Study On The Molecular Targets And Mechanism Of Arctigenin Against Fishmonogeneans

Monogenean are commonly occurring fish parasites which can be found all around the world,resulting in considerable economic losses in aquaculture due to high parasitism and mixed infection by other pathogens.The most pathogenic monogeneans belong to the genus Dactylogyrus and Gyrodactylus,they both contain rich species.However,the frequently used of chemicals often accompanied by drug resistance,the search for alternative compounds aroused wide interest in recent study.Natural products present a bewildering diversity of chemical structures and may represent a novel source of chemotherapeutic agents,including anthelmintic drugs.In our previous study,a lot of bioactive molecules had been isolated from medicinal plants by using bioassay-guided method,among which arctigenin?ARG?displayed high anthelmintic activity and low host toxify,making it as the ideal lead compound.However,the investigations about the targets and mechanism of ARG against monogeneans remain unknown,which in turn,hindered the improvements of structure of the lead compound.In this study,species distribution of Gyrodactylus on goldfish were firstly determined,the model of Gyrodactylus kobayashii infected goldfish was established;further,the targets of ARG against G.kobayashii were explored by morphological studies,RNA-seq based transcriptomics and iTRAQ based proteomics and using gold nanoprobe.The results obtained in this work were as follows:1.Establishment of the experimental model of Gyrodactylus kobayashii infected goldfish.G.kobayashii displayed highest proportion?70.8%?among Gyrodactylus on naturally infected goldfish by using the method of morphological identification.Then,the model of G.kobayashii infected goldfish was established through the following steps:obtaining ectoparasite-free goldfish by praziquantel consecutive baths,caudal fin infection by artificial infection,species identification and maintenance of population.Moreover,Gyrodactylus species in the system were isolated and determined with the help of the enzyme Apo I,results found that 144 parasites sampled randomly?12 parasites each time?were all G.kobayashii.Finally,G.kobayashii populations and immune response of host were also explored,results showed that G.kobayashii numbers increased rapidly during the first 8 days post exposure,with a peak at day 8?1103±212?,then decreasing sharply and remained low infection in the late stages of infection;Besides,G.kobayashii infection caused increased expression of the pro-inflammatory cytokines including IL-1?,IFN-?,TNF-?and iNOS,among which iNOS has the highest transcript level accompanied with increased nitric oxide concentration in the serum of all infected goldfish.These results indicated adding parasite-free fish was critical to keep high G.kobayashii population and can be used for the research of drug target identification.2.Safety assessment of ARG and morphological studies against G.kobayashii.Anthelminthic efficacy and safety assessment of ARG were firstly studied,results showed that in vitro exposure to 8 mg/L ARG resulted in all parasites dying in 33 min.ARG at 4.0 mg/L could kill all G.kobayashii within 4 h exposure in vivo,and 24 h and 48 h EC₅₀values of ARG against G.kobayashii were 1.85 mg/L and 1.58 mg/L,respectively.Besides,the 96 h LC₅₀ of ARG against goldfish was 11.63 mg/L?95%confidence interval was 11.29-11.99 mg/L?;In the liver of goldfish,we observed different regulation of four stress related genes?cyp1a?hsp70?gst and sod?during the first 24 h after treatment with 1.85 mg/L ARG,while no differences were found in 48 h and 96 h.There is no difference of sod between 4 mg/L ARG treated goldfish in 96 h,while other three genes showed the tendency to recover,suggesting ARG has low toxicity for fish.Finally,a noticeable antagonistic effect was soon observed on G.kobayashii attached to fin clips following the addition of ARG,worms were found to move around rapidly,after which time their movements slowed down and stopped quickly.After exposure to ARG,SEM analysis revealed morphological alterations on the tegument,TEM analysis found obvious ultrastructural damages of muscle layers and mitochondria,immunofluorescence analysis also found wide spread muscle damages.These findings revealed the targets of ARG against G.kobayashii might be exist in muscle or mitochondria,the death of parasites may be related to tegumental musculature damages or respiratory depression.3.Transcriptome analysis reveals molecular anthelmintic effects