Targeted Metabolic Profiling and PRM Analysis of Proteins Revealed Impaired Polyunsaturated Fatty Acid Metabolism and GTP Metabolism in the Brainstem of Spontaneously Hypertensive Rats.

An untargeted multi-omics study implicated the potential dysregulation of fatty acid, nucleotide, and energy metabolism in the brainstems of spontaneously hypertensive rats (SHRs). A further quantitative exploration of the alterations in the metabolic pathways is necessary for a deep understanding of the central nervous system in SHRs. Targeted metabolic profiling of 40 fatty acids (PeptideAtlas: PASS01671) and 32 metabolites of nucleotides and energy metabolism (PeptideAtlas: PASS01672) and parallel reaction monitoring analysis of 5 proteins (PeptideAtlas: PASS01673) were performed on the brainstems of SHRs (n = 8, 11 weeks old) and normotensive Wistar rats (n = 8, age-matched) using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem MS. The targeted profiling results of metabolites and proteins revealed decreased polyunsaturated fatty acid (PUFA) synthesis with a significant downregulation of cis-11,14-eicosadienoic acid, cis-13,16-docosadienoic acid, and docosatetraenoate and impaired PUFA oxidation with the accumulation of γ-linolenate induced by the significantly downregulated expression of 2,4-dienoyl-CoA reductase (p < 0.05). Dysregulated GTP and ATP metabolism was observed, with significantly decreased GDP and ADP (p < 0.05) correlated with reduced GTPases of guanine nucleotide-binding protein subunit beta-1 (GNB1), transforming protein RhoA (RHOA), and Rho-related GTP-binding protein RhoB (RHOB) in the brainstem of SHRs. In addition, protein-arginine deiminase type-2 was significantly reduced in the brainstems of SHRs (p < 0.05). The aberrant PUFA and energy metabolism might help to explain the alterations in the brainstem of SHRs. The findings on both metabolites and proteins could provide systemic insights into the pathology basis of altered PUFA and energy metabolism in hypertension, especially in the central nervous system.

Targeted metabolomics in the cell culture media reveals increased uptake of branched amino acids by breast cancer cells.

In addition to the altered amino acids in many cancer cells for their uncontrolled growth, targeted metabolomics in cell culture media could display a dynamic interaction between cancer cells and their micro-environments. Methodology for cell culture medium samples is different from that of cell lysates on sampling points, calculation and statistical analysis. Targeted profiling method of 40 amino acid and derivatives was validated and performed on cell culture medium samples from cell lines of HCC 1806 (breast cancer cell) and MCF-10A (normal breast epithelial cell). Different from the common up-regulation of amino acids in cancer cell lysates, significantly increased uptake (>2.5-fold, VIP>1 and p < 0.001) of branched amino acids was observed in the cell culture media from the breast cancer cells while acetylmethionine, cysteine-glutathione, glutathione, cysteine and glutamic acid were excreted significantly more by the cancer cells to their media. The characteristic metabolic changes of amino acid and derivatives in the cell culture media provide a dynamic portrayal for the interaction of the breast cancer cells, normal breast cells with their micro-environments, which helps to understand the underlying proliferation mechanism of breast cancer cells.

Integrative Metabolic and Proteomic Profiling of the Brainstem in Spontaneously Hypertensive Rats.

The brainstem, the core of the central nervous system, plays a vital role in controlling arterial blood pressure and its elevation of hypertension subtypes, especially essential hypertension. Integrative metabolic and proteomic profiling was performed on the brainstem samples of 11 week old spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar rats, using hydrophilic interaction liquid chromatography quadrupole/time-of-flight mass spectrometry (HILIC-Q/TOFMS) (PeptideAtlas: PASS01621) and nano-liquid chromatography-high-resolution-MS (nano-LC-high-resolution) combined with quantitative tandem mass tags (ProteomeXchange: PXD021210). The results showed a potentially significant measure of metabolic disorders in the brainstem of SHRs, including purine and pyrimidine metabolism and carnitine and acylcarnitine deficiency. By integrating the differential metabolites (VIP > 1 and p < 0.1) with the differentially expressed proteins (>1.2-fold and p < 0.05), the results revealed aberrant insulin signaling in the brainstem of SHRs, including reduced carnitine and acetylcarnitine; increased arginine; and increased flotillin-1 (FLOT1), hemoglobin subunit alpha-1/2, and hemoglobin subunit beta-2 proteins verified by the parallel reaction monitoring analysis (PeptideAtlas: PASS01622). The aberrant insulin signaling pathway in the brainstem of SHRs might help explain the correlation between essential hypertension and insulin resistance. These findings on the brainstem of SHRs could provide new insights into the dysregulation of the central nervous system in hypertension, especially as it relates to metabolite and protein levels.

