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. 2024 Apr 27;13(5):533.
doi: 10.3390/antiox13050533.

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-1β Signaling Pathway

Cheng Shen  1 , Zhen Luo  1 , Sheng Ma  1 , Chengbing Yu  1 , Ting Lai  1 , Shangshang Tang  1 , Hongcai Zhang  1 , Jing Zhang  1 , Weina Xu  1 , Jianxiong Xu  1
Affiliations

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-1β Signaling Pathway

Cheng Shen et al. Antioxidants (Basel). .

Abstract

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.

Keywords: Nrf2/ROS/NLRP3/IL-1β; inflammatory response; microbe-derived antioxidants; oxidative stress; tight junction proteins.

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Conflict of interest statement

All the authors declare that they have no conflicts of interest related to this study.

Figures

Figure 1
Figure 1
MAs demonstrate anti-inflammatory properties in H2O2-induced IPEC-1 cells. (A) The viability of IPEC-1 cells was determined using the CCK-8 assay. (B) The gene expression levels of TNF-α, IL-1β, IL-6, and IL-18 in IPEC-1 cells after treatment with 100 mg/L of MAs for 12 h, followed by stimulation with 1 mM H2O2 for 1 h. (C) The protein expression levels of IL-1β and IL-18. (D) The protein levels of TNF-α, IL-1β, IL-6, and IL-18. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
MAs mitigate the inflammatory effects induced by H2O2 by inhibiting the NLRP3 inflammasome. (A) The gene expression levels of NLRP3, ASC and Caspase-1. (B) The protein expression levels of NLRP3, ASC, and Caspase-1. (C) The gene expression level of NLRP3 after siRNA treatment. The cells underwent a sequential process starting with transfection with siRNA for 48 h, followed by exposure to 100 mg/L of MAs for 12 h, and concluding with treatment with 1 mM H2O2 for 1 h. All subsequent experimental cell treatments depicted in this figure follow the same procedure as this particular cell treatment. (D) The gene expression levels of IL-1β and IL-18 after siRNA treatment. (E) The gene expression levels of NLRP3, ASC, and Caspase-1 after siRNA treatment. (F) The protein level of IL-1β after siRNA treatment. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
MAs attenuate H2O2-induced barrier damage in IPEC-1 cells. (A) The gene expression levels of ZO-1 and Occludin. (B) The protein expression levels of ZO-1 and Occludin. (C) The gene expression levels of ZO-1 and Occludin after siRNA treatment. (D) The protein expression levels of ZO-1 and Occludin after siRNA treatment. Data are shown as mean ± standard error representation from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
MAs attenuate H2O2-induced oxidative stress in IPEC-1 cells. The cells were first exposed to 10 μM SFN for 1 h and/or 100 mg/L of MA for 12 h, followed by stimulation with 1 mM H2O2 for 1 h. (A) The intracellular ROS levels were assessed by quantifying DCF fluorescence employing an enzyme marker. (B) The concentration of MDA was assessed using 2-thiobarbituric, while the activities of antioxidant enzymes such as SOD, GSH-Px, and CAT were measured utilizing ELISA kits. ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
MAs alleviate H2O2-induced cellular inflammation via the Nrf2/NLRP3/IL-1β signaling pathway. The cells were first exposed to 10 μM SFN for 1 h and/or 100 mg/L of MAs for 12 h, followed by stimulation with 1 mM H2O2 for 1 h. (A) The gene expression levels of NLRP3 and IL-1β. (B) The protein expression levels of NLRP3 and IL-1β. (C) The protein level of IL-1β. (D) The protein expression level of N-Nrf2 (Nuclear-Nrf2). (E) The gene expression levels of Nrf2, NQO1 and HO-1. (F) The protein expression levels of Nrf2, NQO1, and HO-1. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
MAs alleviate H2O2-induced cellular inflammation via the Nrf2/NLRP3/IL-1β signaling pathway. The cells underwent a sequential process: they were initially transfected with siRNA for 48 h, then exposed to 100 mg/L of MAs for 12 h, and subsequently subjected to treatment with 1 mM of H2O2 for 1 h. (A) The gene expression level of Nrf2 after siRNA treatment. (B) The protein expression level of Nrf2 after siRNA treatment. (C) The gene expression levels of NLRP3 and IL-1β after siRNA treatment. (D) The protein expression levels of NLRP3 and IL-1β after siRNA treatment. (E) The protein level of IL-1β after siRNA treatment. (F) The gene expression levels of Nrf2, HO-1, and NQO1 after siRNA treatment. (G) The protein expression levels of Nrf2, HO-1, and NQO1 after siRNA treatment. (H) The concentration of MDA was assessed using 2-thiobarbituric, while the activities of antioxidant enzymes such as SOD, GSH-Px, and CAT were measured utilizing ELISA kits. (I) The intracellular ROS levels were assessed by quantifying DCF fluorescence employing an enzyme marker. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 7
Figure 7
MAs attenuate H2O2-induced barrier damage by activating the Nrf2/NLRP3/IL-1β signaling pathway. (A) The gene expression levels of ZO-1 and Occludin with SFN treatment. (B) The protein expression levels of ZO-1 and Occludin with SFN treatment. (C) The gene expression levels of ZO-1 and Occludin after siRNA treatment. (D) The protein expression levels of ZO-1 and Occludin after siRNA treatment. * p < 0.05.
Figure 8
Figure 8
MAs attenuate H2O2-induced intestinal epithelial tight junction protein disruption by reducing IL-1β. The cells were treated with 10 ng/mL IL-1β and/or 100 ng/mL IL-1β mAb for 48 h. (A) The gene expression levels of ZO-1 and Occludin with IL-1β/IL-1β AB treatment. (B) The gene expression levels of ZO-1, Occludin and MLCK with H2O2/IL-1β AB treatment. The cells were treated with 1 mM H2O2 and/or 100 ng/mL IL-1β mAb for 1 h. (C) The protein expression levels of Occludin with IL-1β/IL-1β AB and H2O2/IL-1β AB treatment. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 9
Figure 9
MAs 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-1β signaling pathway. The dashed lines represent conjectures derived from prior studies.
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