Multiple-matrix metabolomics analysis for the distinct detection of colorectal cancer and adenoma.

OBJECTIVES: Although colorectal cancer (CRC) is the leading cause of cancer-related morbidity and mortality, current diagnostic tests for early-stage CRC and colorectal adenoma (CRA) are suboptimal. Therefore, there is an urgent need to explore less invasive screening procedures for CRC and CRA diagnosis. METHODS: Untargeted gas chromatography-mass spectrometry (GC-MS) metabolic profiling approach was applied to identify candidate metabolites. We performed metabolomics profiling on plasma samples from 412 subjects including 200 CRC patients, 160 CRA patients and 52 normal controls (NC). Among these patients, 45 CRC patients, 152 CRA patients and 50 normal controls had their fecal samples tested simultaneously. RESULTS: Differential metabolites were screened in the adenoma-carcinoma sequence. Three diagnostic models were further developed to identify cancer group, cancer stage, and cancer microsatellite status using those significant metabolites. The three-metabolite-only classifiers used to distinguish the cancer group always keeps the area under the receiver operating characteristic curve (AUC) greater than 0.7. The AUC performance of the classifiers applied to discriminate CRC stage is generally greater than 0.8, and the classifiers used to distinguish microsatellite status of CRC is greater than 0.9. CONCLUSION: This finding highlights potential early-driver metabolites in CRA and early-stage CRC. We also find potential metabolic markers for discriminating the microsatellite state of CRC. Our study and diagnostic model have potential applications for non-invasive CRC and CRA detection.

Nanoplastics Shape Adaptive Anticancer Immunity in the Colon in Mice.

The impact of nanoplastics (NPs) on human health is still not well understood, and more research is needed to better understand the risks associated with these particles. In this study, we found that oral administration of polyethylene (PE) NPs in a mice model significantly disrupted the intestinal microenvironment, which shapes adaptive immune response and favors the established in situ colorectal tumor growth. Using single-cell RNA sequencing technology, we show that NPs triggered colon IL-1ß-producing macrophages by inducing lysosome damage, leading to colonic Treg and Th17 differentiation associated with T cell exhaustion, which creates a colon environment that favors the tumor initiation and progress. A similar effect is also observed in polystyrene NPs. Our result provides insight into the potential link between NPs ingestion and colon tumorigenesis, and the urgency of addressing plastic pollution worldwide.

The colonic metabolism differences of main alkaloids in normal and colitis mice treated with Coptis chinensis Franch. and Sophora flavescens Ait. herbal pair using liquid chromatography-high resolution mass spectrometry method combined with chemometrics.

Coptis chinensis Franch. and Sophora flavescens Ait. is a herbal pair frequently used in treating ulcerative colitis. However, the bio-disposition profile of the major components in the inflamed gut remains unclear, which is essential to understand the pharmacological material basis of this herb pair. Here we established an integral quantitative and chemometric method to deduce the colonic metabolism differences of this herbal pair in normal and colitis mice. With this LC-MS method, a total of 41 components have been found in the Coptis chinensis Franch. and Sophora flavescens Ait. extract, and 28 metabolites were found in the colon after oral administration. Alkaloid and its phase I metabolites were the main components in the colon of normal and colitis mice. The results of principal component analysis at 6 h after oral administration showed significant colonic metabolism differences between normal and colitis mice. Heamap results showed that colitis induced significant changes in the colonic bio-disposition of this herbal pair extract. In particular, in the context of colitis, the phase I metabolism of berberine, coptisine, jatrorrhizine, palmatine,and epiberberine has been inhibited. These results may provide a basis for understanding the pharmacological material basis of Coptis chinensis Franch. and Sophora flavescens Ait. in treating ulcerative colitis.

[Guidelines for prevention and treatment of colorectal adenoma with integrated Chinese and western medicine].