The Effect of Exhaustive Exercise on Plasma Metabolic Profiles of Male and Female Rats.

The objective of the study was to evaluate the alteration in biochemical composition and gender difference within exhaustive exercise in male and female rats using a metabolomics strategy. Sixty male and female rats were randomly assigned to control, exhaustive exercise and one-week recovery groups, respectively. The metabolic profiles of plasma were investigated by gas chromatograph-mass spectrometry (GC-MS) and data further underwent orthogonal partial least-squares (OPLS) analysis. The current study found that gender was a significant determinant of the effects of exhaustive exercise on the cortisol, blood urea nitrogen, creatine kinase, and the ratio of reduced glutathione to oxidized glutathione, whereas, no significant interaction effects between gender and exhaustive exercise were found on the levels of testosterone, malonaldehyde, reduced glutathione, oxidized glutathione and lactic dehydrogenase. In male rats, the altered metabolites within exhaustive exercise included increased tricarboxylic acid cycle intermediates (citric acid, fumaric acid, butanedioic acid), branch-chain amino acids (valine, leucine), fatty acids and metabolite (oleic acid, linoleic acid, 3-hydroxybutyric acid), phosphate and decreased glucose, lactic acid, serine, and glutamic acid. In female rats, the levels of fatty acids and metabolite (linoleic acid, oleic acid, arachidonic acid, 3-hydroxybutyric acid), amino acids (valine, leucine, glutamic acid, 5-oxo-proline, methionine, ornithine), other metabolites urea, myo-inositol and phosphate were increased. The results indicated that exhaustive exercise increased the rates of energy metabolism, glucose metabolism, amino acid catabolism and fatty acid metabolism in male rats, whereas, female rats showed an increased propensity to oxidize lipid and conserve carbohydrate and protein metabolism against physical stress. Disordered urea cycle and inositol metabolism also occurred in female rats with exhaustive exercise. Exhaustive exercise affected the balance of hormone adjustment and caused oxidative stress, subsequent cell membrane damage both in male and female rats. A significant gender-related difference in the metabolic profiles was also found between male and female rats within exhaustive exercise.

Identification and characterization of in vitro and in vivo metabolites of steroidal alkaloid veratramine.

Veratramine, a steroidal alkaloid originating from Veratrum nigrum L., has demonstrated distinct anti-tumor and anti-hypertension effects, however, its metabolism has rarely been explored. The objective of the current study was to provide a comprehensive investigation of its metabolic pathways. The in vitro metabolic profiles of veratramine were evaluated by incubating it with liver microsomes and cytosols. The in vivo metabolic profiles in plasma, bile, urine and feces were monitored by UPLC-MS/MS after oral (20 mg/kg) and i.v. (50 µg/kg) administration in rats. Meanwhile, related P450s inhibitors and recombinant P450s and SULTs were used to identify the isozymes responsible for its metabolism. Eleven metabolites of veratramine, including seven hydroxylated, two sulfated and two glucuronidated metabolites, were characterized. Unlike most alkaloids, the major reactive sites of veratramine were on ring A and B instead of on the amine moiety. CYP2D6 was the major isozyme mediating hydroxylation, and substrate inhibition was observed with a Vmax , Ki and Clint of 2.05 ± 0.53 nmol/min/mg, 33.08 ± 10.13 µ m and 13.58 ± 1.27 µL/min/mg. SULT2A1, with Km , Vmax and Clint values of 19.37 ± 0.87 µ m, 1.51 ± 0.02 nmol/min/mg and 78.19 ± 8.57 µL/min/mg, was identified as the major isozyme contributing to its sulfation. In conclusion, CYP2D6 and SULT2A1 mediating hydroxylation and sulfation were identified as the major biotransformation for veratramine.

