基于氨基酸代谢及网络药理探讨益肾活血法抗糖尿病肾脏肥大的分子机制

Early diabetic nephropathy is characterized by hypertrophy of both glomerular and tubular elements. Amino acids are closely related with diabetic renal hypertrophy. However, earlier studies have not yet to identify the specific amino acids involved in mediating diabetic renal hypertrophy, and its underlying molecular mechanisms remain to be elucidated. From the TCM perspective, kidney deficiency and blood stasis are the pathological foundations of diabetic nephropathy. Hence, the corresponding therapeutic principles, kidney tonification and blood circulation promotion, are central concepts in TCM diabetic nephropathy treatment. Astragalus membranaceus and the earth worm are representative herbs used in kidney tonification and blood circulation promotion. Previous studies have shown that these herbs can decrease microalbuminuria, as well as reverse diabetic renal hypertrophic changes. Therefore, how these herbs can effect anti-hypertrophic changes, especially on the molecular level, is a topic of vast interest. Metabolomics, network pharmacology and bioinformatics are powerful modalities that can systematically explore the intricate mechanisms between Chinese herbs and regulatory functions of amino acids in diabetic renal hypertrophy. In our previous studies, we have demonstrated lager kidneys in diabetic rat/mouse as compared to control group. Clinical investigation on serum amino acids and amino acid end products concentrations have also revealed marked elevations in diabetic nephropathy patients. In our network pharmacology study, we identified multiple target proteins of Astragaloside IV and lumbrokinase, can affect in amino acid metabolism and cellular hypertrophy regulation. In our animal model study, reversed diabetic renal hypertrophy was detected when treated with Astragaloside IV and lumbrokinase. We thereby propose this study to illuminate the specific amino acids, including their relevant signaling pathways and molecular targets, which participate in diabetic renal hypertrophy. We also aim to investigate the role of the pivotal amino acid and its relevant pathway in mediating Astragaloside Ⅳ and lumbrokinase treatment of diabetic nephropathy.

肾脏肥大是糖尿病肾病（diabetic nephropathy,DN）早期形态学特征表现。氨基酸与糖尿病肾脏肥大密切相关。然而，具体哪些氨基酸参与DN早期肾脏肥大，通路/靶点如何，目前尚不清楚。中医辨治消渴肾病以肾虚血瘀立论，益肾活血为法，代表中药黄芪、地龙可降低蛋白尿，保护肾脏，但机制尚需深入研究。代谢组学、网络药理和生物信息分析技术为系统评估中药及氨基酸调控糖尿病肾脏肥大机制研究提供平台。本项目基于前期工作：糖尿病动物模型早期存在明显肾脏肥大；DN患者血清氨基酸代谢水平异常；黄芪甲苷和蚓激酶有诸多调控氨基酸代谢、细胞肥大的药物靶点；黄芪甲苷和蚓激酶可减少糖尿病肾小球和肾小管肥大，改善肾脏损伤。本研究拟应用代谢组学，网络药理和生物信息分析及体内外实验，旨在明确：哪些氨基酸通过哪些通路/靶点参与DN早期肾脏肥大；关键差异氨基酸及下游通路/靶点在黄芪甲苷和/或蚓激酶抗肾脏肥大中的作用。

肾脏肥大是引起肾脏结构不可逆改变的始动因素，氨基酸对糖尿病肾脏肥大有重要的调节作用。.通过临床人群及动物模型的代谢组学检测，筛选与疾病进展相关的差异氨基酸；借助网络药理技术，选定特定差异氨基酸，借助代谢检测技术探讨其深入介导肾脏肥大的可能机制以及明确黄芪甲苷抗糖尿病肾损伤是否依赖调控氨基酸代谢抗肾脏肥大实现。.综合人群及动物模型的代谢结果，本研究发现疾病早期缬氨酸、异亮氨酸、亮氨酸、脯氨酸显著上调，疾病进展中，色氨酸、犬尿氨酸，精氨酸、瓜氨酸，蛋氨酸，苯丙氨酸、酪氨酸随病情进展密切相关，色氨酸的糖基化产物2-(α-D-Mannopyranosyl) -L-tryptophan与肾病进展密切相关，并且可以较肌酐更早预测肾功能进展，兼顾反应ACR异常。该指标与GFR的相关系数可达0.918，其每上调表达1倍SD，ACR进展的风险达2.5倍，GFR和ACR同时进展的风险达6.16倍。我们的研究还发现人群的代谢特征较动物模型的代谢特征更丰富，且与氨基酸代谢相关的核酸代谢物异常在糖尿病肾病进展过程中显著变化。通过构建上述氨基酸及黄芪甲苷药靶分子网络，对共有核心基因靶点富集分析提示，我们选取了色氨酸/犬尿氨酸代谢通路，氧化还原反应失衡，SOD2， catalasse，Akt mTOR通路探究色氨酸代谢通路参与肾脏肥大损伤的分子机制，发现色氨酸/犬尿氨酸代谢通过氧化还原反应失衡，调节Akt/mTOR通路参与肾脏肥大损伤。黄芪甲苷可以通过逆转mTOR上调，改善氧化还原反应失衡，调节机体氨基酸稳态失衡，抵抗糖尿病肾损伤发生。.该研究从氨基酸代谢角度探究了糖尿病肾脏损伤的机制，并尝试整合代谢与分子生物学技术，探究代谢物、分子蛋白相互作用，明确氨基酸，尤其是色氨酸下游代谢物，在糖尿病肾损伤中的作用机制，以及中药黄芪甲苷的疗效机制评估，为后续中药抗糖尿病肾损伤提供依据。研究还筛选出可以预测临床人群疾病进展的生物标志物，该指标可以兼顾反应GFR和ACR的进展情况。目前，临床上还没有可以兼顾以上两方面综合评估预测DKD病情的指标。该指标对DKD早期筛查及时治疗有很好的临床使用意义。