of ARG in G.kobayashii.Transcriptome analysis was performed in G.kobayashii after treatment with ARG,differential expression genes?DEGs?were filtered for bioinformatics analysis to explore the anthelmintic mechanism and promising targets of ARG against G.kobayashii.The results obtained were as follows:?1?When comparing to the control,exposure to 4 mg/L ARC for0.5 h resulted in 234 DEGs,for 1.85 mg/L ARC,118 genes were different regulated at 0.5 h post-drug exposure;when extending the bath time to 4 h,the number of DEGs increased to4936,of which 4181 genes were inhibited.?2?GO term enrichment analysis results found that DEGs were mainly involved in“extracellular matrix structural constituent”,“obsolete electron transport”,“macromolecule transmembrane transporter activity”and“energy metabolic process”.?3?“protein digestion and absorption”,“ECM-receptor interaction”,“cardiac muscle contraction”,“oxidative phosphorylation”and“glycolysis/gluconeogenesis”were significantly enriched KEGG pathways of the DEGs.?4?Above results suggested the molecular mechanisms of ARG against G.kobayashii were related to the damages of tegument and the down-expression of critical gene involved in energy metabolism pathway,resulting in parasites death.?4?iTRAQ proteomic analysis of G.kobayashii in response to ARG.iTRAQ-LC-MS/MS was used to analyze the protein expression profile of G.kobayashii treated with or without ARG,the function of differentially expressed proteins?DEPs?was explored by bioinformatics analysis methods.Then the molecular mechanisms and promising targets of ARC against G.kobayashii were investigated through combined proteomics and transcriptomics.Results are as follows:?1?A total of 2850 proteins were identified;Comparing with the control group,335,223 and 313 DEPs were found respectively in three treatment groups?4 mg/L,0.5 h;1.85 mg/L,0.5 h;1.85 mg/L,4 h?,a total of 82 DEPs were detected between all the three ARC treatment groups in comparison with control group.?2?Bioinformatics analysis results showed that these DEPs were mainly related to the important processed such as“microtubule associated complex”,“ion transport”,“regulation of muscle contraction”.?3?Association analysis results of transcriptome and proteome data revealed 19 proteins exhibited the same expression tendency,including gelsolin and ATP synthase E chain,both of them were the most promising targets of ARG.KEGG functional classification results indicated that corrected proteins were involved in regulation of actin cytoskeleton,endocytosis,oxidative phosphorylation and focal adhesion.?4?qRT-PCR analysis results found that gene expressions of the different subunits of ATP synthase and other complexes of the respiratory chain were all downregulated after ARG bath treatment.Furthermore,in vitro and in vivo exposure of worms with ARC induced a rapid dose-dependent depletion of the intracellular ATP pool.?5?Target identification of ARG against G.kobayashii using gold nanoprobes.Using the nanoprobes,the binding proteins of ARG were pulled down from the parasitic proteome.?1?In order to obtain negative control for excluding nonspecific proteins,compound 2 was chosen because the similar structure but different effect with ARG.?2?ARG and compound 2 were firstly modified to be assembled onto the nanoparticle surface with Au-S bond,GNP-6 and ANP-10 were obtained.?3?High resolution TEM found that GNP-6 was spherical with mean grain size 3.58 nm;besides,a significant number of gold nanoparticles were seen in nucleus and high anthelmintic effects were still remained.?4?After they were proven to be active in live parasites,the nanoprobe attached with ARG?GNP-6?and another nanoprobe attached inactive compound 2?GNP-10?were incubated with G.kobayashii lysates to identify the target proteins.According to the comparison with negative control GNP-10,two proteins were selected as the specific target proteins of ARG.Using MALDI-TOF MS and Mascot search engine,the target proteins of ARG were identified to be Myosin-?and Muscle M-line assembly protein.In summary,based on the experimental model of G.kobayashii-goldfish,researches of morphology,transcriptome analysis and iTRAQ proteomic analysis,isolation and identification of targets,Myosin?and UNC-89 were confirmed initially.They are both related to myosin in tegumental musculature,suggesting the molecular mechanisms of ARG against monogeneans were related to the myosin damage and resulting in poor energy production.Overall,these results provided reference and new approach for the structure optimization of ARG.