Frog Skin Research Articles

Biochemical parameters in skin and muscle of Pelophylax kl. esculentus frogs: Influence of a cyanobacterial bloom in situ

There is little information in scientific literature as to how conditions created by a microcystin (MC) producing cyanobacterial bloom affect the oxidant/antioxidant, biotransformation and neurotoxicity parameters in adult frogs in situ. We investigated biochemical parameters in the skin and muscle of Pelophylax kl. esculentus from Lake Ludaš (Serbia) by comparing frogs that live on the northern bloom side (BS) of the lake with those that inhabit the southern no-bloom side (NBS). A higher protein carbonylation level and lower antioxidant defense system capability in the skin of frogs living in conditions of the cyanobacterial bloom were observed. Inhibition of glutathione-dependent machinery was the major mechanism responsible for the induction of cyanobacterial bloom-mediated oxidative stress in frog skin. On the other hand, the detected higher ability of muscle to overcome bloom prooxidant toxicity was linked to a higher efficiency of the biotransformation system through glutathione-S-transferase activity and/or was the consequence of indirect exposure of the tissue to the bloom. Our results have also revealed that the cyanobacterial bloom conditions induced the cholinergic neurotransmitter system in both tissues. This study provides a better understanding of the ecotoxicological impact of the MC producing cyanobacterial bloom on frogs in situ. However, further investigations of the complex mechanism involved in cyanobacterial bloom toxicity in real environmental conditions are required.

Antibiofilm Activities in Skin Secretions of Malaysian Frogs

Biofilms are extracellular structures formed by many species of bacteria that attached on various surfaces. The formation of biofilms is responsible for many health problems as they are very difficult to treat effectively due to enhanced resistant to antibiotics. Frog skin secretions contain bioactive compounds that may exhibit antibiofilms properties against biofilms infection. This study aims to screen the presence of antibiofilm activities from lyophilized skin secretions collected from Malaysian frogs. In this study, lyophilized skin secretions from four species of Malaysian frogs were tested for biofilm inhibition activities against the bacteria Staphylococcus epidermidis, ATCC 35984. Antibiofilm activity was tested at three different stages of biofilm formation which were biofilm maturation, biofilm attachment and biofilm dispersion. Biofilm treated with skin secretion from Fejervarya multistriata showed the presence of antibiofilm activities of 45 % which was able to reduce biofilm formation to 55 % at the attachment stage. Similarly, the biofilm formation at the maturation stage was reduced to 65.8 % only. No antibiofilm activities of Fejervarya multistriata was detected at the dispersion stage. The sample showing positive results was fractionated using column chromatography and further purified by C18 reverse phase high-performance liquid chromatography column (HPLC). Mass spectroscopy revealed the presence of a single peptide. In conclusion, frog skin secretion of F. multistriata contains peptide that inhibits biofilm at both attachment and maturation stages.

Expressions of TLR4, MyD88 and IRAK4 in the Skin of Chinese Brown Frog (Rana Dybowskii) During the Breeding and Pre-Hibernation Periods

Toll-Like Receptors (TLRs) are membrane-bound proteins that recognize invading organisms with Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Related Molecular Patterns (DAMPs)...

Toxicity and Alkaloid Profiling of the Skin of the Golfo Dulcean Poison Frog Phyllobates vittatus (Dendrobatidae).

Frogs in the genus Phyllobates are known for the presence of batrachotoxin, a highly toxic alkaloid, in their skin. Nevertheless, Phyllobates frogs from Costa Rica and Panama (P. lugubris and P. vittatus) are considered non-toxic, as they have been reported to harbor low concentrations of this alkaloid. However, the potential toxicity of Central American Phyllobates has not been assessed experimentally. Our goal was to determine the toxicity of the whole skin of P. vittatus, an endemic species from the Southeastern Pacific region of Costa Rica. We performed median lethal dose (LD50) tests in mice to determine general toxicity, and an irritant assay based on the behavioral responses of mice to subcutaneous injection, to determine differences in irritability, as a measure of toxicity, among three study localities. Using UPLC-ESI-QTOF, we obtained chemical profiles of the methanolic extract of frog skins. Due to the absence of mortality at the studied doses, we were unable to estimate LD50. However, we recorded a list of toxicity symptoms in mice that are consistent with cardiotoxic effects, and found that mice presented more symptoms at higher concentrations of skin extracts during the first hour of the LD50 assays, recovering completely at all doses by the end of the assay. On the other hand, we did not detect differences in irritability among studied localities. Additionally, we putatively identified three toxic alkaloids (Batrachotoxinin A, DHQ 251A and Lehm 275A). This study provides the first experimental data on the toxicity and associated symptoms in mice, as well as the chemical profile of the skin of P. vittatus. We suggest that the skin alkaloids of P. vitattus may confer a chemical defense towards predators.

