本文選題：丹參莖葉 + 丹參��； 參考：《南京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：一、文獻(xiàn)研究較為系統(tǒng)地總結(jié)和歸納了國內(nèi)外關(guān)于慢性腎功能損傷在中西醫(yī)上的發(fā)病機(jī)制、診斷標(biāo)準(zhǔn)及防治手段等研究方面取得的進(jìn)展,以期為慢性腎功能損傷的深入認(rèn)識(shí)及臨床治療提供一定的線索。同時(shí)歸納和總結(jié)了丹參中主要活性成分,并分析了國內(nèi)外學(xué)者近年來在丹參用于慢性腎功能損傷的藥理學(xué)作用與機(jī)制及其在慢性腎功能損傷的臨床應(yīng)用等方面取得的研究進(jìn)展,以期為丹參治療慢性腎功能損傷作用機(jī)制的深入研究提供參考,為丹參資源的綜合利用與開發(fā)提供依據(jù)。二、丹參莖葉及丹參提取制備與成分分析對(duì)回流、冷浸、超聲的提取方法及提取溶劑進(jìn)行考察,發(fā)現(xiàn)采用回流提取法時(shí),丹參提取液中總丹酚酸、總丹參酮及丹參莖葉提取液中總丹酚酸、總黃酮含量最高。提取溶劑為80%乙醇時(shí),丹參醇提取液中總丹參酮含量及丹參莖葉提取液中總黃酮含量最高。采用水提醇沉法制備的丹參水提物中總酚酸含量為16.74%;水提醇沉法制備的丹參莖葉水提物中總酚酸含量為15.26%,蘆丁與異槲皮苷總含量為0.41%。丹參80%乙醇提取物中總酚酸含量為11.62%,總丹參酮含量為3.79%;丹參莖葉80%乙醇提取物中總酚酸含量為11.21%,蘆丁與異槲皮苷總含量為1.21%。三、丹參莖葉對(duì)糖尿病腎病大鼠的腎功能改善作用及機(jī)制研究1、采用高糖高脂飼料伴隨5%葡萄糖水喂養(yǎng),加小劑量STZ腹腔注射建立糖尿病大鼠模型,以丹參莖葉及丹參提取物進(jìn)行干預(yù),待模型組大鼠出現(xiàn)蛋白尿時(shí),收集血清、尿液、腎組織等生物樣品,通過測定生化指標(biāo)、分析病理切片及測定腎組織中相關(guān)蛋白的表達(dá)等評(píng)價(jià)丹參莖葉及丹參對(duì)糖尿病腎病模型大鼠的保護(hù)作用。結(jié)果發(fā)現(xiàn),模型組大鼠空腹血糖可達(dá)29 mmol·L-1,且出現(xiàn)明顯蛋白尿、腎小管損傷及腎小球基底膜略微增厚,給藥組大鼠空腹血糖明顯下降,腎組織病理結(jié)構(gòu)趨向正常組,與模型組存在顯著性差異。說明丹參莖葉與丹參能改善糖尿病癥狀,且可有效預(yù)防其繼發(fā)性腎損傷,可能是通過抑制腎組織中wnt4、β-catenin、TGF-β等相關(guān)蛋白的表達(dá)來實(shí)現(xiàn)。2、采用代謝組學(xué)分析方法,借助UPLC-QTOF/MS技術(shù)并結(jié)合多變量統(tǒng)計(jì)學(xué)分析,探討糖尿病腎病大鼠血清、尿液、腎組織的代謝輪廓特征,并對(duì)丹參莖葉及丹參提取物干預(yù)后的生物效應(yīng)與作用機(jī)制進(jìn)行研究。結(jié)果表明,在血清、尿液、腎組織中分別發(fā)現(xiàn)并鑒定了 21個(gè)、16個(gè)、22個(gè)潛在生物標(biāo)志物,涉及磷脂代謝、花生四烯酸代謝、嘧啶代謝紊亂;正常組、模型組、丹參莖葉及丹參的提取物給藥組在PLS-DA圖上區(qū)分明顯,給藥組均向正常狀態(tài)轉(zhuǎn)歸。3、采用16SrRNA技術(shù)研究正常大鼠、糖尿病腎病大鼠、丹參水提物與醇提物、丹參莖葉水提物與醇提物給藥組大鼠糞便中菌群的多樣性,并采用LEfSe分析尋找差異菌群,并利用PICRUSt軟件對(duì)腸道菌群的生物學(xué)功能進(jìn)行預(yù)測。結(jié)果發(fā)現(xiàn),糖尿病腎病大鼠糞便中腸道菌群的豐富度及多樣性較正常組低,各給藥組大鼠腸道菌群生物多樣性接近正常組。在科及屬分類的菌群中尋找到的差異菌群包括雙歧桿菌屬、傳代菌屬、消化鏈球菌科、脫硫弧菌屬、SMB53、乳酸桿菌屬、梭菌屬、理研菌科、瘤胃菌科。大鼠糞便中腸道菌群涉及的代謝通路有43條,主要包括膜轉(zhuǎn)運(yùn)、氨基酸代謝、核苷酸代謝、輔酶和維生素的代謝、碳水化合物代謝、能量代謝、基因復(fù)制與修復(fù)、基因轉(zhuǎn)錄與翻譯等。4、用高糖建立腎小球系膜細(xì)胞損傷模型,以丹參莖葉及丹參水提物與醇提物、丹酚酸B、丹參酮IIA進(jìn)行干預(yù),利用Weatern blotting檢測各細(xì)胞中Wnt-4、β-catenin、TGF-β的表達(dá)量,發(fā)現(xiàn)丹參莖葉及丹參均能顯著抑制細(xì)胞中該蛋白的表達(dá)。