Determination of six short-chain fatty acids in rat feces using headspace solid-phase dynamic extraction coupled with GC-MS.

Short-chain fatty acids (SCFAs) are organic acids with carbon atoms less than six, released through fermentation products by intestinal microbiome, having multiple physiological activities. Considering weak acidity and high volatility, derivatization or liquid-liquid extraction is essential, which is time consuming. Headspace-solid-phase dynamic extraction (HS-SPDE) coupled with gas chromatography-mass spectrometry is automated and effortless to determine SCFAs in rat feces. The extraction procedure is performed by aspirating and discharging the headspace cyclically through a steel needle, coated with an inner polyethylene glycol sorbent. The key parameters of SPDE were optimized including coating type, incubation time and temperature, and number of extraction strokes. Besides, salting-out was conducted. Then, a method by HS-SPDE-GC-MS was established and validated. It only took 3-min incubation time, 4.5 min extraction time, and 13 min chromatographic separation in a run. The recovery, linearity, limit of quantification, and stability were evaluated. Then, the proposed method was applied to analyze rat feces including 18 rats with liver injury and 23 normal controls. Mann-Whitney U test indicated that the concentrations of six SCFAs in normal rat feces were higher than those with liver injury. This method provides a choice for fast, solvent-free, automated, and high-throughput analysis of SCFAs.

Serum metabolomics strategy for investigating the hepatotoxicity induced by different exposure times and doses of Gynura segetum (Lour.) Merr. in rats based on GC-MS.

Gynura segetum (Lour.) Merr. (GS), has been widely used in Chinese folk medicine and can promote circulation, relieve pain and remove stasis. In recent years, the hepatotoxicity caused by GS has been reported, however its mechanism is not fully elucidated. Metabolomic techniques are powerful means to explore the toxicological mechanism and therapeutic effects of traditional Chinese medicine. The purpose of this study was to establish a serum metabolomics method based on Gas Chromatography-Mass Spectrometry (GC-MS) to explore the hepatotoxicity mechanism of different exposure times and doses of GS in rats. Sprague Dawley (SD) rats were administered daily with distilled water, 7.5 g kg-1 GS, or 15 g kg-1 GS by intragastrical gavage for either 10 or 21 days. The methods adopted included enzyme-linked immunosorbent assay (ELISA), Hematoxylin and Eosin (H&E) staining and GC-MS-based serum metabolomics. Serum biochemistry analysis showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total bilirubin (TBIL) and total bile acid (TBA) significantly (P < 0.05) increased while the levels of albumin (ALB) and high-density lipoprotein (HDL) significantly (P < 0.05) decreased in GS-treated groups, compared with the control group. Interestingly, the ALT, AST, TG and ALB levels changed in a time- and dose-dependent manner. The results of H&E staining showed the degree of liver damage after administration of GS gradually deepened with the extension of administration time and the increase of the dose. According to the results of metabolomics analysis, 26 differential metabolites were identified, which were involved in 8 metabolic pathways including phenylalanine metabolism, glyoxylic acid and dicarboxylic acid metabolism and so on. Meanwhile, the number of differential metabolites in different GS-treated groups was associated with GS exposure time and dose. Therefore, we concluded that GS might induce hepatotoxicity depending on the exposure time and dose.

Determination and tissue distribution comparisons of five xanthones after orally administering crude and processed gamboge.

Caged polyprenylated xanthones are the main active ingredients isolated from the resin of Garcinia hanburyi, which has been reported to exhibit potential anticancer and anti-inflammatory activities. This study aimed to develop sensitive and specific ultra-performance liquid chromatography coupled with the triple quadrupole mass spectrometry method for investigating the tissue distribution of five xanthones in rats: ß-morellic acid, isogambogenic acid, gambogenic acid, R-gambogic acid and S-gambogic acid. All tissue samples were prepared using the liquid-liquid extraction method and separated on a C8 column with a gradient system. Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction monitoring using positive ionization. The method established in this assay was successfully applied to the tissue distribution study of the five selected xanthones after orally administering crude and processed gamboge in rat tissues. The results indicated that these five xanthones were distributed to rat tissues rapidly and could be detected in all of the selected tissues after oral administration. After processing, the contents of R-gambogic acid and S-gambogic acid in the gastrointestinal tract were significantly reduced. The findings of this study might be helpful in further understanding the processing mechanism of gamboge and providing references for its reasonable clinical application.

