Revealing active constituents within traditional Chinese Medicine used for treating bacterial pneumonia, with emphasis on the mechanism of baicalein against multi-drug resistant Klebsiella pneumoniae.

ETHNOPHARMACOLOGICAL RELEVANCE: The emergence of antibiotic-resistant bacteria has rendered it more challenging to treat bacterial pneumonia. Traditional Chinese medicine (TCM) has superior efficacy in the treatment of pneumonia, and it has the unique advantage of antibacterial resistance against multi-drug resistant (MDR) bacteria, but the medication rule and pharmacological mechanism of its antibacterial activity are not clear. AIM OF THE STUDY: This study aims to reveal Chinese medication patterns in treating bacterial pneumonia to select bioactive constituents in core herbs, predict their pharmacological mechanisms and further explore their antibacterial ability against clinically isolated MDR Klebsiella pneumoniae (KP) and their antibacterial mechanisms. MATERIALS AND METHODS: The high-frequency medicinal herbs to treat lung diseases were first screened from Pharmacopoeia of the People's Republic of China (ChP.), and then bioactive compounds in core herbs and targets for compounds and disease were collected. Potential targets, signaling pathways, and drugs' core components were determined by constructing protein-protein interaction network, enrichment analysis and "component-target-pathway-disease" network were mapped by Cytoscape 3.8.2, and the potential therapeutic value of selected core components was verified by comparing the disease targets in the GEO database with the herbal component targets in the ITCM database. The clinically isolated KP were screened by drug sensitivity tests with meropenem (MEM), polymyxin E (PE), and tigecycline and biofilm-forming assay; broth microdilution, chessboard methods and biofilm morphology and permeability experiments were employed to determine the antibacterial, bactericidal and biofilm inhibition ability of selected bioactive constituents alone and in combination with antibiotics; The mechanism of bioactive components on quorum sensing (QS) genes LuxS and LuxR was predicted by molecular docking and tested by RT-PCR. RESULTS: The 13 core Chinese medicines were obtained by mining ChP., and 615 potential targets of core herbal medicine were screened, and the PI3K-Akt signaling pathway might play crucial roles in the therapeutic process. In-vitro experiments revealed that the selected core compounds, including forsythoside B, baicalin, baicalein, and forsythin, all have antibacterial activity, in which baicalein had the strongest ability and a synergistic effect in combination with MEM or PE. Their synergy exhibited a stronger effect on biofilms of MDR KP, inhibiting biofilm formation, disrupting formed biofilms, and removing the residual structures of dead bacteria. Baicalein was predicted to have stable binding capacity to LuxS and LuxR genes by molecular docking, and RT-PCR results verified that the combination of baicalein with MEM or PE was effective in inhibiting the expression of QS genes (LuxS and LuxR) and consequently suppressing biofilm formation. CONCLUSION: The core Chinese herbal medicine in the ChP. to treat lung diseases has a multi-component, multi-target, and multi-pathway synergy to improve bacterial pneumonia. Experimental studies have confirmed that the bioactive compound baicalein was able to combat MDR KP alone and synergistic with MEM or PE, inhibited and disrupted biofilms via regulating LuxS and LuxR genes, and further disturbed quorum sensing system to promote the therapeutic efficacy, which provides a new pathway and rationale for treating MDR KP-induced bacterial pneumonia.

[Comparison of distribution of eight components from Liangxue Tuizi Mixture between normal and Henoch-Schonlein purpura rats].

This study used UPLC-TQ-MS technology to replicate a Henoch-Schonlein purpura(HSP) model in rats by administering warm drugs by gavage and injecting ovalbumin with Freund's complete adjuvant emulsion. The distribution differences and characteristics of eight major components(ferulic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, benzoyl oxypaeoniflorin, tracheloside, loganin, and paeoniflorin) in rat liver, lung, heart, spleen, and kidney tissues were determined after oral administration of the Liangxue Tuizi Mixture at a dose of 42 g·kg~(-1) in both normal physiological and HSP states at 0.5, 1, 2, 6, and 12 hours. The results showed that the distribution patterns of the eight components of Liangxue Tuizi Mixture in the tissues of normal and HSP model rats were different. The main component, paeoniflorin, in Moutan Cortex and Paeoniae Radix Alba had higher content in all tissues. The eight components were predominantly distributed in the liver, lung, and kidney tissues, followed by spleen and heart tissues.

