Safety Toxicological Assessment Of Gelsemium Elegans And Its Application In Megalobrama Amblycephala

As a traditional herbal medicine,Gelsemium elegans has many biological functions,especially in anti-inflammatory,anti-neuropathic pain,anti-anxiety,anti-cancer and immune regulation.G.elegans already used in livestock and poultry production for promote the growth,reduce feed consumption and enhance immunity.G.elegans has high application value,but no report on its application in aquaculture.Therefore,it is necessary to study its application in aquaculture.G.elegans has a certain toxicity,and its toxicity is seldom studied.The study of G.elegans toxicology can promote its application.In this study,the toxic effects of G.elegans on aquatic animals were studied by T.thermophila and zebrafish,and the effects of G.elegans alkaloids used as feed additives on the growth and immune performance were studied in Megalobrama amblycephala.We used the ciliate T.thermophila as a model to evaluate the toxic effects of koumine and gelsemine in eukaryotic microorganisms.Koumine and gelsemine inhibited T.thermophila growth and viability in a dose-dependent manner.Moreover,this drugs produced oxidative stress in T.thermophila cells and expressions of antioxidant enzymes were significantly elevated at high koumine and gelsemine levels(P < 0.05).Koumine also caused significant levels of apoptosis(P < 0.05),and koumine and gelsemine induced DNA damage in a dose-dependent manner.Mitophagic vacuoles were present in cells indicating induction of autophagy by this drugs.Expression of ATG7,MTT2/4,CYP1 and HSP70 as well as the MAP kinase pathway gene MPK1 and MPK3 were significantly altered after exposed to koumine and gelsemine.To further study the mechanism of toxicity,we carried out transcriptome analysis.Transcriptome results showed the differentially expressed genes in koumine group were mainly enriched in amino acid metabolism pathways,while the differentially expressed genes in gelsemine group were mainly enriched in the glutathione metabolism pathways.Therefore,koumine may affect the amino acids metabolism in T.thermophila,and the abnormal amino acids metabolism leads to oxidative stress.Oxidative stress leads to a series of toxic reactions such as apoptosis and DNA damage of T.thermophila cells.The oxidative stress induced by gelsemine may be related to the abnormal glutathione metabolism.This study represents a preliminary toxicologicalevaluation of koumine and gelsemine in the single celled eukaryote T.thermophila.Although the mechanism of koumine toxicity has been preliminarily explored,but there are great differences in physiology between single celled eukaryote T.thermophila and fish.Therefore,it is necessary to study the mechanism of koumine and gelsemine toxicity with zebrafish as research object.We used transcriptome and metabolome to study koumine and gelsemine toxicity.In transcriptome,KEGG enrichment analysis revealed the differentially expressed genes were mainly enriched in Pyrimidine metabolism.We also found the amino acid and lipid metabolism pathways were also enriched in KEGG.In metabolome,KEGG enrichment of differential metabolites revealed that differential metabolites were mainly enriched in some pathways related to amino acid metabolism.In addition,lipid metabolism and pyrimidine metabolism pathways are also enriched.Therefore,We speculate koumine may affect the amino acid and pyrimidine metabolism in zebrafish.Abnormal amino acid metabolism may lead to abnormal lipid metabolism.And abnormal amino acid metabolism also affects the glutathione metabolism,which leads to oxidative stress.Finally,oxidative stress leads to a series of toxic reactions.The fish culture experiment study aim to investigate the effects of dietary G.elegans alkaloids supplementation in M.amblycephala.The study indicated that dietary G.elegans alkaloids supplementation could significantly improve final body weight(FBW),weight gain rate(WGR),specific growth rate(SGR),feed conversion ratio(FCR)and protein efficiency ratio(PER)(P < 0.05).The 20 mg/kg and 40 mg/kg G.elegans alkaloids groups shown significantly higher whole body and muscle crude protein and crude lipid contents compared to the control group(P < 0.05).Dietary various levels G.elegans alkaloids had a significant effect on the contents of LDH,AST,ALT,ALP,TG,TC,LDL-C,HDL-C,ALB and TP in M.amblycephala(P < 0.05).Fish fed 20 mg/kg and 40 mg/kg dietary G.elegans alkaloids showed significant increase in complement 3,complement 4 and immunoglobulin M contents.Liver antioxidant enzymes(SOD,CAT and T-AOC)and MDA content significantly increased at 20 mg/kg and 40 mg/kg G.elegans alkaloids supplement(P <0.05).The m RNA levels of immune-related genes IL-1?,IL8,TNF-? and IFN-? were significantly up-regulated,whereas TGF-? and IL10 genes were significantly down-regulated in liver,spleen and head kidney of fish fed dietary supplementation with 20mg/kg and 40 mg/kg G.elegans alkaloids.After challenge with Aeromonas hydrophila,significant higher survival rate was observed at 20 mg/kg and 40 mg/kg G.elegans alkaloids supplement(P < 0.05).Therefore,these results indicated that M.amblycephala fed a diet supplemented with 20 mg/kg and 40 mg/kg G.elegans alkaloids couldsignificantly promote its growth performance,lipids and amino acids deposition,immune ability and resistance to Aeromonas hydrophila.We found that dietary G.elegans alkaloids at 40 mg/kg improved intestinal morphology by increasing villus length,muscle thickness and villus number in the foregut and midgut and muscle thickness in the hindgut(P < 0.05).This suggested that G.elegans alkaloids enhance intestinal digestion and absorption.These alkaloids also significantly improved intestinal antioxidant capabilities by increasing superoxide dismutase,catalase,total antioxidant capacity and malondialdehyde levels and up-regulated intestinal Cu/Zn-SOD and Mn-SOD(P < 0.05).Dietary G.elegans alkaloids improved intestinal immunity via up-regulating the pro-inflammatory cytokines IL-1?,IL-8,TNF-? and IFN-?and down-regulating expression of the anti-inflammatory cytokines IL-10 and TGF-?(P <0.05).The expression of Toll-like receptors TRL1,3,4 and 7 were also up-regulated in intestine of M.amblycephala(P < 0.05)indicating G.elegans alkaloids may activate intestinal immune responses via TLRs.Furthermore,this study demonstrated that the intestinal microbiota of M.amblycephala was altered after feeding the G.elegans alkaloids.The abundance of Proteobacteria was increased while the Firmicutes abundance was decreased at phylum level(P < 0.05).The alkaloids also increased the abundance of the probiotic Rhodobacter and decreased the abundance of the pathogenic Staphylococcus at genus level(P < 0.05)indicating a positive role on the intestinal microbiota.We also found that predicted metabolic pathways that were affected by the alkaloids were related to amino acid,fatty acid and carbohydrate metabolism suggesting G.elegans alkaloids may affect the metabolic capacity of gut microbiota.In addition,antioxidant-related pathways were also enriched using the KEGG database as well as the Bug Base phenotype prediction indicated the alkaloids improve the antioxidant capacity of the intestinal microbiota.In conclusion,dietary G.elegans alkaloid supplementation promoted intestine health by improving intestine morphology,immunity,antioxidant abilities and intestinal microbiota in M.amblycephala.