Integrated transcriptome and proteome analysis reveals the unique molecular features and nutritional components on the muscles in Chinese Taihe black-bone silky fowl chicken.

The Taihe Black-Bone silky fowl chicken (BB-sfc) is a renowned dietary and medicinal chicken globally recognized for its high nutritional and medicinal value. Compared to the local Black-Bone black-feathered chicken (BB-bfc), the Taihe silky fowl chicken has higher levels of amino acids, trace elements, and unsaturated fatty acids in their muscles, which offer anti-aging, anti-cancer, and immune enhancing benefits. Despite this, the unique nutritional components, genes, and proteins in Taihe silky fowl chicken muscles are largely unknown. Therefore, we performed a comprehensive transcriptome and proteome analysis of muscle development between BB-sfc and BB-bfc chickens using RNA-Seq and TMT-based quantitative proteomics methods. RNA-Seq analysis identified 286 up-regulated genes and 190 down-regulated genes in BB-sfc chickens, with oxidoreductase activity and electron transfer activity enriched in up-regulated genes, and phospholipid homeostasis and cholesterol transporter activity enriched in down-regulated genes. Proteome analysis revealed 186 significantly increased and 287 significantly decreased proteins in Taihe BB-sfc chicken muscles, primarily affecting mitochondrial function and oxidative phosphorylation, crucial for enhancing muscle antioxidant capacity. Integrated transcriptome and proteome analysis identified 6 overlapped up-regulated genes and 8 overlapped down-regulated genes in Taihe silky fowl chicken, related to improved muscle antioxidant status. Taken together, this research provides a comprehensive database of gene expression and protein information in Taihe Black-Bone silky fowl chicken muscles, aiding in fully exploring their unique economic value in the future.

Pexidartinib hydrochloride exposure induces developmental toxicity and immunotoxicity in zebrafish embryos via activation of Wnt signaling.

Pexidartinib, a macrophage colony-stimulating factor receptor (CSF-1R) inhibitor, is indicated for the treatment of tendon sheath giant cell tumor (TGCT). However, few studies on the toxicity mechanisms of pexidartinib for embryonic development. In this study, the effects of pexidartinib on embryonic development and immunotoxicity in zebrafish were investigated. Zebrafish embryos at 6 h post fertilization (6 hpf) were exposed to 0, 0.5, 1.0, and 1.5 µM concentrations of pexidartinib, respectively. The results showed that different concentrations of pexidartinib induced the shorter body, decreased heart rate, reduced number of immune cells and increase of apoptotic cells. In addition, we also detected the expression of Wnt signaling pathway and inflammation-related genes, and found that these genes expression were significantly upregulated after pexidartinib treatment. To test the effects of embryonic development and immunotoxicity due to hyperactivation of Wnt signaling after pexidartinib treatment, we used IWR-1, Wnt inhibitor, for rescue. Results show that IWR-1 could not only rescue developmental defects and immune cell number, but also downregulate the high expression of Wnt signaling pathway and inflammation-related caused by pexidartinib. Collectively, our results suggest that pexidartinib induces the developmental toxicity and immunotoxicity in zebrafish embryos through hyperactivation of Wnt signaling, providing a certain reference for the new mechanisms of pexidartinib function.

Hepatic lipid metabolism disorders and immunotoxicity induced by cysteamine in early developmental stages of zebrafish.

Cysteamine, a sulfhydryl compound, is an intermediate in the metabolism of coenzyme A to taurine in living organisms. However, the potential side effects of cysteamine such as hepatotoxicity in pediatric patients have been reported in some studies. To evaluate the impact of cysteamine on infants and children, larval zebrafish (a vertebrate model) were exposed to 0.18, 0.36 and 0.54 mM cysteamine from 72 hpf to 144 hpf. Alterations in general and pathological evaluation, biochemical parameters, cell proliferation, lipid metabolism factors, inflammatory factors and Wnt signaling pathway levels were examined. Increased liver area and lipid accumulation were observed in liver morphology, staining and histopathology in a dose-dependent manner with cysteamine exposure. In addition, the experimental cysteamine group exhibited higher alanine aminotransferase, aspartate aminotransferase, total triglyceride and total cholesterol levels than the control group. Meanwhile, the levels of lipogenesis-related factors ascended whereas lipid transport-related factors descended. Oxidative stress indicators such as reactive oxygen species, MDA and SOD were upregulated after cysteamine exposure. Afterwards, transcription assays revealed that biotinidase and Wnt pathway-related genes were upregulated in the exposed group, and inhibition of Wnt signaling partially rescued the abnormal liver development. The current study found that cysteamine-induced hepatotoxicity in larval zebrafish is due to inflammation and abnormal lipid metabolism, which is mediated by biotinidase (a potential pantetheinase isoenzyme) and Wnt signaling. This provides a perspective on the safety of cysteamine administration in children and identifies potential targets for protection against adverse reactions.