Identification of a novel necroptosis-associated miRNA signature for predicting the prognosis in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most aggressive malignancies that have a poor prognosis. Necroptosis has been demonstrated in recent years to be a form of inflammatory cell death occurring in multicellular organism, which plays complex roles in cancer. However, the expression of necroptosis-related miRNAs and genes in HNSCC and their correlations with prognosis remain unclear. In this study, R software was used to screen differentially expressed miRNAs downloaded from The Cancer Genome Atlas. A prognostic model containing six necroptosis-related miRNAs (miR-141-3p, miR-148a-3p, miR-331-3p, miR-543, miR-425-5p, and miR-7-5p) was generated, whose risk score was validated as an independent prognostic factor for HNSCC. Target genes of the key miRNAs were obtained from TargetScan, miRDB, and miRTarBase, and 193 genes in the intersection of the three databases were defined as consensus genes. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses indicated that the composition of the tumor microenvironment as well as specific pathways may be closely related to necroptosis in HNSCC. Nine key genes were also obtained by the MCODE and cytoHubba plug-ins of Cytoscape: PIK3CD, NRAS, PTK2, IRS2, IRS1, PARP1, KLF4, SMAD2, and DNMT1. A prognostic model formed by the key gene was also established, which can efficiently predict the overall survival of HNSCC patients. In conclusion, necroptosis-related miRNAs and genes play important roles in tumor development and metastasis and can be used to predict the prognosis of HNSCC.

Investigation of Gynura segetum root extract (GSrE) induced hepatotoxicity based on metabolomic signatures and microbial community profiling in rats.

In recent years, many reports focus on the hepatotoxicity of Gynura segetum root extract (GSrE), but the interaction between GSrE and the gut microbiota is still unclear. This study investigated the mechanism of GSrE-induced hepatotoxicity of different doses and exposure durations by combining metabolomics and gut microbiota analysis. SD rats were divided into 3 groups: blank, low-dose (7.5 g/kg), and high-dose (15 g/kg) groups. Urine and feces samples were collected on day 0, day 10, and day 21. Metabolomics based on gas chromatography-mass spectrometry (GC-MS) was carried out to identify metabolites and metabolic pathways. 16S rDNA gene sequencing was applied to investigate the composition of gut microbiota before and after GSrE-induced hepatotoxicity. Finally, a correlation analysis of metabolites and gut microbiota was performed. Differential metabolites in urine and feces involved amino acids, carbohydrates, lipids, organic acids, and short chain fatty acids. Among them, L-valine, L-proline, DL-arabinose, pentanoic acid, D-allose, and D-glucose in urine and D-lactic acid and glycerol in fecal metabolites depended on the exposure of time and dose. In addition, 16S rDNA sequencing analysis revealed that GSrE-induced hepatotoxicity significantly altered the composition of gut microbiota, namely, f_Muribaculaceae_Unclassified, Lactobacillus, Bacteroides, Lachnospiraceae_NK4A136_group, f_Ruminococcaceae_Unclassified, Prevotellaceae_Ga6A1_group, and Escherichia-Shigella. The correlation analysis between gut microbiota and differential metabolites showed the crosstalk between the gut microbiota and metabolism in host involving energy, lipid, and amino acid metabolisms. In summary, our findings revealed that peripheral metabolism and gut microbiota disorders were time- and dose-related and the correlation between gut microbiota and metabolites in GSrE-induced hepatotoxicity.

[Simultaneous determination of 11 neurotransmitters in brain microdialysis samples from rats by UPLC-MS/MS].