Phellinus linteus polysaccharides: A review on their preparation, structure-activity relationships, and drug delivery systems.

Phellinus linteus polysaccharides exhibit antitumor, immunomodulatory, anti-inflammatory, and antioxidant properties, mitigate insulin resistance, and enhance the diversity and abundance of gut microbiota. However, the bioactivities of P. linteus polysaccharides vary owing to the complex structure, thereby, limiting their application. Various processing strategies have been employed to modify them for improving the functional properties and yield. Herein, we compare the primary modes of extraction and purification employed to improve the yield and purity, review the structure-activity relationships, and discuss the application of P. linteus polysaccharides using nano-carriers for the encapsulation and delivery of various drugs to improve bioactivity. The limitations and future perspectives are also discussed. Exploring the bioactivity, structure-activity relationship, processing methods, and delivery routes of P. linteus polysaccharides will facilitate the development of functional foods and dietary supplements rich in P. linteus polysaccharides.

Angelica gigas polysaccharide induces CR3-mediated macrophage activation and the cytotoxicity of natural killer cells against HCT-116 cells via NF-κB and MAPK signaling pathways.

Angelica gigas (A. gigas) is traditional medicinal herb that mainly exists in Korea and northeastern China. There have been relatively few studies conducted thus far on its polysaccharides and their bioactivities. We purified and described a novel water-soluble polysaccharide derived from A. gigas and investigated its immunoenhancing properties. The basic components of crude and purified polysaccharides (F1 and F2) were total sugar (41.07% - 70.55%), protein (1.12-10.33%), sulfate (2.9-5.5%), and uronic acids (0.5-31.05%) in total content. Our results demonstrated that the crude and fractions' molecular weights (Mw) varied from 42.2 to 285.2 × 103 g/mol. As the most effective polysaccharide, F2 significantly stimulated RAW264.7 cells to release nitric oxide (NO) and express several cytokines. Furthermore, F2 increased the expression of tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-É£), natural killer cytotoxicity receptors (NKp44), and granzyme-B in NK-92 cells and enhanced the cytotoxicity against HCT-116 cells. In our experiments, we found that F2 stimulated RAW264.7 cells and NK-92 cells via MAPK and NF-κB pathways. The monosaccharide and methylation analysis of the high immunostimulant F2 polysaccharide findings revealed that the polysaccharide was primarily composed of 1 â†’ 4, 1 â†’ 6, 1 â†’ 3, 6, 1 â†’ 3 and 1 â†’ 3, 4, 6 galactopyranose residues, 1 â†’ 3 arabinofuranose residues, 1 â†’ 4 glucopyranose residues. These results demonstrated that the F2 polysaccharide of A. gigas which possesses potential immunostimulatory attributes, could be used to create a novel functional food.

Preparation and characterization of soluble dietary fiber from tiger nut residues, showing enhanced antioxidant activity and metal-ion-binding properties.

To improve the utilization of soluble dietary fiber (SDF) from tiger nut residues, the response surface methodology was used to optimize the conditions of superfine grinding to produce SDF with antioxidant and metal-ion-binding properties. The yield was increased (30.56%) and the average particle diameter of SDF was decreased (D50: 32.80 µm) under the optimal conditions (a proportion of grinding medium of 100%, a feeding mass of 0.90 kg, a grinding time of 20 min, and a moisture content of 8.00%). In addition, superfine grinding substantially modified the surface morphology and increased the SDF content and the proportion of monosaccharides by decreasing the molecular weight. Moreover, superfine grinding remarkably enhanced the in vitro antioxidant activities (ABTS+, DPPH, and ·OH) of the SDF, which also exhibited favorable metal-ion-binding properties (Ca2+, Zn2+, and Co2+). These results suggest that superfine grinding can be used as a technique to modify dietary fiber to manufacture functional SDF.