Early diagnosis of cassava frog skin disease in powdered tissue samples using near-infrared spectroscopy

Cassava frog skin disease (CFSD) causes significant yield losses in cassava (Manihot esculenta Crantz). One issue with control is late diagnosis. The objective of this work was to test near-infrared spectrometry (NIRS) and compare calibration and classification models for early detection of CFSD. NIRS analysis was performed on 238 cassava accessions (120 healthy and 118 with CFSD). Six classification models were used: a support vector machine with a linear kernel (SVM), a Bayesian generalized linear model (BGLM), a parallel random forest (PRANDF), an extreme learning machine (ELM), a high dimensional discriminant analysis (HDDA), and a partial least squares (PLS) model. Their predictive abilities were evaluated based on their accuracy and agreement (based on Cohen’s Kappa coefficient). The models demonstrated high efficiency at distinguishing diseased and healthy accessions, with an overall accuracy >80%. The Cohen’s Kappa coefficient values (0.83–0.98) for four of the models (the SVM, BGLM, PRANDF and PLS) indicated that there was almost perfect agreement between their classification results. The SVM and BGLM models exhibited high accuracy (99.07 and 98.92%, respectively) and reproducibility (both 0.98) in classifying the accessions according to their CFSD status. NIRS is a viable alternative for the detection of CFSD with the advantages of early and accurate detection, high speed and low cost compared to traditional diagnostic methods.

Percutaneous absorption between frog species: Variability in skin may influence delivery of therapeutics.

Frogs have permeable skin, so transdermal delivery provides a practical alternative to traditional dosing routes. However, little is known about how frogskin permeability differs interspecifically, and there are different reported clinical outcomes following topical application of the same chemical in different frog species. This study collated in vitro absorption kinetic data previously reported for two frog species: the green tree frog (Litoria caerulea) and the cane toad (Rhinella marina), and used linear mixed-effects modelling to produce a model of absorption. Histology of skin samples from each species was performed to observe morphological differences that may affect absorption. Absorption kinetics differed significantly between species, with the logP of the applied chemical a better predictor of permeability than molecular weight. Application site also influenced permeability, with dorsal permeability consistently higher in cane toads. Ventral permeability was more consistent between species. Skin thickness differed between species and skin regions, and this may explain the differences in absorption kinetics. Guidelines for selecting chemicals and dosing site when treating frogs are presented. The permeability differences identified may explain the poor reproducibility reported in the treatment of disease across frog species, and reinforces the importance of considering interspecies differences when designing therapeutic treatments for frogs.

Alan William Cuthbert. 7 May 1932—27 August 2016

Alan Cuthbert carried out ground-breaking work on epithelial ion transport. He used radiolabelled amiloride and benzamil to measure the sodium channel density in epithelia from frog skin and toad bladder, tissues that are good models for the distal section of the mammalian kidney tubule. This work shed important new light on how the properties of these channels are modified by hormones such as aldosterone and antidiuretic hormone, and increased our understanding of how diuretics affect kidney function. Later, he focused on the ion transport deficits that underlie cystic fibrosis (CF), and was a member of the team that showed that the ion transport defect could be corrected in CF transgenic mice by gene therapy. Alan was Sheild Professor of Pharmacology and Head of the Department of Pharmacology at the University of Cambridge from 1979 until his retirement in 1999. During this time he was instrumental in moving the Department from the Addenbrooke's Hospital site to a new building in the centre of town. He was also Master of Fitzwilliam College from 1991 until 1999. Alan made major contributions to pharmacology nationally and internationally, serving as chairman of the editorial board of the British Journal of Pharmacology for eight years, and as President of the Federation of European Pharmacological Societies for two years. In recognition of his contributions to the subject, the British Pharmacological Society awarded him their Wellcome Gold Medal in 2005. After his retirement, he continued his research in the Department of Medicine, pursuing novel pharmacological approaches to the treatment of CF.

