Huang Bowen, Mo Shuanghao, He Chengguang, Zhu Junhui, Tan Zining, Chen Linlin, Wang Yiming, Ma Hongxia
College of Animal Science and Technology, College of Veterinary Medicine, Jilin Agricultural University, Changchun 130118, China; Jilin Provincial Key Laboratory of New Veterinary Drug R&D and Creation, Changchun 130118, China.
College of Life Sciences, Jilin Agricultural University, Changchun 130118, China; Jilin Agricultural University Bioreactor and Drug Development Engineering Research Center of the Ministry of Education, Changchun 130118, China.
Poult Sci. 2025 May 31;104(9):105383. doi: 10.1016/j.psj.2025.105383.
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.
禽致病性大肠杆菌（APEC）感染对家禽业构成重大挑战，严重威胁家禽健康和产业发展。传统治疗中抗生素耐药性的出现凸显了对新型替代疗法的迫切需求。发酵中草药残渣溶液作为一种潜在替代品，富含生物活性成分，并具有多方面作用，包括抗炎、抗氧化和免疫调节特性。本研究在APEC感染模型中探究发酵中草药残渣溶液（BY3）的抗炎机制和关键活性化合物。基于细胞毒性试验和qPCR分析，建立了体外APEC感染的HD11细胞模型，确定最佳感染条件为感染复数（MOI）为0.1且感染持续时间为6小时。结果表明，BY3干预显著下调炎症细胞因子的表达。APEC感染显著上调PI3K/AKT和NF-κB途径中关键靶基因的表达，而BY3处理显著降低其表达。蛋白质免疫印迹分析进一步证实，BY3显著降低AKT、P65和IκB蛋白的磷酸化水平以及总PI3K蛋白含量。这些发现表明，BY3通过调节PI3K/AKT和NF-κB途径减轻APEC诱导的炎症。为阐明BY3的活性成分，采用非靶向代谢组学测序、数据库比对和分子对接，鉴定出四种关键生物活性化合物：陈皮素、牛蒡子苷元、大黄酸和根皮素。所有测试化合物均显著降低APEC诱导的炎症细胞因子表达。qPCR和蛋白质免疫印迹分析揭示了对PI3K/AKT和NF-κB途径的不同调节作用。在PI3K/AKT途径中，陈皮素表现出最全面的抑制作用，显著降低AKT磷酸化和总PI3K水平。在NF-κB途径中，除大黄酸外的所有化合物均显著降低P65和IκB的磷酸化。总体而言，BY3及其鉴定出的化合物通过调节PI3K/AKT和NF-κB途径对APEC诱导的HD11细胞损伤发挥保护作用，表明发酵中草药残渣溶液可能是家禽APEC相关疾病的一种有前景的治疗方法。
