ABSTRACT
Panax notoginseng saponins (PNS) extracted from the root of Panax notoginseng (Burk.) F. H. Chen are the main biologically active substances. However, PNS have low permeability and degradation in the acid gastric environment, leading to their limited bioavailability. This study aimed to determine the acute toxicity of PNS in zebrafish larvae and prepare enteric-coated pellets of PNS to enhance their permeability and stability. The survival proportion, the morphological abnormalities, and the heartbeat of zebrafish embryos were performed at treated doses of PNS ranging from 0.01 to 50 µg/ml. Administration of PNS at doses greater than 10 µg/ml may result significantly in morphological alterations such as pericardial edema and curved tail. The core pellets containing PNS, polyethylene glycols, and absorption enhancers were fabricated by the dripping method. Then, the dripping pellets were coated using Eudragit L100 as an enteric coating polymer. The characterizations of core pellets and enteric-coated pellets were evaluated. The produced dripping pellets showed advantageous features, such as ease of preparation, excellent uniformity, notable friability, high dissolution rate, and good stability. In addition, absorption enhancers were successfully incorporated into the core pellets to improve the permeability of PNS. Differential scanning calorimetry, X-ray powder diffraction, and Fourier-transform infrared spectroscopy profiles revealed that the drug was maintained in an amorphous state and did not interact with the excipients during the pellet preparation process. Histological evaluation showed that dripping pellets caused a minor effect on the surface of the jejunal mucosa. The enteric-coated pellets displayed smooth surface morphology, desirable sphericity, good flowability, uniform content, and stability in an acidic pH 1.2 environment and rapid drug release in a pH 6.8 buffer solution. Based on the findings, the enteric-coated pellets may improve the absorption and stability of PNS.
ABSTRACT
Background: Angiogenesis plays a role in both physiological and pathological processes such as wound healing, embryonic development, and cancer metastasis, and it is the process by which new blood vessels are formed from existing ones. Diosgenin is a naturally occurring steroidal sapogenin found in plants such as yam and fenugreek, which is a compound of interest because of its pharmacological properties, including anti-carcinogenic effects. Zebrafish embryos are used due to their translucent nature, which facilitates the direct observation of vascular development. Aims and Objectives: The purpose of this study is to utilize zebrafish embryos to examine the impact of diosgenin on angiogenesis. It specifically aims to determine whether diosgenin could inhibit the development of inter-segmental vessels (ISV) in zebrafish embryos and also to investigate its effect on vascular endothelial growth factor (VEGF-A), a key regulator of angiogenesis. Materials and Methods: To explore the anti-angiogenic properties of diosgenin, we performed in vivo experiments using the zebrafish angiogenesis model. This involved staining red blood cells with O-dianisidine and conducting semi- quantitative real-time polymerase chain reaction analysis to assess VEGF-A mRNA expression levels. Results: Diosgenin was shown to hinder the growth and formation of ISV in dosage-dependent development using a red blood cell staining assay when compared to the positive control, SU5416. In addition, there was a significant decrease in VEGF-A expression. Conclusion: In the presence of diosgenin, there is an evidence of inhibition of ISV development and suppression of VEGF mRNA expression. These findings underscore the therapeutic potential of diosgenin for targeting angiogenesis in cancer. However, further research is needed to understand the mechanism underlying diosgenin’s effect and its clinical relevance.
ABSTRACT
Objective To explore the effects of spermary on sexual behavior,gonadal development and sperm count production.Methods Fifty-six male zebrafishes of 10-month-old AB strain after fertilization were randomly divided into 7 tanks and set up as the normal group,model group,positive control group(clomiphene citrate group,Wuzi Yanzong Pills group),and Spermary low,medium and high concentration groups,8 fishes in each group.Except for the normal group,cyclophosphamide was given in water solution to establish the zebrafish oligospermia model,respectively.On 9 d of modeling,the positive control group was ad-ministered at a concentration of 0.1 μg/mL in the clomiphene citrate group and 6 μg/mL in the Wuzi Yanzong Pill group;the spermary low,medium and high concentration groups were administered at 200,300,900 μg/mL,respectively.On 16 d of modeling,the normal female zebrafishes were placed in the culture tank of each group in a 1∶1 ratio,and the frequency of tail-chasing was observed and recorded in male and female fishes.The body length,weight,testis weight and sperm count were measured and the statistical analysis was performed by comparing the differences among the groups before and after drug administration(one-way ANOVA).Results Com-pared with the normal group,the frequency of tail-chasing of zebrafishes,body length,body weight,testis weight and sperm count in the model group were significantly decreased(P＜0.05).Compared with the model group,the frequency of tail-chasing of zebrafishes,body length,body weight,testis weight and sperm count in the spermary low,medium and high concentration groups all were significantly increased(P＜0.05),in which the vitality change of sexual behavior in the spermary medium concentration group was most obvious,and the increase of sperms count was most obvious.Conclusion A certain dose of spermary could effectively promote the sexual behavior,gonadal development and sperm count production in male zebrafish with oligospermia.The efficacy of spermary in enhancing the male sexual function and treating oligozoospermia and infertility de-serves to be studied.