A review of Ganoderma lucidum polysaccharides: Health benefit, structure-activity relationship, modification, and nanoparticle encapsulation.

Ganoderma lucidum polysaccharides possess unique functional properties. Various processing technologies have been used to produce and modify G. lucidum polysaccharides to improve their yield and utilization. In this review, the structure and health benefits were summarized, and the factors that may affect the quality of G. lucidum polysaccharides were discussed, including the use of chemical modifications such as sulfation, carboxymethylation, and selenization. Those modifications improved the physicochemical characteristics and utilization of G. lucidum polysaccharides, and made them more stable that could be used as functional biomaterials to encapsulate active substances. Ultimate, G. lucidum polysaccharide-based nanoparticles were designed to deliver various functional ingredients to achieve better health-promoting effects. Overall, this review presents an in-depth summary of current modification strategies and offers new insights into the effective processing techniques to develop G. lucidum polysaccharide-rich functional foods or nutraceuticals.

Gallic Acid Treats Hypertrophic Scar in Rabbit Ears via the TGF-ß/Smad and TRPC3 Signaling Pathways.

Hypertrophic scars (HSs) develop due to excessive collagen deposition and abnormal fibroblast proliferation during wound healing, significantly impacting patient quality of life. Three dosages of GA ointments were administered to rabbit ear HS models to investigate the potential efficacy and mechanism of gallic acid (GA) on HS. Daily application of ointment was performed on the matrix group, the GA ointment groups, and the silicone gel group for 28 days. (No drug treatment was performed on the skin and model groups as a blank group and vehicle group, and silicone gel ointment was topically administered to the silicone gel group as a positive control group.) Scar specimens were collected for histopathology analysis, RNA sequencing analysis, real-time quantitative polymerase chain reaction, and Western blot analysis at the first, second, and fourth weeks after the treatment. Low-dose and medium-dose GA effectively suppressed HS formation and markedly decreased fibroblast infiltration levels and scar thickness. Moreover, decreased expression of TRPC3 mRNA and TGF-ß1, p-Smad2/3, and Smad2/3 protein was observed in the low- and medium-dose GA groups and the silicone gel group. This study provides evidence for the efficacy of GA in treating HS and sheds light on its potential underlying pharmacological mechanisms.

A novel sulfated mannan-carboxymethyl-5-fluorouracil-folic acid conjugates for targeted anticancer drug delivery.

CFP2 is a sulfated polysaccharide isolated from Codium fragile that shows excellent immunomodulatory activity. To reduce the side effects of 5-fluorouracil (5-FU), CFP2 was used as a macromolecular carrier to react with carboxymethyl-5-fluorouracil (C-5-FU) to form CFP2-C-5-FU, which further reacted with folic acid (FA) via an ester bond to form novel conjugates (CFP2-C-5-FU-FA). CFP2-C-5-FU-FA was confirmed by nuclear magnetic resonance (NMR) analysis. In vitro drug release results showed that the cumulative release rate of C-5-FU was 49.9% in phosphate buffer (pH 7.4) after 96 h, which was much higher than that of the other groups, indicating that CFP2-C-5-FU-FA showed controlled drug release behavior. CFP2-C-5-FU-FA also exhibited enhanced apoptosis and cellular uptake in vitro. Further, intravenous administration of CFP2-C-5-FU-FA in an HCT-116 cell-bearing xenograft mouse showed that the conjugates were safe and effective drug delivery systems. These results suggest that folate-targeted conjugates can be used effectively for efficient chemotherapy of colorectal cancer.

Cnidium officinale polysaccharide enhanced RAW 264.7 cells activation and NK-92 cells cytotoxicity against colon cancer via NF-κB and MAPKs signaling pathways.

