Integrated Systems Pharmacology, Urinary Metabonomics, and Quantitative Real-Time PCR Analysis to Uncover Targets and Metabolic Pathways of the You-Gui Pill in Treating Kidney-Yang Deficiency Syndrome

Kidney-yang deficiency syndrome (KYDS) is a metabolic disease caused by a neuro-endocrine disorder. The You-gui pill (YGP) is a classic traditional Chinese medicine (TCM) formula for the treatment of KYDS and has been widely used to warm and recuperate KYDS clinically for hundreds of years in China. However, it is unknown whetherthe corresponding targets and metabolic pathways can also be found via using metabonomics based on one platform (e.g., 1H NMR) to study different biological samples of KYDS. At the same time, relevant reports on further molecular verification (e.g., RT-qPCR analysis) of these targets associated with biomarkers and metabolic pathways have not yet, to our knowledge, been seen in KYDS’s research. In the present study, a comprehensive strategy integrating systems pharmacology and 1H NMR-based urinary metabonomics analysis was proposed to identify the target proteins and metabolic pathways that YGP acts on KYDS. Thereafter, further validation of target proteins in kidney tissue was performed through quantitative real-time PCR analysis (RT-qPCR). Furthermore, biochemical parameters and histopathological analysis were studied. As a result, seven target proteins (L-serine dehydratase; phosphoenolpyruvate carboxykinase; spermidine synthase; tyrosyl-tRNA synthetase, glutamine synthetase; 3-hydroxyacyl-CoA dehydrogenase; glycine amidinotransferase) in YGP were discovered to play a therapeutic role in KYDS via affecting eight metabolic pathways (glycine, serine and threonine metabolism; butanoate metabolism; TCA cycle, etc.). Importantly, three target proteins (i.e., 3-hydroxyacyl-CoA dehydrogenase; glutamine synthetase; and glycine amidinotransferase) and two metabolic pathways (butanoate metabolism and dicarboxylate metabolism) related to KYDS, to our knowledge, had been newly discovered in our study. The mechanism of action mainly involved energy metabolism, oxidative stress, ammonia metabolism, amino acid metabolism, and fatty acid metabolism. In short, our study demonstrated that targets and metabolic pathways for the treatment of KYDS by YGP can be effectively found via combining with systems pharmacology and urinary metabonomics. In addition to this, common and specific targets and metabolic pathways of KYDS treated by YGP can be found effectively by integration with the analysis of different biological samples (e.g., serum, urine, feces, and tissue). It is; therefore, important that this laid the foundation for deeper mechanism research and drug-targeted therapy of KYDS in future.

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Metabolomics Analysis of Seminal Plasma in Infertile Males with Kidney-Yang Deficiency: A Preliminary Study

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/892930 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Xiang Chen ◽

Chao Hu ◽

Jican Dai ◽

Lei Chen

Keyword(s):

Male Infertility ◽

Seminal Plasma ◽

Metabolic Pathways ◽

Tricarboxylic Acid Cycle ◽

Principal Component ◽

Metabolite Identification ◽

Deficiency Syndrome ◽

Sphingolipid Metabolism ◽

Kidney Yang Deficiency ◽

Metabolomics Analysis

Traditional Chinese medicine (TCM) is an important treatment for male infertility, and its application to therapy is dependent on differentiation of TCM syndromes. This study aims to investigate the changes in metabolites and metabolic pathways in infertile males with Kidney-Yang Deficiency syndrome (KYDS) via metabolomics approaches. Seminal plasma samples were collected from 18 infertile males with KYDS and 18 fertile males. Liquid chromatography and mass spectrometry were used to characterize metabolomics profiles. Principal component analysis (PCA), partial least squares-discriminate analysis (PLS-DA), and pathway analysis were used for pattern recognition and metabolite identification. PCA and PLS-DA results differentiated the two groups of patients. Forty-one discriminating metabolites (18 in positive mode and 23 in negative mode) were identified. Seven metabolites were related to five potential metabolic pathways associated with biosynthesis and metabolism of aromatic amino acids, tricarboxylic acid cycle, and sphingolipid metabolism. The changes in metabolic pathways may play an important role in the origin of KYDS-associated male infertility. Metabolomics analysis of seminal plasma may be used to differentiate TCM syndromes of infertile males, but further research must be conducted.

