Investigation of the anti-inflammatory mechanisms of fermented Chinese herbal residue solution in an APEC-Infected HD11 cell model through the PI3K/AKT and NF-κB pathways

Abstract

Avian pathogenic Escherichia coli (APEC) infection poses a significant challenge to the poultry industry, severely threatening poultry health and industrial development. The emergence of antibiotic resistance in conventional treatments underscores the urgent need for novel alternative therapies. Fermented Chinese herbal residue solution, as a potential substitute, is rich in bioactive components and exhibits multifaceted effects, including anti-inflammatory, antioxidant, and immunomodulatory properties. This study investigates the anti-inflammatory mechanisms and key active compounds of the fermented herbal residue solution (BY3) in an APEC infection model. An in vitro APEC-infected HD11 cell model was established, with optimal infection conditions determined as a multiplicity of infection (MOI) of 0.1 and an infection duration of 6 hours, based on cytotoxicity assays and qPCR analysis. Results demonstrated that BY3 intervention significantly downregulated the expression of inflammatory cytokines. APEC infection markedly upregulated the expression of key target genes in the PI3K/AKT and NF-κB pathways, whereas BY3 treatment significantly reduced their expression. Western blot analysis further confirmed that BY3 significantly decreased the phosphorylation levels of AKT, P65, and IκB proteins, as well as the total PI3K protein content. These findings suggest that BY3 mitigates APEC-induced inflammation by modulating the PI3K/AKT and NF-κB pathways. To elucidate the active components of BY3, non-targeted metabolomics sequencing, database comparison, and molecular docking were employed, identifying four key bioactive compounds: Tangeretin, Arctigenin, Rhein, and Phloretin. All tested compounds significantly reduced APEC-induced inflammatory cytokine expression. qPCR and Western blot analyses revealed differential regulatory effects on the PI3K/AKT and NF-κB pathways. In the PI3K/AKT pathway, Tangeretin exhibited the most comprehensive inhibitory effect, significantly reducing both AKT phosphorylation and total PI3K levels. In the NF-κB pathway, all compounds except Rhein markedly decreased the phosphorylation of P65 and IκB. Collectively, BY3 and its identified compounds exert protective effects against APEC-induced HD11 cell damage by regulating the PI3K/AKT and NF-κB pathways, suggesting that the fermented Chinese herbal residue solution may represent a promising therapeutic approach for APEC-related diseases in poultry.

Keywords: Anti-inflammatory; Avian Pathogenic Escherichia coli (APEC); Fermented Chinese herbal residue solution; HD11 cell.

Fig. 1

Effects of different MOI and durations of APEC infection on HD11 cell viability;(A) HD11 cell viability after 2 hours of APEC infection;(B) HD11 cell viability after 4 hours of APEC infection;(C) HD11 cell viability after 6 hours of APEC infection;(D) HD11 cell viability after 8 hours of APEC infection; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the NC group.

Fig. 2

Effects of different MOI and durations of APEC infection on inflammatory cytokine transcription levels in HD11 cells; (A) IL-1β; (B) IL-2; (C) IL-6; (D) IL-8; (E) TNF-α; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the NC group.

Fig. 3

Effects of BY3 fermentation broth on HD11 cell viability; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the NC group.

Fig. 4

Effects of different treatment groups (NC, IG, AE, 25, 50, GM) on inflammatory cytokine transcription levels in HD11 cells; (A) IL-1β; (B) IL-2; (C) IL-6; (D) IL-8; (E) TNF-α; #P < 0.05 and ##P < 0.01 indicate a statistically significant difference from the NC group; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the IG group.

Fig. 5

Effects of different treatment groups (NC, IG, AE, 25, 50, GM) on transcription levels of key target genes in the PI3K/AKT and NF-κB pathways in HD11 cells; (A-D) PI3K/AKT pathway; (E-H) NF-κB pathway; #P < 0.05 and ##P < 0.01 indicate a statistically significant difference from the NC group; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the IG group.

Fig. 6

Effects of different treatment groups (NC, IG, AE, 25, 50, GM) on expression levels of key target proteins in the PI3K/AKT and NF-κB pathways in HD11 cells; (A-C) PI3K/AKT pathway; (A,D,E) NF-κB pathway; #P < 0.05 and ##P < 0.01 indicate a statistically significant difference from the NC group; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the IG group.

Fig. 7

The molecular docking analysis results of monomeric compounds from traditional Chinese medicine; (A) RHE; (B) ARC; (C) PHL; (D) TAN.

Fig. 8

Effects of monomeric compounds from traditional Chinese medicine on HD11 cell viability; (A) TAN; (B) ARC; (C) RHE; (D) PHL; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the NC group.

Fig. 9

Effects of different treatment groups (NC, IG, TAN, ARC, RHE, PHL) on inflammatory cytokine transcription levels in HD11 cells; (A) IL-1β; (B) IL-2; (C) IL-6; (D) IL-8; (E) TNF-α; #P < 0.05 and ##P < 0.01 indicate a statistically significant difference from the NC group; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the IG group.

Fig. 10

Effects of different treatment groups (NC, IG, TAN, ARC, RHE, PHL) on transcription levels of key target genes in the PI3K/AKT and NF-κB pathways in HD11 cells; (A-D) PI3K/AKT pathway; (E-H) NF-κB pathway; #P < 0.05 and ##P < 0.01 indicate a statistically significant difference from the NC group; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the IG group.

Fig. 11

Effects of different treatment groups (NC, IG, TAN, ARC, RHE, PHL) on expression levels of key target proteins in the PI3K/AKT and NF-κB pathways in HD11 cells; (A-C) PI3K/AKT pathway; (A,D,E) NF-κB pathway; #P < 0.05 and ##P < 0.01 indicate a statistically significant difference from the NC group; *P < 0.05 and **P < 0.01 indicate a statistically significant difference from the IG group.