In this study, Cnidium officinale-derived polysaccharides were isolated and investigated for their immune enhancing and anticancer activities. The isolated crude and its fractions, such as F1 and F2, contain carbohydrates (51.3-63.1%), sulfates (5.4-5.8%), proteins (1.5-7.1%), and uronic acids (2.1-26.9%). The molecular weight (Mw) of the polysaccharides ranged from 59.9 to 429.0 × 103 g/mol. The immunostimulatory activity of the polysaccharides was tested on RAW 264.7 cells, and the results showed that the F2 treatment notably enhanced pro-inflammatory activity in RAW 264.7 cells by increasing NO production and the expression of various cytokines. Furthermore, the influence of polysaccharide treatment on natural killer cells (NK-92) anticancer activities was investigated using a colon cancer cell line (HCT-116). Crude polysaccharide and its fractions showed no direct cytotoxicity to NK-92 and HCT-116 cells. However, the treatment of F2 showed an enhancement of NK-92 cells cytotoxicity against HCT-116 cells by upregulating the mRNA expression of IFN-γ, TNF-α, NKGp44, and granzyme-B. The western blot results showed that the induced RAW 264.7 cells activation and NK-92 cells cytotoxicity occur via NF-κB and MAPK signaling pathways. Overall, C. officinale-derived polysaccharides show potential as immunotherapeutic agents capable of enhancing pro-inflammatory macrophage signaling and activating NK-92 cells; thus, they could be useful for biomedical applications.

Metabonomic variations associated with AOM-induced precancerous colorectal lesions and resveratrol treatment.

Resveratrol (Res), 3,5,4'-trihydroxy-trans-stilbene, is an antioxidant found in the skin of red grapes and in several other plants. This phenolic compound has been recently reported to possess cancer chemopreventive activity that inhibits the process of carcinogenesis. However, the mechanisms underlying its anticancer effects remain largely unresolved. In this study, we investigated the chemoprotective effects of dietary Res in an azoxymethane (AOM) induced precancerous colorectal lesion model in male Wistar rats. The metabolic alterations in urine, sera, and colonic tissues of experimental rats perturbed by AOM intervention as well as the Res treatment were measured by a gas chromatography time-of-flight mass spectrometry (GC-TOFMS) analysis. Significant alterations of metabolites were observed in AOM group in urine, sera, and colonic tissues, which were attenuated by Res treatment and concurrent with the histopathological improvement with significantly decreased aberrant crypt foci (ACF) incidence. Representative metabolites include depleted glucose, ß-hydroxybutyrate (ketone body), hypoxanthine, and elevated branched chain amino acids (isoleucine and valine) and tryptophan in colonic tissue, as well as elevated serum aminooxyacetate and urinary 4-hydroxyphenylacetate and xanthurenate. These metabolic changes suggest that the preventive effect of Res is associated with attenuation of impaired glucose and lipid metabolism and elevated protein breakdown in colonic tissues from AOM-exposed rats. It also appears that Res induced significant metabolic alterations independent of the AOM-induced metabolic changes. The significantly altered metabolites identified in Res-AOM group relative to AOM group include arachidonate, linoleate, glutamate, docosahexaenoate, palmitelaidate, 2-aminobutyrate, pyroglutamate, and threonate, all of which are involved in inflammation and oxidation processes. This suggests that Res exerts the chemopreventive effects on ACF formation by anti-inflammatory and antioxidant mechanisms in addition to amelioration of AOM-induced mitochondrial disruption.

Traditional Chinese medicine for hypertrophic scars-A review of the therapeutic methods and potential effects.

Hypertrophic scar (HS) is a typical pathological response during skin injury, which can lead to pain, itching, and contracture in patients and even affect their physical and mental health. The complexity of the wound healing process leads to the formation of HS affected by many factors. Several treatments are available for HS, whereas some have more adverse reactions and can even cause new injuries with exacerbated scarring. Traditional Chinese Medicine (TCM) has a rich source, and most botanical drugs have few side effects, providing new ideas and methods for treating HS. This paper reviews the formation process of HS, the therapeutic strategy for HS, the research progress of TCM with its relevant mechanisms in the treatment of HS, and the related new drug delivery system of TCM, aiming to provide ideas for further research of botanical compounds in the treatment of HS, to promote the discovery of more efficient botanical candidates for the clinical treatment of HS, to accelerate the development of the new drug delivery system and the final clinical application, and at the same time, to promote the research on the anti-HS mechanism of multiherbal preparations (Fufang), to continuously improve the quality control and safety and effectiveness of anti-HS botanical drugs in clinical application.

