Salivary Metabolites in Patients with Mucopolysaccharidosis

ABSTRACT Objective: To identify the salivary metabolites profile of Mucopolysaccharidosis (MPS) types I, II, IV, and VI patients. Material and Methods: The participants were asked to refrain from eating and drinking for one hour before sampling, performed between 7:30 and 9:00 a.m. Samples were centrifuged at 10.000 × g for 60 min at 4°C, and the supernatants (500µl) were stored at −80°C until NMR analysis. The salivary proton nuclear magnetic resonance (1H-NMR) spectra were acquired in a 500 MHz spectrometer, and TOCSY experiments were used to confirm and assign metabolites. Data were analyzed descriptively. Results: Differences in salivary metabolites were found among MPS types and the control, such as lactate, propionate, alanine, and N-acetyl sugar. Understanding these metabolite changes may contribute to precision medicine and early detection of mucopolysaccharidosis and its monitoring. Conclusion: The composition of low molecular weight salivary metabolites of mucopolysaccharidosis subjects may present specific features compared to healthy controls.

Research Progress of Metabolomics Techniques Combined with Machine Learning Algorithm in Wound Age Estimation / 法医学杂志

Wound age estimation is the core content in the practice of forensic medicine. Accurate estimation of wound age is a scientific question that needs to be urgently solved by forensic scientists at home and abroad. Metabolomics techniques can effectively detect endogenous metabolites produced by internal or external stimulating factors and describe the dynamic changes of metabolites in vivo. It has the advantages of strong operability, high detection efficiency and accurate quantitative results. Machine learning algorithm has special advantages in processing high-dimensional data sets, which can effectively mine biological information and truly reflect the physiological, disease or injury state of the body. It is a new technical means for efficiently processing high-throughput big data. This paper reviews the status and advantages of metabolomic techniques combined with machine learning algorithm in the research of wound age estimation, and provides new ideas for this research.

Metabonomics Analysis of Brain Stem Tissue in Rats with Primary Brain Stem Injury Caused Death / 法医学杂志

OBJECTIVES@#To explore the potential biomarkers for the diagnosis of primary brain stem injury (PBSI) by using metabonomics method to observe the changes of metabolites in rats with PBSI caused death.@*METHODS@#PBSI, non-brain stem brain injury and decapitation rat models were established, and metabolic maps of brain stem were obtained by LC-MS metabonomics method and annotated to the HMDB database. Partial least square-discriminant analysis (PLS-DA) and random forest methods were used to screen potential biomarkers associated with PBSI diagnosis.@*RESULTS@#Eighty-six potential metabolic markers associated with PBSI were screened by PLS-DA. They were modeled and predicted by random forest algorithm with an accuracy rate of 83.3%. The 818 metabolic markers annotated to HMDB database were used for random forest modeling and prediction, and the accuracy rate was 88.9%. According to the importance in the identification of cause of death, the most important metabolic markers that were significantly up-regulated in PBSI group were HMDB0038126 (genipinic acid, GA), HMDB0013272 (N-lauroylglycine), HMDB0005199 [(R)-salsolinol] and HMDB0013645 (N,N-dimethylsphingosine).@*CONCLUSIONS@#GA, N-lauroylglycine, (R)-salsolinol and N,N-dimethylsphingosine are expected to be important metabolite indicators in the diagnosis of PBSI caused death, thus providing clues for forensic medicine practice.

Mechanism of Danggui Sini Decoction in improving kidney injury caused by blood stasis syndrome based on metabolomics and network pharmacology / 中国中药杂志