Seven new 3,4-dihydro-furanocoumarin derivatives from Angelica dahurica.

Objective: To study the chemical constituents of the roots of Angelica dahurica, a well-known Chinese herbal medicine named Baizhi in Chinese. Methods: Compounds were separated by various chromatographies, and the structures of new compounds were elucidated based on the analysis of their spectroscopic and spectrometric data (1D, 2D NMR, HRESI MS, IR, and UV). The absolute configurations of new compounds were determined by the calculated electronic circular dichroism and chemical derivatization. The inhibitory activities of all isolates against nitric oxide (NO) production were evaluated using lipopolysaccharide-activated RAW 264.7 macrophage cells. Results: Seven new 3,4-dihydro-furanocoumarin derivatives (1a/1b, 2a/2b, 3a/3b, 4) together with a known furanocoumarin (5) were isolated from the roots of A. dahurica. The new compounds included three pairs of enantiomers, (4S, 2''R)-angelicadin A (1a)/(4R, 2''S)-angelicadin A (1b), (4S, 2''S)-angelicadin A (2a)/(4R, 2''R)-angelicadin A (2b), and (4S, 2''S)-secoangelicadin A (3a)/(4R, 2''R)-secoangelicadin A (3b), together with (4R, 2''R)-secoangelicadin A methyl ester (4). The known xanthotoxol (5) inhibited the NO production with the half-maximal inhibitory concentration (IC50) value of (32.8 ± 0.8) µmol/L, but all the new compounds showed no inhibitory activities at the concentration of 100 µmol/L. Conclusion: This is the first report of the discovery of 3,4-dihydro-furanocoumarins from A. dahurica. The results are not only meaningful for the understanding of the chemical constituents of A. dahurica, but also enrich the reservoir of natural products.

Yin/Yang associated differential responses to Psoralea corylifolia Linn. In rat models: an integrated metabolomics and transcriptomics study.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoralea corylifolia Linn. (BGZ) is a commonly used traditional Chinese medicine (TCM) for the treatment of kidney-yang deficiency syndrome (Yangsyn) with good curative effect and security. However, BGZ was also reported to induce liver injury in recent years. According to TCM theory, taking BGZ may induce a series of adverse reactions in patients with kidney-yin deficiency syndrome (Yinsyn), which suggests that BGZ-induced liver damage may be related to its unreasonable clinical use. AIM OF THE STUDY: Liver injury caused by TCM is a rare but potentially serious adverse drug reaction, and the identification of predisposed individuals for drug-induced liver injury (DILI) remains challenging. The study aimed to investigate the differential responses to BGZ in Yangsyn and Yinsyn rat models and identify the corresponding characteristic biomarkers. MATERIALS AND METHODS: The corresponding animal models of Yangsyn and Yinsyn were induced by hydrocortisone and thyroxine + reserpine respectively. Body weight, organ index, serum biochemistry, and Hematoxylin and Eosin (HE) staining were used to evaluate the liver toxicity effect of BGZ on rats with Yangsyn and Yinsyn. Transcriptomics and metabonomics were used to screen the representative biomarkers (including metabolites and differentially expressed genes (DEGs)) changed by BGZ in Yangsyn and Yinsyn rats, respectively. RESULTS: The level changes of liver organ index, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), suggested that BGZ has liver-protective and liver-damaging effects on Yangsyn and Yinsyn rats, respectively, and the results also were confirmed by the pathological changes of liver tissue. The results showed that 102 DEGs and 27 metabolites were significantly regulated related to BGZ's protective effect on Yangsyn, which is mainly associated with the glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate, and coenzyme A (CoA) biosynthesis pathways. While 28 DEGs and 31 metabolites, related to the pathway of pantothenate and CoA biosynthesis, were significantly regulated for the BGZ-induced liver injury in Yinsyn. Furthermore, 4 DEGs (aldehyde dehydrogenase 1 family member B1 (Aldh1b1), solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), out at first homolog (Oaf)) and 4 metabolites (phosphatidate, phosphatidylcholine, N-Acetylleucine, biliverdin) in the Yangsyn group and 1 DEG [galectin 5 (Lgals5)] and 1 metabolite (5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate) in Yinsyn group were significantly correlated to the ALT and AST levels of BGZ treated and untreated groups (receiver operating characteristic (ROC) ≥ 0.9). CONCLUSIONS: Yinsyn and Yangsyn are the predisposed syndromes for BGZ to exert liver damage and liver protection respectively, which are mainly related to the regulation of amino acid metabolism, lipid metabolism, energy metabolism, and metabolism of cofactors and vitamins. The results further suggest that attention should be paid to the selection of predisposed populations when using drugs related to the regulation of energy metabolism, and the Yinsyn/Yangsyn animal models based on the theory of TCM syndromes may be a feasible method for identifying the susceptible population to receive TCM.

