Wu Bei, Shen Meng-Yuan, Chen Hong-Ping, Liu You-Ping, Wang Fu, Chen Lin
State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine Chengdu 611137, China.
Zhongguo Zhong Yao Za Zhi. 2022 Aug;47(15):4136-4147. doi: 10.19540/j.cnki.cjcmm.20220218.701.
The present study analyzed the effect of Citri Reticulatae Pericarpium on endogenous metabolites in spleen deficiency and phlegm dampness syndrome by metabolomics, and explored the underlying mechanism of Citri Reticulatae Pericarpium in the treatment of spleen deficiency and phlegm dampness syndrome.The model of spleen deficiency and phlegm dampness syndrome was induced in rats by the multi-factor modeling method.The intervention effects of Citri Reticulatae Pericarpium on rats with spleen deficiency and phlegm dampness syndrome were preliminarily evaluated by observing the pathological changes of rat liver tissues and measuring the plasma content of pathological and biochemical indexes such as triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C).Immunohistochemistry was used to detect the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland, and the effect of Citri Reticulatae Pericarpium on aquaporin expression in rats with spleen deficiency and phlegm dampness syndrome was evaluated.Furthermore, UHPLC-ESI-MS/MS was used to analyze the metabolic profiles of rat plasma samples.Multiple methods, such as principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were used for pattern recognition.Differential metabolites were screened out by t-test and variable importance in projection(VIP), followed by pathway analysis based on MetaboAnalyst 5.0.As revealed by experimental results, Citri Reticulatae Pericarpium could improve the pathological changes of liver tissues, increase the levels of HDL-C in the plasma, reduce the levels of TC, TG, and LDL-C, and enhance the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland of rats with spleen deficiency and phlegm dampness syndrome.In addition, 87 differential metabolites of spleen deficiency and phlegm dampness syndrome were screened out by UHPLC-ESI-MS/MS(the levels of 39 metabolites increased significantly and the levels of 48 metabolites decreased significantly), with the representatives of glycine, L-isoleucine, N-acetyl-L-tyrosine, xanthine, hypoxanthine, and trigonelline.The differential metabolites were mainly enriched in the pathways of steroid hormone biosynthesis, linoleic acid metabolism, and purine metabolism.This study distinguished and revealed the characteristic metabolic pattern of spleen deficiency and phlegm dampness syndrome by metabolomics.The preliminary construction of the OPLS-DA model provides an objective basis for the differentiation of spleen deficiency and phlegm dampness syndrome in traditional Chinese medi-cine(TCM), as well as ideas and methods for exploring the biological basis of TCM syndrome from the molecular level and the overall level.
本研究采用代谢组学方法分析陈皮对脾虚痰湿证内源性代谢产物的影响，探讨陈皮治疗脾虚痰湿证的潜在机制。采用多因素造模法诱导大鼠建立脾虚痰湿证模型。通过观察大鼠肝组织病理变化及检测血浆中甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）和高密度脂蛋白胆固醇（HDL-C）等病理生化指标含量，初步评价陈皮对脾虚痰湿证大鼠的干预作用。采用免疫组化法检测肾组织中AQP2、结肠组织中AQP3及下颌下腺组织中AQP5的表达，评价陈皮对脾虚痰湿证大鼠水通道蛋白表达的影响。此外，采用超高效液相色谱-电喷雾串联质谱（UHPLC-ESI-MS/MS）分析大鼠血浆样本的代谢谱。运用主成分分析（PCA）和正交偏最小二乘法判别分析（OPLS-DA）等多种方法进行模式识别。通过t检验和变量投影重要性（VIP）筛选差异代谢物，随后基于MetaboAnalyst 5.0进行通路分析。实验结果表明，陈皮可改善肝组织病理变化，提高血浆中HDL-C水平，降低TC、TG和LDL-C水平，并增强脾虚痰湿证大鼠肾组织中AQP2、结肠组织中AQP3及下颌下腺组织中AQP5的表达。此外，通过UHPLC-ESI-MS/MS筛选出脾虚痰湿证差异代谢物87个（其中39个代谢物水平显著升高，48个代谢物水平显著降低），代表物质有甘氨酸、L-异亮氨酸、N-乙酰-L-酪氨酸、黄嘌呤、次黄嘌呤和胡芦巴碱。差异代谢物主要富集在类固醇激素生物合成、亚油酸代谢和嘌呤代谢通路中。本研究通过代谢组学区分并揭示了脾虚痰湿证的特征性代谢模式。OPLS-DA模型的初步构建为中医脾虚痰湿证的辨证提供了客观依据，也为从分子水平和整体水平探索中医证候的生物学基础提供了思路和方法。