This article analyzed the mechanism of Danggui Sini Decoction(DSD) in improving kidney injury caused by blood stasis syndrome(BSS) in rats. Firstly, 32 female SD rats were randomly divided into the following four groups: a normal group and a BSS group, both receiving an equal amount of distilled water by gavage; a normal+DSD group and a BSS+DSD group, both receiving 5.103 g·kg~(-1) DSD orally for a total of 14 days. Daily cold water bath was given to establish the BSS model, and on the 14th day, BSS rats were subcutaneously injected with 0.8 mg·kg~(-1) adrenaline. Normal rats were subjected to the water bath at 37 ℃ and injected with an equal volume of distilled water. After the experiment, 24-hour urine, serum, and kidney samples were collected for metabolomic analysis, biochemical measurements, and hematoxylin-eosin(HE) staining. The study then employed ~1H-NMR metabolomic technology to reveal the metabolic network regulated by DSD in improving BSS-induced kidney injury and used network pharmacology to preliminarily elucidate the key targets of the effectiveness of DSD. Pathological and biochemical analysis showed that DSD intervention significantly reduced inflammation and abnormal levels of blood creatinine, blood urea nitrogen, and urine protein in the kidneys. Metabolomic analysis indicated that DSD attenuated BSS-induced kidney injury primarily by regulating 10 differential metabolites and three major metabolic pathways(taurine and hypotaurine metabolism, citrate cycle, and acetaldehyde and dicarboxylic acid metabolism). Network pharmacology analysis suggested that the protective effect of DSD against BSS-induced kidney injury might be related to two key genes, ATP citrate lyase(ACLY) and nitric oxide synthase 2(NOS2), and two main metabolic pathways, i.e., arginine biosynthesis, and arginine and proline metabolism. This study, from the perspective of network regulation, provides initial insights and evidence into the mechanism of DSD in improving kidney injury induced by BSS, offering a basis for further investigation into the molecular mechanisms underlying its efficacy.

Different processed products of Polygonati Rhizoma treat Alzheimer's disease in rats: urine metabolomics based on UPLC-Q/TOF-MS / 中国中药杂志

The study investigated the effects of different processed products of Polygonati Rhizoma(black bean-processed Polygonati Rhizoma, BBPR; stewed Polygonati Rhizoma, SPR) on the urinary metabolites in a rat model of Alzheimer's disease(AD). Sixty SPF-grade male SD rats were randomized into a control group, a model group, a donepezil group, a BBPR group, and a SPR group, with twelve rats in each group. Other groups except the control group were administrated with D-galactose injection(100 mg·kg~(-1)) once a day for seven weeks. The control group was administrated with an equal volume of normal saline once a day for seven consecutive weeks. After three weeks of D-galactose injection, bilateral hippocampal Aβ_(25-35) injections were performed for modeling. The rats were administrated with corresponding drugs(10 mL·kg~(-1)) by gavage since week 2, and the rats in the model and control group with an equal volume of double distilled water once a day for 35 continuous days. The memory behaviour and pathological changes in the hippocampal tissue were observed. The untargeted metabolites in the urine were detected by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF-MS). Principal component analysis(PCA) and orthogonal partial least square-discriminant analysis(OPLS-DA) were employed to characterize and screen differential metabolites and potential biomarkers, for which the metabolic pathway enrichment analysis was conducted. The results indicated that BBPR and SPR increased the new object recognition index, shortened the escape latency, and increased the times of crossing the platform of AD rats in the Morris water maze test. The results of hematoxylin-eosin(HE) staining showed that the cells in the hippocampal tissue of the drug administration groups were closely arranged. Moreover, the drugs reduced the content of interleukin-6(IL-6, P<0.01) and tumor necrosis factor-α(TNF-α) in the hippocampal tissue, which were more obvious in the BBPR group(P<0.05). After screening, 15 potential biomarkers were identified, involving two metabolic pathways: dicoumarol pathway and piroxicam pathway. BBPR and SPR may alleviate AD by regulating the metabolism of dicoumarol and piroxicam.

Metabolomics of nasal lavage fluid in patients with allergic rhinitis treated by Xiaoqinglong Decoction / 中国中药杂志

This study used nasal lavage fluid for metabolomics to explore its feasibility, and applied it to the clinical metabolomics study of Xiaoqinglong Decoction in the treatment of allergic rhinitis(AR), aiming to investigate the molecular mechanism of Xiaoqing-long Decoction in the treatment of AR through differential changes in local nasal metabolism. AR patients were selected as the research subjects, and nasal lavage fluid was collected as the sample. Metabolomics analysis using liquid chromatography-mass spectrometry was performed on normal group, AR group, and Xiaoqinglong Decoction group. The differences in metabolic profiles among the groups were compared using principal component analysis and partial least squares discriminant analysis, and differential metabolites were identified and subjected to corresponding metabolic pathway analysis. The results showed that Xiaoqinglong Decoction significantly improved the symptoms of AR patients. The metabolomics analysis revealed 20 differential metabolites between AR group and Xiaoqinglong Decoction group. The core metabolite with a trending return in comparison to normal group was trimethyladipic acid. The metabolites were involved in multiple pathways, including β-alanine metabolism, glutathione metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. The feasibility of applying nasal lavage fluid in nasal metabolomics was preliminarily demonstrated. Differential metabolites and enriched pathways in the treatment of AR patients with Xiaoqinglong Decoction were identified, indicating that it may improve rhinitis symptoms through the regulation of various metabolites, including antioxidant effects and correction of Th1/Th2 imbalance.

Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology / 中国中药杂志

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

Urine metabolomics study of intervention of Xihuang Pills in rats with hyperplasia of mammary gland / 中国中药杂志

This study aimed to investigate the mechanism of Xihuang Pills in improving hyperplasia of mammary gland(HMG) in rats based on urine metabolomics using ultra-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry(UPLC-Q-Orbitrap-MS). The HMG rat model was established by intramuscular injection of estradiol benzoate solution(0.5 mg·kg~(-1), 25 days) followed by progesterone injection(5 mg·kg~(-1), 5 days). UPLC-Q-Orbitrap-MS technology was used to establish the endogenous small-molecule metabolic profiles in urine samples of rats in the blank group, the HMG model group, and Xihuang Pills group. Multivariate statistical analysis was performed for pattern recognition, t test and variable importance in the projection(VIP) were used to screen potential biomarkers. The significantly changed differential metabolites were identified using the online database Human Metabolome Database(HMDB). Metabolic pathway enrichment analysis was conducted using the MetaboAnalyst 5.0 database. The results showed that 90 differential metabolites with significant changes(P<0.05) were identified between the blank group and the HMG model group using the HMDB. Among them, 48 metabolites significantly reverted(P<0.05) after administration of Xihuang Pills, which may be related to the regulatory effect of Xihuang Pills. Thirteen metabolic pathways significantly associated with HMG were identified when the differential metabolites were imported into the MetaboAnalyst 5.0 database, and Xihuang Pills could modulate seven of these pathways. These metabolic pathways mainly involved histidine metabolism, arginine and proline metabolism, β-alanine metabolism, glycine, serine and threonine metabolism, tryptophan metabolism, pyrimidine metabolism, and amino sugar and nucleotide sugar metabolism. This study utilized UPLC-Q-Orbitrap-MS and urine metabolomics technology to analyze the mechanism of Xihuang Pills in improving HMG, laying the foundation for further in-depth research.

Efficacy and mechanism of Xuefu Zhuyu Decoction on model rats of coronary heart disease with heart blood stasis syndrome based on metabolomics / 中国中药杂志

This study investigated the effects of Xuefu Zhuyu Decoction on myocardial metabolites in a rat model of coronary heart disease with heart blood stasis syndrome and explored the therapeutic mechanism of blood circulation-promoting and blood stasis-removing therapy. SD rats were randomly divided into a sham operation group, a model group, a Xuefu Zhuyu Decoction group(14.04 g·kg~(-1)), and a trimetazidine group(5.4 mg·kg~(-1)). The sham operation group underwent thread insertion without ligation, while the other groups underwent coronary artery left anterior descending branch ligation to induce a model of coronary heart disease with heart blood stasis syndrome. Three days after modeling, drug intervention was performed, and samples were taken after 14 days of intervention. General conditions were observed, and electrocardiogram and cardiac ultrasound indices were measured. Hematoxylin-eosin(HE) staining and Masson staining were used to observe tissue pathological morphology. The enzyme linked immunosorbent assay(ELISA) was used to measure the levels of triglyceride(TG) and total cholesterol(TC) in the serum. Ultra high performance liquid chromatography-quantitative exactive-mass spectrometry(UHPLC-QE-MS) technology was used to screen differential metabolites in myocardial tissue and conduct metabolic pathway enrichment analysis. The results showed that Xuefu Zhuyu Decoction significantly improved the general condition of the model rats, reduced heart rate and ST segment elevation in the electrocardiogram, increased left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), and decreased left ventricular internal diameter in diastole(LVIDd) and left ventricular internal diameter in systole(LVIDs). HE staining and Masson staining showed that Xuefu Zhuyu Decoction effectively alleviated myocardial tissue structural disorders, inflammatory cell infiltration, and collagen fiber deposition in the model rats. ELISA results showed that Xuefu Zhuyu Decoction effectively regulated serum TG and TC levels in the model rats. There were significant differences in the metabolic phenotypes of myocardial samples in each group. Fourteen differential metabolites were identified in the Xuefu Zhuyu Decoction group, involving five metabolic pathways, including arginine and proline metabolism, glycerophospholipid metabolism, aminoacyl-tRNA biosynthesis, ether lipid metabolism, and alanine, aspartate, and glutamate metabolism. Xuefu Zhuyu Decoction improved cardiac function and myocardial structural damage in the rat model of coronary heart disease with heart blood stasis syndrome, and its biological mechanism involved the regulation of lipid metabolism, choline metabolism, amino acid metabolism, energy metabolism, and protein synthesis pathways.

