Oxidative stress can induce inflammation and tight junction disruption in enterocytes. The initiation of inflammation is thought to commence with the activation of the ROS/NLRP3/IL-1β signaling pathway, marking a crucial starting point in the process. In our previous studies, we found that microbe-derived antioxidants (MAs) showed significant potential in enhancing both antioxidant capabilities and anti-inflammatory effects. The main aim of this research was to investigate the ability of MAs to protect cells from oxidative stress caused by H₂O₂, to reduce inflammatory responses, and to maintain the integrity of tight junction proteins by modulating the ROS/NLRP3/IL-1β signaling pathway. IPEC-1 cells (1 × 10⁴ cells/well) were initially exposed to 100 mg/L of MAs for 12 h, after which they were subjected to 1 mM H₂O₂ treatment for 1 h. We utilized small interfering RNA (siRNA) to inhibit the expression of NLRP3 and Nrf2. Inflammatory factors such as IL-1β and antioxidant enzyme activity levels were detected by ELISA. Oxidative stress marker ROS was examined by fluorescence analysis. The NLRP3/IL-1β signaling pathway, Nrf2/HO-1 signaling pathway and tight junction proteins (ZO-1 and Occludin) were detected by RT-qPCR or Western blotting. In our research, it was observed that MA treatment effectively suppressed the notable increase in H₂O₂-induced inflammatory markers (TNF-α, IL-1β, and IL-18), decreased ROS accumulation, mitigated the expression of NLRP3, ASC, and caspase-1, and promoted the expression of ZO-1 and Occludin. After silencing the NLRP3 gene with siRNA, the protective influence of MAs was observed to be linked with the NLRP3 inflammasome. Additional investigations demonstrated that the treatment with MAs triggered the activation of Nrf2, facilitating its translocation into the nucleus. This process resulted in a notable upregulation of Nrf2, NQO1, and HO-1 expression, along with the initiation of the Nrf2-HO-1 signaling pathway. Consequently, there was an enhancement in the activities of antioxidant enzymes like SOD, GSH-Px, and CAT, which effectively mitigated the accumulation of ROS, thereby ameliorating the oxidative stress state. The antioxidant effectiveness of MAs was additionally heightened in the presence of SFN, an activator of Nrf2. The antioxidant and anti-inflammatory functions of MAs and their role in regulating intestinal epithelial tight junction protein disruption were significantly affected after siRNA knockdown of the Nrf2 gene. These findings suggest that MAs have the potential to reduce H₂O₂-triggered oxidative stress, inflammation, and disruption of intestinal epithelial tight junction proteins in IPEC-1 cells. This reduction is achieved by blocking the ROS/NLRP3/IL-1β signaling pathway through the activation of the Nrf2 pathway.

Berberine (BBR) ameliorates cellular oxidative stress, apoptosis and autophagy induced by lipid metabolism disorder, however, the molecular mechanism associated with it is not well known. To study the mechanism, we started with m⁶A methylation modification to investigate its role in lipid deposition zebrafish hepatocytes (ZFL). The results showed that BBR could change the cellular m⁶A RNA methylation level, increase m⁶A levels of Camk1db gene transcript and alter Camk1db gene mRNA expression. Via knockdown of the Camk1db gene, Camk1db could promote cellular ERK phosphorylation levels. Berberine regulated the expression level of Camk1db mRNA by altering the M⁶A RNA methylation of the Camk1db gene, which further affected the synthesis of calmodulin-dependent protein kinase and activated ERK signaling pathway resulting in changes in downstream physiological indicators including ROS production, cell proliferation, apoptosis and autophagy. In conclusion, berberine could regulate cellular oxidative stress, apoptosis and autophagy by mediating Camk1db m⁶A methylation through the targeting of the Camk1db/ERK pathway in zebrafish-hepatocyte.