Predicting Lapatinib Dose Regimen Using Machine Learning and Deep Learning Techniques Based on a Real-World Study.

Lapatinib is used for the treatment of metastatic HER2(+) breast cancer. We aim to establish a prediction model for lapatinib dose using machine learning and deep learning techniques based on a real-world study. There were 149 breast cancer patients enrolled from July 2016 to June 2017 at Fudan University Shanghai Cancer Center. The sequential forward selection algorithm based on random forest was applied for variable selection. Twelve machine learning and deep learning algorithms were compared in terms of their predictive abilities (logistic regression, SVM, random forest, Adaboost, XGBoost, GBDT, LightGBM, CatBoost, TabNet, ANN, Super TML, and Wide&Deep). As a result, TabNet was chosen to construct the prediction model with the best performance (accuracy = 0.82 and AUC = 0.83). Afterward, four variables that strongly correlated with lapatinib dose were ranked via importance score as follows: treatment protocols, weight, number of chemotherapy treatments, and number of metastases. Finally, the confusion matrix was used to validate the model for a dose regimen of 1,250 mg lapatinib (precision = 81% and recall = 95%), and for a dose regimen of 1,000 mg lapatinib (precision = 87% and recall = 64%). To conclude, we established a deep learning model to predict lapatinib dose based on important influencing variables selected from real-world evidence, to achieve an optimal individualized dose regimen with good predictive performance.

Pretreatment with millet-derived selenylated soluble dietary fiber ameliorates dextran sulfate sodium-induced colitis in mice by regulating inflammation and maintaining gut microbiota balance.

Inflammatory activation and intestinal flora imbalance play key roles in the development and progression of inflammatory bowel disease (IBD). Soluble dietary fiber (SDF) and selenium have been proven to be effective for preventing and relieving IBD. This study investigated and compared the therapeutic efficacy of millet-derived selenylated-soluble dietary fiber (Se-SDF) against dextran sulfate sodium (DSS)-induced colitis in mice alone or through the synergistic interaction between selenium and SDF. In female mice, Se-SDF markedly alleviated body weight loss, decreased colon length, reduced histological damage scores, and enhanced IL-10 expression to maintain the barrier function of intestinal mucosa compared to male mice. The 16S rRNA sequence analysis further indicated that pretreatment with Se-SDF restored the gut microbiota composition in female mice by increasing the relative abundance of Lactobacillus and the Firmicutes/Bacteroidetes ratio. In conclusion, these findings demonstrated that Se-SDF can protect against DSS-induced colitis in female mice by regulating inflammation and maintaining gut microbiota balance. This study, therefore, provides new insights into the development of Se-SDF as a supplement for the prevention and treatment of colitis.

Targeted metabolomics reveals dynamic portrayal of amino acids and derivatives in triple-negative breast cancer cells and culture media.

Triple-negative breast cancer (TNBC) is well-known for its metastatic aggressiveness and poor survival prognosis, accounting for nearly a quarter of cases in breast cancer. We performed intra- and extra-cellular profiling of 40 amino acids and derivatives on three cell lines and their culture media, including TNBC, non-TNBC and normal breast epithelial cells, using HILIC-MS/MS. Characteristic metabolic alteration of amino acids and derivatives was observed in TNBC cells, compared to non-TNBC cells, especially in correlated intra- and extra-cellular metabolic pathways. Intra-cellularly, quantified glutamic acid, ß-alanine, aspartic acid, glutathione, N-acetyl-serine and N-acetyl-methionine were most significantly increased (>2-fold, p < 0.01 and VIP > 1) in TNBC cells. Extra-cellularly, significantly increased uptake of glutamine, serine, ß-alanine, and lysine and elevated excretion of glutamic acid and l-cysteine-glutathione (p < 0.01 and VIP > 1) were observed by TNBC cells from or to their cell culture media. This study depicted a novel dynamic portrayal of metabolic dysregulation between TNBC and non-TNBC cells, correlated in both intra- and extra-cellular amino acid profiles. Quantification of these distinctive metabolites of TNBC cells might offer advanced understanding and new treatment targets for TNBC.

