ß-Carboline alkaloids from Picrasma quassioides and their 3D-QSAR study on anti-inflammation in LPS-induced RAW 264.7 cells.

Two new ß-carboline alkaloids (1-2), 1-pyrrolidone propionyl-ß-carboline (1) and 1-(3-hydroxy-2-oxopiperidine-1-ethyl)-4,8-dimethoxyl-ß-carboline (2), named kumujantine W and J respectively, together with ten known compounds (3-12) were isolated from the stems of Picrasma quassioides (D. Don) Benn. Their structures were elucidated from spectral data including 1D and 2D NMR, UV, IR, HR-ESI-MS spectroscopic analysis and ECD calculations as well as by comparison to the reference databases or literature. The anti-inflammatory effects of these alkaloids (1-12) and six other ß-carboline alkaloids (13-18) in LPS-induced RAW 264.7 cells were evaluated by measuring nitric oxide (NO) concentrations. Among them, compounds 1, 3, 6, 15, and 17 could inhibit the secretion of NO, displaying significant anti-inflammatory activity without affecting cell viability in vitro, and 3D-QSAR analysis further revealed the influence of groups on the activity in ß-carboline alkaloids.

A comparative study of Liandan Xiaoyan Formula metabolic profiles in control and colitis rats by UPLC-Q-TOF-MS combined with chemometrics.

Liandan Xiaoyan Formula (LDXYF) is a traditional Chinese medicine prescription (TCMP) consisting of Herba Andrographis (dried herb of Andrographis paniculata) and Picrasmae ramulus et folium (dried twiggeries and leaves of Picrasma quassioides). It is used to treat diarrhea, acute gastroenteritis, colitis, and dysentery, among other inflammatory gastrointestinal diseases. However, because of less research on the in vitro chemical composition and holistic metabolism of LDXYF, in vivo mechanisms of action and quality control of LDXYF have not yet been fully assessed due to the lack of studies into its bioactive components. In this study, ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was established for comprehensive analysis of chemical compounds of LDXYF and their metabolites in serum and urine samples of control and colitis rats. As a result, totally 94 compounds in LDXYF were unambiguously identified or tentatively characterized. And a total of 91 LDXYF-related xenobiotics were characterized, including 61 (16 prototypes and 45 metabolites) in serum, and 72 (26 prototypes and 46 metabolites) in urine. Besides, we compared the exposure of metabolites in normal and colitis rats by chemometrics and summarize similarities and differences of metabolic pathways of mainly compounds in normal and colitis conditions, and found that in control and colitis conditions, alkaloids predominantly went through phase I reaction combined phase II reaction (hydroxylation and sulfation, hydroxylation and glucuronidation, demethylation and glucuronidation), while the major metabolic reaction of diterpene lactones were phase Ⅱ reactions (glucuronidation, sulfation). And there were no significant differences in metabolic pathways between control and colitis groups, just the exposure of prototype and their metabolites absorbed into serum or excreted through the urine were different, and 17 alkaloids and 6 diterpene lactone prototypes and their metabolites in serum could be considered as potential pharmacodynamic substances. A comprehensive analysis of the compounds and metabolic characteristics of LDXYF was conducted in our study, and the results laid the chemical foundation for further research into effective substances and the action mechanism of LDXYF.

Diterpenoid alkaloids isolated from Delphinium trichophorum alleviate pulmonary fibrosis via the TGF-ß/Smad pathway in 3T6 and HFL-1 cells.

Delphinium trichophorum Franch (DTF), a species endemic to China, has been widely used for centuries in Tibet as an indigenous medicine for treating cough, pneumonia, and pulmonary fibrosis. Hetisine-type C20-diterpenoid alkaloids have been reported to be characteristic and active ingredients. Herein, five ones with relatively high contents in D. trichophorum, including 2α,11α,13ß-triacetylhetisine (DTF1), trichodelphinine A (DTF2), trichodelphinine D (DTF3), 2α-acetyl-11α,13ß-dihydroxyhetisine (DTF4), and trichodelphinine C (DTF5), were investigated for anti-fibrosis effects using fibroblasts induced by TGF-ß1 or LPS for the first time. The results showed that all five tested compounds decreased hydroxyproline (HYP) levels and inhibited the abnormal proliferation of 3T6 and HFL-1 cells induced by either TGF-ß1 or LPS. Moreover, DTF1 and DTF2 attenuated the production of collagen (Col-1 and Col-3) at relatively low doses, suggesting their higher efficiency among the five alkaloids. Based on large-scale ligand-based pharmacophore modeling, TGFBR1 was screened as a potential target for these tested alkaloids. The molecular docking results also exhibited high-affinity interactions between TGFBR1 and five alkaloids, especially DTF1 and DTF2. Further experiments revealed that DTF1 and DTF2 could inhibit the expression of TGF-ß1 and α-SMA and the phosphorylation of Smad3 and Smad4 while restoring the expression of Smad7 protein. Overall, DTF1 and DTF2 may reduce collagen generation and delay the development of pulmonary fibrosis by inhibiting the activation of the TGF-ß/Smad signaling pathway. Our results provide experimental and theoretical evidence for DTF1 and DTF2 as superior candidates for further development of anti-fibrotic drugs.