四、丹參莖葉對(duì)慢性腎衰竭大鼠的腎功能改善作用及機(jī)制研究1、以腺嘌呤建立慢性腎衰竭模型,不同劑量丹參莖葉及丹參提取物進(jìn)行干預(yù)后,發(fā)現(xiàn)丹參莖葉及丹參提取物均能降低慢性腎衰竭大鼠體內(nèi)血清肌苷、血清尿素氮、尿蛋白的含量,抑制腎組織中α-SMA、FN、NOX1、NOX2、NOX4、p-ERK、TGF-β的表達(dá),改善腎功能。2、采用代謝組學(xué)分析方法,借助UPLC-QTOF/MS技術(shù)并結(jié)合多變量統(tǒng)計(jì)學(xué)分析,探討慢性腎衰竭大鼠血清、尿液、腎組織的代謝輪廓特征,并對(duì)丹參提取物干預(yù)后的生物效應(yīng)與作用機(jī)制進(jìn)行研究。結(jié)果表明,在血清、尿液、腎組織中分別發(fā)現(xiàn)并鑒定了 13個(gè)、27個(gè)、19個(gè)潛在生物標(biāo)志物,其涉及的代謝通路包括色氨酸代謝、嘌呤代謝、嘧啶代謝、苯丙氨酸代謝。丹參水提物及丹參醇提物能顯著調(diào)控模型大鼠體內(nèi)的代謝紊亂。3、采用16SrRNA技術(shù)研究腺嘌呤誘導(dǎo)的慢性腎衰竭大鼠糞便中菌群的多樣性,采用LEfSe分析在科與屬分類水平上發(fā)現(xiàn)了多個(gè)差異菌群,包括Mucispirillum、Kurthia、Clostridium、Blautia、Butyrivibrio、Shuttleworthia、Peptococcus、Ruminococcus、Bradyrhizobium、Methylobacterium、Azospirillum、Thalassospira、Methylophilus、Pseudomonas、Peptostreptococcaceae、S24-7等。丹參水提物與醇提物能在一定程度上改善腺嘌呤誘導(dǎo)的慢性腎衰竭大鼠糞便菌群多樣性。其中,丹參醇提物可顯著調(diào)節(jié)Clostridium、Pseudomonas及Peptostreptococcaceae的相對(duì)豐度,丹參水提物可顯著調(diào)節(jié)Peptococcus 及 Peptostreptococcaceae。4、通過活化正常大鼠與慢性腎衰竭大鼠糞便中的菌群,將其與丹參中效應(yīng)成分進(jìn)行共培養(yǎng),借助UPLC/Q-TOF/MS技術(shù)鑒定代謝產(chǎn)物,并分析代謝途徑。發(fā)現(xiàn)正常與模型大鼠腸道菌群對(duì)丹參中丹參酮ⅡA、隱丹參酮、丹參酮Ⅰ、二氫丹參酮Ⅰ的代謝,以甲基化、脫甲基化、氧化、加氫化和羥基化等代謝途徑為主,且4種丹參酮在腸道菌群的作用下可以相互轉(zhuǎn)化;對(duì)丹酚酸B、丹酚酸C、迷迭香酸的代謝途徑以葡糖醛酸化、甲基化及加氫化等代謝途徑為主。5、建立硫酸吲哚酚誘導(dǎo)的腎小管上皮細(xì)胞(HK-2)損傷模型,以丹參莖葉及丹參水提物與醇提物進(jìn)行干預(yù),利用Weatern blotting檢測各細(xì)胞中相關(guān)蛋白的表達(dá)量,發(fā)現(xiàn)丹參莖葉及丹參能顯著抑制細(xì)胞中α-SMA、Fibronectin、p-ERK、NOX1、NOX2、NOX4、TGF-β、TGF-βRⅠ、TGF-βRⅡ、Smad2、Smad3 的相對(duì)表達(dá),從而對(duì) HK-2 細(xì)胞NADPH/ROS/ERK及TGF-β/smad信號(hào)通路起調(diào)控作用。五、丹參多組分對(duì)腎功能損傷的作用靶點(diǎn)-信號(hào)通路網(wǎng)絡(luò)分子機(jī)制以丹參中10個(gè)化合物為研究對(duì)象,運(yùn)用網(wǎng)絡(luò)藥理學(xué)研究方法,分析丹參多成分-多靶點(diǎn)-多途徑之間的相互關(guān)系,為進(jìn)一步明確丹參治療慢性腎功能損傷的分子作用機(jī)制提供依據(jù)。發(fā)現(xiàn)結(jié)果表明,丹參中主要酚酸類成分與丹參酮類成分涉及的靶點(diǎn)有195個(gè),信號(hào)通路有108條,如嘌呤代謝、嘧啶代謝、苯丙氨酸代謝、TGF-β信號(hào)通路、Wnt信號(hào)通路、色氨酸代謝、花生四烯酸代謝等。其中與腎功能損傷疾病直接相關(guān)的信號(hào)通路有15條,如TGF-β信號(hào)通路、Wnt信號(hào)通路、嘌呤代謝等,與實(shí)驗(yàn)研究結(jié)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.

【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R284;R285.5

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3 劉s，

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