ABSTRACT
Objective:To study on anti-osteoporosis effect of different extracts of Polygoni Multiflori Radix Praeparata on zebrafish.Methods:Three kinds extracts of Polygoni Multiflori Radix Praeparata, anthraquinone and stilbene glycosides and the removal of anthraquinone and stilbene glycosides were prepared. Prednisolone was used to construct the osteoporosis model of young zebrafish. Normal control group, model group, disodium etidronate group and low-, medium- and high-dosage groups of different extracts of Polygoni Multiflori Radix Praeparata were set up. Alizarin red staining was used to investigate the mineralized skull area and bone density of juvenile zebrafish in each group. Alkaline phosphatase (AKP) and tartrate-resistant acid phosphatase (TRACP) kits were used to detect the activity of osteoblast and osteoclast enzymes in zebra larvae. The qRT PCR method was used to detect the mRNA expressions of osteoporosis related genes Runx2b, col1a2, sparc, and vdrb in each group of zebrafish.Results:Compared with model group, the skull mineralized area and bone mineral density in different extracts of Polygoni Multiflori Radix Praeparata significantly increased ( P<0.01). Polygoni Multiflori Radix Praeparata, anthraquinone and stilbene glycosides and the removal of anthraquinone and stilbene glycosides (medium- and high-dosage) could significantly increase the AKP activity of zebrafish ( P<0.01), and lower the TRAP activity of zebrafish ( P<0.01); the mRNA expression levels of Runx2b, col1a2, sparc and vdrb in juvenile zebrafish osteoporosis model were significantly up-regulated by different extracts of Polygoni Multiflori Radix Praeparata. ( P<0.01). Conclusion:Polygoni Multiflori Radix Praeparata, Anthraquinone and stilbene glycosides and the removal of anthraquinone and stilbene glycosides show better anti-osteoporosis effects. The comparison of the efficacy of three extracts from Polygonum multiflorum shows that in addition to anthraquinone and stilbene glycosides, other chemical components of Polygonum multiflorum have anti-osteoporosis effects.
ABSTRACT
Circadian rhythm is one of the biorhythms formed by organisms in the process of evolution to adapt to the rotation of the earth,which is manifested as a cyclical biorhythm of about 24 hours produced by the body under the control of the internal biological clock,coordinating sleep/wakefulness,body temperature regulation,endocrine time and other activities.Long-term circadian rhythm disorders can cause increased risk of metabolic disorders,gastrointestinal diseases,neurodegenerative diseasesand other illnesses.As a typical model animal,the aquatic organism zebrafish(Danio rerio)has been widely used in experimental studies of circadian rhythm.This paper introduces in detailthe operating mechanism of zebrafish circadian clock,the influencing factors of the input system,the genes and pathways of the circadian clock,and the physiological output,summarizes the application and advantages in circadian rhythm research,finally looks forward to future research and development,in order to provide theoretical support for circadian rhythm regulation mechanism research,related drug development and disease treatment strategies.