Shikonin Inhibits Fin Regeneration in Zebrafish Larvae.

Shikonin is a naphthoquinone compound extracted from Chinese comfrey for treating cancer. However, there are few reports on its research on vertebrate tissue regeneration. Zebrafish is an ideal model for studying organ regeneration. In this study, we found that 3-dpf of zebrafish larvae exposed to shikonin at concentrations of 0.2, 0.3, and 0.4 mg/L showed increasingly inhibited regeneration of the tail fin. Immunohistochemical staining showed that shikonin exposure from 6 to 12 hpa increased the number of apoptotic cells in the caudal fin wound of larvae and decreased the number of proliferating cells. Shikonin exposure was found to up-regulate oxidative stress, increase ROS levels, and reduce neutrophil recruitment in the early stage of wound repair. Moreover, shikonin exposure caused disordered expression of fin regeneration blastemal-related genes. The use of astaxanthin to down-regulate oxidative stress was found to significantly reduce the inhibition of caudal fin regeneration. Mixed exposure of AMPK inhibitors or fullerenes (C60) with shikonin also showed the similar rescue effect. Collectively, our study showed that shikonin inhibited fin regeneration in zebrafish larvae by the upregulation of oxidative stress level and AMPK signaling pathway. This research provides valuable information on the mechanism of action of shikonin for its safe application.

Cysteamine affects skeletal development and impairs motor behavior in zebrafish.

Cysteamine is a kind of feed additive commonly used in agricultural production. It is also the only targeted agent for the treatment of cystinosis, and there are some side effects in clinical applications. However, the potential skeletal toxicity remains to be further elucidated. In this study, a zebrafish model was for the first time utilized to synthetically appraise the skeletal developmental defects induced by cysteamine. The embryos were treated with 0.35, 0.70, and 1.05 mM cysteamine from 6 h post fertilization (hpf) to 72 hpf. Substantial skeletal alterations were manifested as shortened body length, chondropenia, and abnormal somite development. The results of spontaneous tail coiling at 24 hpf and locomotion at 120 hpf revealed that cysteamine decreased behavioral abilities. Moreover, the level of oxidative stress in the skeleton ascended after cysteamine exposure. Transcriptional examination showed that cysteamine upregulated the expression of osteoclast-related genes but did not affect osteoblast-related genes expression. Additionally, cysteamine exposure caused the downregulation of the Notch signaling and activating of Notch signaling partially attenuated skeletal defects. Collectively, our study suggests that cysteamine leads to skeletal developmental defects and reduces locomotion activity. This hazard may be associated with cysteamine-mediated inhibition of the Notch signaling and disorganization of notochordal cells due to oxidative stress and apoptosis.

Pamiparib Induces Neurodevelopmental Defects and Cerebral Haemorrhage in Zebrafish Embryos via Inhibiting Notch Signalling.

Pamiparib is a poly ADP-ribose polymerase (PARP) inhibitor used in clinical studies, which can penetrate the blood-brain barrier efficiently. At present, there are few studies on its effect on vertebrate neurodevelopment. In this study, we exposed zebrafish embryos to 1, 2 and 3 µM of Pamiparib from 6 to 72 h post-fertilisation (hpf). Results showed that pamiparib can specifically induce cerebral haemorrhage, brain atrophy and movement disorders in fish larvae. In addition, pamiparib exposure leads to downregulation of acetylcholinesterase (AChE) and adenosine triphosphate (ATPase) activities, and upregulation of oxidative stress which then leads to apoptosis and disrupts the gene expression involved in the neurodevelopment, neurotransmitter pathways and Parkinson's disease (PD) like symptoms. Meanwhile, astaxanthin can partially rescue neurodevelopmental defects by downregulating oxidative stress. After exposure to pamiparib, the Notch signalling is downregulated, and the use of an activator of Notch signalling can partially rescue neurodevelopmental toxicity. Therefore, our research indicates that pamiparib may induce zebrafish neurotoxicity by downregulating Notch signalling and provides a reference for the potential neurotoxicity of pamiparib during embryonic development.

