[Immune regulation mechanism of Saposhnikoviae Radix polysaccharide based on zebrafish model].

The immunomodulatory effect of Saposhnikoviae Radix polysaccharide(SRP) was evaluated based on the zebrafish mo-del, and its mechanism was explored by transcriptome sequencing and real-time fluorescence-based quantitative PCR(RT-qPCR). The immune-compromised model was induced by navelbine in the immunofluorescence-labeled transgenic zebrafish Tg(lyz: DsRed), and the effect of SRP on the density and distribution of macrophages in zebrafish was evaluated. The effect of SRP on the numbers of macrophages and neutrophils in wild-type AB zebrafish was detected by neutral red and Sudan black B staining. The content of NO in zebrafish was detected by DAF-FM DA fluorescence probe. The content of IL-1ß and IL-6 in zebrafish was detected by ELISA. The differentially expressed genes(DEGs) of zebrafish in the blank control group, the model group, and the SRP treatment group were analyzed by transcriptome sequencing. The immune regulation mechanism was analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment, and the expression levels of key genes were verified by RT-qPCR. The results showed that SRP could significantly increase the density of immune cells in zebrafish, increase the number of macrophages and neutrophils, and reduce the content of NO, IL-1ß, and IL-6 in immune-compromised zebrafish. The results of transcriptome sequencing analysis showed that SRP could affect the expression level of immune-related genes on Toll-like receptor pathway and herpes simplex infection pathway to affect the release of downstream cytokines and interferon, thereby completing the activation process of T cells and playing a role in regulating the immune activity of the body.

Kunxian Capsule Extract Inhibits Angiogenesis in Zebrafish Embryos via PI3K/AKT-MAPK-VEGF Pathway.

OBJECTIVE: To investigate the anti-angiogenic activity of Kunxian Capsule (KX) extract and explore the underlying molecular mechanism using zebrafish. METHODS: The KX extract was prepared with 5.0 g in 100 mL of 40% methanol followed by ultrasonication and freeze drying. Freeze dried KX extract of 10.00 mg was used as test stock solution. Triptolide and icariin, the key bioactive compounds of KX were analyzed using ultra-high performance liquid chromatography. The transgenic zebrafish Tg(flk1:GFP) embryos were dechorionated at 20-h post fertilization (hpf) and treated with PTK 787, and 3.5, 7, 14 and 21 µg/mL of KX extract, respectively. After 24-h post exposure (hpe), mortality and malformation (%), intersegmental vessels (ISV) formation, and mRNA expression level of angiogenic pathway genes including phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular signal-regulated kinases (ERKs), mitogen-activated protein kinase (MAPK), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) were determined. Further, the embryos at 72 hpf were treated with KX extract to observe the development of sub-intestinal vein (SIV) after 24 hpe. RESULTS: The chromatographic analysis of test stock solution of KX extract showed that triptolide and icariin was found as 0.089 mg/g and 48.74 mg/g, respectively, which met the requirements of the national drug standards. In zebrafish larvae experiment, KX extract significantly inhibited the ISV (P<0.01) and SIV formation (P<0.05). Besides, the mRNA expression analysis showed that KX extract could significantly suppress the expressions of PI3K and AKT, thereby inhibiting the mRNA levels of ERKs and MAPK. Moreover, the downstream signaling cascade affected the expression of VEGF and its receptors (VEGFR and VEGFR-2). FGF-2, a strong angiogenic factor, was also down-regulated by KX treatment in zebrafish larvae. CONCLUSION: KX extract exhibited anti-angiogenic effects in zebrafish embryos by regulating PI3K/AKT-MAPK-VEGF pathway and showed promising potential for RA treatment.

Radix Panacis quinquefolii Extract Ameliorates Inflammatory Bowel Disease through Inhibiting Inflammation.

OBJECTIVE: To investigate the anti-inflammatory activity of Radix Panacis quinguefolii root extract (RPQE) and its therapeutic effects on inflammatory bowel disease (IBD). METHODS: The 72-hour post-fertilization zebrafish was used to generate the local and systematic inflammation models through tail-amputation and lipopolysaccharide (LPS)-induction (100 µ g/mL), respectively. The Tg(zlyz:EGFP) zebrafish was induced with 75 µ g/mL 2,4,6-trinitrobenzene sulfonic acid (TNBS) for establishing the IBD model. The tail-amputated, LPS-, and TNBS-induced models were subjected to RPQE (ethanol fraction, 10-20 µ g/mL) administration for 12 and 24 h, respectively. Anti-inflammatory activity of RPQE was evaluated by detecting migration and aggregation of leukocytes and expression of inflammation-related genes. Meanwhile, TNBS-induced fish were immersed in 0.2% (W/V) calcein for 1.5 h and RPQE for 12 h before photographing to analyze the intestinal efflux efficiency (IEE). Moreover, the expression of inflammation-related genes in these fish was detected by quantitative polymerase chain reaction. RESULTS: Subject to RPQE administration, the migration and aggregation of leukocytes were significantly alleviated in 3 zebrafish models (P<0.01). Herein, RPQE ameliorated TNBS-induced IBD with respect to a significantly reduced number of leukocytes, improved IEE, and inhibited gene expression of pro-inflammatory factors (P<0.05 or P<0.01). CONCLUSION: RPQE exhibited therapeutic effects on IBD by inhibiting inflammation.