The Guidelines for prevention and treatment of colorectal adenoma with integrated Chinese and western medicine are put forward by Nanjing University of Chinese Medicine and approved by China Association of Chinese Medicine. According to the formulation processes and methods of relevant clinical practice guidelines, the experts in clinical medicine and methodology were organized to discuss the key problems to be addressed in the clinical prevention and treatment of colorectal adenoma(CRA) and provided answers following the evidence-based medicine method, so as to provide guidance for clinical decision-making. CRA is the major precancerous disease of colorectal cancer. Although the prevention and treatment with integrated Chinese and western medicine have been applied to the clinical practice of CRA, there is still a lack of high-quality guidelines. Four basic questions, 15 clinical questions, and 10 outcome indicators were determined by literature research and Delphi questionnaire. The relevant randomized controlled trial(RCT) was retrieved from CNKI, Wanfang, VIP, SinoMed, PubMed, EMbase, Cochrane Library, Web of Science, and 2 clinical trial registries, and finally several RCTs meeting the inclusion criteria were included. The data extracted from the RCT was imported into RevMan 5.3 for evidence synthesis, and the evidence was evaluated based on the Grading of Recommendations, Assessment, Development, and Evaluations(GRADE). The final recommendations were formed by the nominal group method based on the evidence summary table. The guidelines involve the diagnosis, screening, treatment with integrated Chinese and western medicine, prevention, and follow-up of colorectal adenoma, providing options for the clinical prevention and treatment of CRA.

Identification of peptides of cinobufacini by gel filter chromatography and peptidomics.

Aqueous extract of toad skin (named as Cinobufacini or Huachansu) provides plentiful sources of bioactive peptides that remain undetected and unidentified. High-resolution mass spectrometry-based peptidomics platforms have developed into a major approach to the discovery of natural peptides, with data-dependent acquisition modes providing a wealth of peptide profiling information. In this study, we used a gel- and HLB (a solid phase extraction cartridge)-based two-dimensional separation and purification system and nano-liquid chromatography-tandem mass spectrometry-based peptidomic studies with homology matching for the identification of peptides from Cinobufacini. We evaluated 232 multi-charged peptides and found several specific peptides, some of which were validated by target parallel reaction monitoring mode. These peptides are the first to be identified in Cinobufacini and are completely different from ones identified in toad venom. So, this mapping provides key peptide information for the quality control of Bufo bufo gargarizans skin and its preparation.

Characterization of the chemical constituents and in vivo metabolic profile of Scutellaria barbata D. Don by ultra high performance liquid chromatography with high-resolution mass spectrometry.

Scutellaria barbata D. Don (S. barbata) is one of the most frequently used anticancer herb medicine in China. Mechanistic understanding of the biological activities of S. barbata is hindered by limited knowledge regarding its components and metabolic profile. In this study, ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (quadrupole time-of-flight mass spectrometry) was used to identify the chemical constituents in S. barbata and their metabolic profiles in rats. By applying cleavage rules and comparison with reference substances, 89 components were identified in S. barbata, which included 45 flavonoids, 28 diterpenoids, 10 phenolics, and 6 others. A total of 110 compounds, including 32 prototype compounds and 78 metabolites, were identified or tentatively characterized in vivo. Methylation, sulfonation, and glucuronidation were the main metabolic pathways, which could be attributed to the fact that several of the compounds in S. barbata have phenolic hydroxyl groups. This is the first systematic study on the chemical constituents and in vivo metabolic profile of S. barbata. The analytical method features a quick and comprehensive dissection of the chemical composition and metabolic profile of S. barbata and provides a basis for exploring its various biological activities.

[Multi-index components of Scutellariae Barbatae Herba according to UHPLC-QTRAP-MS coupled with chemometrics].