ZIF-8 nanoparticles induce neurobehavioral disorders through the regulation of ROS-mediated oxidative stress in zebrafish embryos.

Zeolite imidazolate framework-8 (ZIF-8) is a nanomaterial of metal-organic frameworks (MOFs), which have various applications in drug delivery and water pollution remediation. However, little is known about its developmental neurotoxicity in aquatic organisms, especially on the low-level exposure. In the present study, we investigated the toxic effects of ZIF-8 NPs on the neuron development, behavioral traits, oxidative stress and gene expression in zebrafish embryos. Firstly, our results showed that ZIF-8 induced significantly embryonic malformations and abnormal development of nervous system in zebrafish embryos with a concentration-dependent manner. Meanwhile, the locomotor behavior was obviously inhibited while the anxiety behavior was greatly increased after ZIF-8 exposure. Secondly, the levels of ROS and antioxidant enzyme activities (CAT, SOD and MDA) together with AChE and ATPase were substantially increased in the ZIF-8 exposed groups. At the molecular level, ZIF-8 NPs could down-regulate the expression profiles of neural development-related genes (gap43, synapsin 2a and neurogenin 1) and PD-like related genes (dj-1, dynactin and parkin), but up-regulate the expression levels of neuro-inflammatory genes (nox-1, glip1a and glip1b) in larval zebrafish. In addition, we further explored the molecular mechanism of neurotoxicity induced by ZIF-8 with pharmacological experiments. The results showed that specific inhibition of ROS-mediated oxidative stress by the astaxanthin could reverse the expression patterns of ATPase, AChE and neurodevelopmental genes. Moreover, astaxanthin can partially rescue the ZIF-8-modulated locomotor behavior. Taken together, our results demonstrated that ZIF-8 had the potential to cause neurotoxicity in zebrafish embryos. These informations presented in this study will help to elucidate the molecular mechanisms of ZIF-8 nanoparticles exposure in zebrafish, which providing a scientific evaluation of its safety to aquatic ecosystems.

Lenvatinib induces cardiac developmental toxicity in zebrafish embryos through regulation of Notch mediated-oxidative stress generation.

Due to an increasing number of abused drugs dumped into the wastewater, more and more drugs are detected in the water environment, which may affect the survival of aquatic organisms. Lenvatinib is a multi-targeted tyrosine kinase inhibitor, and is clinically used to treat differentiated thyroid cancer, renal epithelial cell carcinoma and liver cancer. However, there are few reports on the effects of lenvatinib in embryos development. In this study, zebrafish embryos were used to evaluate the effect of lenvatinib on cardiovascular development. Well-developed zebrafish embryos were selected at 6 h post fertilization (hpf) and exposed to 0.05 mg/L, 0.1 mg/L and 0.2 mg/L lenvatinib up to 72 hpf. The processed embryos demonstrated cardiac edema, decreased heart rate, prolonged SV-BA distance, inhibited angiogenesis, and blocked blood circulation. Lenvatinib caused cardiac defects in the whole stage of cardiac development and increased the apoptosis of cardiomyocyte. Oxidative stress in the processed embryos was accumulated and inhibiting oxidative stress could rescue cardiac defects induced by lenvatinib. Additionally, we found that lenvatinib downregulated Notch signaling, and the activation of Notch signaling could rescue cardiac developmental defects and downregulate oxidative stress level induced by lenvatinib. Our results suggested that lenvatinib might induce cardiac developmental toxicity through inducing Notch mediated-oxidative stress generation, raising concerns about the harm of exposure to lenvatinib in aquatic organisms.

Chlorogenic acid alleviates thioacetamide-induced toxicity and promotes liver development in zebrafish (Danio rerio) through the Wnt signaling pathway.