Metabolomics study on the intervention effect of Radix Salviae Miltiorrhizae extract in exercise-induced exhaustion rat using gas chromatography coupled to mass spectrometry.

The metabolomics approach based on the gas chromatography coupled to mass spectrometry (GC-MS) was adopted to explore the underlying mechanism of the anti-fatigue effect of Radix Salviae Miltiorrhizae (RSM), a famous herbal medicine in China used for multiple biological functions, in load-weighted swimming test in rat, combined with biochemical parameters evaluations. As a result, the metabolomics study followed by orthogonal partial least-square (OPLS) analysis could differentiate metabolic profiling between the control and exhaustive exercise group, showing the rats underwent an obvious metabolic perturbation, whereas RSM treatment restored scores plot close to normal and showed regulatory effects on the muscle metabolic profiles. The changed metabolic pathways of the potential biomarkers in response to the effect of RSM treatment for exhaustive exercise rats included in glucose metabolism (glucose, lactic acid, alanine), glutathione metabolism (glycine, glutamate, 5-oxo-proline), TCA cycle (succinic acid), arginine biosynthesis (glutamine, ornithine, urea), glyoxylate and dicarboxylate metabolism (serine, glycine), oxidative stress (taurine) and purine metabolism (inosine). In addition, intervention of RSM increased hepatic glycogen, muscle glycogen and serum glucose, and decreased triglyceride and blood urea nitrogen levels, indicating RSM treatment may regulate energy metabolism by increasing the rate of fat utilization, decrease the protein and carbohydrate utilization. Furthermore, RSM reduced exhaustive exercise-induced accumulation of the lipid peroxidation byproduct malonaldehyde and elevated antioxidants' levels, including reduced glutathione and superoxide dismutase, which might be a positive reflection of improved oxidant-antioxidant balance. Moreover, RSM could protect against exercise-induced muscle damage by attenuating creatine kinase release. In summary, RSM provided a good anti-fatigue effect by regulating energy metabolism, oxidant-antioxidant balance, and the endogenous metabolites in the exercising muscle. This study demonstrates that metabolomics is an effective tool for the estimation of the potential anti-fatigue effect of RSM and for the illustration of its pharmacological mechanism.

Metabolic mechanism and anti-inflammation effects of sinomenine and its major metabolites N-demethylsinomenine and sinomenine-N-oxide.

AIMS: Sinomenine (SIN) is clinically used as an anti-rheumatic drug. However, the metabolic and pharmacological mechanisms of SIN combined with its metabolites are unclear. This study aims to explore the cyclic metabolic mechanism of SIN, the anti-inflammation effects of SIN and its major metabolites (N-demethylsinomenine (DS) and sinomenine-N-oxide (SNO)), and the oxidation property of SNO. MATERIALS AND METHODS: SIN was administrated to rats via gavage. Qishe pills (a SIN-containing drug) were orally administrated to humans. The bio-samples were collected to identify SIN's metabolites. Enzymatic and non-enzymatic incubations were used to reveal SIN's metabolic mechanism. Impacts of SIN, SNO and DS on the inflammation-related cytokine's levels and nuclear translocation of NF-κB were evaluated in LPS-induced Raw264.7 cells. ROS induced by SNO (10 µM) was also assessed. KEY FINDINGS: CYP3A4 and ROS predominantly mediated the formation of SNO, and CYP3A4 and CYP2C19 primarily mediated the formation of DS. Noteworthily, SNO underwent N-oxide reduction both enzymatically, by xanthine oxidase (XOD), and non-enzymatically, by ferrous ion and heme moiety. The levels of IL-6 and TNF-α and nuclear translocation of NF-κB were ameliorated after pretreatment of SIN in LPS-induced Raw264.7 cells, while limited attenuations were observed after pretreatment of DS (SNO) even at 200 µM. In contrast, SNO induced ROS production. SIGNIFICANCE: This study elucidated that SIN underwent both enzymatic and non-enzymatic cyclic metabolism and worked as the predominant anti-inflammation compound, while SNO induced ROS production, suggesting more studies of SIN combined with SNO and DS are necessary in case of DDI and potential toxicities.