Antimicrobial Peptides: Amphibian Host Defense Peptides.

Antimicrobial Peptides (AMPs) are one of the most common components of the innate immune system that protect multicellular organisms against microbial invasion. The vast majority of AMPs are isolated from the frog skin. Anuran (frogs and toads) skin contains abundant AMPs that can be developed therapeutically. Such peptides are a unique but diverse group of molecules. In general, more than 50% of the amino acid residues form the hydrophobic part of the molecule. Normally, there are no conserved structural motifs responsible for activity, although the vast majority of the AMPs are cationic due to the presence of multiple lysine residues; this cationicity has a close relationship with antibacterial activity. Notably, recent evidence suggests that synthesis of AMPs in frog skin may confer an advantage on a particular species, although they are not essential for survival. Frog skin AMPs exert potent activity against antibiotic-resistant bacteria, protozoa, yeasts, and fungi by permeating and destroying the plasma membrane and inactivating intracellular targets. Importantly, since they do not bind to a specific receptor, AMPs are less likely to induce resistance mechanisms. Currently, the best known amphibian AMPs are esculentins, brevinins, ranacyclins, ranatuerins, nigrocin-2, magainins, dermaseptins, bombinins, temporins, and japonicins-1 and -2, and palustrin-2. This review focuses on these frog skin AMPs and the mechanisms underlying their antimicrobial activity. We hope that this review will provide further information that will facilitate further study of AMPs and cast new light on novel and safer microbicides.

Development of Amphipathic Antimicrobial Peptide Foldamers Based on Magainin 2 Sequence.

Magainin 2 (Mag 2), which is isolated from the skin of frogs, is a representative antimicrobial peptide (AMP), exerts its antimicrobial activity via microbial membrane disruption. It has been reported that both the amphipathicity and helical structure of Mag 2 play an important role in its antimicrobial activity. In this study, we revealed that the sequence of 17 amino acid residues in Mag 2 (peptide 7) is required to exert sufficient activity. We also designed a set of Mag 2 derivatives, based on enhancement of helicity and/or amphipathicity, by incorporation of α,α-disubstituted amino acid residues into the Mag 2 fragment, and evaluated their preferred secondary structures and their antimicrobial activities against both Gram-positive and Gram-negative bacteria. As a result, peptide 11 formed a stable helical structure in solution, and possessed potent antimicrobial activities against both Gram-positive and Gram-negative bacteria without significant cytotoxicity.

Unravelling the Skin Secretion Peptides of the Gliding Leaf Frog, Agalychnis spurrelli (Hylidae).

Frog skin secretions contain medically-valuable molecules, which are useful for the discovery of new biopharmaceuticals. The peptide profile of the skin secretion of Agalychnis spurrelli has not been investigated; therefore, the structural and biological characterization of its compounds signify an inestimable opportunity to acquire new biologically-active chemical scaffolds. In this work, skin secretion from this amphibian was analysed by molecular cloning and tandem mass spectrometry. Although the extent of this work was not exhaustive, eleven skin secretion peptides belonging to five peptide families were identified. Among these, we report the occurrence of two phyllokinins, and one medusin-SP which were previously reported in other related species. In addition, eight novel peptides were identified, including four dermaseptins, DRS-SP2 to DRS-SP5, one phylloseptin-SP1, and three orphan peptides. Phylloseptin-SP1 and dermaseptins-SP2 were identified in HPLC fractions based on their molecular masses determined by MALDI-TOF MS. Among the antimicrobial peptides, dermaseptin-SP2 was the most potent, inhibiting Escherichia coli, Staphylococcus aureus, and ORSA with a minimum inhibitory concentration (MIC) of 2.68 μM, and Candida albicans with an MIC of 10.71 μM, without haemolytic effects. The peptides described in this study represent but a superficial glance at the considerable structural diversity of bioactive peptides produced in the skin secretion of A. spurrelli.