Chlorogenic acid (CGA) is a phenylpropanoid compound that is well known to improve the antioxidant capacity and other biological activities. However, the roles of CGA in the liver development of organisms are unclear. In the present study, we aimed to investigate the function of CGA in the hepatic development in thioacetamide (TAA)-induced zebrafish embryos. We found that CGA exerted certain beneficial effects on zebrafish larvae from TAA-exposed zebrafish embryos, such as increasing the liver size, body length, heart rate, acetylcholinesterase activity, and motor ability. In addition, CGA displayed an antioxidant effect on TAA-induced zebrafish embryos by enhancing the activities of superoxide dismutase (SOD), catalase (CAT), and glucose-6-phosphate dehydrogenase (G6PDH), and decreasing of the contents of malondialdehyde (MDA), reactive oxygen species (ROS), and nitric oxide (NO). The results of western blotting analysis showed that CGA inhibited cell apoptosis by increasing the levels of Bcl2 apoptosis regulator and decreasing the levels of Bcl2 associated X (Bax), apoptosis regulator and tumor protein P53. Moreover, CGA promoted cell proliferation in TAA-induced zebrafish larvae, as detected using proliferating cell nuclear antigen fluorescence immunostaining. In addition, CGA inhibited the expression of Wnt signaling pathway genes Dkk1 (encoding Dickkopf Wnt signaling pathway inhibitors), and promoted the expression of Lef1 (encoding lymphoid enhancer binding factor 1) and Wnt2bb (encoding wingless-type MMTV integration site family, member 2Bb). When the Wnt signal inhibitor IWR-1 was added, there was no significant change in liver development in the IWR-1 + TAA group compared with the IWR-1 + TAA + CGA group (p <0.05), which suggested that CGA regulates liver development via Wnt signaling pathway. Overall, our results suggested that CGA might alleviate TAA-induced toxicity in zebrafish and promote liver development through the Wnt signaling pathway, which provides a basis for the therapeutic effect of CGA on liver dysplasia.

Lenvatinib exposure induces hepatotoxicity in zebrafish via inhibiting Wnt signaling.

Lenvatinib is a multi-kinase inhibitor for widely treating thyroid cancer. However, little studies have been done about it or its toxicity on embryonic development of vertebrate. In this study, we used zebrafish to assess the effect of lenvatinib on early embryonic development. Exposure of zebrafish embryos to 58, 117, 176 nM lenvatinib induced abnormal embryonic development, such as decreased heart rate, pericardial edema, delayed yolk absorption, and bladder atrophy. Lenvatinib exposure reduced liver area and down-regulated liver developmental related genes. The proliferation of hepatocytes and the expression of apoptosis-related genes were significantly reduced.by Lenvatinib. Furthermore, the imbalance of liver metabolism and abnormal liver tissue structure were observed in adult zebrafish after Lenvatinib exposure. Oxidative stress was up-regulated by lenvatinib and astaxanthin partially rescued hepatic developmental defects via downregulating oxidative stress. After lenvatinib exposure, Wnt signaling was down-regulated, and activation of Wnt signaling partially rescued hepatic developmental defects. Therefore, these results suggested that lenvatinib might induce zebrafish hepatotoxicity by down-regulating Wnt signaling related genes and inducing oxidative stress. This study provides a reference for the potential hepatotoxicity of lenvatinib during embryonic development and raises health concern about the potential harm of exposure to lenvatinib for foetuses.

Effects of lincomycin hydrochloride on the neurotoxicity of zebrafish.

Lincomycin hydrochloride is one of the commonly used drugs in clinic. However, it has many side effects on patients, and its mechanism is still poorly understood. In this study, 6 h post-fertilization (6 hpf) zebrafish embryos were exposed to several concentrations of lincomycin hydrochloride (15, 30, 60 µg/mL) for up to 24 or 96 hpf to detect their developmental toxicity and neurotoxicity, and to 6 days post-fertilization (6 dpf) to detect their behavioral toxicity. Our results showed that lincomycin hydrochloride could lead to embryonic head deformities (unclear ventricles, smaller ventricles, fewer new neurons). The studies showed that the frequency of spontaneous tail flick of zebrafish embryo increased at 24 hpf, and the lincomycin hydrochloride exposed zebrafish embryos showed increased heart rate, shorter body length, and yolk sac edema with severe pericardial edema at 96 hpf. The studies also showed that lincomycin hydrochloride increased oxidative stress level, Acetylcholinesterase (AChE) activity, ATPase activity and apoptosis in zebrafish larvae. In addition, the swimming behavior of zebrafish larvae decreased with the increase of lincomycin hydrochloride concentration, but the angular velocity and meandering degree increased, which might be due to the decreased activity of AChE and ATPase, as well as the decreased expression of genes related to neurodevelopment and neurotransmitter system, leading to the change of their motor behaviors. In summary, we found that lincomycin hydrochloride induced developmental toxicity and neurotoxicity in zebrafish larvae, contributing to a more comprehensive evaluation of the safety of the drug.

