Lentinula edodes Cultured Extract Intake at Puberty Mitigates Inflammatory Signals at the Mammary Glands by the Involvement of Epigenetic Mechanisms.

Exposure to immune stress or lipopolysaccharide (LPS) during critical developmental stages, particularly puberty, may result in gut microbiome dysbiosis and disturb epigenetic regulation in mammary glands. This disruption can affect gene expression and potentially increase breast cancer susceptibility later in life. While the adverse effects of LPS on intestinal and brain function are documented, its impact on mammary glands is not well understood. An immunocompetent murine model (BALB/c mice) was administered an acute dosage of LPS during the pubertal stage. The study aimed to comprehensively evaluate the enduring effects of LPS exposure, as well as LPS exposure in combination with AHCC (Lentinula Edodes Cultured Extract), on various parameters, including DNA methylation patterns, cytokine profiles, and microRNA expression within the mammary gland during adulthood, at the age of 9 weeks. Analysis was carried out using DNA methylation sequencing, multiplex immunoassays, quantitative PCR, and image processing.Our findings revealed significant and enduring dysregulation in DNA methylation patterns, cytokine levels, and microRNA expression in the mammary glands exposed to LPS during puberty. These dysregulations significantly impacted critical signaling pathways related to mammary gland growth and potential breast cancer development. Notably, we observed hypermethylation of the transcriptional start sites (TSS), promoters, and 5' UTRs of critical genes, including Vav3, Pdgfa, Pdgfc, Jag2, Hras, Ksr1, Il2rb, Il17b, and Il17rb in the LPS group, whereas hypomethylation was evident in the prebiotic+LPS group. The multiplex immunoassays and quantitative PCR confirmed these findings. LPS exposure during puberty causes long-lasting epigenetic and inflammatory alterations in the mammary glands, which could influence breast cancer risk. The use of AHCC as a mitigating factor suggests potential for dietary interventions to prevent or reduce these effects. This study investigates the long-term effects of pubertal exposure to lipopolysaccharide (LPS) and prebiotic supplementation on the gut microbiota and immune system in female BALB/c mice. A total of 36 mice were divided into two dietary intervention groups: the prebiotic group, which received AHCC (2 g/kg BW/day) in drinking water, and the control group, which received regular drinking water. At the onset of puberty, defined by vaginal opening at five weeks of age, half of the mice in each group were intraperitoneally injected with LPS (1.5 mg/kg body weight) to induce inflammation and gut dysbiosis, while the remaining half received sterile PBS as a control. This resulted in four experimental groups, with nine mice per group: control + PBS, control + LPS, prebiotic + PBS, and prebiotic + LPS. The dietary intervention continued for one week after injection, followed by a standard diet until early adulthood. At nine weeks of age, the mice were sacrificed, and mammary gland tissue samples were collected for molecular analysis. DNA methylation profiling was performed using the Infinium™ Mouse Methylation BeadChip (Illumina), cytokine expression was measured using a multiplex Luminex immunoassay, and gene expression analysis was conducted via RT-qPCR for microRNA profiling. The study includes biological replicates (n=9 per group) and was designed to evaluate the impact of LPS exposure and prebiotic supplementation on immune modulation and epigenetic alterations. Control and experimental conditions were carefully structured to enable comparisons of differential methylation, cytokine expression, and miRNA regulation across the treatment groups.