[Potential components and mechanism of Liangxue Tuezi Mixture in treating Henoch-Schönlein purpura based on network pharmacology and metabolomics].

Ultra-performance liquid chromatography-quadrupole time of fight/mass spectrometry(UPLC-Q-TOF-MS) and UNIFI were employed to rapidly determine the content of the components in Liangxue Tuizi Mixture. The targets of the active components and Henoch-Schönlein purpura(HSP) were obtained from SwissTargetPrediction, Online Mendelian Inheritance in Man(OMIM), and GeneCards. A "component-target-disease" network and a protein-protein interaction(PPI) network were constructed. Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were performed for the targets by Omishare. The interactions between the potential active components and the core targets were verified by molecular docking. Furthermore, rats were randomly assigned into a normal group, a model group, and low-, medium-, and high-dose Liangxue Tuizi Mixture groups. Non-targeted metabolomics was employed to screen the differential metabolites in the serum, analyze possible metabolic pathways, and construct the "component-target-differential metabolite" network. A total of 45 components of Liangxue Tuizi Mixture were identified, and 145 potential targets for the treatment of HSP were predicted. The main signaling pathways enriched included resistance to epidermal growth factor receptor tyrosine kinase inhibitors, phosphatidylinositol 3-kinase/protein kinase B(PI3K-AKT), and T cell receptor. The results of molecular docking showed that the active components in Liangxue Tuizi Mixture had strong binding ability with the key target proteins. A total of 13 differential metabolites in the serum were screened out, which shared 27 common targets with active components. The progression of HSP was related to metabolic abnormalities of glycerophospholipid and sphingolipid. The results indicate that the components in Liangxue Tuizi Mixture mainly treats HSP by regulating inflammation and immunity, providing a scientific basis for rational drug use in clinical practice.

[LC-MS analysis of 2-(2-phenylethyl) chromones in sodium chloride-treated suspension cells of Aquilaria sinensis].

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 µm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.

Isoandrographolide from Andrographis paniculata ameliorates tubulointerstitial fibrosis in ureteral obstruction-induced mice, associated with negatively regulating AKT/GSK-3ß/ß-cat signaling pathway.

Tubulointerstitial fibrosis (TIF) is a prominent pathological manifestation for the progression of almost all chronic kidney diseases (CKDs) to end-stage renal failure. However, there exist few efficient therapies to cure TIF. Our recent results showed that (8R, 12S)-isoandrographolide (ISA), a diterpenoid lactone ingredient of traditional Chinese herbal Andrographis paniculata (Burm.f.) Nees, exhibited anti-pulmonary fibrosis in silica-induced mice. Herein, we investigated the therapeutic effect of ISA on TIF, using mice subjected to unilateral ureteral obstruction (UUO) and human kidney proximal tubular epithelial (HK-2) cells treated with transforming growth factor-ß1 (TGF-ß1) or tumor necrosis factor-α (TNF-α). The pathological changes and collagen deposition results displayed that ISA administration significantly attenuated inflammatory response, ameliorated TIF, and protected the kidney injury. Interestingly, ISA revealed much lower cytotoxicity on HK-2 cells, but exhibited stronger inhibitory effect on tubular epithelial mesenchymal transformation (EMT) and inflammation, as compared to andrographolide (AD), the major ingredient of A. paniculata extract that has been reported to ameliorate TIF in diabetic nephropathy mice. It was further clarified that the amelioration of TIF by ISA was associated with suppressing the aberrant activation of AKT/GSK-3ß/ß-catenin pathway through network pharmacology analysis and experimental validation. Taken together, these findings indicate that ISA is a promising lead compound for development of anti-TIF, and even broad-spectrum anti-fibrotic drugs.