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Exploring the biomarkers and therapeutic mechanism of kidney-yang deficiency syndrome treated by You-gui pill using systems pharmacology and serum metabonomics

RSC Advances ◽

10.1039/c7ra12451a ◽

2018 ◽

Vol 8 (2) ◽

pp. 1098-1115 ◽

Cited By ~ 8

Author(s):

Ruiqun Chen ◽

Jia Wang ◽

Chengbin Liao ◽

Lei Zhang ◽

Qian Guo ◽

...

Keyword(s):

Molecular Targets ◽

Deficiency Syndrome ◽

Systems Pharmacology ◽

Therapeutic Mechanism ◽

Kidney Yang Deficiency

In this study, systems pharmacology was used to predict the molecular targets of You-gui pills (YGP) and explore the therapeutic mechanism of Kidney-Yang Deficiency Syndrome (KYDS) treated with YGP.

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Transcriptomic comparison between beetle strains selected for short and long durations of death feigning

Scientific Reports ◽

10.1038/s41598-019-50440-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Hironobu Uchiyama ◽

Ken Sasaki ◽

Shogo Hinosawa ◽

Keisuke Tanaka ◽

Kentarou Matsumura ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Pathways ◽

Antipredator Behavior ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Differentially Expressed ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Death Feigning ◽

Encoding Genes

Abstract The molecular basis of death feigning, an antipredator behavior that has received much attention recently, was analyzed. We compared the gene expression profiles of strains with different behaviors, i.e., different durations of death feigning, in the beetle Tribolium castaneum. Beetles artificially selected for short (S) and long (L) durations of death feigning for many generations were compared thoroughly by RNA sequencing. We identified 518 differentially expressed genes (DEGs) between the strains. The strains also showed divergence in unexpected gene expression regions. As expected from previous physiological studies, genes associated with the metabolic pathways of tyrosine, a precursor of dopamine, were differentially expressed between the S and L strains; these enzyme-encoding genes were expressed at higher levels in the L strain than in the S strain. We also found that several genes associated with insulin signaling were expressed at higher levels in the S strain than in the L strain. Quantitative real-time PCR analysis showed that the relative expression levels of Tchpd (encoding 4-hydroxyphenylpyruvate dioxygenase, Hpd) and Tcnat (encoding N-acetyltransferase, Nat) were significantly higher in the L strain than in the S strain, suggesting the influence of these enzymes on the supply of dopamine and duration of death feigning.

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A Novel Strategy Facilitates Reference Gene Selection by RT-qPCR Analysis in Kidney Yang Deficiency Syndrome Mice Infected with the Influenza A (H1N1) Virus

BioMed Research International ◽

10.1155/2020/9075165 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Yepei Fu ◽

Jia Yang ◽

Shanshan Fan ◽

Shaozhe Zhao ◽

Syed Muhammad Ali Shah ◽

...

Keyword(s):

Reference Gene ◽

Reference Genes ◽

Influenza A ◽

Gene Selection ◽

Quantitative Polymerase Chain Reaction ◽

Deficiency Syndrome ◽

Pcr Analysis ◽

Influenza A H1n1 ◽

Kidney Yang Deficiency ◽

The Difference

In reverse transcription-quantitative polymerase chain reaction (RT-qPCR) studies, endogenous reference genes are routinely used to normalize the expression of target gene studies. In order to precisely evaluate the relative expression of genes in the cells of mice suffering from Kidney Yang Deficiency Syndrome (KYDS) in response to influenza A virus (IAV) H1N1 using RT-qPCR, it is crucial to identify reliable reference genes. In the present study, 15 candidate reference genes (Actb, β2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhae, 18S rRNA, Rpl13, Ubc, Rpl32, and Ppia) were investigated in lung cells from KYDS mice infected with IAV H1N1. NormFinder, BestKeeper, and GeNorm were used to assess the stability of reference genes. The results were authenticated over extended experimental settings by a group of 10 samples. In the present study, we explored a novel method using dual-gene combinations; the difference in gene expression between the model and normal control groups was statistically analyzed by an independent-samples t-test, and the difference in the mean value between the two groups was compared. A P value > 0.05 and the lowest absolute value of the difference indicated the optimal reference two-gene combination. Four additional host innate immune system-related genes (TLR3, TLR4, TLR7, and RIG-I) were analyzed together with the two treatment datasets to confirm the selected reference genes. Our results indicated that none of these 15 candidate reference genes can be used as reference gene individually for relative quantitative fluorescence PCR analysis; however, the combination of Grcc10 and Ppia, based on the process of calculating the higher P value and lower difference values between groups, was the best choice as a reference gene for the lung tissue samples in KYDS mice infected with IAV. This technique may be applied to promote the selection process of the optimal reference gene in other experiments.

