Bialorastins A-F, highly oxygenated and polycyclic andrastin-type meroterpenoids with proangiogenic activity from the deep-sea cold-seep-derived fungus Penicillium bialowiezense CS-283.

Six new highly oxygenated and polycyclic andrastin-type meroterpenoids, namely, bialorastins A-F (1-6), were discovered from the culture of Penicillium bialowiezense CS-283, a fungus isolated from the deep-sea cold seep squat lobster Shinkaia crosnieri. The planar structures and absolute configurations of these compounds were determined by detailed analysis of spectroscopic data, single crystal X-ray diffraction, and TDDFT-ECD calculations. Structurally, bialorastin A (1) represents a rare 17-nor-andrastin that possesses an unusual 2-oxaspiro[4.5]decane-1,4-dione moiety with a unique 6/6/6/6/5 polycyclic system, while bialorastin B (2) is also a 17-nor-andrastin featuring a gem-propane-1,2-dione moiety. Additionally, bialorastins C-E (3-5) possess a 6/6/6/6/5/5 fused hexacyclic skeleton, characterized by distinctive 3,23-acetal/lactone-bridged functionalities. All isolated compounds were evaluated for their proangiogenic activities in transgenic zebrafish. Compound 3 exhibited significant proangiogenic activity, which notably increased the number and length of intersegmental blood vessels in model zebrafish in a dose-dependent manner at concentrations of 20 and 40 µM. On a molecular scale, the tested compounds were modeled through molecular docking to have insight into the interactions with the possible target VEGFR2. Mechanistically, RT-qPCR results revealed that compound 3 could promote angiogenesis via activating VEGFR2 and subsequently activating the downstream PI3K/AKT and MAPK signaling pathways. These findings indicate that 3 could be a potential lead compound for developing angiogenesis agents.

A cross-sectional study for prevalence and risk factors of peri-implant marginal bone loss.

STATEMENT OF PROBLEM: Progressive peri-implant marginal bone loss and peri-implantitis have become a growing problem, but cross-sectional studies on their prevalence and risk factors are sparse. PURPOSE: The purpose of this cross-sectional clinical study was to investigate the prevalence of peri-implant marginal bone loss (MBL) and to identify systemic and local risk factors. MATERIAL AND METHODS: All adult patients who had received dental implants at the National Taiwan University Hospital (NTUH) during 2009 or 2010 were included. Their medical records were collected from the NTUH-integrative Medical Database. Consecutive follow-up radiographs were accessed for severity of MBL. The influence of each factor on MBL was estimated by using generalized estimating equations (GEEs). RESULTS: A total of 732 participants with 1873 implants were analyzed (mean follow-up: 5.30 years). The prevalence of MBL was 59.15% at the individual level and 49.55% at the implant level. The risk indicators identified for the presence of MBL were follow-up period of more than 2 years, diagnosis of diabetes within 12 months, radiation therapy (2 years after implant placement), implant location at maxillary canine (compared with mandibular molar), and implants from the Nobel Biocare brands (Brånemark System and NobelActive). A second multivariate GEE model confirmed the association of progressive MBL with implant location at the maxillary canine and mandibular incisor and implant brand or design. CONCLUSIONS: The identified risk indicators for MBL were longer follow-up period, diagnosis of diabetes, radiation therapy, implant location at maxillary canine, and implant brand or design.

[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.

The molecular mechanism of three novel peptides from C-phycocyanin alleviates MPTP-induced Parkinson's disease-like pathology in zebrafish.

Previous studies have shown that peptides isolated from C-phycocyanin (C-PC) possess various functions including antioxidant and anticancer activities. However, there is little research on C-PC peptides applied for the neuroprotective effect against a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) model. In this study, twelve novel peptides from C-PC were isolated, purified and identified, and the anti-PD effect of the synthesized peptides was evaluated in a zebrafish PD model. As a result, three of these peptides (MAAAHR, MPQPPAK, and MTAAAR) significantly reversed the loss of dopamine neurons and cerebral vessels, and reduced the locomotor impairment in PD zebrafish. In addition, three novel peptides could inhibit the MPTP-induced decrease of antioxidant enzymes (SOD, CAT, and GSH-Px) and increase the ROS and protein carbonylation content. In addition, they can also alleviate apoptosis of brain regions and acetylcholinesterase (AChE) activity in zebrafish. Further studies elucidated the potential molecular mechanism of peptides' anti-PD effects in the larvae. The results showed that C-PC peptides could modulate multiple genes associated with oxidative stress, autophagy and apoptosis signaling pathways, and thereby alleviate the occurrence of PD symptoms. Overall, our results highlight the neuroprotective effects of three novel peptides and provide valuable mechanistic insights and a promising drug target for the treatment of PD.

