Sinomenine Hydrochloride Ameliorates Fish Foodborne Enteritis via α7nAchR-Mediated Anti-Inflammatory Effect Whilst Altering Microbiota Composition

Abstract

Foodborne intestinal inflammation is a major health and welfare issue in aquaculture. To prevent enteritis, various additives have been incorporated into the fish diet. Considering anti-inflammatory immune regulation, an effective natural compound could potentially treat or prevent intestinal inflammation. Our previous study has revealed galantamine’s effect on soybean induced enteritis (SBMIE) and has highlighted the possible role of the cholinergic anti-inflammatory pathway in the fish gut. To further activate the intestinal cholinergic related anti-inflammatory function, α7nAchR signaling was considered. In this study, sinomenine, a typical agonist of α7nAChR in mammals, was tested to treat fish foodborne enteritis via its potential anti-inflammation effect using the zebrafish foodborne enteritis model. After sinomenine’s dietary inclusion, results suggested that there was an alleviation of intestinal inflammation at a pathological level. This outcome was demonstrated through the improved morphology of intestinal villi. At a molecular level, SN suppressed inflammatory cytokines’ expression (especially for tnf-α) and upregulated anti-inflammation-related functions (indicated by expression of il-10, il-22, and foxp3a). To systematically understand sinomenine’s intestinal effect on SBMIE, transcriptomic analysis was done on the SBMIE adult fish model. DEGs (sinomenine vs soybean meal groups) were enriched in GO terms related to the negative regulation of lymphocyte/leukocyte activation and alpha-beta T cell proliferation, as well as the regulation of lymphocyte migration. The KEGG pathways for glycolysis and insulin signaling indicated metabolic adjustments of α7nAchR mediated anti-inflammatory effect. To demonstrate the immune cells’ response, in the SBMIE larva model, inflammatory gatherings of neutrophils, macrophages, and lymphocytes caused by soybean meal could be relieved significantly with the inclusion of sinomenine. This was consistent within the sinomenine group as CD4+ or Foxp3+ lymphocytes were found with a higher proportion at the base of mucosal folds, which may suggest the Treg population. Echoing, the sinomenine group’s 16s sequencing result, there were fewer enteritis-related TM7, Sphingomonas and Shigella, but more Cetobacterium, which were related to glucose metabolism. Our findings indicate that sinomenine hydrochloride could be important in the prevention of fish foodborne enteritis at both immune and microbiota levels.

Dietary Inclusion of Seabuckthorn (Hippophae rhamnoides) Mitigates Foodborne Enteritis in Zebrafish Through the Gut-Liver Immune Axis

“…Then, 6-week feeding trials of experimental diets, including FM (negative control), SBM (positive control), as well as the drug (seabuckthorn) groups (SBM +35ppm seabuckthorn fruit powder) ( Table 1 ), were carried through feeding twice a day. The feeding trials for the SBMIE larvae model followed our recently published protocol ( Xie et al, 2021 ). In brief, a 4-day feeding trial of experimental diets from 5 to 8 dpf using larvae of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) was completed for examining the response of innate immune cells.…”

“…The composition of bacterioplankton was analyzed using NovaSeq sequencing of 16S rRNA gene amplicons ( n = 6) as previously published protocol ( Xie et al, 2021 ). In brief, the V3V4 regions of the 16S rRNA gene were amplified.…”

“…Following previously published methods for zebrafish larvae imaging ( Bravo-Tello et al, 2017 ; Coronado et al, 2019 ; Ikeda-Ohtsubo et al, 2020 ), in vivo imaging of intestinal immune cells in zebrafish was conducted according to our recently published protocol ( Li et al, 2021 ; Xie et al, 2021 ). As the innate cell’s response could be examined at a very early stage, the feeding trial was done from 5 dpf to 9 dpf, using the neutrophil and macrophage labeled Tg lines.…”

“…To analyze innate immune cells, between 5 and 8 dpf, the larvae of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) were kept in Danieau’s solution and fed experimental diets. Upon imaging of innate immune cells, including neutrophils (lyz:DsRED2 labeled) and macrophages (mpeg1:EGFP labeled), was performed in the hindgut (posterior part) of the larvae of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) at 9 dpf ( Xie et al, 2021 ). Meanwhile, to test the cellular response of adaptive immune cells, the immature (rag2:DsRed labeled) ( Zhang et al, 2018 ) and mature lymphocytes (lck-eGFP labeled) ( Langenau and Zon, 2005 ), in the intestine (posterior part) of Tg(rag2:DsRed) and Tg(lck:lck-eGFP) larvae ( Xie et al, 2021 ), were imaged.…”

“…Upon imaging of innate immune cells, including neutrophils (lyz:DsRED2 labeled) and macrophages (mpeg1:EGFP labeled), was performed in the hindgut (posterior part) of the larvae of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) at 9 dpf ( Xie et al, 2021 ). Meanwhile, to test the cellular response of adaptive immune cells, the immature (rag2:DsRed labeled) ( Zhang et al, 2018 ) and mature lymphocytes (lck-eGFP labeled) ( Langenau and Zon, 2005 ), in the intestine (posterior part) of Tg(rag2:DsRed) and Tg(lck:lck-eGFP) larvae ( Xie et al, 2021 ), were imaged. After 12 days (5–16 dpf) feeding of Larval AP100 Diet (Zeigler) as well as 10 days of experimental diets feeding from 17 dpf, the intestinal lymphocyte in larvae were imaged at 27 dpf.…”