The present study developed an ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap composite mass spectrometry(UHPLC-QTRAP-MS) to simultaneously determine the content of potential active components in Scutellariae Barbatae Herba and also to provide a reference approach for screening out the differential quality control components among different batches of Scutellariae Barbatae Herba. Chromatographic separations were conducted on a Thermo Acclaim~(TM) RSLC 120 C_(18) column(3.0 mm×100 mm, 2.2 µm) in a gradient program. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile, and the column temperature was maintained at 40 ℃. The flow rate was 0.4 mL·min~(-1) and the injection volume was 2 µL. The targeted compounds were monitored in the multiple reaction monitoring(MRM) mode. The acquired data were processed by hierarchical cluster analysis(HCA) and partial least square discriminant analysis(PLS-DA). Sixteen compounds all showed good linear relationship within the corresponding linear ranges and the R~2 values were all higher than 0.993 2. The RSDs of precision, repeatability, and stability were less than or equal to 3.7%. Mean recovery rates were in the range of 95.67% and 104.8% with RSDs≤3.2%. According to HCA and PLS-DA, all samples were clustered into four categories. Scutellarin, acteoside, scutellarein, and scutebarbatine X(VIP>1) were considered as differential chemical markers in the four categories. In conclusion, the developed method can be used for the simulta-neous determination of the multiple components and quality control of Scutellariae Barbatae Herba.

Bruceine A induces cell growth inhibition and apoptosis through PFKFB4/GSK3ß signaling in pancreatic cancer.

Pancreatic cancer is one of the most aggressive cancers with a poor prognosis and 5-year low survival rate. In the present study, we report that bruceine A, a quassinoid found in Brucea javanica (L.) Merr. has a strong antitumor activity against human pancreatic cancer cells both in vitro and in vivo. Human proteome microarray reveals that 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is the candidate target of bruceine A and both fluorescence measurement and microscale thermophoresis suggest bruceine A binds to PFKFB4. Bruceine A suppresses glycolysis by inhibiting PFKFB4, leading to cell cycle arrest and apoptosis in MIA PaCa-2 cells. Furthermore, glycogen synthase kinase-3 ß (GSK3ß) is identified as a downstream target of PFKFB4 and an PFKFB4-interacting protein. Moreover, bruceine A induces cell growth inhibition and apoptosis through GSK3ß, which is dysregulated in pancreatic cancer and closely related to the prognosis. In all, these findings suggest that bruceine A inhibits human pancreatic cancer cell growth via PFKFB4/GSK3ß-mediated glycolysis, and it may serve as a potent agent for curing human pancreatic cancer.

Worenine reverses the Warburg effect and inhibits colon cancer cell growth by negatively regulating HIF-1α.

BACKGROUND: Some natural compounds inhibit cancer cell growth in various cancer cell lines with fewer side effects than traditional chemotherapy. Here, we explore the pharmacological effects and mechanisms of worenine (isolated from Coptis chinensis) against colorectal cancer. METHODS: The effects of worenine on colorectal cancer cell proliferation, colony formation and cell cycle distribution were measured. Glycolysis was investigated by examining glucose uptake and consumption, lactate production, and the activities and expressions of glycolysis enzymes (PFK-L, HK2 and PKM2). HIF-1α was knocked down and stimulated in vitro to investigate the underlying mechanisms. RESULTS: Worenine somewhat altered the glucose metabolism and glycolysis (Warburg effect) of cancer cells. Its anti-cancer effects and capability to reverse the Warburg effect were similar to those of HIF-1α siRNA and weakened by deferoxamine (an HIF-1α agonist). CONCLUSION: It is suggested that worenine targets HIF-1α to inhibit colorectal cancer cell growth, proliferation, cell cycle progression and the Warburg effect.

Lobetyolin induces apoptosis of colon cancer cells by inhibiting glutamine metabolism.

The purpose of the present study was to evaluate the anti-cancer property of Lobetyolin on colorectal cancer and explore its potential mechanism. Lobetyolin was incubated with HCT-116 cells in the absence or presence of ASCT2 inhibitor Benser or p53 inhibitor Pifithrin-α. The levels of glutamine, glutamic acid, α-ketoglutarate, ATP and GSH were determined to measure the glutamine metabolism. Annexin V-FITC/PI staining and TUNEL assay were applied to estimate the apoptotic condition. The levels of ASCT2 were examined by RT-qPCR, Western blot and immunofluorescence staining. The expressions of cleaved-caspase-3, caspase-3, cleaved-caspase-7, caspase-7, cleaved-PARP, PARP, p53, p21, bax and survivin were detected using Western blot analysis. As a result, the treatment with Lobetyolin effectively induced apoptosis and glutamine metabolism in HCT-116 cells through ASCT2 signalling. The inhibition of ASCT2 reduced the glutamine-related biomarkers and augmented the apoptotic process. We further found that the effect of Lobetyolin on HCT-116 was related to the expressions of p21 and bax, and transportation of p53 to nucleus. The inhibition of p53 by Pifithrin-α promoted the inhibitory effect of Lobetyolin on ASCT2-mediated apoptosis. Lobetyolin also exerted anti-cancer property in nude mice. In conclusion, the present work suggested that Lobetyolin could induce the apoptosis via the inhibition of ASCT2-mediated glutamine metabolism, which was possibly governed by p53.