ABSTRACT
Objective:To conduct a comparative analysis of the radiation damage to zebrafish embryos and the associated biological mechanism after ultra-high dose rate (FLASH) and conventional dose rate irradiation.Methods:Zebrafish embryos at 4 h post-fertilization were exposed to conventional and FLASH irradiation (9 MeV electron beam). The mortality and hatchability of zebrafish after radiation exposure were recorded. Larvae at 96 h post-irradiation underwent morphological scoring, testing of reactive oxygen species (ROS) levels, and analysis of changes in oxidative stress indicators.Results:Electron beam irradiation at doses of 2-12 Gy exerted subtle effects on the mortality and hatchability of zebrafish embryos. However, single high-dose irradiation (≥ 6 Gy) could lead to developmental malformation of larvae, with conventional irradiation showing the most significant effects ( t = 0.87-9.75, P < 0.05). In contrast, after FLASH irradiation (≥ 6 Gy), the ROS levels in zebrafish and its oxidative stress indicators including superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were significantly reduced ( t = 0.42-15.19, P < 0.05). There was no statistically significant difference in ROS levels in incubating solutions after conventional and FLASH irradiation ( P > 0.05). Conclusions:Compared to conventional irradiation, FLASH irradiation can reduce radiation damage to zebrafish embryos, and this is in a dose-dependent manner. The two irradiation modes lead to different oxidative stress levels in zebrafish, which might be a significant factor in the reduction of radiation damage with FLASH irradiation.
ABSTRACT
@#Objective: To evaluate the effect of the ethyl acetate fraction derived from Sargassum pallidum extract against particulate matter (PM)-induced oxidative stress and inflammation in HaCaT cells and zebrafish. Methods: HaCaT cells and zebrafish were used to evaluate the protective effects of the ethyl acetate fraction of Sargassum pallidum extract against PM-induced oxidative stress and inflammation. The production of nitric oxide (NO), intracellular ROS, prostaglandin E2 (PGE2), and pro-inflammatory cytokines, and the expression levels of COX-2, iNOS, and NF-κB were evaluated in PM-induced HaCaT cells. Furthermore, the levels of ROS, NO, and lipid peroxidation were assessed in the PM-exposed zebrafish model. Results: The ethyl acetate fraction of Sargassum pallidum extract significantly decreased the production of NO, intracellular ROS, and PGE2 in PM-induced HaCaT cells. In addition, the fraction markedly suppressed the levels of pro-inflammatory cytokines and inhibited the expression levels of COX-2, iNOS, and NF-κB. Furthermore, it displayed remarkable protective effects against PM-induced inflammatory response and oxidative stress, represented by the reduction of NO, ROS, and lipid peroxidation in zebrafish. Conclusions: The ethyl acetate fraction of Sargassum pallidum extract exhibits a protective effect against PM-induced oxidative stress and inflammation both in vitro and in vivo and has the potential as a candidate for the development of pharmaceutical and cosmeceutical products.
ABSTRACT
Background Nano-alumina (nano-Al2O3) is a widely utilized nanomaterial. Its impacts on the environment and biological systems have garnered significant attention. Zebrafish serves as a common model organism in scientific research due to its high homology with the human genome and is extensively used in toxicity studies. Objective To investigate the developmental toxicity and neurotoxicity of nano-Al2O3 exposure in zebrafish and the corresponding mechanisms of action. Method Zebrafish embryos at 6 h post-fertilization (hpf) were randomly assigned to a control group and five dose groups exposed to nano-Al2O3 at concentrations of 200, 400, 600, 800, and 1000 μg·mL−1, respectively. Thirty embryos were included in each group, and the culture medium was replaced every 24 h until 144 hpf. The hatching rates at 48 and 72 hpf and the cumulative malformation rates up to 144 hpf were calculated. At 144 hpf, a zebrafish behavior analyzer was used to record the movement trajectories of the zebrafish, and the total distance traveled and average speed were analyzed for each group. At 144 hpf, the development of dopaminergic neurons in transgenic zebrafish expressing vmat2: GFP, brain vessels in those expressing vegf: GFP, and central nervous system neurons in those expressing elavl3: EGFP were observed under a fluorescence microscope, and statistical analysis was conducted using Image Pro Plus. Real-time quantitative PCR was employed to detect the expression levels of neuron development-related genes (syn2α, gap43, dat), Lewy body formation-related gene (α-syn), and autophagy-related genes (pink1, parkin) at 144 hpf. Results Compared to the control group, the nano-Al2O3 exposed groups exhibited reduced hatching rates at 48 hpf and increased cumulative malformation rates (P<0.05), with phenomena such as delayed development, absence of the swim bladder, and body curvature. The autonomous behavioral tests revealed that the nano-Al2O3 exposed zebrafish showed a decrease in the total distance swum (P<0.001) and a significant reduction in average speed compared to the control group. The fluorescence observations indicated that the length of dopaminergic neurons in vmat2: GFP transgenic zebrafish was reduced in the nano-Al2O3 exposed groups (P<0.001). Additionally, vegf: GFP transgenic zebrafish exhibited a significant absence of brain vessels, while elavl3: EGFP transgenic zebrafish showed a weakened fluorescence intensity of central nervous system neurons (P<0.001) and a decreased length of these neurons (P<0.01). The real-time quantitative PCR analysis revealed that compared to the control group, the gene expression levels of α-syn, syn2α, dat, and gap43 were upregulated in the zebrafish exposed to nano-Al2O3 (except for the 400 μg·mL−1 exposure group) (P<0.01), while the expression levels of parkin were downregulated in the 600 and 800 μg·mL−1 nano-Al2O3 exposed groups, and the expression levels of pink1 were downregulated in all exposure groups except for the 200 μg·mL−1 group (P<0.05). Conclusion Exposure to nano-Al2O3 exhibits developmental toxicity in zebrafish larvae and induces Parkinsonism-like symptoms in zebrafish. The preliminary speculation of the mechanism suggests that it may be related to nano-Al2O3-induced mitochondrial autophagy impairment.
