of Salvia miltiorrhiza resources Two, extraction preparation and composition analysis of Salvia miltiorrhiza and Salvia miltiorrhiza, the extraction methods of reflux, cold soaking, ultrasonic extraction and extraction solvent were investigated. It was found that the total salvianolic acid in Salvia miltiorrhiza extract, total tanshinone and Salvia miltiorrhiza extracted from Salvia miltiorrhiza extract was the highest. The content of total flavonoids was the highest. The extraction solvent was 80% B. The content of total Tanshinone in tanshinol extract and the total flavonoids in the extract of Salvia miltiorrhiza are the highest. The content of total phenolic acid in Salvia miltiorrhiza water extract by water extraction and alcohol precipitation method is 16.74%. The content of total phenolic acid in the water extract of Salvia miltiorrhiza by water extraction and alcohol precipitation method is 15.26%, and the total content of rutin and isoquercetin is 0.41%. Salvia miltiorrhiza 80% ethanol extraction The content of total phenolic acid in the extract was 11.62%, the content of total tanshinone was 3.79%, the content of total phenolic acid in the 80% ethanol extract of Salvia miltiorrhiza was 11.21%. The total content of rutin and isoquercetin was 1.21%. three. The effect and mechanism of the stem and leaf of Salvia miltiorrhiza on the renal function of diabetic nephropathy rats were studied 1, with high fat diet with 5% glucose water and small dosage. The diabetic rat model was established by intraperitoneal injection of STZ, and the stem leaves of Salvia miltiorrhiza and the extract of Salvia miltiorrhiza were intervened. When the rats in the model group appeared proteinuria, the biological samples were collected, such as serum, urine and kidney tissue. By measuring biochemical indexes, analyzing the pathological sections and determining the expression of the phase proteins in the kidney tissue, the salvia miltiorrhiza leaves and Salvia miltiorrhiza were evaluated for diabetes. The results showed that the fasting blood glucose of the rats in the model group could reach 29 mmol. L-1, with obvious proteinuria, renal tubule injury and a slight thickening of the glomerular basement membrane. The fasting blood glucose of the rats in the administration group decreased obviously. The pathological structure of the kidney tissue tended to be in the normal group, and there was a significant difference between the model group and the model group. And Salvia miltiorrhiza can improve the symptoms of diabetes and effectively prevent secondary renal injury. It is possible to realize.2 by inhibiting the expression of Wnt4, beta -catenin, TGF- beta and other related proteins in renal tissue. By metabolic analysis, the serum, urine and kidney of diabetic nephropathy rats were studied with the help of UPLC-QTOF/MS technique combined with multivariable analysis. The biological effects and mechanisms of the stem and leaf of Salvia miltiorrhiza and Salvia miltiorrhiza extract were studied. The results showed that 21, 16, 22 potential biomarkers were identified and identified in serum, urine and kidney tissue, involving the metabolism of phospholipids, the metabolism of peanut four enoic acid, the disorder of pyrimidine metabolism, and the normal group, model. Group, the extracts of Salvia miltiorrhiza and Salvia miltiorrhiza were distinguished on the PLS-DA map, and all the drug groups were transferred to.3 in normal state. The diversity of the normal rats, diabetic nephropathy rats, Salvia miltiorrhiza water extract and alcohol extract, the water extract of Salvia miltiorrhiza and alcohol extract in the feces of rats were studied, and LEfSe analysis was used. The biological function of intestinal flora was predicted by PICRUSt software. The results showed that the abundance and diversity of intestinal microflora in the feces of diabetic nephropathy rats were lower than those of the normal group. The biological diversity of intestinal flora in each group was close to the normal group. Including the genus Bifidobacterium, genera, Streptococcus, SMB53, Lactobacillus, Clostridium, lactidaceae, and rumen. The metabolic pathways involved in the intestinal flora of rats are 43, mainly including membrane transport, amino acid metabolism, nucleotides metabolism, coenzyme and vitamin metabolism, carbohydrate metabolism, energy metabolism, Gene replication and repair, gene transcription and translation, and so on.4, use high sugar to establish mesangial cell damage model, using Salvia miltiorrhiza and Salvia miltiorrhiza water extract and alcohol extract, salvianolic acid B, tanshinone IIA, and Weatern blotting to detect the expression of Wnt-4, beta -catenin, and TGF- beta in each cell, and found that Salvia miltiorrhiza stem and leaf and Salvia miltiorrhiza can be significantly inhibited. The expression of this protein in the cells. Four, the effect and mechanism of the stem and leaf of Salvia miltiorrhiza on the renal function of rats with chronic renal failure. 