Immunomodulatory effects of ultrasonically lysed Bifidobacterium breve on Pam3CSK4-stimulated J774A.1 macrophages via SIRT1/NRF2 activation and NF-κB suppression

BACKGROUND: Bacterial lysates, which contain cell wall components and intracellular metabolites, have emerged as important modulators of macrophage immune function through interactions with pattern recognition receptors (PRRs). Ultrasonication is an effective technique for extracting these bioactive constituents. The goal of this work was to examine the immunomodulatory and cytoprotective effects of ultrasonically lysed Bifidobacterium breve (BB-UL) in J774A.1 macrophages activated by Pam3CSK4. METHODS: J774A.1 macrophages were pre-exposed to BB-UL (equivalent to 10⁸ CFU/mL) for 12 h before stimulation with Pam3CSK4 (500 ng/mL). Cell viability was determined via the MTT assay, intracellular reactive oxygen species (ROS) were quantified using the DCFDA assay, and apoptosis was assessed with Annexin V-FITC/PI staining. Gene expression levels of Sirt1, Nfe2l2, Hmox1, Rela, Il1b, Il6, Tnf, Bax, and Bcl2 were analyzed by RT-qPCR, while secreted cytokines were measured using ELISA. Statistical significance was evaluated using one-way ANOVA followed by Tukey's post hoc test (n = 3). RESULTS: Pam3CSK4 exposure significantly decreased cell viability to approximately 50% of control levels, whereas BB-UL pretreatment restored viability to around 80-85% (p < 0.01 vs. Pam3CSK4), indicating a protective effect. Pam3CSK4 also induced a substantial increase in intracellular ROS (~ 3-fold vs. control), which was reduced by approximately 40% following BB-UL treatment (p < 0.05-0.01). Similarly, late apoptosis increased from ~ 3-4% in control cells to ~ 15% upon stimulation, while BB-UL pretreatment decreased this value to approximately 8% (p < 0.01). At the molecular level, Pam3CSK4 downregulated Sirt1 (~ 0.5-fold) and Nfe2l2 (~ 0.55-fold), while upregulating Rela (~ 2.5-fold). BB-UL partially restored Sirt1 (~ 0.7-fold) and Nfe2l2 (~ 0.75-fold) expression and reduced NF-κB activation (~ 1.8-fold vs. control, p < 0.05). Additionally, BB-UL increased Hmox1 expression (~ 0.9-fold) and favorably regulated apoptosis-related genes by decreasing Bax (~ 1.5-fold) and increasing Bcl2 (~ 0.8-fold), thereby improving the Bax/Bcl2 ratio. Following BB-UL pretreatment, elevated levels of pro-inflammatory cytokines (Il1b, Il6, and Tnf) caused by Pam3CSK4 were considerably decreased at both gene and protein levels (p < 0.05-0.01). CONCLUSION: In Pam3CSK4-stimulated macrophages, ultrasonically lysed B. breve had notable cytoprotective, antioxidant, and anti-inflammatory properties. Reduced oxidative stress, apoptosis, and inflammatory cytokine production were associated with modulation of SIRT1, NRF2, HO-1, and NF-κB-related gene expression. These findings suggest that BB-UL may represent a promising postbiotic candidate for modulating macrophage-mediated inflammatory responses.