Identification of the immunoprotective activity of Mori Cortex Radicis ethanol extract against aquaculture pathogens (Aeromonas veronii, Pseudomonas aeruginosa).

OBJECTIVE: Mori Cortex Radicis (MCR) is an important Chinese herbal medicine with antitussive, anti-inflammatory, and analgesic effects. It is abundant in resources and low in cost, but its application in the control of fish pathogens has been insufficiently studied. METHODS: We employed an ethanol extraction method to obtain the MCR ethanol extract (MCR-EE), and analyzed its small-molecule and macromolecular components using LC-MS and commercial kits, respectively. Subsequently, using a crucian carp (Carassius auratus) model fed with MCR-EE and challenged with aquaculture pathogens, the immunoprotective activities of MCR-EE were evaluated through a combination of survival rate monitoring, leukocyte phagocytosis assays, immune factor activities, qRT-PCR detection of mRNA expression of inflammatory cytokines and antioxidant pathway factors, histopathological examination of visceral tissues, and immunofluorescence analysis of the expression of the apoptotic factor (p53) and the DNA damage marker (γH2AX). RESULTS: The MCR-EE was prepared. The contents of proteins, polysaccharides, and polyphenols of MCR-EE were 0.7%, 0.44%, and 1.21%, respectively. A total of 646 small substances were identified in MCR-EE, and the five most abundant small-molecule compounds were L(+)-Arginine, 1-Deoxynojirimycin, 9,12,13-Todea, 4-Guanidinobutanoic acid, and Kelampayoside A. The MCR-EE enhanced the general phagocytic activity of fish blood leukocytes and the activity of immune factors. The immune protection rates of MCR-EE against Aeromonas veronii and Pseudomonas aeruginosa were 46.67% and 40% (p < 0.05), respectively. MCR-EE reduced the bacterial loads of A. veronii and P. aeruginosa in kidney tissues, as well as the mRNA expression of inflammatory cytokines (IL-6, TNF-α, and IL-1β) and key factors of oxidative stress pathway (Nrf2, HO-1, Keap1) (p < 0.05), and decreased the levels of antioxidant factors (p < 0.05). Histopathological examination revealed that MCR-EE maintained the structural integrity of the spleen, kidney, and intestine, and decreased the expression of p53 and γH2AX (p < 0.05) in kidney. CONCLUSION: MCR-EE exhibits immunomodulatory activity and holds promise as an immunostimulant for the prevention and control of fish pathogens (A. veronii and P. aeruginosa).