This study established a method for simultaneous determination of 11 neurotransmitters, such as acetylcholine, glutamic acid, glycine, and norepinephrine from rat brain microdialysis samples using UPLC-MS/MS. A total of 20 µL of rat brain dialysate was diluted with 60 µL of acetonitrile-water(4∶1) and centrifuged for 10 min at 10 000 r·min~(-1),and 5 µL was injected into UPLC-MS/MS system for assay. Chromatographic separation was performed on a Waters ACQUITY BEH amide column(2.1 mm×100 mm, 1.7 µm) with gradient elution using acetonitrile/0.2% formic acid-water as mobile phases with a flow rate of 0.35 mL·min~(-1) and column temperature of 35 ℃. The eluate was detected by multiple-reaction monitoring(MRM) scanning with an electrospray ionization source operating in the positive ionization mode with an analysis duration of 3.5 min. The relationship between the recovery rate of 11 neurotransmitters and the perfusion rate or the concentration of neurotransmitters was investigated. Furthermore, the effects of puerarin alone or combined with borneol on the content of 11 neurotransmitters in the striatum of rats were investigated. The results showed the calibration curves displayed good linear regression with coefficients all greater than 0.99 and the lower limit of quantification(LLOQ) less than 12.5 nmol·L~(-1) for each analyte. The within-run and between-run precision(RSD) of the 11 neurotransmitters at low, medium, and high levels was less than 9.3%, and the relative error of the accuracy ranged from-8.4% to 9.5%. The stability, recovery, and matrix effects were in line with the biological sample analysis requirements. As revealed by experimental results, the levels of most neurotransmitters in the brain striatum changed significantly after rats were treated with puerarin as compared with the conditions in the blank group. Except for dopamine and norepinephrine, the degree of changes of other neurotransmitters in the combination group(borneol and puerarin) was less than that of the puerarin group. The established UPLC-MS/MS method could be applied to the quantitative determination of 11 neurotransmitters in microdialysis samples, providing an efficient and useful tool to study neurotransmitter changes in animal models of health and diseases.

Porphyromonas gingivalis Promotes Colorectal Carcinoma by Activating the Hematopoietic NLRP3 Inflammasome.

Porphyromonas gingivalis (P. gingivalis) is a keystone periodontal pathogen associated with various digestive cancers. However, whether P. gingivalis can promote colorectal cancer and the underlying mechanism associated with such promotion remains unclear. In this study, we found that P. gingivalis was enriched in human feces and tissue samples from patients with colorectal cancer compared with those from patients with colorectal adenoma or healthy subjects. Cohort studies demonstrated that P. gingivalis infection was associated with poor prognosis in colorectal cancer. P. gingivalis increased tumor counts and tumor volume in the ApcMin/+ mouse model and increased tumor growth in orthotopic rectal and subcutaneous carcinoma models. Furthermore, orthotopic tumors from mice exposed to P. gingivalis exhibited tumor-infiltrating myeloid cell recruitment and a proinflammatory signature. P. gingivalis promoted colorectal cancer via NLRP3 inflammasome activation in vitro and in vivo. NLRP3 chimeric mice harboring orthotopic tumors showed that the effect of NLRP3 on P. gingivalis pathogenesis was mediated by hematopoietic sources. Collectively, these data suggest that P. gingivalis contributes to colorectal cancer neoplasia progression by activating the hematopoietic NLRP3 inflammasome. SIGNIFICANCE: This study demonstrates that the periodontal pathogen P. gingivalis can promote colorectal tumorigenesis by recruiting myeloid cells and creating a proinflammatory tumor microenvironment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2745/F1.large.jpg.

Dihydrokaempferol (DHK) ameliorates severe acute pancreatitis (SAP) via Keap1/Nrf2 pathway.

Severe acute pancreatitis (SAP) is a non-bacterial inflammatory disease that clinically causes a very high rate of mortality. Dihydrokaempferol (DHK) is a natural flavonoid extracted from Bauhinia championii. Our research aimed to establish the treatment function of DHK on SAP-induced pancreas injury and delve into its potential mechanism. In this study, SAP was induced by caerulein (CER) and Lipopolysaccharide (LPS). DHK was administered orally at different doses of 20, 40, or 80 mg/kg. Results from serum amylase/lipase, pancreas hematoxylin-eosin staining technique, pancreas malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) showed the therapeutic effect of DHK in a mice SAP model. MTT revealed DHK alleviated CER + LPS induced cytotoxicity in a dose-dependent manner in the pancreatic acinar cells of mice. Next, we verified DHK suppressed the level of Keap1 and promoted transcriptional activation of nuclear Nrf2 in the presence of CER + LPS. The molecular docking study suggested that there is a potential interaction between DHK and Keap1. To further look at the role of Keap1 using in vitro and in vivo models, Keap1 overexpression adenovirus (ad-Keap1) was performed. The results revealed that ad-Keap1suppressed the nuclear translocation of Nrf2 which is enhanced by DHK, and suppressed the antioxidative functionality of DHK both in mice and cell models. Collectively, this research demonstrated that DHK bettered the SAP induced pancreas injury by regulating the Keap1/Nrf2 pathway and regulating oxidative stress injury.