Potential skin protective effects after UVB irradiation afforded by an antioxidant peptide from Odorrana andersonii

With increasing demand, the development of new natural antioxidants has become a primary direction of scientific research. We previously identified a short gene-encoded peptide (OA-VI12) from Odorrana andersonii frog skin secretions that exerted direct scavenging capacity against free radicals, suggesting a possible function in protecting skin against photodamage caused by their high-altitude habitat. In the current research, we examined the effects of OA-VI12 on both UVB-irradiation and hydrogen peroxide-induced oxidative stress models established with human immortalized keratinocytes. In addition, we identified the differentially expressed genes (DEGs) in the oxidative stress and OA-VI12 groups and further performed transcriptome as well as functional and pathway enrichment analyses. Results showed that OA-VI12 protected cell viability, promoted the release of catalase, and decreased the levels of lactate dehydrogenase and reactive oxygen species. Moreover, the peptide promoted the production of superoxide dismutase and glutathione, alleviated epidermis and dermis thickness, and decreased the production of light spots and collagen fibers in skin from the photo-injured mouse model. Kyoto Encyclopedia of Genes and Genomes analysis showed mitogen-activated protein kinase (MAPK) to be the most abundant signaling pathway. Gene Ontology (GO) analysis indicated that the top 55 significantly enriched GO terms mainly involved cellular processes, parts, and binding. These results revealed the beneficial role of the small molecule gene-encoding antioxidant peptide (OA-VI12) and its potential application as a protective agent against photodamage.

Fighting a fungal scourge

Researchers trying to rescue amphibians from a global fungus epidemic are finding that bacteria may be their best allies . More than a decade ago, amphibian microbial ecologist Reid Harris watched a mother salamander as she marched in a figure-eight pattern through her clutch of soft, jellylike eggs. He knew that her strange walk, rubbing up against her brood, transferred beneficial bacteria from her skin onto the eggs to fight off fungal infection. Then something clicked. Harris wondered if she might also be showing him the solution to a scourge threatening hundreds of other amphibian species around the world. To fight the deadliest wildlife disease on record, a fungus responsible for the decline or disappearance of hundreds of amphibian species including the Panamanian golden frog, researchers are looking to the bacteria that live on frog skin. Image credit: Flickr/Brian Gratwicke. Then, as now, amphibians were dying in droves. At least one-third of species face extinction, with at least 90 confirmed gone and another 500 in decline (1, 2). The major culprit is the skin disease chytridiomycosis, caused by the waterborne fungus Batrachochytrium dendrobatidis , or Bd for short. Under a microscope, fungal bodies anchor themselves in the translucence of frog skin and often group together to form a collection of small spheres. Chytridiomycosis kills by disrupting ion and fluid transport, eventually stopping the host’s heart (3). A particularly deadly Bd lineage arose in the Korean peninsula sometime in the last century, the source of a pandemic that fanned out across the world through the global amphibian trade, infecting about 700 species in six global regions over the past five decades (4). Naive immune systems (mostly those of frogs) couldn’t fight off the highly contagious and aggressive pathogen, and chytridiomycosis spread like a leveling wave, becoming the most devastating …

Effect of gelatin-based coating containing frog skin oil on the quality of persimmon and its characteristics

The effect of gelatin-based coating containing frog skin oil (FSO) at different concentrations (5%, 25%, and 50%, w/w base in gelatin) on the quality of persimmon and its characteristics were investigated. Gelatin-based coating containing FSO at high concentrations significantly influenced the uniformity of the distribution of the emulsion system and smoothness of the film surface. Among persimmon fruit, the coated fruit showed the significantly (P < 0.05) reduced weight loss, pH, total soluble solids (°Brix), and firmness loss compared with uncoated samples. Notably, no differences in color between uncoated and coated fruit were observed. Lower respiration rate in the coated samples confirmed the effectiveness of gelatin-based coating containing FSO. Therefore, the results of the present study suggest that gelatin-based coating containing FSO can effectively be used as an alternative the natural coating agents.

Cinobufacini ameliorates experimental colitis via modulating the composition of gut microbiota.

BackgroundCinobufacini, the sterilized hot water extraction of dried toad skin, has been widely used in the treatment of inflammation and cancers. Recently we found cinobufacini could ameliorate dextran sulfate sodium (DSS)-induced colitis in mice, but the underlying mechanism was not fully understood. In current study, we explored the effect of cinobufacini on gut microbiota in DSS-induced acute colitic mouse model by pyrosequencing of colonic contents.MethodsC57BL/6 mice were supplied with normal or 3.0% DSS containing drinking water. DSS-treat mice were gavaged daily either with vehicle (water) or cinobufacini (10.0 or 30.0 mg/kg) for 7 days. The composition of the gut microbiota was assessed by analyzing 16S rRNA gene sequences.ResultsOur data indicated that cinobufacini reversed DSS-induced gut dysbiosis and enhanced intestinal barrier integrity. Moreover, changing of some specific microbial groups such as Proteobacteria and Bacteroides was closely correlated with the re-establishment of intestinal equilibrium and the recovery of intestinal function.ConclusionCinobufacini prevents colitis in mice by modifying the composition and function of gut microbiota. The current study provides additional mechanistic insight in the therapeutic effect of cinobufacini treatment and may pave the way for clinical application of cinobufacini in colitis therapy.