Tripterygium wilfordii multiglycoside-induced hepatotoxicity via inflammation and apoptosis in zebrafish.

Tripterygium wilfordii multiglycoside (GTW) is a commonly used compound for the treatment of rheumatoid arthritis (RA) and immune diseases in clinical practice. However, it can induce liver injury and the mechanism of hepatotoxicity is still not clear. This study was designed to investigate GTW-induced hepatotoxicity in zebrafish larvae and explore the mechanism involved. The 72 hpf (hours post fertilization) zebrafish larvae were administered with different concentrations of GTW for three days and their mortality, malformation rate, morphological changes in the liver, transaminase levels, and histopathological changes in the liver of zebrafish larvae were detected. The reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the levels of microRNA-122 (miR-122) and genes related to inflammation, apoptosis, cell proliferation and liver function. The results showed that GTW increased the mortality of zebrafish larvae, while significant malformations and liver damage occurred. The main manifestations were elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), significant liver atrophy, vacuoles in liver tissue, sparse cytoplasm, and unclear hepatocyte contours. RT-PCR results showed that the expression of miR-122 significantly decreased by GTW; the mRNA levels of inflammation-related genes il1ß, il6, tnfα, il10, cox2 and ptges significantly increased; the mRNA level of tgfß significantly decreased; the mRNA levels of apoptosis-related genes, caspase-8 and caspase-9, significantly increased; the mRNA level of bcl2 significantly decreased; the mRNA levels of cell proliferation-related genes, top2α and uhrf1, significantly reduced; the mRNA levels of liver function-related genes, alr and cyp3c1, significantly increased; and the mRNA level of cyp3a65 significantly decreased. In zebrafish, GTW can cause increased inflammation, enhanced apoptosis, decreased cell proliferation, and abnormal expression of liver function-related genes, leading to abnormal liver structure and function and resulting in hepatotoxicity.

Pyriproxyfen induced impairment of reproductive endocrine homeostasis and gonadal histopathology in zebrafish (Danio rerio) by altered expression of hypothalamus-pituitary-gonadal (HPG) axis genes.

Pyriproxyfen (PPF), a broad-spectrum insecticide known to cause reproductive and endocrine disruption in invertebrates, while the data is scarce in aquatic vertebrates. The goal of this study is to investigate the impact of PPF on reproductive endocrine system of male and female zebrafish along hypothalamus-pituitary-gonadal (HPG) axis. In brain, PPF caused significant alteration in the transcripts of erα, lhß, and cyp19b genes in male and fshß, lhß, and cyp19b genes in female zebrafish. The downstream genes of steroidogenic pathway like, star, 3ßhsd, 17ßhsd, and cyp19a expression were significantly altered in gonad of both sexes. Subsequent changes in circulatory steroid hormone levels lead to imbalance in hormone homeostasis as revealed from estradiol/testosterone (E2/T) ratio. Further, the vitellogenin transcript level was enhanced in hepatic tissues and their blood plasma content was increased in male (16.21%) and declined in female (21.69%). PPF also induced histopathological changes in gonads such as, reduction of mature spermatocytes in male and vitellogenic oocytes in female zebrafish. The altered E2/T ratio and gonadal histopathology were supported by the altered transcript levels of HPG axis genes. Overall, these findings provide new insights of PPF in zebrafish reproductive system and highlights for further investigations on its potential risks in aquatic environment.