ABSTRACT
Objective To study the microscopic structure and morphological characteristics of Zebrafish eyeball and retina at different developmental stages, and to lay a foundation for visual research model. Methods Select eight groups of zebrafish at different ages, with six fish in each group, 48 fish in total. Optical microscopy and transmission electron microscopy were used to observe the eyeball structure of Zebrafish at different developmental stages, and the thickness of retinal each layer was measured to analyze the temporal and spatial development pattern. The morphological characteristics of various cells in the retina and the way of nerve connection were observed from the microscopic and ultrastructural aspects, especially the structural differences between rod cells and cone cells. Results The retina of Zebrafish can be divided into ten layers including retinal pigment epithelial layer, rod cells and cone cells layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, inner limiting membrane. Rod cells had a smaller nucleus and a higher electron density than cone cells. Photoreceptor terminals were neatly arranged in the outer plexiform layer, forming neural connections with horizontal cells and bipolar cells, and several synaptic ribbons are clearly visible within them. In Zebrafish retina, ganglion cell layer and inner plexiform layer are the earliest developed. With the growth and development of Zebrafish, the thickness of rod cells and cone cells layer and retinal pigment epithelial layer gradually increases, and the retinal structure was basically developed in about 10 weeks. Conclusion The retinal structure of Zebrafish is typical, with obvious stratification and highly differentiated nerve cells. There are abundant neural connections in the outer plexiform layer. The ocular development characteristics of Zebrafish are similar to those of most mammals.
ABSTRACT
Morphine is a potent analgesic opiate used to treat chronic pain, mostly in cancer patients. In addition, it is widely used as a drug of abuse. Due to the continuous rise of morphine-associated addiction, there is an urgent need to develop pre-clinical animal models to understand the behavioural pattern of drug dependence and its withdrawal. Recently, the experimental use of zebrafish has attained significance in behavioural neuroscience studies. The literature on zebrafish is conflicting with regard to morphine withdrawal symptoms. Unfortunately, no single model provides comprehensive details to evaluate zebrafish behaviour on opiate exposure. Further, the current models have various limitations, such as short duration, complexity of phenotypes, intricate quantification, and difficulty in studying withdrawal symptoms. Consequently, a firm standardization of the protocol to understand the influence of opiates on physiological and psychological behaviours is required. In this study, we have tried to overcome the shortcomings associated with the existing models and to optimize the protocols involving an array of parameters. We observed that the administration of morphine caused a significant increase in zebrafish behavioural patterns of spiral movements, circular movements, erratic movements, upper transitions, water surface transitions, wall licking, wall licking with upper transitions, wall licking with lower transitions, absolute angle changes, and time spent in the upper compartment. A decline in the freezing bouts and time spent in the lower compartment were noticed. In essence, this study offers a zebrafish model to comprehensively examine changes in behaviour of animals on opiate dependence and its withdrawal. The present study also reported that in zebrafish, the influence of chronic exposure of morphine modulates key gene targets involved in behaviour, neuroinflammation, and autophagy, which directly or indirectly are associated with morphine addiction in a chronic morphine model.