Metabolomics analysis reveals the protective effect of quercetin-3-O-galactoside (Hyperoside) on liver injury in mice induced by acetaminophen.

We investigated the protective effect of Hyperoside (HPS) on liver injury induced by acetaminophen (APAP) in C57 mice. HPS was administered orally for 7 days and APAP was administered orally on the 7th day. Serum and liver samples were then collected for biochemical analyses, histopathology assessments, and metabolomics studies. Metabolites were assessed using a UHPLC-MS system. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to process the data. Pathway analyses were performed using Metaboanalyst 4.0. Western blot and qRT-PCR were used to determine the protein and mRNA levels, respectively. HPS interacted with active sites in CYP2E1 and caused protein degradation. In conclusion, our results suggested that HPS prevented the oxidative stress-induced liver injury caused by APAP. PRACTICAL APPLICATIONS: Hyperoside was shown to have potential protective and therapeutic effects against liver diseases. Male C57 mice were used to perform pharmacodynamic, pharmacology, and metabolomics evaluations. At a dose of 60 mg/kg, HPS prevented oxidative stress-induced liver injury caused by APAP by regulating the glutathione-related metabolites and enzymes through the inhibition of CYP2E1.

[Analysis of metabolites of 4,5-dicaffeoylquinic acid in rat plasma and urine based on LC-MS].

Ultra high performance liquid chromatography tandem high field orbital trap mass spectrometry(UPLC-Orbitrap Elite-MS/MS) method was applied in this paper to analyze the metabolites of 4,5-dicaffeoylquinic acid in rat plasma and urine after oral administration. A gradient elution was performed by using Thermo C_(18) column(2.1 mm×100 mm, 1.9 µm), with 0.1% formic acid solution-acetonitrile as the mobile phase. Mass spectral data of biological samples were collected in negative ion mode. The data were extracted by Compound Discovery 2.1 software. Then the blank group samples and the drug samples were compared for exact molecular weight and the mass fragmentation information, and the secondary fragment fitting ratio was calculated to finally attribute the metabolites. As a result, 15 metabolites were detected in rat plasma, and 16 metabolites were detected in urine. The involving metabolic reactions included methylation, hydration, dehydration, reduction, glucuronide conjugation, and sulfation reaction. The metabolites in plasma and urine complemented each other and initially revealed the migration and excretion patterns of this compound in the body. A method for pre-processing biological samples, high-resolution LC-MS instrumentation data, and qualitative software was established in this study to identify metabolite structures, laying the foundation for the study of the active ingredients and in vivo pharmacodynamics forms of Chinese medicines.

Intracellular Porphyromonas gingivalis Promotes the Proliferation of Colorectal Cancer Cells via the MAPK/ERK Signaling Pathway.

Porphyromonas gingivalis (P. gingivalis) is a keystone pathogen in periodontitis. However, several clinical studies have revealed an enrichment of P. gingivalis in the stool samples and colorectal mucosa of colorectal cancer patients. Thus, the goal of this study was to determine whether P. gingivalis can promote colorectal cancer progression in vitro. We established an acute infection model (24 h, multiplicity of infection =100) of P. gingivalis invasion of colorectal cancer cells to study the alterations induced by P. gingivalis in the proliferation and cell cycle of colorectal cancer cells. We observed that P. gingivalis can adhere and invade host cells a few hours after infection. Once invaded, P. gingivalis significantly promoted colorectal cancer cell proliferation, and the percentage of S phase cells was increased in the cell cycle assay. However, KDP136, a gingipain-deficient mutant of P. gingivalis 33277, showed a decreased ability to promote colorectal cancer cell proliferation, indicating that gingipain is associated with colorectal cancer cell proliferation. Furthermore, we extracted RNA from colorectal cancer cells for high-throughput sequencing analysis and reconfirmed the results by quantitative polymerase chain reaction and western blot analyses. The results suggested that the MAPK/ERK signaling pathway is significantly activated by P. gingivalis, while these changes were not observed for KDP136. In conclusion, P. gingivalis can invade cells and promote the proliferation of colorectal cancer cells by activating the MAPK/ERK signaling pathway. Gingipain is an essential virulence factor in this interaction.