Characterization of a coumarin C-/O-prenyltransferase and a quinolone C-prenyltransferase from Murraya exotica.

Prenyltransferases (PTs) play important roles in the biosynthesis and structural diversification of natural products. In the present study, two new PTs were characterized from a medicinal plant Murraya exotica. MePT1 unprecedentedly catalyses the formation of two C-geranylated products 8/6-C-geranylumbelliferone together with a trace product 7-O-geranylumbelliferone from umbelliferone. MePT2 regio-specifically catalyses the formation of C-3 dimethylallylated products from quinolone alkaloids. This is the first report that a plant PT catalyses the simultaneous formation of C- and O-prenylated products, and a plant PT specifically utilizes quinolone alkaloids as prenyl acceptors. The results not only provide important insight into the functional diversity of plant PTs and the biosynthesis of the prenylated coumarins, quinolone and carbazole alkaloids in Murraya plants, but also pave the way for the overproduction of the prenylated coumarins and alkaloids using metabolic engineering approaches.

Effective co-treatment of synthetic acid mine drainage and domestic sewage using multi-unit passive treatment system supplemented with silage fermentation broth as carbon source.

A multi-unit passive treatment system was constructed for co-treatment of synthetic acid mine drainage (AMD) and domestic sewage supplemented with silage fermentation broth as carbon source. AMD and domestic sewage mixing pretreatment (unit 1) improved influent quality with pH increase, metals removal and nutrients supplement. The generated metal-rich sludge in unit 1 retained the metals (69.95% of Fe, 97.36% of Cu, 96.53% of Cd, 72.52% of Zn, and 8.59% of Mn) of influent prior to entering subsequent bioreactors. Silage fermentation broth performed well to promote bacterial sulfate reduction in sulfate reducing bioreactor system (unit 2). Residual metals (Mn) and organic/nutrient pollutants were further polished in surface-flow aerobic wetland (unit 3), where relatively high pH (7.4-8.6), aerobic condition, potential Mn-oxidizing bacteria, limestone layer and low concentrations of Fe(II) (0.04-3.5 mg/L) favored the efficient removal of Mn. After 210-day continuous flow-through experiment, this passive treatment system demonstrated the efficient performance, increasing pH from 2.5 to 8.0 with removal of metals (99%), sulfate and organic/nutrient pollutants. Diverse sulfate reducing bacteria including complete organic oxidizers (e.g. Desulfobacter) and incomplete organic oxidizers (e.g. Desulfovibrio) promoted sulfate reduction and organic/nutrient pollutants removal. Ammonia oxidizing bacteria (e.g. Nitrosomonas) and nitrite oxidizing bacteria (e.g. unidentified_Nitrospiraceae) were the potential nitrifiers for ammonia removal. Collaboration of anaerobic denitrifiers (e.g. Denitratisoma) and potential heterotrophic nitrifying and aerobic denitrifiers (HN-AD) achieved effective nitrate removal. This multi-unit treatment system with domestic sewage and silage fermentation broth as stimulation substrates provided an attractive option for AMD treatment.

[Study on alleviating effect of Glycyrrhizae Radix et Rhizoma on Psoraleae Fructus-induced liver injury based on network pharmacology and cell experiments].