Daphnetin ameliorates experimental colitis by modulating microbiota composition and Treg/Th17 balance.

Inflammatory bowel diseases (IBDs) are characterized by chronic pathologies associated with extensive gut dysbiosis and intestinal inflammation. Hence, endeavors to improve the inflammatory pathology by manipulating gut microbiota are ongoing. Daphnetin (DAPH) is a coumarin derivative extracted from Daphne odora var with anti-inflammatory and immune-regulatory properties that has been widely used in treating inflammatory disorders. Herein, we showed that DAPH remarkably alleviated experimental colitis by reducing colonic inflammation, improving colonic integrity, and reestablishing immune and metabolic homeostasis in the inflicted intestines. Our analysis showed that DAPH modified the composition of gut microbiota and altered the metabolic profiles in dextran sulfate sodium-treated mice. In particular, this agent significantly elevated the abundance of short-chain fatty acid (SCFA)-producing gut microbiota, causatively related with the enhanced development of Treg cells and the reduced proinflammatory Th17 cell differentiation. More critically, the protective effect of DAPH was shown to be transmissible among colitic mice through cohousing or fecal microbiota transplantation, further substantiating the importance of SCFA-producing gut microbiota in DAPH action. We thus for the first time reveal the potential of DAPH in resetting the gut microbiome and reestablishing immune homeostasis in colitic mice, which may have clinical implications for treating IBD.-Ji, J., Ge, X., Chen, Y., Zhu, B., Wu, Q., Zhang, J., Shan, J., Cheng, H., Shi, L. Daphnetin ameliorates experimental colitis by modulating microbiota composition and Treg/Th17 balance.

Functionalized reduced graphene oxide with aptamer macroarray for cancer cell capture and fluorescence detection.

An integrated aptamer macroarray functionalized with reduced graphene oxide (rGO) to specifically capture and sensitively detect cancer cells is reported. The capture for cancer cells is based on effective recognition of the modified rGO surface through the aptamer against epithelial cell adhesion molecule (EpCAM). The rough structure of rGO enhances morphologic interactions between rGO film interface and the cancer cells, while super-hydrophilicity of modified rGO hinders nonspecific cell capture. The synergistic interactions offer the aptamer macroarray high efficiency of cancer cell capture. By means of a turn-on fluorescence strategy based on the conformation change of the aptamer induced by the target recognition, the enriched cancer cells can be directly read out at excitation/emission wavelengths of 550/680 nm without washing, separation, and dying steps. The working range is 1 × 102 to 2 × 104 cells per mL with a detection limit of 22 cells per mL. The results indicate that the aptamer macroarray has a considerable foreground for early diagnosis, therapy, and monitoring of cancer. Graphical abstract.

Automatic Recognition of Sucker-Rod Pumping System Working Conditions Using Dynamometer Cards with Transfer Learning and SVM.