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Plasma Metabolomics Analysis Based on GC-MS in Infertile Males with Kidney-Yang Deficiency Syndrome

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/6270195 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

Piao Zheng ◽

Yun Wang ◽

Hongmei Lu ◽

Xinyi Zhou ◽

Tao Tang ◽

...

Keyword(s):

Chinese Medicine ◽

Male Infertility ◽

Metabolic Pathways ◽

Characteristic Curve ◽

Deficiency Syndrome ◽

Gas Chromatography Mass Spectrometry ◽

Healthy Controls ◽

Chinese Medicine Syndrome ◽

Kidney Yang Deficiency ◽

Potential Biomarkers

Introduction. Chinese medicine syndrome diagnosis is the key requisite in the treatment of male infertility with traditional Chinese medicine (TCM). Kidney-Yang deficiency syndrome (KYDS) is the critical Chinese medicine syndrome of male infertility. To explore the modernized mechanisms of KYDS in male infertility, this study aims to investigate the metabolomics of males with KYDS. Methods. The gas chromatography-mass spectrometry method was applied to analyze the plasma samples of 67 infertile males with KYDS compared with 55 age-matched healthy controls. The chemometric methods including principal component and partial least squares-discriminate analyses were employed to identify the potential biochemical patterns. With the help of the variable importance for the projection and receiver operating characteristic curve analyses, the potential biomarkers were extracted to define the clinical utility. Simultaneously the high-quality KEGG metabolic pathways database was used to identify the related metabolic pathways. Results. The metabolomics profiles of infertile males with KYDS including 10 potential biomarkers and six metabolic pathways were identified. They precisely distinguished infertile males with KYDS from healthy controls. Conclusions. These potential biomarkers and pathways suggest the substantial basis of infertile males with KYDS. The metabolomics profiles highlight the modernized mechanisms of infertile males with KYDS.

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1H NMR studies on serum metabonomic changes over time in a kidney-Yang deficiency syndrome model

RSC Advances ◽

10.1039/c7ra04057a ◽

2017 ◽

Vol 7 (54) ◽

pp. 34251-34261 ◽

Cited By ~ 7

Author(s):

Ruiqun Chen ◽

Jia Wang ◽

Chengbin Liao ◽

Na Ma ◽

Lei Zhang ◽

...

Keyword(s):

1H Nmr ◽

Metabolic Pathways ◽

Deficiency Syndrome ◽

Nmr Studies ◽

Kidney Yang Deficiency ◽

Changes Over Time ◽

Over Time

The central aim of this study was to investigate metabolite changes in metabolic pathwaysviametabonomic approaches in rats suffering from Kidney-Yang Deficiency Syndrome (KYDS) induced by hydrocortisone.

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Understanding Radix Angelica sinensis Blood Replenishing mechanisms on Blood Deficiency Rats Based on a UPLC-Q/TOF-MS Metabolomics and Network Pharmacology

Records of Natural Products ◽

10.25135/rnp.224.21.01.1950 ◽

2021 ◽

Vol 15 (4) ◽

pp. 267-280

Author(s):

Xiujuan Y ang ◽

Yi Deng ◽

Zhijun Y ang ◽

Shuo Li ◽

Yunxiang Hai ◽

...

Keyword(s):