Protective effects and molecular mechanisms of baicalein on thioacetamide-induced toxicity in zebrafish larvae.

Baicalein is a flavonoid that is widely found in plants. Studies have shown that baicalein has anti-inflammatory, anti-cancer, and liver-protective effects. However, the effects of baicalein on TAA-induced toxicity and the underlying molecular mechanisms in zebrafish larvae are still unknown. Here, we investigated the effects of baicalein on liver development and its anti-inflammatory effects in zebrafish larvae. The results showed that baicalein has significant anti-embryonic developmental toxicity and significant antioxidant and anti-inflammatory capabilities in TAA-induced zebrafish larvae and promotes liver development and cell proliferation, reduces the expression of apoptotic proteins, and induces the expression of anti-apoptotic proteins. At the molecular level of TAA-treated zebrafish larvae, there was a decrease in the relative expression levels of mRNAs of three subfamilies, P38, ERK1, and ERK2, of the MAPK-signaling pathway and of the products of peroxisome proliferator-activated receptor (PPAR)α. Compared with TAA-treated zebrafish larvae, zebrafish larvae treated with baicalein showed an increase in the relative expression levels of P38, ERK1, and ERK2 mRNAs and the downstream products of PPARα. When MAPK signal inhibitor (SB203580) was added, it was found that liver development was inhibited and baicalin had no protective effect on TAA induced hepatotoxicity in zebrafish larvae. The results showed baicalein can protect the zebrafish larvae against toxicity induced by TAA through MAPK signal pathway. Several molecular mechanisms discovered in this study may help in the development of new drugs.

Graphene oxide nanoparticles induce hepatic dysfunction through the regulation of innate immune signaling in zebrafish (Danio rerio).

Graphene oxide (GO) is an increasingly important nanomaterial that exhibits great promise in the area of bionanotechnology and nanobiomedicine. However, the toxic effects of GO on the vertebrate developmental system are still poorly understood. Here, we aimed to investigate the toxic effects and molecular mechanisms of GO exposure in larval and adult zebrafish. The results showed that the major hepatotoxic phenotype induced by GO in zebrafish embryos was a significant decrease in liver area and a dose-dependent decrease in the hepatocytes. Moreover, the number of macrophages and neutrophils in zebrafish embryos were reduced but the expressions of pro-inflammatory cytokines were increased after GO treatment. High through-put RNA-Seq identified 314 differentially expressed genes (DEGs) in GO-induced zebrafish embryos including 192 up-regulated and 122 down-regulated. KEGG and GO functional analysis revealed that steroid hormone biosynthesis, lipoprotein metabolic process, and PPAR signaling pathway were significantly enriched. Most of the lipid metabolism genes were down-regulated while majority of the immune genes were up-regulated after GO treatment. Moreover, GO induced NF-κB p65 into the nucleus and increased the protein levels of NF-κB p65, JAK2, STAT3, and Bcl2 in adult zebrafish liver. In addition, pharmacological experiments showed that inhibition of ROS and blocking the MAPK signaling could rescue the hepatotoxic phenotypes induced by GO exposure. On the contrary, pharmacological activation of PPAR-α expression have increased the hepatotoxic effects in GO-induced larval and adult zebrafish. Taken together, these informations demonstrated that GO induced hepatic dysfunction mainly through the ROS and PPAR-α mediated innate immune signaling in zebrafish.

Immunotoxicity and transcriptome analysis of zebrafish embryos in response to glufosinate-ammonium exposure.