Research progress of metabolomics in psoriasis / 中华医学杂志(英文版)

Psoriasis is a chronic inflammatory skin disease with significant physical and psychological burdens. The interplay between the innate and adaptive immune systems is thought to contribute to the pathogenesis; however, the details of the pathogenesis remain unclear. In addition, reliable biomarkers for diagnosis, assessment of disease activity, and monitoring of therapeutic response are limited. Metabolomics is an emerging science that can be used to identify and analyze low molecular weight molecules in biological systems. During the past decade, metabolomics has been widely used in psoriasis research, and substantial progress has been made. This review summarizes and discusses studies that applied metabolomics to psoriatic disease. These studies have identified dysregulation of amino acids, carnitines, fatty acids, lipids, and carbohydrates in psoriasis. The results from these studies have advanced our understanding of: (1) the molecular mechanisms of psoriasis pathogenesis; (2) diagnosis of psoriasis and assessment of disease activity; (3) the mechanism of treatment and how to monitor treatment response; and (4) the link between psoriasis and comorbid diseases. We discuss common research strategies and progress in the application of metabolomics to psoriasis, as well as emerging trends and future directions.

Mechanism of famous classical formula Huaihua Powder in treatment of ulcerative colitis based on metabonomics / 中国中药杂志

Ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry(UHPLC-Q-TOF-MS) was employed in this study to observe the effect of Huaihua Powder on the serum metabolites of mice with ulcerative colitis and reveal the mechanism of Huaihua Powder in the treatment of ulcerative colitis. The mouse model of ulcerative colitis was established by dextran sodium sulfate salt(DSS). The therapeutic effect of Huaihua Powder on ulcerative colitis was preliminarily evaluated based on the disease activity index(DAI), colon appearance, colon tissue morphology, and the content of inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β). UHPLC-Q-TOF-MS was employed to profile the endogenous metabolites of serum samples in blank control group, model group, and low-, medium-, and high-dose Huaihua Powder groups. Multivariate analyses such as principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and orthogonal partial least squares discriminant analysis(OPLS-DA) were performed for pattern recognition. Potential biomarkers were screened by Mass Profiler Professional(MPP) B.14.00 with the thresholds of fold change≥2 and P<0.05. The metabolic pathways were enriched by MetaboAnalyst 5.0. The results showed that Huaihua Powder significantly improved the general state and colon tissue morphology of mice with ulcerative colitis, reduced DAI, and lowered the levels of TNF-α, IL-6, and IL-1β in serum. A total of 38 potential biomarkers were predicted to be related to the regulatory effect of Huaihua Powder, which were mainly involved in glycerophospholipid metabolism, glycine, serine, and threonine metabolism, mutual transformation of glucuronic acid, and glutathione metabolism. This study employed metabolomics to analyze the mechanism of Huaihua Powder in the treatment of ulcerative colitis, laying a foundation for the further research.