Linoleic Acid ◽

Chinese Medicine ◽

Hemolytic Anemia ◽

Metabolic Pathways ◽

Acid Metabolism ◽

Deficiency Syndrome ◽

Network Pharmacology ◽

Angelica Sinensis ◽

Proline Metabolism ◽

Linoleic Acid Metabolism

Radix Angelica sinensis (RAS) is a famous Chinese medicine with hematinic effects and has been applied for the treatment of blood deficiency syndrome for many years. Previous studies have indicated that RAS has beneficial effects in the treatment of hemolytic anemia. The hemolytic anemia/blood deficiency syndrome is a common syndrome that is often presented in most traditional Chinese medicine (TCM) clinics. Based on routine blood indicators, metabolomics analysis was conducted to investigate the mechanism of RAS in the treatment of hemolytic anemia. Multivariate and univariate statistical analysis were used to identify potential biomarkers in the serum. On administering RAS to the haemololitic anaemic rat, the levels of WBC, RBC, HGB, and PLT in AG tended to shift toward that of the control group. Additionally, all the 26 metabolites such as cholic acid, succinic acid and orotate which are regulated by blood deficiency appeared normal through the five metabolic pathways, such as linoleic acid metabolism, alanine, aspartic acid and glutamate metabolism, pyrimidine metabolism, arginine and proline metabolism. Thus, three metabolic pathways predicted by the network pharmacology were consistent with the metabolism pathway of Angelica sinensis: linoleic acid metabolism, arginine and proline metabolism, tryptophan metabolism. The integrated metabolomics and network pharmacology comprehensively improved the understanding of the physiological and metabolic state of an organism. The possible hematopoietic effects and underlying mechanism of action on hemolytic anemia rats after lavage with RAS water extracts, could potentially be elucidated by combining pharmacology with untargeted metabolomics. These pointed out the significance of metabolomics as a valuable tool for studying the essence of Chinese medicine’s syndrome theory and the mechanism of RAS under anti-blood deficiency syndrome.

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A 1232 bp upstream sequence of glutamine synthetase 1b from Eichhornia crassipes is a root-preferential promoter sequence

BMC Plant Biology ◽

10.1186/s12870-021-02832-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yanshan Zhong ◽

Xiaodan Lu ◽

Zhiwei Deng ◽

Ziqing Lu ◽

Minghui Fu

Keyword(s):

Glutamine Synthetase ◽

Eichhornia Crassipes ◽

Invasive Plant ◽

Promoter Sequence ◽

Regulatory Mechanisms ◽

Pcr Analysis ◽

Regulation Mechanism ◽

Upstream Sequence ◽

Specific Expression ◽

N Metabolism

Abstract Background Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism. Results A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5′-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of β-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves. Conclusions EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

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Comprehensive transcriptome and metabolome profiling reveal metabolic mechanisms of Nitraria sibirica Pall. to salt stress

Scientific Reports ◽

10.1038/s41598-021-92317-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Huanyong Li ◽

Xiaoqian Tang ◽

Xiuyan Yang ◽

Huaxin Zhang

Keyword(s):

Salt Stress ◽

Amino Acid ◽

Salt Tolerance ◽

Metabolic Pathways ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Sulfur Metabolism ◽

Enrichment Analysis ◽

Extracellular Region ◽

Nitraria Sibirica

AbstractNitraria sibirica Pall., a typical halophyte that can survive under extreme drought conditions and in saline-alkali environments, exhibits strong salt tolerance and environmental adaptability. Understanding the mechanism of molecular and physiological metabolic response to salt stress of plant will better promote the cultivation and use of halophytes. To explore the mechanism of molecular and physiological metabolic of N. sibirica response to salt stress, two-month-old seedlings were treated with 0, 100, and 400 mM NaCl. The results showed that the differentially expressed genes between 100 and 400 mmol L−1 NaCl and unsalted treatment showed significant enrichment in GO terms such as binding, cell wall, extemal encapsulating structure, extracellular region and nucleotide binding. KEGG enrichment analysis found that NaCl treatment had a significant effect on the metabolic pathways in N. sibirica leaves, which mainly including plant-pathogen interaction, amino acid metabolism of the beta alanine, arginine, proline and glycine metabolism, carbon metabolism of glycolysis, gluconeogenesis, galactose, starch and sucrose metabolism, plant hormone signal transduction and spliceosome. Metabolomics analysis found that the differential metabolites between the unsalted treatment and the NaCl treatment are mainly amino acids (proline, aspartic acid, methionine, etc.), organic acids (oxaloacetic acid, fumaric acid, nicotinic acid, etc.) and polyhydric alcohols (inositol, ribitol, etc.), etc. KEGG annotation and enrichment analysis showed that 100 mmol L−1 NaCl treatment had a greater effect on the sulfur metabolism, cysteine and methionine metabolism in N. sibirica leaves, while various amino acid metabolism, TCA cycle, photosynthetic carbon fixation and sulfur metabolism and other metabolic pathways have been significantly affected by 400 mmol L−1 NaCl treatment. Correlation analysis of differential genes in transcriptome and differential metabolites in metabolome have found that the genes of AMY2, BAM1, GPAT3, ASP1, CML38 and RPL4 and the metabolites of L-cysteine, proline, 4-aminobutyric acid and oxaloacetate played an important role in N. sibirica salt tolerance control. This is a further improvement of the salt tolerance mechanism of N. sibirica, and it will provide a theoretical basis and technical support for treatment of saline-alkali soil and the cultivation of halophytes.

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