Exploration of Anti-inflammatory Mechanism of Forsythiaside A and Forsythiaside B in CuSO4-Induced Inflammation in Zebrafish by Metabolomic and Proteomic Analyses
Abstract Background: Inflammation is a general pathological phenomenon during severe disturbance of homeostasis. Forsythiaside A (FA) and Forsythiaside B (FB) are phenylethanoid components isolated from dried fruits of Forsythia suspensa (Thunb.) Vahl., which show a significant effect against inflammation. However, the anti-inflammatory properties and therapeutic mechanisms of FA and FB have not yet been systematically clarified. Methods: In this study, the anti-inflammatory effects of FA and FB were investigated in CuSO4-induced inflammation in zebrafish. Intracellular ROS and NO generation in zebrafish were investigated using fluorescent probes. Metabolomics and proteomics analyses using liquid chromatography mass spectrometry were carried out to identify the expressions of metabolites and proteins associated with the treatment of FA and FB in CuSO4-induced inflammation in zebrafish larvae. Quantitative polymerase chain reaction (PCR) was performed to detect the progressive changes in genes. Results: FA and FB inhibited neutrophils migration to the damaged neuromasts and remarkably reduced CuSO4-induced ROS and NO generation in zebrafish. Metabolomic analysis indicated that Nicotinate and nicotinamide metabolism, Energy metabolism, Pyrimidine metabolism and Purine metabolism were mainly involved. Proteomic analysis identified 146 differentially expressed proteins between control and model group. The changed expressions of Collagen (collagen type II alpha 1b precursor (col2a1b), collagen alpha-2(IX) chain precursor (col9a2), collagen type IX alpha I precursor (col9a1b)), nucleoside diphosphate kinase 3 isoform X1 (Nme3), WD repeat-containing protein 3 (Wdr3), 28S ribosomal protein S7 mitochondrial precursor (Mrps7) in model group were all reversed after being treated by FA or FB. FA and FB could reverse the abnormal expressions of potential metabolite and protein biomarkers to alleviate CuSO4 damage to the neuromasts in the lateral line of zebrafish. Conclusions: Our results indicated that FA and FB possessed remarkable anti-inflammatory activities against CuSO4-induced neuromasts damage in zebrafish larvae, and suggested a multi-component and multi-regulatory therapeutic mechanism of FA and FB.