Sucker-rod pumping systems are the most widely applied artificial lift equipment in the oil and gas industry. Accurate and intelligent working condition recognition of pumping systems imposes major impacts on oilfield production benefits and efficiency. The shape of dynamometer card reflects the working conditions of sucker-rod pumping systems, and different conditions can be indicated by their typical card characteristics. In traditional identification methods, however, features are manually extracted based on specialist experience and domain knowledge. In this paper, an automatic fault diagnosis method is proposed to recognize the working conditions of sucker-rod pumping systems with massive dynamometer card data collected by sensors. Firstly, AlexNet-based transfer learning is adopted to automatically extract representative features from various dynamometer cards. Secondly, with the extracted features, error-correcting output codes model-based SVM is designed to identify the working conditions and improve the fault diagnosis accuracy and efficiency. The proposed AlexNet-SVM algorithm is validated against a real dataset from an oilfield. The results reveal that the proposed method reduces the need for human labor and improves the recognition accuracy.

Traditional Chinese medicine as targeted treatment for epithelial-mesenchymal transition-induced cancer progression.

The epithelial-mesenchymal transition (EMT) program, which loosens cell-cell adhesion complexes, endows cells with enhanced migratory and invasive properties. Furthermore, this process facilitates both the development of drug resistance and immunosuppression by tumor cells, which preclude the successful treatment of cancer. Recent research has demonstrated that many signaling pathways are involved in EMT progression. In addition, cancer stem cells (CSCs), vasculogenic mimicry (VM) and the tumor-related immune microenvironment all play important roles in tumor formation. However, there are few reports on the relationships between EMT and these factors. In addition, in recent years, traditional Chinese medicine (TCM) has developed a unique system for treating cancer. In this review, we summarize the crucial signaling pathways associated with the EMT process in cancer patients and discuss the interconnections between EMT and other molecular factors (such as CSCs, VM, and the tumor-related immune microenvironment). We attempt to identify common regulators that might be potential therapeutic targets to thereby optimize tumor treatment. In addition, we outline recent research on TCM approaches that target EMT and thereby provide a foundation for further research on the exact mechanisms by which TCMs affect EMT in cancer.

LC-MS/MS analysis and evaluation of the anti-inflammatory activity of components from BushenHuoxue decoction.

CONTEXT: BushenHuoxue decoction (BSHXD) is a Chinese medicine prescription, which is composed of nine Chinese medical materials, used to treat osteoarthritis (OA). OBJECTIVE: This study develops sensitive and convenient LC-MS/MS methods to analyze chemical components from BSHXD, and assess the anti-inflammatory activities thereof. MATERIALS AND METHODS: The chemical composition from BSHXD water extract was qualitative analyzed by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS). Twelve reference compounds were analyzed by UPLC-ESI-MS/MS. Anti-inflammatory activities of target components were assessed by ELISA at 20 and 100 µg/mL. RESULTS: It is the first time that 88 compounds were qualitatively identified from BSHXD, of which 12 with potential in treating OA according to the literature were quantified. Within BSHXD the contents of quercetin, isopsoralen, icarisideII, osthole, and isoimperatorin increased remarkably compared with those in single herb which make up BSHXD, the contents were 0.1999, 0.4634, 0.0928, 0.5364, and 0.1487 mg/g. ELISA data displayed that BSHXD and the five compounds mentioned inhibited the expressions of TNF-α, IL-6 and NO released from LPS-stimulated RAW264.7 cell, with maximum inhibition rates of 104.05% (osthole, 100 µg/mL), 100.03% (osthole, 100 µg/mL), and 93.46% (isopsoralen, 20 µg/mL), respectively. DISCUSSION AND CONCLUSION: Content changes of 12 compounds in BSHXD and single herbs which comprise the prescription were measured and analyzed. Contents of five compounds increased may be explained by solubilization between drugs and chemical reaction. ELISA results reported that the increased contents of the five compounds could inhibit expression of the inflammatory factors.

Quality assessment of Fructus Ligustri Lucidi by the simultaneous determination of six compounds and chemometric analysis.