1, the model of chronic renal failure was established with adenine, and the stem and leaf of Salvia miltiorrhiza and the extract of Salvia miltiorrhiza were used for the treatment. The results showed that the stem and leaf of Salvia miltiorrhiza and the extract of Salvia miltiorrhiza could reduce the serum inosine in the rats with chronic renal failure. The content of serum urea nitrogen and urine protein inhibit the expression of alpha -SMA, FN, NOX1, NOX2, NOX4, p-ERK, TGF- beta in renal tissue, improve the renal function.2, use the metabolic analysis method, with the help of UPLC-QTOF/MS technique combined with multivariate statistical analysis, to explore the metabolic profile of serum, urine and kidney tissue in rats with chronic renal failure and to extract Salvia miltiorrhiza. The results showed that 13, 27 and 19 potential biomarkers were identified and identified in serum, urine, and kidney tissue, including tryptophan metabolism, purine metabolism, pyrimidine metabolism, phenylalanine metabolism, and Salvia miltiorrhiza extract and Salvia alcohol extract. The metabolic disorder of the model rats was controlled by.3, and the diversity of the bacteria in the feces of rats with chronic renal failure induced by adenine was studied by 16SrRNA technique. A number of different groups were found on the level of family and genera by LEfSe analysis, including Mucispirillum, Kurthia, Clostridium, Blautia, Butyrivibrio, Shuttleworthia, Peptococcus, Ruminoco. CCUs, Bradyrhizobium, Methylobacterium, Azospirillum, Thalassospira, Methylophilus, Pseudomonas, Peptostreptococcaceae, S24-7 and so on. Salvia miltiorrhiza water extract and alcohol extract can improve the diversity of fecal flora of rats induced by adenine induced chronic renal failure to a certain extent. In this case, the alcohol extract of Salvia miltiorrhiza can significantly regulate Clostridium, Pseudomonas and The relative abundance of Peptostreptococcaceae, Salvia miltiorrhiza water extract can significantly regulate the Peptococcus and Peptostreptococcaceae.4, through the activation of normal rats and the feces of chronic renal failure rats, the effect components of Salvia miltiorrhiza were co cultured, the metabolic products were determined with the help of UPLC/Q-TOF/MS technology and the metabolic pathway was analyzed. The metabolism of tanshinone II A, cryptotanshinone, tanshinone I, two hydrogen tanshinone I in Salvia miltiorrhiza and the metabolic pathways of methylation, demethylation, oxidation, hydrogenation and hydroxylation, and the conversion of 4 Tanshinone in the intestinal flora, and the metabolic pathways of salvianolic acid B, salvianolic acid C, and rosemary acid in Salvia miltiorrhiza The damage model of renal tubular epithelial cell (HK-2) induced by indolonol sulfate was established with glucuronization, methylation and hydrogenation of.5, and the water extract of Salvia miltiorrhiza and Salvia miltiorrhiza and alcohol extract were intervened. The expression of the related egg white in the cells was detected by Weatern blotting. It was found that the stem and leaf of Salvia miltiorrhiza and Salvia miltiorrhiza could be significantly inhibited. Alpha -SMA, Fibronectin, p-ERK, NOX1, NOX2, NOX4, TGF- beta, TGF- beta R I, TGF- beta R II, Smad2, and beta signaling pathways play a regulatory role. Five, the target signal pathway network molecular mechanism of the multiple components of Salvia miltiorrhiza on renal function damage is studied by 10 compounds in Salvia miltiorrhiza In order to further clarify the molecular mechanism of the treatment of chronic renal damage by Salvia miltiorrhiza, the results show that there are 195 targets involved in the main phenolic acids in Salvia miltiorrhiza and the signal pathway of the Danshen ketone component. There are 108 items, such as purine metabolism, pyrimidine metabolism, phenylalanine metabolism, TGF- beta signaling pathway, Wnt signaling pathway, tryptophan metabolism, and peanut four enaconic acid metabolism. There are 15 signal pathways directly related to renal dysfunction, such as TGF- beta signaling pathway, Wnt signaling pathway, purine metabolism, and other experimental results.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285.5

【参考文献】