Glufosinate-ammonium (Gla) is a broad-spectrum and non-selective herbicide that widely used in many countries worldwide, but the biological safety including potentially negative effects on aquatic organisms remains largely unknown. In this study, we investigated the immunotoxic effects of Gla exposure on zebrafish embryos. Firstly, Gla markedly decreased the survival rate and caused a series of morphological malformations in a dose-dependent manner. Meanwhile, the number of macrophages and neutrophils was substantially reduced upon Gla exposure. In addition, the levels of oxidative stress were changed and the antioxidant enzyme activities such as CAT and SOD were elevated with the increase of Gla concentrations. Secondly, comparative transcriptome analysis identified 1, 366 differentially expressed genes (DEGs) including 789 up-regulated and 577 down-regulated in zebrafish embryos after Gla exposure. KEGG pathway analysis revealed that metabolic pathways such as drug metabolism-cytochrome P450 was markedly regulated and proteolysis, oxidation-reduction process, and peptidase activity were significantly enriched by the GO analysis. Besides, 55 immunity-related genes were identified in the DEGs, and we found that the genes in the metabolism, redox and immunity display an unique expression profilings by clustering analysis. Finally, 8 inflammatory cytokines and chemokines were further confirmed and they were differentially regulated after Gla exposure. In summary, a global survey of zebrafish defense against glufosinate was performed, and a large number of gene expression levels regarding metabolism, redox, and immunity-related genes were acquired from RNA-Seq. This study provides valuable informations for future elucidating the molecular mechanism of herbicide induced immunotoxicity in aquatic ecosystems.

CTL14, a recognition receptor induced in late stage larvae, modulates anti-fungal immunity in cotton bollworm Helicoverpa armigera.

C-type lectin (CTL) is usually considered as pattern recognition receptors in insect innate immunity. Here we found that CTL14 of Helicoverpa armigera was only activated in the fifth instar larvae not in the second instar by entomopathogen Beauveria bassiana infection. Recombinant CTL14 protein was found to form aggregates with zymosan and B. bassiana in vitro. Immunoprecipitation studies demonstrated that CTL14 interacted with serine proteinases (SP), serine proteinase inhibitor (serpin), prophenoloxidases (PPO) and vitellogenin (Vg) in the larval hemolymph. Furthermore, depletion of CTL14 using dsRNA led to dramatic decrease in the expression level of PPO1. Additionally, CTL14 depleted H. armigera decreased the resistance to fungal challenge. Taken together, our study showed the direct involvement of CTL14 in the anti-fungal immunity of H. armigera, which further explained the stronger immune responses in the fifth instar compared to the second instar larvae.

Identification of potential diagnostic biomarkers of cerebral infarction using gas chromatography-mass spectrometry and chemometrics.

Cerebral infarction (CI) is one of the most common cerebrovascular diseases and remains a major health problem worldwide. In this study, we evaluated the potential diagnostic biomarkers and important relevant metabolic pathways associated with CI. Metabolomics based on gas chromatography-mass spectrometry coupled with the multivariate pattern recognition technique were used to characterize the potential serum metabolic profiles of CI. Forty healthy controls and thirty-three cerebral infarction patients were recruited for the nontargeted global metabolites' study and subsequent targeted fatty acid analysis. Overall, thirty-four endogenous metabolites were found in serum from the untargeted global study, four of which were detected to be significantly different between the CI group and healthy controls, including l-lysine, octadecanoic acid (fatty acid), l-tyrosine and lactic acid. Additionally, fourteen free fatty acids were identified by the subsequent targeted fatty acid analysis, and seven of them were detected to be significantly different between the CI group and healthy controls, which were mainly associated with arachidonic acid metabolism and fatty acid metabolism. Our results suggest several potential diagnostic biomarkers, and serum metabolism research is demonstrated as a powerful tool to explore the pathogenesis of CI.

Benzophenone glycosides from the flower buds of Aquilaria sinensis.

Four new benzophenone glycosides named as aquilaside A-D (1-4) along with five known compounds (5-9) were isolated from the methanol extract of the flower buds of Aquilaria sinensis. Their structures were elucidated on the basis of 1D and 2D NMR and mass spectroscopic analyses. All purified compounds were evaluated for their anti-inflammatory and cytotoxic activities. Aquilasides B and C displayed moderate cytotoxicity against SK-MEL cells with IC50 of 17.0 and 12.0µM and weak NF-κB inhibitive activity at 100µM with 30% and 60%, respectively.