Phakellisins A-E, cyclopeptides from a marine sponge Phakellia sp. guided by LC-MS.

A chemical investigation of the marine sponge Phakellia sp. from the South China Sea yielded five new cyclopeptides, phakellisins A-E (1-5). Structures of these compounds were determined by comprehensive analysis of 1D/2D NMR, HRESIMS/MS spectroscopic data and the advanced Marfey's method. All compounds were evaluated for their cytotoxic activity. Compound 1 showed a strong inhibitory activity against WSU-DLCL-2 cells with an IC50 value of 5.25 ± 0.2 µM by induction of G0/G1 cell cycle arrest and apoptosis.

Secondary metabolites from the marine-derived fungus Penicillium chrysogenum Y20-2, and their pro-angiogenic activity.

A systematic chemical study of the secondary metabolites of the marine fungus, Penicillium chrysogenum (No. Y20-2), led to the isolation of 21 compounds, one of which is new (compound 3). The structures of the 21 compounds were determined by conducting extensive analysis of the spectroscopic data. The pro-angiogenic activity of each compound was evaluated using a zebrafish model. The results showed that compounds 7, 9, 16, and 17 had strong and dose-dependent pro-angiogenic effects, with compound 16 demonstrating the strongest pro-angiogenic activity, compounds 6, 12, 14, and 18 showing moderate activity, and compounds 8, 13, and 19 exhibiting relatively weak activity.

Management-induced changes in soil organic carbon and related crop yield dynamics in China's cropland.

Enhancing soil organic carbon (SOC) sequestration and food supply are vital for human survival when facing climate change. Site-specific best management practices (BMPs) are being promoted for adoption globally as solutions. However, how SOC and crop yield are related to each other in responding to BMPs remains unknown. Here, path analysis based on meta-analysis and machine learning was conducted to identify the effects and potential mechanisms of how the relationship between SOC and crop yield responds to site-specific BMPs in China. The results showed that BMPs could significantly enhance SOC and maintain or increase crop yield. The maximum benefits in SOC (30.6%) and crop yield (79.8%) occurred in mineral fertilizer combined with organic inputs (MOF). Specifically, the optimal SOC and crop yield would be achieved when the areas were arid, soil pH was ≥7.3, initial SOC content was ≤10 g kg-1 , duration was >10 years, and the nitrogen (N) input level was 100-200 kg ha-1 . Further analysis revealed that the original SOC level and crop yield change showed an inverted V-shaped structure. The association between the changes in SOC and crop yield might be linked to the positive role of the nutrient-mediated effect. The results generally suggested that improving the SOC can strongly support better crop performance. Limitations in increasing crop yield still exist due to low original SOC level, and in regions where the excessive N inputs, inappropriate tillage or organic input is inadequate and could be diminished by optimizing BMPs in harmony with site-specific conditions.

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.

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.

Sinudenoids A-E, C19-Norcembranoid Diterpenes with Unusual Scaffolds from the Soft Coral Sinularia densa.

Five new furanobutenolide-derived C19-norcembranoid diterpenes, sinudenoids A-E (1-5, respectively), were isolated from the soft coral Sinularia densa. Sinudenoid A (1) possesses an uncommon 5/5/11-fused tricyclic ring system. Sinudenoids B-D (2-4, respectively) share the same tetracyclic 5/5/6/6 ring system but represent two kinds of new skeletons. Sinudenoid E (5) is the second compound with the rare 8/8 bicyclic carbon core. A plausible biosynthesis pathway for compounds 1-6 is proposed. Compound 5 exhibits strong anti-inflammatory activity in the zebrafish model.

Chevalinulins A and B, Proangiogenic Alkaloids with a Spiro[bicyclo[2.2.2]octane-diketopiperazine] Skeleton from Deep-Sea Cold-Seep-Derived Fungus Aspergillus chevalieri CS-122.

Chevalinulins A (1) and B (2), two indole diketopiperazine alkaloids containing an unprecedented spiro[bicyclo[2.2.2]octane-diketopiperazine] skeleton, together with a known analogue neoechinulin B (3), were isolated from the deep-sea cold-seep-derived fungus Aspergillus chevalieri CS-122. Their structures were determined by spectroscopic analysis, single-crystal X-ray diffraction, specific rotation (SR), and NMR calculations. Compounds 1 and 2 exhibited significant in vivo proangiogenic activity in transgenic zebrafish.