Diterpenoid Alkaloids Isolated from Delphinium brunonianum and Their Inhibitory Effects on Hepatocytes Lipid Accumulation.

This research aimed to excavate compounds with activity reducing hepatocytes lipid accumulation from Delphinium brunonianum. Four novel diterpenoid alkaloids, brunodelphinine B-E, were isolated from D. brunonianum together with eleven known diterpenoid alkaloids through a phytochemical investigation. Their structures were elucidated by comprehensive spectroscopy methods including HR-ESI-MS, NMR, IR, UV, CD, and single-crystal X-ray diffraction analysis. The inhibitory effects of a total of 15 diterpenoid alkaloids on hepatocytes lipid accumulation were evaluated using 0.5 mM FFA (oleate/palmitate 2:1 ratio) to induce buffalo rat liver (BRL) cells by measuring the levels of triglyceride (TG), total cholesterol (TC), alanine transaminase (ALT), aspartate transaminase (AST), and the staining of oil red O. The results show that five diterpenoid alkaloids-brunodelphinine E (4), delbruline (5), lycoctonine (7), delbrunine (8), and sharwuphinine A (12)-exhibited significant inhibitory effects on lipid accumulation in a dose-dependent manner and without cytotoxicity. Among them, sharwuphinine A (12) displayed the strongest inhibition of hepatocytes lipid accumulation in vitro. Our research increased the understanding on the chemical composition of D. brunonianum and provided experimental and theoretical evidence for the active ingredients screened from this herbal medicine in the treatment of the diseases related to lipid accumulation, such as non-alcoholic fatty liver disease and hyperlipidemia.

Filamentous GLS1 promotes ROS-induced apoptosis upon glutamine deprivation via insufficient asparagine synthesis.

GLS1 orchestrates glutaminolysis and promotes cell proliferation when glutamine is abundant by regenerating TCA cycle intermediates and supporting redox homeostasis. CB-839, an inhibitor of GLS1, is currently under clinical investigation for a variety of cancer types. Here, we show that GLS1 facilitates apoptosis when glutamine is deprived. Mechanistically, the absence of exogenous glutamine sufficiently reduces glutamate levels to convert dimeric GLS1 to a self-assembled, extremely low-Km filamentous polymer. GLS1 filaments possess an enhanced catalytic activity, which further depletes intracellular glutamine. Functionally, filamentous GLS1-dependent glutamine scarcity leads to inadequate synthesis of asparagine and mitogenome-encoded proteins, resulting in ROS-induced apoptosis that can be rescued by asparagine supplementation. Physiologically, we observed GLS1 filaments in solid tumors and validated the tumor-suppressive role of constitutively active, filamentous GLS1 mutants K320A and S482C in xenograft models. Our results change our understanding of GLS1 in cancer metabolism and suggest the therapeutic potential of promoting GLS1 filament formation.

Rapid Identification of Pollen- and Anther-Specific Genes in Response to High-Temperature Stress Based on Transcriptome Profiling Analysis in Cotton.

Anther indehiscence and pollen sterility caused by high temperature (HT) stress have become a major problem that decreases the yield of cotton. Pollen- and anther-specific genes play a critical role in the process of male reproduction and the response to HT stress. In order to identify pollen-specific genes that respond to HT stress, a comparative transcriptome profiling analysis was performed in the pollen and anthers of Gossypium hirsutum HT-sensitive Line H05 against other tissue types under normal temperature (NT) conditions, and the analysis of a differentially expressed gene was conducted in the pollen of H05 under NT and HT conditions. In total, we identified 1111 pollen-specific genes (PSGs), 1066 anther-specific genes (ASGs), and 833 pollen differentially expressed genes (DEGs). Moreover, we found that the late stage of anther included more anther- and pollen-specific genes (APSGs). Stress-related cis-regulatory elements (CREs) and hormone-responsive CREs are enriched in the promoters of APSGs, suggesting that APSGs may respond to HT stress. However, 833 pollen DEGs had only 10 common genes with 1111 PSGs, indicating that PSGs are mainly involved in the processes of pollen development and do not respond to HT stress. Promoters of these 10 common genes are enriched for stress-related CREs and MeJA-responsive CREs, suggesting that these 10 common genes are involved in the process of pollen development while responding to HT stress. This study provides a pathway for rapidly identifying cotton pollen-specific genes that respond to HT stress.

Degradation of de-esterified pctin/homogalacturonan by the polygalacturonase GhNSP is necessary for pollen exine formation and male fertility in cotton.

The pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map-based cloning and confirmed by genetic analysis (co-segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de-esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de-esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de-esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de-esterified homogalacturonan in pollen exine formation.

A UPLC-MS/MS-based metabolomics analysis of the pharmacological mechanisms of rabdosia serra against cholestasis.

BACKGROUND: Rabdosia Serra, the dried aerial parts of Rabdosia serra (Maxim.) Hara (RS) from the Labiatae family, is a traditional Chinese herbal medicine called Xihuangcao. Although RS has been found to exert a therapeutic effect on cholestasis, the underlying molecular mechanism remains unclear. PURPOSE: This study was designed to investigate the pharmacological effect and mechanism of RS on cholestatic rats using metabolomics platform. METHODS: Histopathology and biochemical evaluations were performed to determine the therapeutic effect of RS and developed a rapid metabolite detection technology method based on UPLC-MS/MS to perform metabolomics research. Further, quantitative real-time polymerase chain reaction (RT-qPCR) was used to study the effect of RS on the bile acid metabolism pathway at the transcriptional level. RESULTS: RS significantly reduced the bile flow rates in cholestatic rats and decreased the levels of ALT, AST, TBA, T-BIL, and LDH, which were increased in the model group. Histological analysis showed that RS alleviated the liver injury induced by ANIT. Serum metabolomics results revealed 33 of the 37 biomarkers were found to be significantly altered by ANIT, and 26 were considerably changed following treatment with RS. Metabolic pathway analysis revealed four pathways such as primary bile acid biosynthesis, biosynthesis of unsaturated fatty acids, and arachidonic acid and tryptophan metabolism. The bile acid secretion process and the inflammation and oxidative stress processes are the major biochemical reactions following treatment with ANIT and RS. Bile acid-targeted metabolomics study showed that TCA, GCA, GCDCA, and GDCA might be sensitive biomarkers that induced liver injury. we found that treatment with RS regulated the levels of bile acid in the serum and liver and restored the proportion of bile acids, especially CA and conjugated bile acids, such as TCA and GCA, in the bile duct. RS increased the mRNA expression levels of FXR, SHP, BSEP, and MRP2 in livers, and IBABP, OST-α, and OST-ß in the ileum. CONCLUSION: In this study, RS was found to protect the liver by regulating multiple metabolic pathways and promoting the excretion of bile acids. Simultaneously, RS played an essential role in reversing the imbalance of bile acids and protected against cholestasis by regulating the expression of transporters associated with bile acids. We demonstrated the correlation between molecular mechanisms and metabolites, provide a reference for the fabrication of extracts that can be used to treat cholestasis.

Investigation of the relevance between biomass pyrolysis polygeneration and washing pretreatment under different severities: Water, dilute acid solution and aqueous phase bio-oil.

Washing pretreatments of rice straw were performed using three different solutions, namely water, dilute hydrochloric acid solution (HCl solution, pH = 2.9), and aqueous phase bio-oil (APBO, pH = 2.9). The raw and pretreated samples were pyrolyzed at 550 °C in a fixed bed reactor. Results showed that among the three pretreatments, washing with APBO had the highest removal efficiency of alkali metal and alkaline earth metals (AAEMs). Among the pyrolysis products, bio-oil from APBO washed sample had the highest mass, energy, and carbon yields, lowest water content of 36.9%, highest HHV of 17.2 MJ/kg, and highest relative content of anhydrosugars of 31.2%. Its biochar had the lowest ash content of 27.3% and highest specific surface area of 98.6 m2/g, and its non-condensable gases had the highest HHV of 11.9 MJ/m3. Therefore, APBO washing was effective in improving the quality of biomass and its subsequent pyrolysis products.

Revealing the Inhibitory Effect of Ginseng on Mitochondrial Respiration through Synaptosomal Proteomics.

Ginseng, the active ingredients of which are ginsenosides, is the most popular herbal medicine and has potential merit in the treatment of cerebral disorders. To better understand the function of Ginseng in the cerebral system, we examined changes in the protein expression profiles of synaptosomes extracted from the cerebral cortical and hippocampal tissues of rats administered a high or low dose of Ginseng for 2 weeks. More than 5000 proteins belonging to synaptosomes were simultaneously identified and quantitated by an approach combining tandem mass tags with 2D liquid chromatography-mass spectrometry (LC-MS). Regarding differentially expressed proteins, downregulated proteins were much more highly induced than upregulators in the cerebral cortical and hippocampal synaptosomes, regardless of the dose of Ginseng. Bioinformatic analysis indicated the majority of the altered proteins to be located in the mitochondria, directly or indirectly affecting mitochondrial oxidative respiration. Further functional experiments using the substrate-uncoupler inhibitor titration approach confirmed that three representative ginsenosides were able to inhibit oxidative phosphorylation in mitochondria. Our results demonstrate that Ginseng can regulate the function of mitochondria and alter the energy metabolism of cells, which may be useful for the treatment of central nervous disorders.