The Application Of Lipopolysaccharide-induced Zebrafish Inflammation Model In Screening Traditional Chinese Medicine With Anti-inflammatory Activity

BackgroudLipopolysaccharide(LPS)is a pathogen-associated pattern of molecules that threaten human health and life.The mortality rate of diseases caused by LPS remains high,yet there is no ideal treatment for them.Therefore,there is an urgent need to find effective drugs to treat them.In recent years,many studies on the use of traditional Chinese medicine(TCM)against endotoxin damage have been reported.Our research group conducted an in-depth study on Liang-Ge-San(LGS)and confirmed that this formulation has significant anti-endotoxin effects.However,what is the material basis of the anti-LPS inflammatory properties of LGS and other herbal medicines or compounds with anti-LPS properties,which are the basis for further development of the the establishment of a screening and evaluation system for the anti-LPS activity of TCM.A model for screening anti-inflammatory activity is the key to solve the primary problem of the application of such prescription drugs.Our research group successfully established a zebrafish inflammation model by using yolk sac microinjection LPS induction.Is the LPS-induced zebrafish inflammation model applicable for a single herbal medicine or compound with numerous factors,especially when the active site,crude extract,and the extract are used as the basis for the formulation?The new question is whether and how the zebrafish model can be applied even when decoctions are the subject of study.ObjectTo improve LPS-induced zebrafish inflammation model and to evaluate the anti-LPS activities of different extracts of Forsythia based on this model.The anti-LPS composition ratio of emodin,phillyrin,baicalin and liquiritin from LGS was also studied to obtain the optimal composition.This work aimed to expand the application of the model in the screening and evaluation of the anti-LPS activity of TCM,and to provide experimental basis for the development and application of LGS.Methods and Results1.Screening of anti-LPS activities of different sxtracts of Forsythia based on the zebrafish model.Healthy zebrafish embryos were selected,soaked and given different extracts of Forsythia,and the abnormity and mortality rates of zebrafish embryos in each experimental group were recorded for 72 h.The non-toxic concentration was chosen as the maximum safe concentration for subsequent experiments.Zebrafish were treated by immersion with drugs after being microinjected 2 nL of LPS(2 mg/mL)into the yolk to induce the lethal LPS-infected model.The treatment group with different extracts of Forsythia and dexamethasone group(5?g/mL,positive control)both improved the survival rate of LPS-infected zebrafish,indicating the reliability of the zebrafish model for anti-LPS screening.2.Formulation study of anti-LPS components from Liang-Ge-San based on zebrafish model.The maximum safe concentrations of emodin,phillyrin,baicalin and liquiritin were determined by soaking and the concentrations were selected as the highest dosage.The dosages of the four components were grouped into orthogonal design groups at three levels to obtain a 9-component dosing group.Observation results for 72 h did not show any toxic effects on zebrafish embryos,and subsequent experiments were carried out.After the microinjection of LPS into the yolk sac,the zebrafish embryos were treated with different groups of components by immersion.The results showed that the survival rate of LPS-infected zebrafish larvae was improved in all drug treatment groups,and there was a significant difference between groups 4-8 and the model group(P<0.05).The results of real-time fluorescence obrsevation,sudan black staining and neutral red staining count indicated that the multicomponent drugs could inhibit the the migration and aggregation of neutrophils and macrophages to the site of inflammation.The results of RT-PCR showed that the expression of the pro-inflammatory mediators IL-6 and TNF-? was inhibited.According to the results,the best combination was group 8.3.Sequencing of circular RNA transcriptome of the optimal proportion components of LGS.Collection of total RNA samples from whole fish tissues after optimal rationing treatment for circular RNA transcriptome sequencing and analysis of results.KEGG and GO analyses showed that the above differentially expressed circular RNAs might be involved in the regulation of signaling pathways such as the apoptosis and MAPK signaling pathways,mainly involving the biological processes,including assembly of protein-DNA complex assembly,carbohydrate transmembrane transport,etc.However,the specific role of each differentially expressed circular RNA needs to be validated.Conclusion1.Anti-LPS effects of different extracts of Forsythia were evaluated by using an immersion delivery method based on the LPS-induced zebrafish inflammation model,which indicate the applicability of the model for screening the anti-LPS activity of crude extracts of TCM.2.The orthogonal design test showed that different combinations of LGS components could protect LPS-infected zebrafish and the best proportion was selected with the significant characteristic of the improvement of the survival rate of zebrafish,the inhibitition of the migration and aggregation of inflammatory cells and the decrease of pro-inflammatory factors TNF-? and IL-6.These results provided experimental evidence for the development and application of the active components of LGS.In addition,the best combination obtained from orthogonal analysis was selected for the sequencing of circular RNA genome.According to the change of circular RNAs,the associated biological pathways and functions were predicted by KEGG and GO analyses.The sequencing data will provide scientific basis for further studies on the anti-LPS mechanism of LGS components.