ABSTRACT
Aims: Obesity is a non-communicable disease whose prevalence continues to increase every year throughout the world. Obesity contributes to the emergence of several diseases such as type-2 diabetes mellitus, hypertension, cardiovascular disorders, cancer, and non-alcoholic fatty liver. A number of studies report that natural ingredients have the potential to be used as a treatment for obesity while reducing a fatty liver. The aim of this research is to evaluate the activity of red spinach ethanol extract in reducing the accumulation of fatty liver in diet-induced obese zebrafish based on its histopathological profile. Methodology: Zebrafish must be adapted for 2 weeks. After 2 weeks, the zebrafish were divided into 6 groups which included: the normal group (or negative control); the obesity group (positive control group); the standard drug (orlistat with dose 4.5 µg/ml); the EERS group (dose of 50 µg/ml); the EERS group (dose of 100 µg/ml); and the EERS group (dose of 200 µg/ml). For a period of 4 weeks, the normal group received a standard diet. A positive control group received Artemia. The treated group received Artemia which was combined with the administration of red spinach (preventive method). The obese group and extract-treated group were given 60 mg/group/fish in the experimental diet. Results: The results showed that EERS at a dose of 100 µg/ml did not show any fatty liver based on their histopathological profile. The EERS dose of 200 µg/ml is more effective in reducing fatty liver when compared to doses of 50 µg/ml and 100 µg/ml on obese zebrafish. Conclusion: Based on the results of the study, it can be concluded that EERS is very prospective for further research and development as a drug for treating obesity and reducing fatty liver.
ABSTRACT
Radiation therapy has emerged as a mainstay therapeutic approach for cancer therapy. Radiation therapy includes beams of intense energy that destroy cancer cells by targeting their genetic material. Radiation treatment is a localized therapy that can be used to shrink the tumor for which it will be eligible for surgery. Chemoradiation combination is often used to inhibit the rapid proliferation and metastasis of cancer. Although radiation therapy is an important therapeutic modality for cancer, its adverse effect on normal cells and unwanted side effects cannot be ignored. Therefore, with the increase in cancer prevalence, the clinical management of radiation therapy has become a major challenge in cancer therapy. The challenges in radiation therapy can be addressed by identifying novel radiation modifiers that can potentiate the low dose of radiation on cancer, protect normal cells from radiation, and suppress radiation-induced side effects. The search for radiation modifiers needs a suitable model system through which potential radiosensitizers and radioprotectors can be screened and validated to be used in the radiation field. Keeping the importance of a suitable model in the clinical management of radiation therapy, we have discussed different models in this review that can be used to screen radiation modifiers.
ABSTRACT
Aim: The present study aimed to evaluate the antinociceptive effect of Triphala extract coated with marine derived chitosan against the formalin-induced adult zebrafish (Danio rerio) model. Methodology: Marine derived chitosan was extracted from Aspergillus niger and the extracted chitosan was coated witha commercial formulation of Triphala by a simple dispersion method. The prepared formulation was administrated to formalin-induced zebra fish model followed by studying changes in various behavioural parameters like swimming pattern, dark-light cycle pattern, antioxidants which include catalase, superoxide dismutase, lipid peroxidase, glutathione S. transferase, glutathione peroxidase and the expression pattern of inflammatory markers genes like tumour necrosis factor (TNF) and induced nitrous oxide synthase (, iNOs). Results: Marine derived chitosan-coated extract reveals notable antinociceptive activity by modulation of behavioural changes and inflammatory marker gene expression. The marine derived chitosan treatment group exhibits normal swimming patterns, dark-light cycle patterns, and morphological parameters as in the control group marine derived chitosan-coated Triphala treatment showed notable changes in the antioxidants. MARINE DERIVED CHITOSAN treatment did not induce any inflammatory signals, confirmed by a less expression pattern of inflammatory marker genes. Interpretation: These findings imply that marine derived chitosan-coated Triphala can be used as an antinociceptive agent through active phyto-principles involved in the molecular mechanism of pain manifestation.