Whole genome sequencing identifies a deletion mutation in the unknown-functional KCNG2 from familial sick sinus syndrome.

Sick sinus syndrome (SSS) is a term used for a variety of disorders defined by abnormal cardiac impulse formation and by abnormal propagation from the heart's sinoatrial node. In this study, we present a case from a Chinese family in which two closely related individuals had the symptoms and electrocardiographic evidence of SSS. We hypothesized that multiple individuals affected by the disease in the family was an indication of its genetic predisposition, and thus performed high-throughput sequencing for the participants from the family to detect potential disease-associated variants. One of the potential variants that was identified was a KCNG2 gene variant (NC_000018.9: g.77624068_77624079del). Further bioinformatic analysis showed that the observed variant may be a pathogenic mutation. The results of protein-protein docking and whole cell patch-clamp measurements implied that the deletion variant in KCNG2 could affect its binding the KV2.1 protein, and finally affect the function of Kv channel, which is an important determinant in regulation of heartbeat. Therefore, we inferred that the variable KCNG2 gene may affect the function of Kv channel by changing the binding conformation of KCNG2 and KV2.1 proteins and then adversely affect propagation from the sinoatrial node and cardiac impulse formation by changing the action potential repolarization of heart cells. In summary, our findings suggested that the dominant KCNG2 deletion variant in the examined Chinese family with SSS may be a potential disease-associated variant.

Impact of airborne total suspended particles (TSP) and fine particulate matter (PM2.5 )-induced developmental toxicity in zebrafish (Danio rerio) embryos.

Airborne total suspended particles (TSP) and particulate matter (PM2.5 ) threaten global health and their potential impact on cardiovascular and respiratory diseases are extensively studied. Recent studies attest premature deaths, low birth weight, and congenital anomalies in the fetus of pregnant women exposed to air pollution. In this regard, only few studies have explored the effects of TSP and PM2.5 on cardiovascular and cerebrovascular development. As both TSP and PM2.5 differ in size and composition, this study is attempted to assess the variability in toxicity effects between TSP and PM2.5 on the development of cardiovascular and cerebrovascular systems and the underlying mechanisms in a zebrafish model. To explore the potential toxic effects of TSP and PM2.5 , zebrafish embryos/larvae were exposed to 25, 50, 100, 200, and 400 µg/ml of TSP and PM2.5 from 24 to 120 hpf (hours post-fertilization). Both TSP and PM2.5 exposure increased the rate of mortality, malformations, and oxidative stress, whereas locomotor behavior, heart rate, blood flow velocity, development of cardiovasculature and neurovasculature, and dopaminergic neurons were reduced. The expression of genes involved in endoplasmic reticulum stress (ERS), Wnt signaling, and central nervous system (CNS) development were altered in a dose- and time-dependent manner. This study provides evidence for acute exposure to TSP and PM2.5 -induced cardiovascular and neurodevelopmental toxicity, attributed to enhanced oxidative stress and aberrant gene expression. Comparatively, the effects of PM2.5 were more pronounced than TSP.

Development of an alternative medium via completely replaces the medium components by mixed wastewater and crude glycerol for efficient production of docosahexaenoic acid by Schizochytrium sp.

Exorbitant substrates for Schizochytrium culture result in the high cost of docosahexaenoic acid (DHA) production. In order to develop a feasible approach that is expected to reduce DHA production cost, Schizochytrium sp. S31 cultivation with a mixture of saline wastewater (SWW) and tofu whey wastewater (TWW) was investigated in this study. Using glucose as the carbon source, the maximum biomass and DHA yield in cultures using mixed wastewater containing 5% SWW reached 19.08 and 2.66 g/L, respectively, which were 2.29 and 2.66 times higher than those of cultures using control medium. Moreover, a good wastewater treatment performance was achieved as approximately 60% of the COD, TN, and TP were reduced in the cultures using mixed wastewater with a SWW ratio of 5%. The mixed wastewater presented better performance on DHA production than control medium using all tested carbon sources including glucose, fructose, and pure and crude glycerol. The components of control medium can be completely replaced by the mixed wastewater and crude glycerol. It is expected to effectively decrease the medium cost for DHA production and reduce the environmental risk of food processing wastewater.

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.

