Meng Yu

Variations in gut microbiota and fecal metabolic phenotype associated with depression by 16S rRNA gene sequencing and LC/MS-based metabolomics

&NA; As a prevalent, life‐threatening and highly recurrent psychiatric illness, depression is characterized by a wide range of pathological changes; however, its etiology remains incompletely understood. Accumulating evidence supports that gut microbiota affects not only gastrointestinal physiology but also central nervous system (CNS) function and behavior through the microbiota‐gut‐brain axis. To assess the impact of gut microbiota on fecal metabolic phenotype in depressive conditions, an integrated approach of 16S rRNA gene sequencing combined with ultra high‐performance liquid chromatography‐mass spectrometry (UHPLC–MS) based metabolomics was performed in chronic variable stress (CVS)‐induced depression rat model. Interestingly, depression led to significant gut microbiota changes, at the phylum and genus levels in rats treated with CVS compared to controls. The relative abundances of the bacterial genera Marvinbryantia, Corynebacterium, Psychrobacter, Christensenella, Lactobacillus, Peptostreptococcaceae incertae sedis, Anaerovorax, Clostridiales incertae sedis and Coprococcus were significantly decreased, whereas Candidatus Arthromitus and Oscillibacter were markedly increased in model rats compared with normal controls. Meanwhile, distinct changes in fecal metabolic phenotype of depressive rats were also found, including lower levels of amino acids, and fatty acids, and higher amounts of bile acids, hypoxanthine and stercobilins. Moreover, there were substantial associations of perturbed gut microbiota genera with the altered fecal metabolites, especially compounds involved in the metabolism of tryptophan and bile acids. These results showed that the gut microbiota was altered in association with fecal metabolism in depressive conditions. These findings suggest that the 16S rRNA gene sequencing and LC–MS based metabolomics approach can be further applied to assess pathogenesis of depression. Graphical abstract Depression not only perturbs the gut microbiota at abundance level but that it also alters the fecal metabolic phenotype. In addition, there was a strong correlation between gut microbiota and fecal metabolites. Figure. No caption available. HighlightsA combined 16S rRNA gene sequencing and MS‐based metabolomics has been established.Depression led to significant changes of gut microbiota at phylum and genus levels.Depression led to significant changes of fecal metabolic phenotype.Strong correlation of gut microbiota, fecal metabolites, and catecholamine levels.

Aberrant purine metabolism in allergic asthma revealed by plasma metabolomics

Asthma is a disease characterized by chronic relapsing airways, and its etiology remains incompletely understood. To better understand the metabolic phenotypes of asthma, we investigated a plasma metabolic signature associated with allergic asthma in ovalbumin (OVA)-sensitized mice by using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Sixteen metabolites were characterized as potential pathological biomarkers related to asthma. Among them, 6 (dodecanoic acid (P1), myristic acid (P2), phytosphingosine (P3), sphinganine (P4), inosine (P13) and taurocholic acid (P15)) were first reported to have potential relevance in the pathogenesis of experimental asthma. The identified potential biomarkers were involved in 6 metabolic pathways and achieved the most entire metabolome contributing to the formation of allergic asthma. Purine metabolism was the most prominently influenced in OVA-induced asthma mice according to the metabolic pathway analysis (MetPA), suggesting that significantly changes in inflammatory responses in the pathophysiologic process of asthma. The metabolites of purine metabolism, especially uric acid (P12) and inosine (P13), may denote their potential as targeted biomarkers related to experimental asthma. The decreased plasma uric acid (P12) suggested that inflammation responses of allergic asthma inhibited the activity of xanthine oxidase in purine metabolism, and manifested the severity of asthma exacerbation. The increased level of inosine (P13) suggests that inflammatory cells induce adenosine triphosphate (ATP) breakdown, resulting in excessive expression of adenosine deaminase (ADA) in the formation of allergic asthma. These findings provided a novel perspective on the metabolites signatures related to allergic asthma, which provided us with new insights into the pathogenesis of asthma, and the discovery of targets for clinical diagnosis and treatment.

Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene expression

Depression is a complex disease characterized by a series of pathological changes. Research on depression is mainly focused on the changes in brain, but not on liver. Therefore, we initially explored the metabolic profiles of hepatic extracts from rats treated with chronic unpredictive mild stress (CUMS) by UPLC-Q-TOF/MS. Using multivariate statistical analysis, a total of 26 altered metabolites distinguishing CUMS-induced depression from normal control were identified. Using two-stage receiver operating characteristic (ROC) analysis, 18 metabolites were recognized as potential biomarkers related to CUMS-induced depression via 12 metabolic pathways. Subsequently, we detected the mRNA expressions levels of apoptosis-associated genes such as Bax and Bcl-2 and four key enzymes including Pla2g15, Pnpla6, Baat and Gad1 involved in phospholipid and primary bile acid biosynthesis in liver tissues of CUMS rats by real-time qRT-PCR assay. The expression levels of Bax, Bcl-2, Pla2g15, Pnpla6 and Gad1 mRNA were 1.43,1.68, 1.74, 1.67 and 1.42-fold higher, and those of Baat, Bax/Bcl-2 ratio mRNA were 0.83, 0.85-fold lower in CUMS rats compared with normal control. Results of liver-targeted metabonomics and mRNA expression demonstrated that CUMS-induced depression leads to variations in hepatic metabolic profile and gene expression, and ultimately results in liver injury.

The Chinese Herbal Medicine Formula mKG Suppresses Pulmonary Fibrosis of Mice Induced by Bleomycin

Pulmonary fibrosis (PF) is a serious progressive lung disease and it originates from inflammation-induced parenchymal injury with excessive extracellular matrix deposition to result in the destruction of the normal lung architecture. Modified Kushen Gancao Formula (mKG), derived from traditional Chinese herbal medicine, has a prominent anti-inflammatory effect. The present study is to explore the inhibitory effects of mKG on bleomycin (BLM)-induced pulmonary fibrosis in mice. mKG significantly decreased pulmonary alveolitis, fibrosis scores, and interleukin-6 (IL-6), interleukin-17 (IL-17), transforming growth factor-β (TGF-β) and hydroxyproline (HYP) levels in lung tissue of mice compared with BLM treatment. It markedly alleviated the increase of HYP content in the lung tissues and pathologic changes of pulmonary fibrosis caused by BLM instillation. In conclusion, mKG has an anti-fibrotic effect and might be employed as a therapeutic candidate agent for attenuating pulmonary fibrosis.

Urinary and Fecal Metabonomics Study of the Protective Effect of Chaihu-Shu-Gan-San on Antibiotic-Induced Gut Microbiota Dysbiosis in Rats

Accumulating evidence suggests that the gut microbiota dysbiosis and their host metabolic phenotype alteration is an important factor in human disease development. A traditional Chinese herbal formula, Chaihu-Shu-Gan-San (CSGS), has been effectively used in the treatment of various gastrointestinal (GI) disorders. The present study was carried out to investigate whether CSGS modulates the host metabolic phenotype under the condition of gut microbiota dysbiosis. The metabonomics studies of biochemical changes in urine and feces of antibiotic-induced gut microbiota dysbiosis rats after treatment with CSGS were performed using UPLC-Q-TOF/MS. Partial least squares-discriminate analysis (PLS-DA) indicated that the CSGS treatment reduced the metabolic phenotype perturbation induced by antibiotic. In addition, there was a strong correlation between gut microbiota genera and urinary and fecal metabolites. Moreover, the correlation analysis and the metabolic pathway analysis (MetPA) identified that three key metabolic pathways including glycine, serine and threonine metabolism, nicotinate and nicotinamide metabolism, and bile acid metabolism were the most relevant pathways involved in antibiotic-induced gut microbiota dysbiosis. These findings provided a comprehensive understanding of the protective effects of CSGS on the host metabolic phenotype of the gut microbiota dysbiosis rats, and further as a new source for drug leads in gut microbiota-targeted disease management.