Investigations on the membrane interaction of C-terminally amidated esculentin-2 HYba1 and 2 peptides against bacteria

The membrane interaction and damage caused by C-terminally amidated esculentin-2 peptides identified from the frog skin is illustrated in the present study using Staphylococcus aureus and Vibrio cholerae. Double staining with fluorescent probes SYTOX and DAPI proved the concentration-dependent bacterial membrane damage induced by the peptides. It was found that the sub-MIC of both peptides induced transient pores on the bacterial membrane. These peptides also caused depolarisation on the bacterial membrane during their interaction. The physical changes on bacterial cells like blebbing, elongation, fusion, and so forth upon peptide treatment were visualized through SEM images. The antimicrobial activity of the peptides against S. aureus and V. cholerae was not altered at physiological concentrations of divalent and monovalent cations, which is advantageous in a therapeutic context. The increase of MIC against V. cholerae at higher concentrations of Mg2+ and Ca2+ (>5 µM) is due to the concentration-dependent antagonism exhibited by these ions for the cation binding sites on the bacterial membrane, which facilitates the process of ‘self-promoted uptake.’ The study emphasizes to utilize the ability of these peptides to produce transient pores at sub-MICs in combinatorial therapy.

The co‐occurrence of loose skin and underwater calling in frogs—further evidence from Amolops ricketti and its implications

AbstractUnderwater vocalizations of frogs are usually inaudible to the human ear and have been poorly documented. Some authors believe that they are more common than currently recognized, yet there have been no new records at the genus level for nearly 30 years. I report on the recordings of underwater calling by the ranid species Amolops ricketti (Boulenger, 1899) from a mountain stream in southern China. Males emitted 2.2–3.5 calls per minute under submerged rocks, which were inaudible to the human ear. Most of the recorded calls lasted 0.73–2.09 s and contained a weak initial note with fewer pulses (1–5) and one to three subsequent notes (3–13 pulses each) repeated at a rate of 0.82–1.99 notes·s−1, with an internote interval lasting 0.36–1.19 s. The dominant frequency of calls ranged from 2.1 to 3.0 kHz, and the notes exhibited increasing intensity and slight upward frequency modulation. Comparisons between individuals of different sexes and breeding stages reveal that the loose skin of the flanks and thighs is a male nuptial trait in A. ricketti. The finding extends the phylogenetic distribution of underwater vocalization to a distinct lineage and constitutes another instance of loose‐skin frogs calling underwater, which can also been identified in Pelobates (Pelobatidae), Leptobrachium (Megophryidae), Telmatobius (Telmatobiidae), Rana italica and Rana sakuraii (Ranidae). These independent cases may allow the proposal of an evolutionary correlation of the two traits in anurans, as a large surface/volume ratio facilitates high levels of cutaneous respiration required for the effective performance of underwater calling. An awareness of this possibility may aid in increasing our understanding of the natural history of anurans.

Peptide and non-peptide mimetics as potential therapeutics targeting oral bacteria and oral biofilms.

The development of the oral biofilm requires a complex series of interactions between host tissues and the colonizing bacteria as well as numerous interspecies interactions between the organisms themselves. Disruption of normal host–microbe homoeostasis in the oral cavity can lead to a dysbiotic microbial community that contributes to caries or periodontal disease. A variety of approaches have been pursued to develop novel potential therapeutics that are active against the oral biofilm and/or target specific oral bacteria. The structure and function of naturally occurring antimicrobial peptides from oral tissues and secretions as well as external sources such as frog skin secretions have been exploited to develop numerous peptide mimetics and small molecule peptidomimetics that show improved antimicrobial activity, increased stability and other desirable characteristics relative to the parent peptides. In addition, a rational and minimalist approach has been developed to design small artificial peptides with amphipathic α‐helical properties that exhibit potent antibacterial activity. Furthermore, with an increased understanding of the molecular mechanisms of beneficial and/or antagonistic interspecies interactions that contribute to the formation of the oral biofilm, new potential targets for therapeutic intervention have been identified and both peptide‐based and small molecule mimetics have been developed that target these key components. Many of these mimetics have shown promising results in in vitro and pre‐clinical testing and the initial clinical evaluation of several novel compounds has demonstrated their utility in humans.