A comprehensive strategy was designed for the quality assessment of Fructus Ligustri Lucidi, a well-known and commonly used herbal medicine in clinical practice in China. First, a simple and stable method of high-performance liquid chromatography was developed for the simultaneous quantitative analysis of six compounds, namely, salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid in Fructus Ligustri Lucidi. The separation of analytes was conducted on a C18 column (200 mm × 4.6 mm, 5 µm) at 30°C, and the wavelength of UV detector was set at 210 nm. In quantitative analysis, all of the calibration curves showed good linear regression (R(2) > 0.9994) within the tested ranges, and the mean recoveries of three different concentrations ranged from 95.21-102.34%. The described method was applied to determine 11 batches of samples collected from different stores in China. Then multiple chemometrics analysis including hierarchical cluster analysis and principal component analysis were performed to classify samples and search significant compounds. Three notable compounds, specnuezhenide, oleanic acid, and acetyl oleanic acid, were discovered for better quality control compared with those stated in the China pharmacopeia. The results demonstrated that this strategy could be readily utilized for the comprehensive quality control of Fructus Ligustri Lucidi.

Hepatoprotective triterpene saponins from the roots of Glycyrrhiza inflata.

Two novel oleanane-type triterpene saponins, licorice-saponin P2 (1) and licorice-saponin Q2 (3), together with nine known compounds 2, 4-11, have been isolated from the water extract of the roots of Glycyrrhiza inflata. The structures of these compounds were elucidated on the basis of spectroscopic analysis, including 2D-NMR experiments (1H-1H COSY, HSQC, HMBC and ROESY). In in vitro assays, compounds 2-4, 6 and 11 showed significant hepatoprotective activities by lowering the ALT and AST levels in primary rat hepatocytes injured by D-galactosamine (D-GalN). In addition, compounds 2-4, 6, 7 and 11 were found to inhibit the activity of PLA2 with IC50 values of 6.9 µM, 3.6 µM, 16.9 µM, 27.1 µM, 32.2 µM and 9.3 µM, respectively, which might be involved in the regulation of the hepatoprotective activities observed.

[Relevance of cancer toxin pathogenesis theory with transformation of inflammation to carcinoma].

The "Cancer Toxin" pathogenesis theory is an innovate theoretical system for cancer pathogenesis of Chinese Medicine, which was built on the basis of "Cancer Toxin" concept initially raised by Professor ZHOU Zhong-ying. The mechanism of the transformation from inflammation to carcinoma has become one of hot-points in the field of cancer research at home and abroad in recent years. We focused on discussing the relevance of the "Cancer Toxin" pathogenesis theory with the transformation mechanism from inflammation to cancer, provided evidence for using "Cancer Toxin" pathogenesis theory in intervening transformation from inflammation to cancer, hoping to guide for Chinese medical prevention and treatment of tumor.

Integrated plasma and urine metabolomics coupled with HPLC/QTOF-MS and chemometric analysis on potential biomarkers in liver injury and hepatoprotective effects of Er-Zhi-Wan.

Metabolomics techniques are the comprehensive assessment of endogenous metabolites in a biological system and may provide additional insight into the molecular mechanisms. Er-Zhi-Wan (EZW) is a traditional Chinese medicine formula, which contains Fructus Ligustri Lucidi (FLL) and Herba Ecliptae (HE). EZW is widely used to prevent and treat various liver injuries through the nourishment of the liver. However, the precise molecular mechanism of hepatoprotective effects has not been comprehensively explored. Here, an integrated metabolomics strategy was designed to assess the effects and possible mechanisms of EZW against carbon tetrachloride-induced liver injury, a commonly used model of both acute and chronic liver intoxication. High-performance chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS) combined with chemometric approaches including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discover differentiating metabolites in metabolomics data of rat plasma and urine. Results indicate six differentiating metabolites, tryptophan, sphinganine, tetrahydrocorticosterone, pipecolic acid, L-2-amino-3-oxobutanoic acid and phosphoribosyl pyrophosphate, in the positive mode. Functional pathway analysis revealed that the alterations in these metabolites were associated with tryptophan metabolism, sphingolipid metabolism, steroid hormone biosynthesis, lysine degradation, glycine, serine and threonine metabolism, and pentose phosphate pathway. Of note, EZW has a potential pharmacological effect, which might be through regulating multiple perturbed pathways to the normal state. Our findings also showed that the robust integrated metabolomics techniques are promising for identifying more biomarkers and pathways and helping to clarify the function mechanisms of traditional Chinese medicine.