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. 2022 Oct 18;23(20):12477.
doi: 10.3390/ijms232012477.

Microbe-Derived Antioxidants Reduce Lipopolysaccharide-Induced Inflammatory Responses by 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 , Qingying Gao  1 , Meijuan Zhang  1 , Hongcai Zhang  1 , Jing Zhang  1 , Weina Xu  1 , Jianbo Yao  2 , Jianxiong Xu  1
Affiliations

Microbe-Derived Antioxidants Reduce Lipopolysaccharide-Induced Inflammatory Responses by Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1β Signaling Pathway

Cheng Shen et al. Int J Mol Sci. .

Abstract

Inflammation plays an important role in the innate immune response, yet overproduction of inflammation can lead to a variety of chronic diseases associated with the innate immune system; therefore, modulation of the excessive inflammatory response has been considered a major strategy in the treatment of inflammatory diseases. Activation of the ROS/NLRP3/IL-1β signaling axis has been suggested to be a key initiating phase of inflammation. Our previous study found that microbe-derived antioxidants (MA) are shown to have excellent antioxidant and anti-inflammatory properties; however, the mechanism of action of MA remains unclear. The current study aims to investigate whether MA could protect cells from LPS-induced oxidative stress and inflammatory responses by modulating the Nrf2-ROS-NLRP3-IL-1β signaling pathway. In this study, we find that MA treatment significantly alleviates LPS-induced oxidative stress and inflammatory responses in RAW264.7 cells. MA significantly reduce the accumulation of ROS in RAW264.7 cells, down-regulate the levels of pro-inflammatory factors (TNF-α and IL-6), inhibit NLRP3, ASC, caspase-1 mRNA, and protein levels, and reduce the mRNA, protein levels, and content of inflammatory factors (IL-1β and IL-18). The protective effect of MA is significantly reduced after the siRNA knockdown of the NLRP3 gene, presumably related to the ability of MA to inhibit the ROS-NLRP3-IL-1β signaling pathway. MA is able to reduce the accumulation of ROS and alleviate oxidative stress by increasing the content of antioxidant enzymes, such as SOD, GSH-Px, and CAT. The protective effect of MA may be due to its ability of MA to induce Nrf2 to enter the nucleus and initiate the expression of antioxidant enzymes. The antioxidant properties of MA are further enhanced in the presence of the Nrf2 activator SFN. After the siRNA knockdown of the Nrf2 gene, the antioxidant and anti-inflammatory properties of MA are significantly affected. These findings suggest that MA may inhibit the LPS-stimulated ROS/NLRP3/IL-1β signaling axis by activating Nrf2-antioxidant signaling in RAW264.7 cells. As a result of this study, MA has been found to alleviate inflammatory responses and holds promise as a therapeutic agent for inflammation-related diseases.