ABSTRACT
ObjectiveTo study the mechanism of matrine in the treatment of inflammatory bowel disease （IBD） based on the zebrafish model and network pharmacology. MethodThe IBD model of zebrafish was established using 2，4，6-trinitro-benzenesulfonicacid （TNBS）， and the intestinal phagocytic function， goblet cell secretion， and neutrophil aggregation were evaluated using neutral red staining， alcian blue staining， and neutrophil number changes. Changes in tumor necrosis factor （TNF）-α and cholecystokinin （CCK） content in zebrafish were determined by using relevant reagent kits. Network pharmacology and molecular docking techniques were used to predict the potential mechanism of matrine in the treatment of IBD. Gene expression of relevant targets was verified through Real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the model group， the matrine administration group can increase the neutral red staining area in a dose-dependent manner and improve intestinal phagocytic function（P<0.05，P<0.01）. It can reduce the staining area of alcian blue and affect the secretion of intestinal goblet cells（P<0.01）. It can reduce the number of neutrophil granulocytes， relieve its aggregation， significantly reduce TNF-α content（P<0.01）， and increase the CCK content. Network pharmacology analysis identifies 28 potential targets for matrine in the treatment of IBD. The top five targets by protein-protein interaction （PPI） network analysis are CHRNA7， DRD1， CHRNA4， SLC6A3， and GRM5. The Kyoto encyclopedia of genes and genomes （KEGG） results show that the treatment of IBD with matrine may be related to neuroactive ligand-receptor interaction， cholinergic synapse， and neutrophil extracellular trap formation. Real-time PCR results show that matrine can affect the expression level of related target genes. Conclusionmatrine has a certain therapeutic effect on IBD and can affect the inflammatory response of IBD. Its therapeutic effect may be related to neuroactive ligand-receptor interaction and other pathways.
ABSTRACT
Objective: To detect the therapeutic efficacy of FGF21 analogues on the zebrafish model of non-alcoholic fatty liver disease. Methods: A zebrafish model of non-alcoholic fatty liver disease was established by providing the normal diet fed to wild-type zebrafish three times daily. PF-05231023 was administered exogenously at a final concentration of 0.5 μmol/L. Body length, body weight, triglycerides, and other indexes were measured after 20 days. Pathological changes were evaluated in liver tissue sections by HE staining. Quantitative PCR was used to identify expressional changes in genes related to lipid metabolism, endoplasmic reticulum stress, and inflammation. Results: QPCR and immunofluorescence staining results showed that FGF21 was highly expressed in the zebrafish model group. The addition of the FGF21 analogue PF-05231023 significantly reduced the body length and body weight (P < 0.01), and the triglyceride content (P < 0.05) in the zebrafish model group. The liver HE staining results showed that PF-05231023 had alleviated the large and tiny bullae fat, lesions, and others in the zebrafish model group. The quantitative PCR results demonstrated that PF-05231023 reduced the expression of lipogenic factors (P < 0.01), inflammatory-related factors (P < 0.001), and genes related to endoplasmic reticulum stress (P < 0.05), but raised lipid-oxidation-related factors (P < 0.05) in the zebrafish model group. The addition of PF-05231023 reduced oleic acid-induced lipid and triglyceride levels in HepG2 cells. Conclusion: FGF21 analogue addition can improve indexes in the zebrafish disease model of non-alcoholic fatty liver disease.
Subject(s)
Animals , Body Weight , Diet, High-Fat , Lipids , Liver/pathology , Non-alcoholic Fatty Liver Disease/pathology , Triglycerides/metabolism , Zebrafish/metabolism , Zebrafish ProteinsABSTRACT
Polyphyllin Ⅰ(PPⅠ)and polyphyllin Ⅱ(PⅡ)are the main active substances in the Paris polyphylla.However,liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated.In this work,we found that PPⅠ and PⅡ exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner.The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepa-totoxicity of PPⅡ and PⅡwas associated with the cholesterol biosynthetic pathway disorders,which were alleviated by the cholesterol biosynthesis inhibitor lovastatin.Additionally,3-hydroxy-3-methy-lglutaryl CoA reductase(HMGCR)and squalene epoxidase(SQLE),the two rate-limiting enzymes in the choles-terol synthesis,selected as the potential targets,were confirmed by the molecular docking,the over-expression,and knockdown of HMGCR or SQLE with siRNA.Finally,the pull-down and surface plasmon resonance technology revealed that PPⅠ could directly bind with SQLE but not with HMGCR.Collectively,these data demonstrated that PPⅠ-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways,thus disturbing the cholesterol biosynthesis pathway.The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.
