Protection of clove extract against Salmonella enteritidis-induced intestinal dysfunction in broilers through JAK2/STAT3-mediated stem cell activation

Salmonella enteritidis(SE) is a major pathogenic bacterium causing intestinal epithelial tissue damage in broiler chickens raised without antibiotic supplementation. Clove extract, as a natural antibacterial agent, possesses activity against bacterial invasion and improves intestinal health. However, the mechanism by which clove extract alleviates SE infection remains unclear. This study aimed to elucidate the protective effects of clove extract and its underlying mechanism against SE-induced intestinal damage. Experiment 1 determined the minimum inhibitory concentration (MIC) of ten plant extracts against SE using the microdilution method and selected effective extracts based on inhibition zone diameter. Experiment 2 established conditions for an intestinal injury model in broilers by gavage with different concentrations of SE. In experiment 3, 160 broilers were distributed into three treatments: Control (CON), SE-infected (SE), SE-infected + low level of clove extract (CE_low: 150 mg/kg diet) and SE-infected + high level of clove extract (CE_high: 300 mg/kg diet). Gut barrier function was assessed by serum lipopolysaccharide (LPS) and diamine oxidase (DAO) levels, while epithelial stem cell dynamics and Janus kinase 2 (JAK2) / Signal transducer and activator of transcription 3 (STAT3) pathway activity were profiled in parallel. This study demonstrated that clove extract, characterized by the lowest MIC and largest inhibition zone, effectively inhibited SE growth. An SE inoculation dose of 10¹⁰ CFU/mL significantly decreased small intestine weight, transmembrane resistance (TEER), villus height, and villus/crypt ratio (P < 0.05), thereby establishing an intestinal injury model. At a dosage of 300 mg/kg, clove extract significantly reduced serum DAO and LPS levels (P < 0.05) while increasing jejunum weight, villus height, villus/crypt ratio, and transmembrane resistance (P < 0.05), effectively reversing SE-induced intestinal damage. Additionally, clove extract significantly upregulated key proteins associated with intestinal stem cells (Lgr5, Olfm4, SOX9), cell proliferation (PCNA), and barrier function (Occludin) (P < 0.05), enhanced organoid formation and stem cell proliferation (P < 0.05), and mitigated the SE-induced excessive activation of the JAK2/STAT3 signaling pathway. Clove extract exhibited strong inhibitory effects against SE proliferation, and effectively attenuated th SE-induced JAK2/STAT3 pathway hyperactivation, thereby boosting the proliferative and differentiative abilities of intestinal stem cells (ISCs), exerting dual antibacterial and anti-inflammatory effects to ameliorate SE-induced intestinal injury. This positions clove extract as a potential phytotherapeutic candidate for antibiotic-free poultry production.