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.

Permeability of frog skin to chemicals: effect of penetration enhancers

Rarely do commercial chemical products contain solely the active chemical/ingredient. It is therefore important to consider whether ingredients other than the active may: 1) alter absorption of the active chemical, or 2) be absorbed themselves, resulting in systemic effects. Frogs have highly permeable skin and are routinely exposed to commercial chemical products in the environment or therapeutically. Ethanol and propylene glycol (PG), which have known penetration-enhancing effects, are commonly included in such products. The current study has therefore investigated the in vitro absorption kinetics through Rh. marina skin of three model chemicals – caffeine, benzoic acid, and ibuprofen – formulated individually as solutions containing: 1%, 10% or 30% v/v ethanol, or 20% v/v PG. Differential scanning calorimetry and histology were used to characterise fresh frog skin, investigate the mechanism of these enhancers in frog skin, and to determine whether these enhancers significantly affected skin structure. Results showed that the extent of absorption enhancement was influenced by chemical, enhancer and skin region, and that enhancement was generally not consistent for individual enhancers or skin regions. The exception was 1% v/v ethanol, which did not significantly alter flux across the skin for any of the chemicals evaluated. Caffeine absorption was not enhanced by any of the investigated penetration enhancers, and was in fact significantly reduced by 30% v/v ethanol and PG. Ethanol caused concentration-dependant changes in skin morphology and should be avoided in concentrations ≥10% v/v. PG, however, caused minimal changes to the skin and consistently improved absorption of benzoic acid and ibuprofen through all skin regions. Owing to the significant changes in skin structure following ≥10% v/v ethanol exposure, it is recommended to avoid its use in frogs. For enhancement of penetration of moderately-to-highly lipophilic chemicals, this study has identified 20% v/v PG should to be the enhancer of choice.

The protection of indolealkylamines from LPS-induced inflammation in zebrafish

Ethnopharmacological relevanceToad skin came from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. As the traditional Chinese medicine, it had the effect of clearing away heat and detoxification. In traditional applications, toad skin was often used for the treatment of cancer and inflammation. Total indolealkylamines (IAAs) from this medicine were proved the main compounds exert anti-inflammatory activity in our previous research. Aim of the studyIn the present study, we aimed to investigate the potential mechanism of anti-inflammatory activity of IAAs on LPS induced zebrafish. Materials and methodsLPS induced zebrafish was applicated as an in vivo inflammation model to clarify the structure-activity relationship of 4 major IAAs (N-methyl serotonin, bufotenine, dehydrobufotenine and bufothionine) from toad skin. Quantitative RT-PCR was applied to detect key cytokines and members of the MyD88-dependent signaling pathway. In addition, the targeted lipidomics was conducted to find out the potential biomarkers in the inflammatory zebrafish. Network pharmacology was used to unveil the main enzymes closely related to the target lipids. ResultsOur results showed that the anti-inflammatory activity of free IAAs (N-methyl serotonin, bufotenine and dehydrobufotenine) was more potent than that of combined IAAs (bufothionine). RT-PCR demonstrated that 4 IAAs exerted antiendotoxin inflammatory effect via suppressing the TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs signaling pathway. A total of 33 possible inflammatory biomarkers, including 14 SM, 6 Cer, 11 PC and 2 GlcCer, triggered by LPS were screened out. The levels of most of candidates could be regulated toward a normal level by IAAs, especially in N-methyl serotonin and dehydrobufotenine groups. Enzymes especially LBP, PLA2, CERK, SMPD and SGMS were found closely associated with the regulation of most lipid markers. ConclusionsOverall, the mechanism underlying the anti-inflammatory activity of IAAs probably attributed to their capability to suppress NF-κB and MAPKs inflammatory pathway. Meanwhile, IAAs could also interfere the metabolism of SM, Cer and PC probably by regulating LBP, PLA2, CERK, SMPD and SGMS.