Asperfloketals A and B, the First Two Ergostanes with Rearranged A and D Rings: From the Sponge-Associated Aspergillus flocculosus 16D-1.

Asperfloketals A (1) and B (2), two 1(10 → 6)-abeo-14,15-secosteroids featuring a novel trioxahexaheterocyclic ring system, were isolated from the sponge-associated fungus Aspergillus flocculosus 16D-1. Their structures were elucidated by extensive spectroscopic analysis and NMR chemical shifts calculations, supported by DP4+ probability analysis, and their absolute configurations were determined by ECD calculations and the modified Mosher's method. Asperfloketals A and B showed strong anti-inflammatory activity in the CuSO4-induced transgenic fluorescent zebrafish but displayed no cytotoxicity against HeLa, HepG2, and SW480 cell lines.

Involvement of dopamine signaling pathway in neurodevelopmental toxicity induced by isoniazid in zebrafish.

AIMS: This study evaluated the neurodevelopmental toxicity of isoniazid (INH) in zebrafish embryos and the underlying mechanism. METHODS: Zebrafish embryos were exposed to different concentrations (2 mM, 4 mM, 8 mM, 16 mM, 32 mM) INH for 120 hpf. During the exposure period, the percentage of embryo/larva mortality, hatching, and morphological malformation were checked every 24 h until 120 hpf. The development of blood vessels in the brain was observed at 72 hpf and 120 hpf, and behavioral capacity and acridine orange (AO) staining were measured at 120 hpf. Alterations in the mRNA expression of apoptosis and dopamine signaling pathway related genes were assessed by real-time quantitative PCR (qPCR). RESULTS: INH considerably inhibited zebrafish embryo hatching and caused zebrafish larval malformation (such as brain malformation, delayed yolk sac absorption, spinal curvature, pericardial edema, and swim bladder defects). High concentration of INH (16 mM, 32 mM) even induced death of zebrafish. In addition, INH exposure markedly restrained the ability of the zebrafish autonomous movement, shortened the length of dopamine neurons and inhibited vascular development in the brain. No obvious apoptotic cells were observed in the control group, whereas considerable numbers of apoptotic cells appeared in the head of INH-treated larvae at 120 hpf. PCR results indicated that INH significantly raised the transcription levels of caspase-3, -8, -9, and bax and significantly decreased bcl-2 and bcl-2/bax in the zebrafish apoptotic signaling pathway. INH also markedly decreased the genes related to dopamine signaling pathway (th1, dat, drd1, drd2a, drd3, and drd4b). CONCLUSIONS: Experimental results indicated that INH had obvious neurodevelopmental toxicity in zebrafish. Persistent exposure to INH for 120 h caused apoptosis, decreased dopaminergic gene expression, altered vasculature, and reduced behaviors.

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.

Particulate matter (PM10) induces cardiovascular developmental toxicity in zebrafish embryos and larvae via the ERS, Nrf2 and Wnt pathways.

Particulate matter (PM10) is one of the most important indicators of the pollution that characterizes air quality. Epidemiological studies have shown that PM10 can cause cardiovascular-related diseases in the population. And, we studied the developmental toxicity of PM10 and the underlying mechanism of its effects on the cardiovascular system of zebrafish embryo/larva. Changes in cardiac morphology, sinus venosus and bulbus arteriosus (SV-BA) distance, heart rate, vascular subintestinalis, blood flow, returned blood volume, and reactive oxygen species (ROS) level were measured, and changes in the expression levels of certain genes were assessed via RT-PCR. The results showed that PM10 caused a significant increase in pericardial sac area and SV-BA distance, a decrease in heart rate, inhibition of vascular subintestinalis growth, blood flow obstruction, reduced venous return, and other cardiovascular toxicities. PM10 induced an increase in the ROS level and significant increases in the expression levels of ERS signalling pathway factors and Nrf2 signalling pathway factors. The expression levels of the Wnt pathway-related genes also showed significant changes. Furthermore, ROS inhibitor N-Acetyl-l-cysteine (NAC) could ameliorate the cardiovascular toxicity of PM10 in zebrafish larvae. It is speculated that PM10 may result in cardiovascular toxicity by inducing higher ROS levels in the body, which could then induce ERS and lead to defects in the expression of genes related to the Wnt signalling pathway. The Nrf2 signalling pathway was activated as a stress compensatory mechanism during the early stage of PM10-induced cardiovascular injury. However, it was insufficient to counteract the PM10-induced cardiovascular toxicity.

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.