ABSTRACT
This study used the zebrafish model to explore the hepatotoxicity of Rhododendri Mollis Flos(RMF). The mortality was calculated according to the number of the survival of zebrafish larvae 4 days after fertilization under different concentration of RMF, and the dose-toxicity curve was fitted to preliminarily evaluate the toxicity of RMF. The liver phenotypes under the sublethal concentration of RMF in the treatment group and the blank control group were observed by hematoxylin-eosin(HE) staining and acridine orange(AO) staining. Meanwhile, the activities of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were determined to confirm the hepatotoxicity of RMF. Real-time quantitative polymerase chain reaction(real-time PCR) and Western blot were used to determine the expressions of genes and proteins in zebrafish larvae. Gas chromatography time-of-flight mass spectrometry(GC-TOF-MS) was used to conduct untargeted metabolomics testing to explore the mechanism. The results showed that the toxicity of RMF to zebrafish larvae was dose-dependent, with 1 100 μg·mL~(-1) of the absolute lethal concentration and 448 μg·mL~(-1) of sublethal concentration. The hepatocyte apoptosis and degeneration appeared in the zebrafish larvae under the sublethal concentration of RMF. The content of ALT and AST in zebrafish larvae at the end of the experiment was significantly increased in a dose-dependent manner. Under the sublethal concentration, the expressions of genes and proteins related to apoptosis in zebrafish larvae were significantly increased as compared with the blank control group. The results of untargeted metabolomics showed that the important metabolites related to the he-patotoxicity of RMF were mainly enriched in alanine, aspartic acid, glutamic acid, and other pathways. In conclusion, it is inferred that RMF has certain hepatotoxicity to zebrafish larvae, and its mechanism may be related to apoptosis.
Subject(s)
Animals , Zebrafish/genetics , Apoptosis , Larva , Chemical and Drug Induced Liver InjuryABSTRACT
Juvenile zebrafish were used to screen the active components of Lycii Fructus for improving osteoporosis. The screening results were further verified by zebrafish adult osteoporosis model and the action mechanism was explored. Prednisolone was used as the inducer to build osteoporosis models of juvenile and adult zebrafish, and 9 groups of samples of different extracts and chemical parts of Lycii Fructus were given. Alizarin red staining was applied for observing the scale matrix mineralization and bone resorption. The activities of osteoblasts and osteoclasts were detected using alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP/TRACP) staining. The expressions of bone metabolism-related genes alp, osteoprotectin (opn), osteoblast specific transcription factor (sp7), cathepsin K (ctsk), tracp, and Runt family transcription factor 2b (runx2b) in each group were determined using quantitative polymerase chain reaction. The results showed that all components of Lycii Fructus improved the formation area of the first vertebrae, the staining light density value, and the number of vertebrae joints in juvenile zebrafish and the Lycium barbarum polysaccharide (LBP) treatment group exerted the best effect. In addition, LBP prevented the formation of bone resorption lacunae in zebrafish scales, increased ALP activity, decreased TRAP activity, up-regulated the alp, sp7, and opn genes, and lowered the expressions of ctsk and tracp genes. In conclusion, LBP regulated the activity of osteoblasts and osteoclasts, reduced bone resorption, promoted bone formation and enhanced bone density, which might be the main anti-osteoporosis active fraction of Lycii Fructus. This study provided modern scientific evidence for the scientific connotation of the traditional effect of "strengthening bones and muscles" of Lycii Fructus, provided the reference for the evaluation of the anti-osteoporosis activity of traditional Chinese medicine based on zebrafish adult model, and provided beneficial enlightenment for the bone health needs of the aging society population.