UPLC-Q-TOF-MS metabolomic study on improvement of acute myocardial ischemia in rats by Dalbergia cochinchinensis heartwood / 中国中药杂志

This paper aimed to study the effect of Dalbergia cochinchinensis heartwood on plasma endogenous metabolites in rats with ligation of the left anterior descending coronary artery, and to analyze the mechanism of D. cochinchinensis heartwood in improving acute myocardial ischemic injury. The stability and consistency of the components in the D. cochinchinensis heartwood were verified by the establishment of fingerprint, and 30 male SD rats were randomly divided into a sham group, a model group, and a D. cochinchinensis heartwood(6 g·kg~(-1)) group, with 10 rats in each group. The sham group only opened the chest without ligation, while the other groups established the model of ligation. Ten days after administration, the hearts were taken for hematoxylin-eosin(HE) staining, and the content of heart injury indexes in the plasma creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH), energy metabolism-related index glucose(Glu) content, and vascular endothelial function index nitric oxide(NO) was determined. The endogenous metabolites were detected by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS). The results showed that the D. cochinchinensis heartwood reduced the content of CK-MB and LDH in the plasma of rats to relieve myocardial injury, reduced the content of Glu in the plasma, improved myocardial energy metabolism, increased the content of NO, cured the vascular endothelial injury, and promoted vasodilation. D. cochinchinensis heartwood improved the increase of intercellular space, myocardial inflammatory cell infiltration, and myofilament rupture caused by ligation of the left anterior descending coronary artery. The metabolomic study showed that the content of 26 metabolites in the plasma of rats in the model group increased significantly, while the content of 27 metabolites decreased significantly. Twenty metabolites were significantly adjusted after the administration of D. cochinchinensis heartwood. D. cochinchinensis heartwood can significantly adjust the metabolic abnormality in rats with ligation of the left anterior descending coronary artery, and its mechanism may be related to the regulation of cardiac energy metabolism, NO production, and inflammation. The results provide a corresponding basis for further explaining the effect of D. cochinchinensis on the acute myocardial injury.

Mechanism of Xiaoer Chiqiao Qingre Granules in clearing heat and removing food stagnation in suckling rats with fever and food accumulation based on metabolomics / 中国中药杂志

Children's fever is often accompanied by food accumulation. Traditional Chinese medicine believes that removing food stagnation while clearing heat of children can effectively avoid heat damage. To systematically evaluate the efficacy of Xiaoer Chiqiao Qingre Granules(XRCQ) in clearing heat and removing food accumulation and explore its potential mechanism, this study combined suckling SD rats fed with high-sugar and high-fat diet with injection of carrageenan to induce rat model of fever and food accumulation. This study provided references for the study on the pharmacodynamics and mechanism of XRCQ. The results showed that XRCQ effectively reduced the rectal temperature of suckling rats, improved the inflammatory environment such as the content of interleukin-1β(IL-1β), interleukin-2(IL-2), interferon-γ(IFN-γ), white blood cells, and monocytes. XRCQ also effectively repaired intestinal injury and enhanced intestinal propulsion function. According to the confirmation of its efficacy of clearing heat, the thermolytic mechanism of XRCQ was further explored by non-targeted and targeted metabolomics methods based on LTQ-Orbitrap MS/MS and UPLC-QQQ-MS/MS. Non-target metabolomics analysis of brain tissue samples was performed by QI software combined with SIMCA-P software, and 22 endogenous metabolites that could be significantly regulated were screened out. MetaboAnalyst pathway enrichment results showed that the intervention mechanism was mainly focused on tyrosine metabolism, tricarboxylic acid cycle, inositol phosphate metabolism, and other pathways. At the same time, the results of targeted metabolomics of brain tissue samples showed that XRCQ changed the vitality of digestive system, and inhibited abnormal energy metabolism and inflammatory response, playing a role in clearing heat and removing food stagnation from multiple levels.

Effect of Uremic Clearance Granules on improvement of chronic kidney disease in rats based on microbiome-metabolomics and its mechanism / 中国中药杂志