5,7,3',4'-flavan-on-ol (taxifolin) protects against acetaminophen-induced liver injury by regulating the glutathione pathway.

Taxifolin (TAX) reportedly exerts protective and therapeutic effects in liver. Herein, the effects of TAX against acetaminophen (APAP)-induced hepatotoxicity were investigated. Pharmacodynamics, pharmacology and metabolomics analyses of TAX were assessed on C57 mice and L-02 cells. TAX was administered for 7 days, and APAP was given on the last day to establish an acute liver injury model. ALT and AST levels were determined, and liver ROS, MDA, GST, GSH and GPX1 were analysed. The expression and protein abundance of GPX1, GPS-Pi, GCLC and GCLM were assessed by PCR and western blotting, and metabolic changes in cells and serum were investigated by UPLC-Q-Orbitrap-MS. Serum ALT and AST, and liver ROS, MDA, GST, GSH and GPX1 levels confirmed the protective effects of TAX. Besides, we found Only treating with TAX decreased the expression of CYP2E1 in mice liver tissue. TAX reversed the APAP-induced decrease in cell viability in L-02 cells, and reduced cellular ROS levels. Furthermore, TAX reversed the APAP-induced decrease in antioxidant enzymes at both mRNA and protein levels. Metabolomics analysis identified metabolites mainly related to glutathione metabolism (36 in vivo and 23 in vitro). The concentration of glutathione, oxidized glutathione, carnitine, succinic acid, pyroglutamic acid, citrulline, taurine, palmitoleic acid, phytoshingosine-1-P and sphingosine-1-P were close to normal levels after treating with TAX. These results indicate that TAX prevents APAP-induced liver injury by inhibiting APAP metabolic activation mediated by CYP450 enzymes, modulating glutathione metabolism, and expression of related antioxidative signals. These properties could be harnessed to prevent or treat hepatotoxicity.

Protective effect of 7,3',4'-flavon-3-ol (fisetin) on acetaminophen-induced hepatotoxicity in vitro and in vivo.

BACKGROUND: Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure in clinic. Fisetin (FST) is a phenolic compound that has been isolated from many natural products. PURPOSE: Our aim is to study the protection effect and mechanisms of FST on APAP-induced hepatotoxicity in endogenous metabolism and metabolomics in vitro and in vivo. METHODS: FST was i.g. administered to mice at 10, 20 and 40 mg/kg for 7 days and a single dose of APAP (400 mg/kg) was given on the last day. Serum and tissue were collected for biochemical analysis. L-02 cells were used to assess cell viability. LC-MS was used to study the metabolic fingerprinting in vivo and vitro. PCA and OPLS-DA were used to search the potential biomarkers (VIP > 1, p < 0.05). The pathway analysis was conducted on Metaboanalyst 4.0. Then liver oxidative stress indices and glutathione markers were examined using PCR and kits. RESULTS: ALT, AST, liver histological observation and cell viability results showed that FST could reverse APAP induced toxicology in mice and L-02 cells. In metabolomics study, 26 metabolites in vitro and 60 metabolites in vivo were identified by searching in the library and most of them decreased to normal level in FST treatment. It is observed in pathway analysis that the most significant pathway was glutathione metabolism. Furthermore, the results of mRNA and immunofluorescence showed that FST suppressed ROS formation in liver tissue and L-02 cells, as well as restored the expression of GPX1, GST and other antioxidative enzymes genes. CONCLUSION: Our results indicate that FST prevented APAP-induced hepatotoxicity by regulating glutathione metabolism and the expression of related antioxidative signals.