This study was designed to explore the alleviating effect and mechanism of Glycyrrhizae Radix et Rhizoma against Psora-leae Fructus-induced liver injury based on network pharmacology and cell experiments. The active components of Glycyrrhizae Radix et Rhizoma and Psoraleae Fructus were first retrieved from the Encyclopedia of Traditional Chinese Medicine(ETCM), Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), Comparative Toxicogenomics Database(CTD), and literature and further screened by SwissADME. The obtained 25 potential toxic components of Psoraleae Fructus and 29 flavonoids in Glycyrrhizae Radix et Rhizoma were input into the SwissTargetPrediction for target predication. A total of 818 targets related to liver injury were screened out based on GeneCards and MalaCards, and 91 common targets of Psoraleae Fructus, Glycyrrhizae Radix et Rhizoma, and liver injury were obtained from Venny. STRING was applied for constructing the PPI network, and Metascape for analyzing the biological processes and signaling pathways that common targets participated in. Cytoscape was used to construct the component-target-disease network and component-target-pathway network for Glycyrrhizae Radix et Rhizoma against Psoraleae Fructus-induced liver injury. The predicted core targets were proto-oncogene tyrosine-protein kinase(SRC), phosphatidylinositol 4,5-bisphosphate 3-kinase subunit alpha(PIK3 CA), RAC-alpha serine/threonine-protein kinase(AKT1), etc, with PI3 K-AKT signaling pathway, MAPK signaling pathway, apoptosis, Toll-like receptor signaling pathway, and NF-κB signaling pathway mainly involved. Following the scree-ning of the main toxic and pharmacodynamic components, the pharmacodynamic effects were investigated by cell experiments. The results showed that licochalcone A was mainly responsible for alleviating coryfolin-induced liver injury, licochalcone B for coryfolin-and psoralidin-induced liver injury, and echinatin for corylifolinin-and bakuchiol-induced liver injury. The preliminary revealing of the alleviating effect of Glycyrrhizae Radix et Rhizoma on Psoraleae Fructus-induced liver injury and the prediction of related mechanisms will provide reference for further mechanism research and reasonable clinical compatibility.

Isoandrographolide inhibits NLRP3 inflammasome activation and attenuates silicosis in mice.

Silicosis is an irreversible occupational disease caused by silica particle exposure. Abundant evidences suggest that NLRP3-mediated inflammation acts an essential role in fibrogenesis and the pathogenesis of silicosis. In the current work, we firstly reported that (8R-12S)-isoandrographolide (ISA), a diterpenoid lactone ingredient of Chinese traditional medicinal plant Andrographis paniculata (Burm.f.) Nees, could reduce pulmonary inflammation and fibrosis by inhibiting NLRP3, and thereby ameliorate silicosis. ISA administration significantly alleviated lung injury, and attenuated inflammatory response, EMT, as well as collagen deposition in the lung of silica-induced mice. Further studies verified that ISA inhibited the expressions of NLRP3 inflammasome-related proteins NLRP3, ASC and caspase-1 in vivo and in vitro, leading to the attenuation of inflammation and EMT. Additionally, the molecular docking assay indicated that ISA possibly interacted with the residues of LYS26 and GLU47 of NLRP3, implying that ISA might directly bond to protein NLRP3. Of note, ISA revealed a lower cytotoxicity but more potent therapeutic effect than andrographolide (AD), the major active extract of A. paniculata, which has been traditionally used to treat inflammation-related diseases. Taken together, our study clarified a novel role of ISA in attenuating inflammation and fibrosis in silicosis, and indicated a bright future of ISA as a lead compound for developing therapeutic drug for silicosis.

[Comparison on distribution of 8 components from ethanol extract of Psoraleae Fructus between normal and lipopolysaccharide-induced model rats].

UPLC-TQ/MS was employed to determine the content of 8 main components(psoralen, isopsoralen, psoralenoside, isopsoralenoside, bavachin, psoralidin, corylin, and neobavaisoflavone) in tissues of normal and lipopolysaccharide(LPS)-induced model rats 0.5, 1, 2, 6, and 12 h after intragastric administration of 3.6 g·kg~(-1) ethanol extract of Psoraleae Fructus. The distribution characteristics of the 8 main components in the different tissues(liver, kidney, spleen, heart, and lung) were studied and compared. The results showed that the distribution behaviors of the components varied among different tissues. At different time points, the components presented wide and uneven distribution in the body. Liver and kidney had higher content of the components, followed by spleen, heart, and lung. In both normal and LPS-induced model rats, the content of the 8 main components was higher in liver and kidney and varied significantly among different tissues. The content of psoralen in the tissues of LPS-induced model rat was significantly higher than that of the normal group 12 h after administration. The reason may be that the modeling slowed down the absorption and distribution of psoralen. The LPS-induced model rats had higher content of psoralenoside and isopsoralenoside in the liver tissue than the normal rats, which indicated that the modeling increased the absorption and distribution of psoralenoside and isopsoralenoside in the liver tissue. Further, it is hypothesized that psoralenoside and isopsoralenoside may be toxic substances of Psoraleae Fructus-induced liver injury.