ABSTRACT
Background Lead is widely distributed. Lead exposure interferes with early life development in zebrafish, but the mechanisms by which lead exposure affects skeletal development and cardiac development are not clear as yet. Objective To investigate the molecular mechanisms of bone development and cardiac development toxicity induced by lead acetate exposure. Methods Zebrafish embryos were exposed to different concentrations of lead acetate (0, 6, 12, 24, and 48 μmol·L−1) for 3 h post-fertilization (3 hpf) until 5 d post-fertilization (5 dpf). The malformation phenotypes of 5 dpf were counted, and the mRNA expressions of spinal development-related genes (bmp2b, bmp4, bmp9, runx2a, runx2b) and heart development-related genes (nkx2.5, myh6, myh7) were detected by quantitative PCR (qPCR). Expressions of genes of development-related regulatory pathways including Wnt/β-catenin pathway (wnt5a, wnt8a, wnt10a, β-catenin) and TGF-β pathway (tgf-β1, tgf-β2) as well as key molecule eph of Eph-Ephrin signaling were analyzed. Results At 5 dpf, the zebrafish in the lead acetate treated groups showed deformed phenotypes including spinal curvature and pericardial sac edema compared to the control group. In the lead acetate groups at 24 and 48 μmol·L−1, the spinal curvature deformity rates reached 26.47% and 71.52% (P<0.01) respectively. The qPCR results revealed that the expression levels of spinal development-related genes bmp2b, bmp4, bmp9, runx2a, and runx2b were downregulated in the 48 μmol·L−1 exposure group compared to the control group by 82.8%, 58.0%, 88.7%, 85.5%, and 69.2%, respectively (P<0.05 or P<0.01); the expression levels of heart development-related genes myh6, myh7, and nkx2.5 were down-regulated by 63.7%, 58.9%, and 55.2%, respectively (P<0.01); the expression levels of wnt8a and β-catenin in the Wnt/β-catenin pathway were down-regulated by 71.5% and 47.3% (P < 0.05 or P < 0.01), respectively; the expression level of tgf- β1 in the TGF-β pathway was down-regulated by 67.5% (P<0.01); the expression level of eph was down-regulated by 86.9% (P<0.01). Conclusion Lead acetate exerts developmental toxic effects on zebrafish heart and bone by down-regulating the expressions of genes related to spinal development and heart development, as well as inhibiting development-related Wnt/β-catenin and TGF-β pathways and Eph-Ephrin signaling, causing malformed phenotypes such as spinal curvature and pericardial sac edema.
ABSTRACT
Background Lead and manganese are heavy metal pollutants widely existing in the environment, which can accumulate in the human body through the food chain, exert neurotoxicity, and cause neurodegenerative disorders. Especially in early childhood, the developing blood-brain barrier and nervous system are highly susceptible to environmental chemical pollutants. Most of the previous studies focused on the toxic effects of single heavy metal such as lead or manganese, while the studies on combined toxic effect are still scarce, and involved mechanisms are still unclear. c-Jun N-terminal kinase (JNK) is involved in neuronal development and regeneration, and some studies have found that JNK is involved in lead or manganese induced neurotoxicity. Its role in the toxicity of combined lead and manganese is unknown. Objective To understand the neurodevelopmental toxicity mechanism and to observe changes of JNK expression in zebrafish induced by combined lead and manganese exposure at environmentlly low concentrations. Methods Zebrafish embryos within 2 h post fertilization (hpf) were divided into four groups: control group, lead exposure group (0.1 mg·L−1 lead acetate), manganese exposure group (0.3 mg·L−1 manganous chloride), and lead-manganese combined exposure group (0.1 mg·L−1 lead acetate +0.3 mg·L−1 manganous chloride) and exposed to lead or/and manganese at designed levels for 7 d. Spontaneous movements and motor locomotion were observed, and mortality rate were calculated. The changes of JNK mRNA expression in zebrafish were evaluated. Results The experimental results showed that no significant effect of lead or/and manganese on spontaneous movements and mortality rate was found in zebrafish compared with the control group (P＞0.05). The results of locomotion analysis showed that compared with the control group, the activity counts and activity distance of zebrafish in the manganese exposure group were slightly increased (P＜0.01); the activity counts and activity distance of zebrafish in the lead exposure group were reduced by 50% and those in the lead-manganese exposure group were reduced by 80% (P＜0.01). Compared with the lead exposure group, the activity counts and activity distance of zebrafish in the lead-manganese combined exposure group decreased significantly by 60% (P＜0.05). The real-time quantitative PCR results showed that the JNK mRNA expression level was significantly increased in the lead-manganese combined exposure group compared with the control group(P＜0.01). Conclusion Lead exposure combined with manganese exposure at environmentlly low concentration can induce neurodevelopmental toxicity to zebrafish. JNK may be involved in neurodevelopmental toxicity induced by the combined exposure to lead and manganese.