Chaihu-Shu-Gan-San regulates phospholipids and bile acid metabolism against hepatic injury induced by chronic unpredictable stress in rat

Chaihu-Shu-Gan-San (CSGS) is a famous classic traditional Chinese medicines (TCM) formula for treatment of liver stagnancy recorded in a famous book of traditional Chinese medicine, Jing Yue Quan Shu published in 1624. It has been extensively accepted as an antidepressant in China and its mechanism of action is still not clear. Previously we have found that hepatic injury happens in chronic unpredicted mild stress (CUMS). Thus, the protection of CSGS against hepatic injury induced by CUMS treatment was explored by metabonomics study and gene expression of the rat liver tissue. The results indicated that CSGS improved 8 of the 18 perturbed potential biomarkers in liver tissues of rats treated with CUMS, and involved in regulating phospholipids and bile acid metabolism against hepatic injury induced by CUMS in rat. The expressions of two apoptosis associated genes (Bcl-2 and Bax) and four genes (Pnpla6, Pla2g15, Baat and Gad1) related to the perturbed metabolic pathways were further investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Both metabonomics and studies of genetic influences on metabolites demonstrated that CSGS inhibited hepatocyte apoptosis, and regulated phospholipids and bile acid metabolism against hepatic injury induced by CUMS in rat. Exploring the protection of CSGS against hepatic injury related to depression further clarify the relationship between CUMS-induced depression and hepatic injury, and also provide a novel insight to understand the underlying antidepressive mechanism of CSGS.

The Effect of Chinese Herbal Medicine Formula mKG on Allergic Asthma by Regulating Lung and Plasma Metabolic Alternations

Asthma is a chronic inflammatory disorder of the airway and is characterized by airway remodeling, hyperresponsiveness, and shortness of breath. Modified Kushen Gancao Formula (mKG), derived from traditional Chinese herbal medicines (TCM), has been demonstrated to have good therapeutic effects on experimental allergic asthma. However, its anti-asthma mechanism remains currently unknown. In the present work, metabolomics studies of biochemical changes in the lung tissue and plasma of ovalbumin (OVA)-induced allergic asthma mice with mKG treatment were performed using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Partial least squares–discriminate analysis (PLS−DA) indicated that the metabolic perturbation induced by OVA was reduced after mKG treatment. A total of twenty-four metabolites involved in seven metabolic pathways were identified as potential biomarkers in the development of allergic asthma. Among them, myristic acid (L3 or P2), sphinganine (L6 or P4), and lysoPC(15:0) (L12 or P16) were detected both in lung tissue and plasma. Additionally, l-acetylcarnitine (L1), thromboxane B2 (L2), 10-HDoHE (L10), and 5-HETE (L11) were first reported to be potential biomarkers associated with allergic asthma. The treatment of mKG mediated all of those potential biomarkers except lysoPC(15:0) (P16). The anti-asthma mechanism of mKG can be achieved through the comprehensive regulation of multiple perturbed biomarkers and metabolic pathways.

Role of Bai-Shao towards the antidepressant effect of Chaihu-Shu-Gan-San using metabonomics integrated with chemical fingerprinting

Chaihu-Shu-Gan-San (CSGS) is a classical traditional Chinese medicine formula for the treatment of depression. As one of the single herbs in CSGS, Bai-Shao displayed antidepressant effect. In order to explore the role of Bai-Shao towards the antidepressant effect of CSGS, the metabolic regulation and chemical profiles of CSGS with and without Bai-Shao (QBS) were investigated using metabonomics integrated with chemical fingerprinting. At first, partial least squares regression (PLSR) analysis was applied to characterize the potential biomarkers associated with chronic unpredictable mild stress (CUMS)-induced depression. Among 46 differential metabolites found in the ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) and (1)H NMR-based urinary metabonomics, 20 were significantly correlated with the preferred sucrose consumption observed in behavior experiments and were considered as biomarkers to evaluate the antidepressant effect of CSGS. Based on differential regulation on CUMS-induced metabolic disturbances with CSGS and QBS treatments, we concluded that Bai-Shao made crucial contribution to CSGS in the improvement of the metabolic deviations of six biomarkers (i.e., glutamate, acetoacetic acid, creatinine, xanthurenic acid, kynurenic acid, and N-acetylserotonin) disturbed in CUMS-induced depression. While the chemical constituents of Bai-Shao contributed to CSGS were paeoniflorin, albiflorin, isomaltopaeoniflorin, and benzoylpaeoniflorin based on the multivariate analysis of the UPLC-Q-TOF/MS chemical profiles from CSGS and QBS extracts. These findings suggested that Bai-Shao played an indispensable role in the antidepressant effect of CSGS.