Prognostic value of immune checkpoint molecules in head and neck cancer: a meta-analysis.

Immune checkpoint molecules are important targets in cancer immunotherapy, but their association with prognosis in patients with head and neck cancer is controversial. In this meta-analysis, we searched for 12 immune checkpoint molecules in the PubMed, Embase and Cochrane Library databases and retrieved 52 studies with 7127 participants. Among the molecules included in the search, indoleamine 2, 3-dioxygenase (IDO), programmed death ligand 1 (PD-L1), and programmed death 1 (PD-1) met the inclusion criteria for further analysis. Higher expression of IDO was associated with poorer overall survival in head and neck cancer patients (P = 0.011), but higher expression of PD-L1 correlated with better overall survival specifically in nasopharyngeal carcinoma patients (P = 0.01). In a sensitivity analysis, higher PD-L1 expression correlated with better progression-free survival (P = 0.043), and was associated with better overall survival in Caucasian subjects (P = 0.02), nasopharyngeal carcinoma patients (P = 0.015), and studies with small sample sizes (P = 0.001). PD-1 had no prognostic significance. There was no publication bias affecting the results. Thus, among the immune checkpoint molecules, IDO and PD-L1 are potential prognostic predictors in head and neck cancer.

Qualitative and quantitative determination of Atractylodes rhizome using ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry with data-dependent processing.

A quick and effective workflow based on ultra-performance liquid chromatography coupled with electron spray ionization and LTQ-Orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS) was established for compositional analysis and screening of the characteristic compounds of three species of Atractylodes rhizome for quality evaluation. This technique was employed to determine the seven main components in Atractylodes rhizome samples. Ultimately, 78 constituents were identified; of these, seven characteristic compounds were selected for species discrimination, comprising atractylodin (63), atractylenolide I (43), atractylenolide II (49), atractylenolide III (53), atractylon (69), methyl-atractylenolide II (54) and (4E,6E,12E)-tetradecadecatriene-8,10-diyne-1,3-diacetate (59). The seven main compounds, including six characteristic compounds, were simultaneously determined in 29 batches of Atractylodes rhizome samples. Thus, the method validation showed acceptable results. Quantitative analysis showed significantly different contents of the seven main components among the three species of Atractylodes rhizome, which indicates possible distinctions in the pharmacological effects. This established method can simultaneously provide qualitative and quantitative results for compositional characterization of Atractylodes rhizomes and for quality control.

New method for quantification of gasotransmitter hydrogen sulfide in biological matrices by LC-MS/MS.

Hydrogen sulfide exists widely in mammalian tissues and plays a vital role in physiological and pathophysiological processes. However, striking differences with orders of magnitude were observed for the detected hydrogen sulfide concentrations in biological matrices among different measurements in literature, which lead to the uncertainty for examination the biological relevance of hydrogen sulfide. Here, we developed and validated a liquid chromatography- mass spectrometry (LC-MS/MS) method for the determination of hydrogen sulfide in various biological matrices by determination of a derivative of hydrogen sulfide and monobromobimane named sulfide dibimane (SDB). 36S-labeled SDB was synthesized and validated for using as an internal standard. This method has been successfully used to measure hydrogen sulfide levels in a broad range of biological matrices, such as blood, plasma, tissues, cells, and enzymes, across different species. Moreover, a novel mode that hydrogen sulfide could loosely and non-covalently bind to human serum protein (HSA) and hemoglobin (HB) was revealed by using the developed method.

Naturally occurring proteinaceous nanoparticles in Coptidis Rhizoma extract act as concentration-dependent carriers that facilitate berberine absorption.