This research aimed to study the effect of Uremic Clearance Granules on chronic kidney disease in SD rats by using the methods of microbial functional genomics combined with metabolomics, and to preliminarily explore its mechanism. The SD rat model of chronic kidney disease was established by the adenine-induced method. After the model was successfully induced, the animals were randomly divided into a negative control group, a Uremic Clearance Granule treatment group, and a normal control group, with 8 rats in each group. After 4 weeks of administration, animal feces and serum were collected, and 16S rDNA sequencing technology was used to analyze the abundance, diversity, and function prediction of intestinal microorganisms. Liquid chromatography-mass spectrometry(LC-MS) technology was used to perform high-throughput sequencing to detect animal serum metabolites. The MetPA database was used to screen out potential biomarkers of chronic kidney disease in rats and conduct the enrichment analysis of metabolic pathways. Spearman's method was used to analyze the correlation between the two omics. The results showed that Uremic Clearance Granules effectively improved the body weight loss and renal function-related biochemical and appearance indicators in rats with chronic kidney disease. The results of 16S rDNA sequencing showed that Uremic Clearance Granules regulated the diversity and composition of the intestinal flora in rats with chronic kidney disease. The changes in the intestinal flora affected functional metabolic pathways such as amino acid biosynthesis and metabolism, lipid metabolism, and carbohydrate metabolism. The results of LC-MS showed that as compared with the negative control group, 15 metabolites were reversed in the Uremic Clearance Granule treatment group, among which 11 potential marker metabolites were significantly up-regulated and 4 potential marker metabolites were significantly down-regulated. Five amino acid metabolic pathways were mainly involved, which were significantly correlated with changes in the intestinal flora. Therefore, Uremic Clearance Granules can improve the renal function of rats with chronic kidney disease, and the mechanism may be related to its effect on the amino acid metabolism pathway by regulating the intestinal flora.

Dryness-alleviating effect of processed Aurantii Fructus from Lingnan based on correlation of fecal metabolomics and dryness / 中国中药杂志

With the approach of untargeted metabolomics and correlation analysis, this study aimed to explore the mechanism of Aurantii Fructus from Lingnan region in alleviating dryness by analyzing the different effects of raw Aurantii Fructus(RAF) and processed Aurantii Fructus(PAF) on fecal endogenous metabolism in normal rats. Eighteen Sprague-Dawley(SD) rats were randomly divided into a control group(C), an RAF group(10 g·kg~(-1)), and a PAF group(10 g·kg~(-1)). After seven days of administration, the effects of RAF and PAF on dryness-related indexes were compared, including water intake, fecal water content, salivary secretion, the expression of AQP5, VIP, and 5-HT in the submandibular gland, as well as the expression of AQP3, VIP, and 5-HT in the colon. The fecal samples in each group were determined by LC-MS. Multivariate statistical analysis and Pearson correlation coefficient were used for screening the differential metabolites and metabolic pathways in alleviating dryness of RAF. The results indicated that both RAF and PAF showed certain dryness, and the dryness of RAF was more significant. Moreover, PAF could alleviate dryness of RAF to a certain extent by reducing the water intake, fecal water content, and the expression of AQP3, VIP, and 5-HT in the colon and increasing the salivary secretion and the levels of AQP5, VIP, and 5-HT in the submandibular gland. According to the analysis of fecal metabolomics, 99 and 58 metabolites related to dryness were found in RAF and PAF respectively, where 16 of them played an important role in alleviating dryness of RAF. Pathway analysis revealed that the mechanism of PAF in alleviating dryness of RAF was presumably related to the regulation of riboflavin metabolism, purine metabolism, arginine biosynthesis, pyrimidine metabolism, alanine metabolism, aspartate metabolism, glutamate metabolism, and retinol metabolism pathways. This study suggested that PAF might alleviate dryness of RAF by affecting the metabolic levels of the body, which provides a new basis for further clarifying the processing mechanism of PAF.

Mechanism of active ingredients in Periploca forrestii compound against rheumatoid arthritis based on integrative metabolomics and network pharmacology / 中国中药杂志