Metabolic profiling of hypoxia/reoxygenation injury in H9c2 cells reveals the accumulation of phytosphingosine and the vital role of Dan-Shen in Xin-Ke-Shu

BACKGROUND Xin-Ke-Shu (XKS), a patent medicine consisting of five commonly used traditional Chinese herbs, is used for the treatment of coronary heart diseases. A previous study showed that XKS has protective effects for ameliorating myocardial ischemia/reperfusion (I/R) injury. PURPOSE This study was aimed to deeply understand the mechanisms and compatible principle of XKS against hypoxia/reoxygenation (H/R) injury and the contribution of each single herb to the efficacy of XKS. METHODS An H/R model in H9c2 cardiomyocytes was applied to mimic I/R injury observed in vivo. The cell viability, the levels of LDH, MDA, SOD, and apoptosis were determined to evaluate the cardioprotection of XKS and its subtracted formula (knocked out one herb) in H/R injury. Cell metabolomics, combined with western blot analysis, was performed to uncover the inert molecular mechanism of XKS against H/R injury. RESULTS Significant protective effects of XKS against oxidative stress and apoptosis induced by H/R injury were found in the pharmacodynamic evaluation. Moreover, the metabolic profile deviation of the H/R group from the control group was mainly ascribed to thirteen metabolites involved in four aberrant pathways, in which sphingolipid metabolism was revealed as the most relevant pathway involved in H/R injury (impact > 0.1). Notably, the accumulation of phytosphingosine (VIP = 5.84) was considered the most likely characteristic in H/R injury, which is well known to promote the opening of the mitochondrial permeability transition pore (mPTP) and activate cell apoptosis. Furthermore, XKS ameliorated all the abnormalities of the metabolic network in response to H/R injury. In agreement with this, a western blot analysis showed that XKS markedly regulated the over-expression of CaMK II and cleaved caspase-3. However, the subtracted formula showed no significant difference in comparison with the XKS group on protecting H/R injury except for QDS (subtracted Dan-Shen from XKS). CONCLUSION The roots of Salvia miltiorrhiza Bge. (Dan-Shen) play an important role in the regulation of Ca2+ overloading, oxidative stress and apoptosis in H/R injury. Our study enabled information from holistic cell metabolomics to be used for mechanism and compatibility rule elucidations of TCMs.

Chemical profiling of Di-Wu-Yang-Gan Granules by ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with MSE technology

Abstract Di-Wu-Yang-Gan Granules is a Traditional Chinese Medicine prescription used for the treatment of HBeAg-negative chronic hepatitis B patients in China. It consists of five commonly used Chinese herbs. However, the chemical constituents of the whole prescription had not been clarified yet. Hence, in this study, the chemical profiling of Di-Wu-Yang-Gan Granules was explored by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which can provide accurate molecular weight within 5-ppm error and sufficient MS/MS fragment ions without the need for precursor ion selection. As a result, 116 compounds were identified, including lignans, triterpenesaponins, flavonoids, coumarins, iridoids, nortriterpenoids, phenolic acids, and sesquiterpenes. All compounds were further assigned to the individual herbs. In conclusion, this established method was reliable and effective for the separation and identification of the constituents in Di-Wu-Yang-Gan Granules. The findings are beneficial for quality control of the prescription during production and provide helpful chemical information for exploring its efficacy and the mechanism of action. The fragmentation regularity summarized in this study also provided important information for the rapid identification of the chemical composition in herbal medicines or their prescription.