Study on the mechanism of ErtongKe granules in the treatment of cough using network pharmacology and molecular docking technology.

BACKGROUND: The etiology and pathogenesis of cough are complex. As a Chinese patent medicine that has been on the market, ErtongKe (ETK) granules have a good effect in treating acute and chronic cough in children. The purpose of this research was to determine the bioactive components and possible action mechanisms of ETK in the treatment of cough using an integrated network pharmacology method. METHODS: The Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Swiss target prediction databases were used to screen the potential components and associated targets of ETK. The Genecards database was then used to gather targets interacting with cough. An analysis of the signaling pathways associated with ETK for cough treatment was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analysis methods. Cytoscape 3.8.1 was used to design the protein-protein interaction (PPI) and compound-target-pathway networks. Finally, the important genes and active components of ETK were confirmed using Auto Dock vina and Discovery studio software. RESULTS: Total 242 active components of ETK were screened, 1,173 potential targets related to the ingredients and 4,400 targets related to cough were collected separately. Moreover, 600 candidate targets and 39 signaling pathways were determined. We also screened out the following core components, including tuberostemonone, quercetin, kaempferol, praeruptorin E, stigmasterol, oroxylin A, and other potentially active ingredients. At the same time, 8 core targets, including JUN, PIK3CA, PIK3R1, MAPK14, EGFR, SRC, AKT1, and MAPK1, and 20 key pathways, including the cAMP signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway among others, were also selected. All the 8 core targets were verified by molecular docking. CONCLUSIONS: This research established that ETK exerts anti-cough activity by modulating several targets and pathways through multiple components. Additionally, the pooled results shed light on ETK compounds being investigated as potential antitussives.

Illumination of Molecular Pathways in Multiple Sclerosis Lesions and the Immune Mechanism of Matrine Treatment in EAE, a Mouse Model of MS.

The etiology of multiple sclerosis (MS) is not clear, and the treatment of MS presents a great challenge. This study aimed to investigate the pathogenesis and potential therapeutic targets of MS and to define target genes of matrine, a quinolizidine alkaloid component derived from the root of Sophorae flavescens that effectively suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To this end, the GSE108000 gene data set in the Gene Expression Omnibus Database, which included 7 chronic active MS lesions and 10 control samples of white matter, was analyzed for differentially expressed genes (DEGs). X cell was used to analyze the microenvironmental differences in brain tissue samples of MS patients, including 64 types of immune cells and stromal cells. The biological functions and enriched signaling pathways of DEGs were analyzed by multiple approaches, including GO, KEGG, GSEA, and GSVA. The results by X cell showed significantly increased numbers of immune cell populations in the MS lesions, with decreased erythrocytes, megakaryocytes, adipocytes, keratinocytes, endothelial cells, Th1 cells and Tregs. In GSE108000, there were 637 DEGs, including 428 up-regulated and 209 down-regulated genes. Potential target genes of matrine were then predicted by the network pharmacology method of Traditional Chinese medicine, and 12 key genes were obtained by cross analysis of the target genes of matrine and DEGs in MS lesions. Finally, we confirmed by RT-PCR the predicted expression of these genes in brain tissues of matrine-treated EAE mice. Among these genes, 2 were significantly downregulated and 6 upregulated by matrine treatment, and the significance of this gene regulation was further investigated. In conclusion, our study defined several possible matrine target genes, which can be further elucidated as mechanism(s) of matrine action, and novel targets in the treatment of MS.

Genome mining and biosynthesis of the Acyl-CoA:cholesterol acyltransferase inhibitor beauveriolide I and III in Cordyceps militaris.