Discovery and identification of antithrombotic chemical markers in Gardenia Fructus by herbal metabolomics and zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Gardenia Fructus (GF), a traditional Chinese medicine for clearing heat and purging fire, has been reported to use to treat thrombotic related diseases, but the antithrombotic components are not clear. AIM OF THE STUDY: To develop efficient research methods for discovering some representative antithrombotic compounds of GF. MATERIALS AND METHODS: AB line zebrafish induced by arachidonic acid (AA) was used as a fast and trace-sample-required valuation model for antithrombptic effect of GF samples. Among nine samples of GF from different production areas, two samples with the largest difference in bioactivity were selected for downstream analysis. High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) was applied to detect compounds in the GF samples. And herbal metabolomics and grey correlation analysis (GCA) were used to identify crucial compounds with potential antithrombotic activity. Then the bioactivity of those important compounds was verified on the zebrafish model. Network pharmacology was used to explore the protein targets and signaling pathways of these compounds. RESULTS: Among the GF samples, S1 (Huoshan City, Anhui Province), and S6 (Jichun City, Hubei Province), significantly differed in thrombus inhibiting bioactivity. HPLC-Q-TOF/MS identified a total of 614 compounds in each GF sample. 19 compounds were selected as important potential variables from metabolomics data by orthogonal partial least squares discriminant analysis (OPLS-DA). And 10 compounds among them were further found to be positively correlated with the antithrombotic bioactivity of GF by GCA. Finally, 3 compounds in them, geniposide, citric acid, and quinic acid, were confirmed as representative antithrombotic chemical markers of GF. Using network pharmacology analysis, some key protein targets, such as proto-oncogene tyrosine-protein kinase Src (SRC) and cyclin-dependent kinase 2 (CDK2), and some signaling pathways were found to supply powerful evidence about antithrombotic mechanisms of three compounds and GF. CONCLUSIONS: This research have succeeded to discover and identify three representative antithrombotic compounds of GF using an efficient integrated research strategy we established, an Omics Discriminant-Grey Correlation-Biological Activity strategy. The antithrombotic chemical makers we found could also contribute to provided more accurate index components for comprehensive quality control of GF.

[Study on liver protection and hepatotoxicity of saikosaponin a based on zebrafish model].

Bupleuri Radix has both liver protection and hepatotoxicity. Saponins are the main pharmacodynamic and toxic components of Bupleuri Radix. Based on zebrafish physical model and the model of alcoholic fatty liver( AFL) pathology,the liver toxic and protective effect of saikosaponin a( SSa) were assessed. The results indicated that 1. 77 µmol·L-1 SSa showed protective effect to AFL zebrafish. 5. 30 µmol·L-1 SSa was hepatotoxic to healthy zebrafish,but it showed protective effect to AFL zebrafish. 5. 62 µmol·L-1 SSa was hepatotoxic to healthy and AFL zebrafish. This study is benefit for clinical safety of saikosaponin a.

[TCM chemical biology--emerging interdiscipline of "TCM chemistry" and "biology"].

Traditional Chinese medicine(TCM) is a research area with highly original innovation features,and is also a Chinese name card to the world. However,TCM owns a unique theoretical system which is quite different from western modern medicine,leading to an awkward situation of deficient modern social identity as well as poor international spread. Therefore,how to establish a research strategy in line with the characteristics of TCM itself to systematically interpret the unique scientific connotation of TCM is always a public hot topic. Based on persistent practical exploration and scientific consideration in TCM,our group firstly promoted the concept of traditional Chinese medicine chemical biology(TCM chemical biology,TCMCB). The major idea of TCMCB is to clarify the nature of TCM regulating life progress to link TCM to modern medicine by using TCM components as chemical tools. Notably,TCMCB mainly focuses on TCM target identification and TCM-guided disease molecular mechanism exploration,further to clarify the basic law of TCM mediating disease process. Finally,TCMCB-guided scientific studies can help explain TCM theory and promote the developmentof modern innovative drugs based on identified targets using TCM active components. Moreover,TCMCB is of vital importance for investigating the scientific nature of biological progress and the pattern of disease occurrence and development,indicating a key significance for modern life science and medicine. This review introduces the definition of TCMCB as well as its academic thought,research method,technology system and scientific significance,for providing new research ideas and scientific thoughts for TCM development.

Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects.

One rare flavonoid-diterpene heterodimer, denticulatain C (1), one modified geranyl-type side chain substituted flavonoid, denticulatain D (2) and one geranylated flavonoid, denticulatain E (3), as well as 11 known compounds (4-14) were isolated from the fronds of Macaranga denticulata. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 4 and 8 inhibited the proliferation of A-549 cell line with IC50 values of 48.6 and 20.2 µg/mL, respectively. Compounds 3, 6, and 8 exhibited significant antiangiogenic activity on a zebrafish model with IC50 values of 9.78, 0.34, and 2.55 µg/mL, respectively.

Cytotoxic prenylated flavonoids from Macaranga indica.

Three new prenylated flavonoids, macarindicins A-C (1-3), as well as seven known compounds (4-10) were isolated from the twigs of Macaranga indica. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 2 and 3 enriched the diversity of prenyl moiety in genus Macaranga especially in the aspect of various lengths of prenyl chain. All the known compounds were isolated from M. indica for the first time and this plant was found to contain large number of ellagic acid. Compounds 1-10 were tested for their cytotoxicity against four cancer cell lines (MCF-7, Hep G2, Hela and P388) and showed IC50 values in the range of 2.61-20.35 µg/mL.

[Chemical constituents of Kadsura oblongifolia and evaluation of their toxicity].