Pharmacological activities of some natural products diminish and even disappear after purification. In this study, we explored the mechanisms underlying the decrease of acute oral toxicity of Coptidis Rhizoma extract after purification. The water solubility, in vitro absorption, and plasma exposure of berberine (the major active compound) in the Coptidis Rhizoma extract were much better than those of pure berberine. Scanning electron microscopy, laser scanning confocal microscopy (LSCM), and dynamic light scattering experiments confirmed that nanoparticles attached to very fine precipitates existed in the aqueous extract solution. The LSCM experiment showed that the precipitates were absorbed with the particles by the mouse intestine. High-speed centrifugation of the extract could not remove the nanoparticles and did not influence plasma exposure or acute oral toxicity. However, after extract dilution, the attached precipitates vanished, although the nanoparticles were preserved, and there were no differences in the acute oral toxicity and plasma exposure between the extract and pure berberine. The nanoparticles were then purified and identified as proteinaceous. Furthermore, they could absorb co-dissolved berberine. Our results indicate that naturally occurring proteinaceous nanoparticles in Coptidis Rhizoma extract act as concentration-dependent carriers that facilitate berberine absorption. These findings should inspire related studies in other natural products.

Long-term effect of early postnatal overnutrition on insulin resistance and serum fatty acid profiles in male rats.

BACKGROUND: Increasing evidence suggests that overnutrition during the early postnatal period, a critical window of development, increases the risk of adult-onset obesity and insulin resistance. In this study, we investigated the impact of overnutrition during the suckling period on body weight, serum biochemistry and serum fatty acid metabolomics in male rats. METHODS: Rats raised in small litters (SL, 3 pups/dam) and normal litters (NL, 10 pups/dam) were used to model early postnatal overnutrition and control, respectively. Serum glucose, triglyceride, high-density lipoprotein-cholesterol, free fatty acid, insulin and leptin concentrations were assayed using standard biochemical techniques. Serum fatty acids were identified and quantified using a gas chromatography-mass spectrometry-based metabolomic approach. mRNA and protein levels of key components of the insulin receptor signaling pathway were measured in epididymal fat and gastrocnemius muscle by quantitative PCR and western blotting. RESULTS: SL rats were 37.3 % and 15.1 % heavier than NL rats at weaning and 16-weeks-old, respectively. They had increased visceral fat mass, adult-onset insulin resistance and glucose intolerance as well as elevated serum levels of free fatty acids and triglycerides. All detectable fatty acids were elevated in the serum of SL pups at weaning compared to NL controls, and significant increases in the levels of four fatty acids (palmitic acid, palmitoleic acid, oleic acid and arachidonic acid) persisted into adulthood. Moreover, a significantly positive correlation was identified between an insulin resistance index (HOMA-IR) and concentrations of myristic, palmitic, palmitoleic and oleic acid in serum at postnatal 16 weeks. Early postnatal overnutrition also resulted in a significant downregulation of insulin receptor substrate-1 (Irs-1), protein kinase B (Akt2) and glucose transporter 4 (Glut4) at the protein level in epididymal fat of SL rats at 16 weeks, accompanied by decreased mRNA levels for Irs-1 and Glut4. In gastrocnemius muscle, Akt2 and Glut4 mRNA and Glut4 protein levels were significantly decreased in SL rats. CONCLUSIONS: This study demonstrates that early postnatal overnutrition can have long-lasting effects on body weight and serum fatty acid profiles and can lead to impaired insulin signaling pathway in visceral white adipose tissue and skeletal muscle, which may play a major role in IR.

Identification and pharmacokinetics of multiple constituents in rat plasma after oral administration of Yinchenzhufu decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Yinchenzhufu decoction (YCZFD) is a classical Chinese herbal formula and has been used to treat severe jaundice in chronic liver injuries since the Qing Dynasty (18th century CE). To identify the components absorbed into the blood in YCZFD and explore their pharmacokinetic profile for understanding the effective ingredients of YCZFD. MATERIALS AND METHODS: After rats were given YCZFD by intragastric administration, the plasma was processed by precipitation of protein. The compounds in YCZFD extract and the plasma were identified by using high-resolution mass spectrometry with a database-directed strategy. The pharmacokinetics of multiple compounds from YCZFD in rat plasma was studied by using the established UPLC-MS/MS method. RESULTS: Forty compounds in YCZFD extract and 21 prototype compounds with 11 metabolites in rat plasma were detected after oral administration. The pharmacokinetic parameters of glycyrrhizic acid, glycyrrhetic acid, cinnamic acid, ononin, atractylenolide III, and liquiritin from YCZFD were obtained in rats. CONCLUSIONS: The identified constituents and the pharmacokinetic features of YCZFD are helpful for understanding the material bases of its therapeutic effects.