Ascomycete fungi Cordyceps are widely used in traditional Chinese medicine, and numerous investigations have been carried out to uncover their biological activities. However, primary researches on the physiological effects of Cordyceps were committed using crude extracts. At present, there are only a few compounds which were comprehensively characterized from Cordyceps, partial owing to the low production. In order to scientifically take advantage of Cordyceps, we used the strategy of genome mining to discover bioactive compounds from Cordyceps militaris. We found the putative biosynthetic gene cluster of the acyl-CoA:cholesterol acyltransferase inhibitor beauveriolides in the genome of C. militaris, and produced the compounds by heterologous expression in Aspergillus nidulans. Production of beauveriolide I and III also was detected in both ferment mycelia and fruiting bodies of C. militaris. The possible biosynthetic pathway was proposed. Our studies unveil the active compounds of C. militaris against atherosclerosis and Alzheimer's disease and provide the enzyme resources for the biosynthesis of new cyclodepsipeptide molecules.

Matrine Treatment Blocks NogoA-Induced Neural Inhibitory Signaling Pathway in Ongoing Experimental Autoimmune Encephalomyelitis.

Myelin-associated inhibitors, such as NogoA, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), play a pivotal role in the lack of neuroregeneration in multiple sclerosis, an inflammatory demyelinating disease of the central nervous system (CNS). Matrine (MAT), a monomer that is used in traditional Chinese medicine as an anti-inflammatory agent, has shown beneficial effects in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, the underlying mechanisms of MAT-induced EAE amelioration are not fully understood. In the present study, we show that MAT treatment suppressed ongoing EAE, and this effect correlated with an increased expression of growth-associated protein 43, an established marker for axonal regeneration. MAT treatment significantly reduced the levels of NogoA, its receptor complex NgR/p75NTR/LINGO-1, and their downstream RhoA/ROCK signaling pathway in the CNS. In contrast, intracellular cyclic AMP (cAMP) levels and its protein kinase (protein kinase A (PKA)), which can promote axonal regrowth by inactivating the RhoA, were upregulated. Importantly, adding MAT in primary astrocytes in vitro largely induced cAMP/PKA expression, and blockade of cAMP significantly diminished MAT-induced expression of PKA and production of BDNF, a potent neurotrophic factor for neuroregeneration. Taken together, our findings demonstrate that the beneficial effects of MAT on EAE can be attributed not only to its capacity for immunomodulation, but also to its directly promoting regeneration of the injured CNS.

Disruption of Gpr45 causes reduced hypothalamic POMC expression and obesity.

A rise in the occurrence of obesity has driven exploration of its underlying genetic basis and potential targets for intervention. GWAS studies have identified obesity susceptibility pathways involving several neuropeptides that control energy homeostasis, suggesting that variations in the genes that regulate food intake and energy expenditure may contribute to obesity. In this study, we identified 5 additional obesity loci, including a neuronal orphan GPCR called Gpr45, in a forward genetic screen of mutant mice generated by piggyBac insertional mutagenesis. Disruption of Gpr45 led to increased adiposity at the time of weaning and increases in body mass, fat content, glucose intolerance, and hepatic steatosis with advancing age. Mice with disruptions in Gpr45 also displayed a reduction in expression of the metabolic regulator POMC and less energy expenditure prior to the onset of obesity. Mechanistically, we determined that GPR45 regulates POMC expression via the JAK/STAT pathway in a cell-autonomous manner. Consistent with this finding, intraventricular administration of melanotan-2, an analog of the POMC derivative α-MSH, suppressed adult obesity in Gpr45 mutants. These results reveal that GPR45 is a regulator of POMC signaling and energy expenditure, which suggests that it may be a potential intervention target to combat obesity.

Iron-Deficiency Anemia After Laparoscopic Roux-en-Y Gastric Bypass in Chinese Obese Patients with Type 2 Diabetes: a 2-Year Follow-Up Study.