In this study, an ultra-performance liquid chromatography-quadrupole time-of-flight high resolution mass spectrometer(UPLC-Q-TOF-HRMS) was used to investigate the effects of the active ingredients in Periploca forrestii compound on spleen metabolism in rats with collagen-induced arthritis(CIA), and its potential anti-inflammatory mechanism was analyzed by network pharmacology. After the model of CIA was successfully established, the spleen tissues of rats were taken 28 days after administration. UPLC-Q-TOF-HRMS chromatograms were collected and analyzed by principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and MetPA. The results showed that as compared with the blank control group, 22 biomarkers in the spleen tissues such as inosine, citicoline, hypoxanthine, and taurine in the model group increased, while 9 biomarkers such as CDP-ethanolamine and phosphorylcholine decreased. As compared with the model group, 21 biomarkers such as inosine, citicoline, CDP-ethanolamine, and phosphorylcholine were reregulated by the active ingredients in P. forrestii. Seventeen metabolic pathways were significantly enriched, including purine metabolism, taurine and hypotaurine metabolism, glycerophospholipid metabolism, and cysteine and methionine metabolism. Network pharmacology analysis found that purine metabolism, glycerophospholipid metabolism, and cysteine and methionine metabolism played important roles in the pathological process of rheumatoid arthritis. This study suggests that active ingredients in P. forrestii compound can delay the occurrence and development of inflammatory reaction by improving the spleen metabolic disorder of rats with CIA. The P. forrestii compound has multi-target and multi-pathway anti-inflammatory mechanism. This study is expected to provide a new explanation for the mechanism of active ingredients in P. forrestii compound against rheumatoid arthritis.

Effective substances and mechanism of Yishen Guluo Mixture in treatment of chronic glomerulonephritis based on metabolomics and serum pharmacochemistry / 中国中药杂志

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.

Network pharmacology approaches for research of Traditional Chinese Medicines / 中国天然药物

Pharmacodynamics material basis and effective mechanisms are the two main issues to decipher the mechnisms of action of Traditional Chinese medicines (TCMs) for the treatment of diseases. TCMs, in "multi-component, multi-target, multi-pathway" paradigm, show satisfactory clinical results in complex diseases. New ideas and methods are urgently needed to explain the complex interactions between TCMs and diseases. Network pharmacology (NP) provides a novel paradigm to uncover and visualize the underlying interaction networks of TCMs against multifactorial diseases. The development and application of NP has promoted the safety, efficacy, and mechanism investigations of TCMs, which then reinforces the credibility and popularity of TCMs. The current organ-centricity of medicine and the "one disease-one target-one drug" dogma obstruct the understanding of complex diseases and the development of effective drugs. Therefore, more attentions should be paid to shift from "phenotype and symptom" to "endotype and cause" in understanding and redefining current diseases. In the past two decades, with the advent of advanced and intelligent technologies (such as metabolomics, proteomics, transcriptomics, single-cell omics, and artificial intelligence), NP has been improved and deeply implemented, and presented its great value and potential as the next drug-discovery paradigm. NP is developed to cure causal mechanisms instead of treating symptoms. This review briefly summarizes the recent research progress on NP application in TCMs for efficacy research, mechanism elucidation, target prediction, safety evaluation, drug repurposing, and drug design.

Study on the dynamic variation of the secondary metabolites in Viscum coloratum using targeted metabolomics / 中国天然药物

Viscum coloratum (Kom.) Nakai is a well-known medicinal plant. However, the optimal harvest time for V. coloratum is unknown. Few studies were performed to analyze compound variation during storage and to improve post-harvest quality control. Our study aimed to comprehensively evaluate the quality of V. coloratum in different growth stages, and determine the dynamic variation of metabolites. Ultra-performance liquid chromatography tandem mass spectrometry was used to quantify 29 compounds in V. coloratum harvested in six growth periods, and the associated biosynthetic pathways were explored. The accumulation of different types of compounds were analyzed based on their synthesis pathways. Grey relational analysis was used to evaluate the quality of V. coloratum across different months. The compound variation during storage was analyzed by a high-temperature high-humidity accelerated test. The results showed that the quality of V. coloratum was the hightest in March, followed by November, and became the lowest in July. During storage, compounds in downstream steps of the biosynthesis pathway were first degraded to produce the upstream compounds and some low-molecular-weight organic acids, leading to an increase followed by a decrease in the content of some compounds, and resulted in a large gap during the degradation time course among different compounds. Due to the rapid rate and large degree of degradation, five compounds were tentatively designated as "early warning components" for quality control. This report provides reference for better understanding the biosynthesis and degradation of metabolites in V. coloratum and lays a theoretical foundation for rational application of V. coloratum and better quality control of V. coloratum during storage.

High expression of VARS promotes the growth of multiple myeloma cells by causing imbalance in valine metabolism / 中南大学学报(医学版)

OBJECTIVES@#Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth.@*METHODS@#The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively.@*RESULTS@#Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05).@*CONCLUSIONS@#MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.