To study the chemical constituents of K. oblongifolia, silica gel column chromatography, MCI and Sephadex LH-20 were used to separate the 70% acetone extract of the stems of K. oblongifolia. The structures of the isolated compounds have been established on the basis of physicochemical and NMR spectroscopic evidence as well as ESI-MS in some cases. Twenty compounds were obtained and identified as heteroclitalignan A (1), kadsulignan F (2), kadoblongifolin C (3), schizanrin F (4), heteroclitalignan C (5), kadsurarin (6), kadsulignan O (7), eburicol (8), meso-dihydroguaiaretic acid (9), kadsufolin A (10), tiegusanin M (11), heteroclitin B (12), (7'S)-parabenzlactone (13), angeloylbinankadsurin B (14), propinquain H (15), quercetin (16), kadsulignan P (17), schizanrin G (18), micrandilactone C (19) and (-)-shikimic acid (20). Compouds 1, 5, 8, 11-15, 18 and 20 were isolated from this plant for the first time. Toxicity of compounds 1-10 were evaluated with zebrafish model to observe the effect on its embryonic development and heart function. The results showed that compounds 7, 9 and 10 caused edema of zebrafish embryo and decreased the heart rate of zebrafish, which exhibited interference effect on heart development of zebrafish.

Cytotoxic prenylated bibenzyls and flavonoids from Macaranga kurzii.

One unique prenylated bibenzyl, kurzphenol A (1), two new prenylated flavonoids, kurzphenols B and C (2 and 3), as well as fourteen known compounds (4-17) were isolated from the twigs of Macaranga kurzii. Compound 1 was the first example of prenylated bibenzyl which possesses a benzofuran ring. All the known compounds were isolated from M. kurzii for the first time. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 1-17 were tested for their cytotoxicity against A-549 and Hep G2 cancer cell lines and showed IC50 values in the range of 9.76-30.14 µg/mL.

Chemical constituents from Euphorbia stracheyi and their biological activities.

Three new diterpenoids, stracheyioids A-C (1-3), as well as 36 known compounds (4-39) were isolated from the whole plants of Euphorbia stracheyi. Compound 1 was a rare 13-deoxy tigliane diterpenoid and compound 2 was an ingenol diterpenoid characterized by an unique 2Z,4Z-decadienoyl acidic moieties. All the known compounds were isolated from E. stracheyi for the first time. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 1-39 were tested for their cytotoxicity against five cancer cell lines (A-549, MCF-7, Hep G2, Hela and P388) and showed IC50 values in the range 6.64-42.86 µM. The antiangiogenic activities of the isolated compounds were also evaluated using a zebrafish model.

Jatropholane-type diterpenes from Euphorbia sikkimensis.

Four new jatropholane-type diterpenes (1-4), named sikkimenoids A-D, were isolated from the aerial parts of Euphorbia sikkimensis. The structural elucidations of 1-4 were accomplished by extensive NMR analyses, and their absolute configurations were established by ECD calculations. Compound 2 exhibited weak antiangiogenic activity with an IC(50) value of 43.0 µM when evaluated using a zebrafish model.

Isolation of antithrombotic phenolic compounds from the leaves of Crataegus pinnatifida.

Four novel phenolic compounds (1-4) were isolated from the leaves of Crataegus pinnatifida, along with three known ones (5-7). Their structures were elucidated as: methyl 4-O-ß-D-glucopyranosyl-3-[(2E,6E)-8-O-ß-D-glucopyranosyl-3,7-dimethyl-2,6-octadienyl] benzoate (1), biphenyl-5-ol-3-O-ß-D-glucoside (2), 3,4'-dimethoxy-biphenyl-5-ol-4-O-ß-D-glucoside (3), (E)-6-(benzoyloxy)-1-hydroxyhex-3-en-2-O-ß-D-glucoside (4), shanyenoside A (5), eriodectyol (6), and 2″-O-rhamnosyl vitexin (7), using a combination of mass spectroscopy, 1D and 2D NMR spectroscopy, and chemical analysis. The antithrombotic activity of the isolated compounds was investigated on the transgenic zebra fish system. Among them, eriodectyol (6) potently inhibited the production of thrombus.

Isolation of chemical constituents from the aerial parts of Verbascum thapsus and their antiangiogenic and antiproliferative activities.

Phytochemical investigation of Verbascum thapsus led to the isolation and identification of one new iridoid compound named verbathasin A, along with ten known compounds. The structure and relative stereochemistry of verbathasin A were elucidated by analysis of spectroscopic data. All the isolates except 10-deoxyeucommiol and ajugol were tested for antiangiogenic and antiproliferative activities, and compounds luteolin and 3-O-fucopyranosylsaikogenin F showed promising antiproliferative activities, with an obvious effect of inducing apoptosis of A549 lung cancer cells.