Chen Ruiqun, Liao Chengbin, Guo Qian, Wu Lirong, Zhang Lei, Wang Xiufeng
School of Basic Courses, Guangdong Pharmaceutical University Guangzhou 510006 P. R. China
Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University Guangzhou 510006 P. R. China.
RSC Adv. 2018 Aug 1;8(48):27448-27463. doi: 10.1039/c8ra04358b. eCollection 2018 Jul 30.
In our study, systems pharmacology was used to predict the molecular targets of and Leech, and explore the therapeutic mechanism of type 2 diabetic nephropathy (T2DN) treated with and Leech. Simultaneously, to reveal the systemic metabolic changes and biomarkers associated with T2DN, we performed H NMR-based metabonomics and multivariate analysis to analyze fecal samples obtained from model T2DN rats. In addition, ELISA kits and histopathological studies were used to examine biochemical parameters and kidney tissue, respectively. Striking differences in the Pearson's correlation of 22 biomarkers and 9 biochemical parameters were also observed among control, T2DN and treated rats. Results of systems pharmacology analysis revealed that 9 active compounds (3,9-di--methylnissolin; (6aR,11aR)-9,10-dimethoxy-6,11-dihydro-6-benzofurano[3,2-]chromen-3-ol; hirudin; l-isoleucine; phenylalanine; valine; hirudinoidine A-C) and 9 target proteins (l-serine dehydratase; 3-hydroxyacyl-CoA dehydrogenase; tyrosyl-tRNA synthetase; tryptophanyl-tRNA synthetase; branched-chain amino acid aminotransferase; acetyl-CoA C-acetyltransferase; isovaleryl-CoA dehydrogenase; pyruvate dehydrogenase E1 component alpha subunit; hydroxyacylglutathione hydrolase) of and Leech were closely associated with the treatment of T2DN. Using fecal metabonomics analysis, 22 biomarkers were eventually found to be closely associated with the occurrence of T2DN. Combined with systems pharmacology and fecal metabonomics, these biomarkers were found to be mainly associated with 6 pathways, involving amino acid metabolism (leucine, valine, isoleucine, alanine, lysine, glutamate, taurine, phenylalanine, tryptophan); energy metabolism (lactate, succinate, creatinine, α-glucose, glycerol); ketone body and fatty acid metabolism (3-hydroxybutyrate, acetate, -butyrate, propionate); methylamine metabolism (dimethylamine, trimethylamine); and secondary bile acid metabolism and urea cycle (deoxycholate, citrulline). The underlying mechanisms of action included protection of the liver and kidney, enhancement of insulin sensitivity and antioxidant activity, and improvement of mitochondrial function. To the best of our knowledge, this is the first time that systems pharmacology combined with fecal metabonomics has been used to study T2DN. 6 metabolites (-butyrate, deoxycholate, propionate, tryptophan, taurine and glycerol) associated with T2DN were newly discovered in fecal samples. These 6 metabolites were mainly derived from the intestinal flora, and related to amino acid metabolism, fatty acid metabolism, and secondary bile acid metabolism. We hope the results of this study could be inspirational and helpful for further exploration of T2DN treatment. Meanwhile, our results highlighted that exploring the biomarkers of T2DN and therapeutic mechanisms of Traditional Chinese Medicine (TCM) formulas on T2DN by combining systems pharmacology and fecal metabonomics methods was a promising strategy.
在我们的研究中，运用系统药理学来预测水蛭和地龙的分子靶点，并探究水蛭和地龙治疗2型糖尿病肾病（T2DN）的作用机制。同时，为揭示与T2DN相关的全身代谢变化和生物标志物，我们进行了基于核磁共振氢谱的代谢组学研究及多变量分析，以分析T2DN模型大鼠的粪便样本。此外，分别使用酶联免疫吸附测定试剂盒和组织病理学研究来检测生化参数和肾脏组织。在对照组、T2DN组和治疗组大鼠中，还观察到22种生物标志物与9种生化参数的皮尔逊相关性存在显著差异。系统药理学分析结果显示，水蛭和地龙的9种活性化合物（3,9 - 二 - 甲基尼索林；(6aR,11aR) - 9,10 - 二甲氧基 - 6,11 - 二氢 - 6 - 苯并呋喃[3,2 - ]色烯 - 3 - 醇；水蛭素；L - 异亮氨酸；苯丙氨酸；缬氨酸；类水蛭素A - C）和9种靶蛋白（L - 丝氨酸脱水酶；3 - 羟酰基辅酶A脱氢酶；酪氨酰 - tRNA合成酶；色氨酰 - tRNA合成酶；支链氨基酸转氨酶；乙酰辅酶A C - 乙酰转移酶；异戊酰辅酶A脱氢酶；丙酮酸脱氢酶E1组分α亚基；羟酰谷胱甘肽水解酶）与T2DN的治疗密切相关。通过粪便代谢组学分析，最终发现22种生物标志物与T2DN的发生密切相关。结合系统药理学和粪便代谢组学研究发现，这些生物标志物主要与6条通路相关，涉及氨基酸代谢（亮氨酸、缬氨酸、异亮氨酸、丙氨酸、赖氨酸、谷氨酸、牛磺酸、苯丙氨酸、色氨酸）；能量代谢（乳酸、琥珀酸、肌酐、α - 葡萄糖、甘油）；酮体和脂肪酸代谢（3 - 羟基丁酸、乙酸、丁酸、丙酸）；甲胺代谢（二甲胺、三甲胺）；以及次级胆汁酸代谢和尿素循环（脱氧胆酸盐、瓜氨酸）。其潜在作用机制包括保护肝肾、增强胰岛素敏感性和抗氧化活性以及改善线粒体功能。据我们所知，这是首次将系统药理学与粪便代谢组学结合用于研究T2DN。在粪便样本中新发现了6种与T2DN相关的代谢物（丁酸、脱氧胆酸盐、丙酸、色氨酸、牛磺酸和甘油）。这6种代谢物主要来源于肠道菌群，与氨基酸代谢、脂肪酸代谢和次级胆汁酸代谢有关。我们希望本研究结果能为进一步探索T2DN的治疗提供启发和帮助。同时，我们的研究结果表明，通过结合系统药理学和粪便代谢组学方法来探索T2DN的生物标志物及中药方剂对T2DN的治疗机制是一种很有前景的策略。