BACKGROUND: This study was conducted to evaluate the prevalence of iron-deficiency anemia (IDA) after Roux-en-Y gastric bypass (RYGB) in Chinese obese patients with type 2 diabetes (T2DM). Furthermore, we evaluate potential predicting factors for onset of IDA after RYGB. METHODS: A total of 184 obese T2DM individuals who underwent RYGB were enrolled in the study. Patients were divided into three groups: male, premenopausal female, and postmenopausal female. Hematologic parameters were obtained prior to and after surgery on standardized time intervals up to 24 months postoperatively. RESULTS: At baseline, 6.0 % of patients were anemic, with similar percentages of anemic patients in each group. The relative decrease in the mean hemoglobin (Hb) level was significantly more pronounced for premenopausal female than for postmenopausal female or male. The percentage of anemia in male group had increased to 15.2 and 17.0 % at 6 and 12 months, respectively, and then decreased to 4.5 % at 24-month visit. In postmenopausal female group, the percentages of anemia constantly increase to 34.0 % at 6-month follow-up. Then, it decreased gradually to 25.0 and 26.7 % at 12- and 24-month visits, respectively. In premenopausal female group, the anemia percentages dramatically increased to 62.5 % at 24-month follow-up. Multiple logistic regression indicated that lower serum ferritin level preoperative and female were associated with higher possibility to suffer IDA 2 years after RYGB. CONCLUSIONS: Iron-deficiency and IDA are extremely frequent after RYGB in Chinese obese patients with T2DM. Premenopausal female presents unexpectedly high incidence of IDA during the 2-year observation.

Preparation of metal-resistant immobilized sulfate reducing bacteria beads for acid mine drainage treatment.

Novel immobilized sulfate-reducing bacteria (SRB) beads were prepared for the treatment of synthetic acid mine drainage (AMD) containing high concentrations of Fe, Cu, Cd and Zn using up-flow anaerobic packed-bed bioreactor. The tolerance of immobilized SRB beads to heavy metals was significantly enhanced compared with that of suspended SRB. High removal efficiencies of sulfate (61-88%) and heavy metals (>99.9%) as well as slightly alkaline effluent pH (7.3-7.8) were achieved when the bioreactor was fed with acidic influent (pH 2.7) containing high concentrations of multiple metals (Fe 469 mg/L, Cu 88 mg/L, Cd 92 mg/L and Zn 128 mg/L), which showed that the bioreactor filled with immobilized SRB beads had tolerance to AMD containing high concentrations of heavy metals. Partially decomposed maize straw was a carbon source and stabilizing agent in the initial phase of bioreactor operation but later had to be supplemented by a soluble carbon source such as sodium lactate. The microbial community in the bioreactor was characterized by denaturing gradient gel electrophoresis (DGGE) and sequencing of partial 16S rDNA genes. Synergistic interaction between SRB (Desulfovibrio desulfuricans) and co-existing fermentative bacteria could be the key factor for the utilization of complex organic substrate (maize straw) as carbon and nutrients source for sulfate reduction.

Utilization of deep eutectic solvents as novel mobile phase additives for improving the separation of bioactive quaternary alkaloids.

Deep eutectic solvents (DESs) were used as novel mobile phase additives to improve chromatographic separation of four quaternary alkaloids including coptisine chloride, sanguinarine, berberine chloride and chelerythrine on a C18 column. DESs as a new class of ionic liquids are renewably sourced, environmentally benign, low cost and easy to prepare. Seven DESs were obtained by mixing different hydrogen acceptors and hydrogen-bond donors. The effects of organic solvents, the concentration of DESs, the types of DESs and the pH values of the buffer solution on the separation of the analytes were investigated. The composition of acetonitrile and 1.0% deep eutectic solvents aqueous solution (pH 3.3, adjusted with hydrochloric acid) in a 32:68 (v/v) ratio was used as optimized mobile phase, with which four quaternary alkaloids were well separated. When a small amount of DESs was added in the mobile phase for the separation of alkaloids on the C18 column, noticeable improvements were distinctly observed such as decreasing peak tailing and improving resolution. The separation mechanism mediated by DESs as mobile phase additives can be attributed to combined effect of both hydrogen acceptors and hydrogen-bond donors. For example, choline chloride can effectively cover the residual silanols on silica surface and ethylene glycol can reduce the retention time of analytes. The proposed method has been applied to determine BerbC in Lanqin Chinese herbal oral solution and BerbC tablet. Utilization of DESs in mobile phase can efficiently improve separation and selectivity of analytes from complex samples.