Keywords: Nrf2; ROS/NLRP3/IL-1β; inflammatory response; microbial-derived antioxidants; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MA exhibits anti-inflammatory effects in LPS-induced RAW264.7 cells. (a) Cell viability was assessed by CCK-8 assay in RAW264.7 cells treated with different concentrations of MA (0-500 mg/L) for 12 h, followed by 1 mg/L LPS stimulation for 24 h. (b) qRT-PCR was performed to evaluate the mRNA expressions of TNF-α, IL-1β, IL-6, and IL-18 in RAW264.7 cells after treatment with 100 mg/L MA for 12 h, followed by stimulation with 1 mg/L LPS for 24 h. (c) Western blot was performed to detect the protein levels of IL-1β and IL-18 in RAW264.7 cells. (d) ELISA was conducted to detect the levels of inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-18 in RAW264.7 cells. Data are shown as mean ± SEM obtained from three independent experiments. ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
MA attenuates LPS-induced inflammatory effects through inhibition of NLRP3. (a) qRT-PCR was performed to evaluate the mRNA expression of NLRP3, ASC and Caspase-1 in RAW264.7 cells. (b) Western blot analysis was performed to detect the protein levels of NLRP3, ASC, and Caspase-1 in RAW264.7 cells. (c) qRT-PCR was performed to evaluate the mRNA expression of NLRP3 in RAW264.7 cells after siRNA treatment. Cells were first transfected with siRNA for 48 h, followed by treatment with 100mg/L MA for 12h, and finally treated with 1mg/L LPS for 24 h. All subsequent experimental cell treatments in this figure are performed in the same way as this cell treatment. (d) Western blot analysis was performed to detect the protein levels of NLRP3 in RAW264.7 cells after siRNA treatment. (e) qRT-PCR was performed to evaluate the mRNA expression of IL-1β and IL-18 in RAW264.7 cells after siRNA treatment. (f) Western blot was performed to detect the protein levels of IL-1β and IL-18 in RAW264.7 cells after siRNA treatment. (g) ELISA was conducted to detect the levels of IL-1β inflammatory cytokines after siRNA treatment. (h) qRT-PCR was performed to evaluate the mRNA expression of NLRP3, ASC, and Caspase-1 in RAW264.7 cells after siRNA treatment. (i) Western blot was performed to detect the protein levels of NLRP3, ASC, and Caspase-1 in RAW264.7 cells after siRNA treatment. Data are shown as mean ± SEM obtained from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
MA mitigates LPS-induced oxidative stress in RAW264.7 cells. Treatment with 100 mg/L MA for 12 h or 10 μM NAC (Positive control) for 30 min, followed by 1 mg/L LPS for 24 h. (a) The level of intracellular ROS was measured by DCF fluorescence using an enzyme marker. (b) MDA concentration was analyzed with 2-thiobarbituric and the activities of antioxidant enzymes including SOD, GSH-Px, and CAT were determined using ELISA kits. Data are shown as mean ± SEM obtained from three independent experiments. *** p < 0.001.
Figure 4
Figure 4
MA attenuates the inflammatory effects induced by LPS through the NLRP3/IL-1β signaling pathway by activating the Nrf2 antioxidant pathway. Cells were pretreated with10 μM SFN for 1 h and/or 100 mg/L MA for 12 h and/or 10 μM NAC (Positive control) for 30 min and then stimulated with LPS (1 mg/L) for 24 h. (a) qRT-PCR was performed to evaluate the mRNA expression of NLRP3 and IL-1β in RAW264.7 cells. (b) Western blot was performed to detect the protein levels of NLRP3 and IL-1β in RAW264.7 cells. (c) ELISA was conducted to detect the levels of inflammatory cytokines IL-1β in RAW264.7 cells. (d) Western blot was performed to detect the protein levels of N-Nrf2 (Nuclear-Nrf2) in RAW264.7 cells. (e) qRT-PCR was performed to evaluate the mRNA expression of Nrf2, NQO1 and HO-1 in RAW264.7 cells. (f) Western blot was performed to detect the protein levels of Nrf2, NQO1, and HO-1 in RAW264.7 cells. (g) MDA concentration was analyzed with 2-thiobarbituric and the activities of antioxidant enzymes including SOD, GSH-Px, and CAT were determined using ELISA kits. (h) The level of intracellular ROS was measured by DCF fluorescence using an enzyme marker. Data are shown as mean ± SEM obtained from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
MA attenuates the inflammatory effects induced by LPS through the NLRP3/IL-1β signaling pathway by activating the Nrf2 antioxidant pathway. Cells were first transfected with siRNA for 48 h, followed by treatment with 100 mg/L MA for 12 h or 10μM NAC for 30 min, and finally treated with 1mg/L LPS for 24 h. (a) qRT-PCR was performed to evaluate the mRNA expressions of Nrf2 after siRNA treatment. (b) Western blot was performed to detect the protein levels of Nrf2 after siRNA treatment. (c) qRT-PCR was performed to evaluate the mRNA expressions of NLRP3 and IL-1β after siRNA treatment. (d) Western blot was performed to detect the protein levels of NLRP3 and IL-1β after siRNA treatment. (e) ELISA was conducted to detect the levels of IL-1β inflammatory cytokines after siRNA treatment. (f) qRT-PCR was performed to evaluate the mRNA expressions of Nrf2, HO-1, and NQO1 after siRNA treatment. (g) Western blot was performed to detect the protein levels of Nrf2, HO-1, and NQO1 after siRNA treatment. (h) MDA concentration was analyzed with 2-thiobarbituric and the activities of antioxidant enzymes including SOD, GSH-Px, and CAT were determined using ELISA kits. (i) The level of intracellular ROS was measured by DCF fluorescence using an enzyme marker after siRNA treatment. Data are shown as mean ± SEM obtained from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
Proposed molecular mechanisms underlying the inhibitory effect of MA on the activation of macrophage induced by LPS. MA activates Nrf2 pathway to increase antioxidant genes and proteins, which in turn reduce inflammation and oxidative stress. The dashed lines are speculations based on previous research.
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