Microbe-Derived Antioxidants Protect IPEC-1 Cells from H2O2-Induced Oxidative Stress, Inflammation and Tight Junction Protein Disruption via Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1ß Signaling Pathway.

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-1ß 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-1ß 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-1ß and antioxidant enzyme activity levels were detected by ELISA. Oxidative stress marker ROS was examined by fluorescence analysis. The NLRP3/IL-1ß 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-1ß, 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-1ß signaling pathway through the activation of the Nrf2 pathway.

Identification of key overlapping DEGs and molecular pathways under multiple stressors in the liver of Nile tilapia (Oreochromis niloticus).

The identification of key genes and molecular pathways that are involved in the response to stressors is crucial for controlling stress in fish and sustainable aquaculture. Environmental stressors can induce stress responses in aquatic animals, resulting in compromised immune function, inhibited growth, and increased mortality rates. mRNA-seq analysis provides a powerful tool to identify key genes and pathways associated with stress response. In the present study, mRNA-seq analysis was employed to identify key overlapping differentially expressed genes (DEGs) and molecular pathways under salinity, nitrite, copper, and pH stress in the liver of Nile tilapia (Oreochromis niloticus). The pathways associated with the immune response, oxygen transport, homeostasis, and oxidative stress were enriched across all stressors. The top KEGG pathways were complement and coagulation cascades, PPAR signaling pathway, and cardiac muscle contraction. The top GO enrichment terms were oxidoreductase activity, aerobic respiration, endopeptidase inhibitor activity, endopeptidase regulator activity, heme binding, and iron ion binding. The complement genes (C3, C4, C5, factor B, and factor H), alpha-2-macroglobulin (A2M), hemoglobin subunit epsilon (HBE), hemoglobin subunit alpha (HBA), coagulation factor genes (XI and X) and the cytochrome c oxidase (COX) gene family (cox1, cox2, cox3, cytochrome P450) were identified as key shared genes across multiple stressors. The discovery of these genes and molecular pathways provided a better understanding of the molecular mechanism underlying the stress response in Nile tilapia. The results of the present study can facilitate the development of stress management strategies in Nile tilapia.

Identification of key immune and stress related genes and pathways by comparative analysis of the gene expression profile under multiple environmental stressors in pacific white shrimp (Litopenaeus vannamei).

Water salinity, pH, and nitrite concentration are considered environmental factors affecting the growth rate, survival, health, and physiological conditions of aquatic animals. The identification of key genes that are involved in the response to environmental stressors is essential for controlling stress in aquatic animals and sustainable aquaculture. In this study, RNA sequencing was performed to identify the differentially expressed genes (DEGs) and biological pathways that are involved in the response of the hepatopancreas to environmental stressors, including low salinity stress, nitrite stress, low pH stress, and high pH stress. The DEGs were enriched in biological pathways related to immune response, energy metabolism, oxidative stress response, hemostasis, and enzymatic activity of the hepatopancreas. In addition to the identification of DEGs related to each stressor, some DEGs were found to be expressed among all groups. The most important overlapping DEGs under multiple stressors were juvenile hormone esterase-like protein 2 (JHE-like), myosin light chain, C-type lectin 2, myosin-9-like, anti-lipopolysaccharide factor 1 (ALF-1), peroxisomal acyl-coenzyme An oxidase 1-like (ACX1), hepatic lectin-like, venom phosphodiesterase 2-like, hemolymph clottable protein-like (CP), cathepsin L, and Ras-like protein 2. The results of the present study provide additional information regarding the transcriptional response of the hepatopancreas to low salinity, nitrite, low pH, and high pH stress. Moreover, the discovery of several overlapping DEGs among different stressors provided a better understanding of the molecular function of the hepatopancreas.

Berberine Regulation of Cellular Oxidative Stress, Apoptosis and Autophagy by Modulation of m6A mRNA Methylation through Targeting the Camk1db/ERK Pathway in Zebrafish-Hepatocytes.

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.