Ex) Article Title, Author, Keywords
Ex) Article Title, Author, Keywords
DTT 2024; 3(2): 105-110
Published online September 30, 2024
https://doi.org/10.58502/DTT.24.0009
Copyright © The Pharmaceutical Society of Korea.
Hami Yu* , Lan Phuong Phan*, Kyung-Sun Heo
Correspondence to:Kyung-Sun Heo, kheo@cnu.ac.kr
*The authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Macrophage plays a critical role in inducing inflammatory response, activated though TLR4 receptor by endotoxin lipopolysaccharide (LPS). It has been reported that cell inflammation is related to the oxidative stress response. However, it is not clear how the LPS-induced inflammatory signaling pathway is mechanistically related to oxidative stress responses. Here, we examined the impact of LPS-induced inflammation and oxidative stress using RAW 264.7 cells. LPS treatment up to 1 µg/ml did not show any cell toxicity. LPS stimulation causes Akt and ERK1/2 activation. However, treatment with LY294002, an Akt signaling inhibitor, completely inhibited Akt activation but not ERK1/2 activation. Interestingly, We found that LPS induces down-regulation of Nrf2 nuclear expression and translocation, whereas inhibition of Akt activation with LY294002 reverses the effects of LPS on Nrf2, as judged by Western blotting with nuclear fraction protein and immunofluorescence analysis. In addition, to investigate the role of Akt signaling in NF-κB activation-induced inflammatory response, NF-κB nuclear translocation and its target gene expression were analyzed by immunofluorescence assay and qRT-PCR analysis. LPS-induced NF-κB nuclear translocation was suppressed by LY294002 treatment. Furthermore, LY294002 inhibited LPS-induced mRNA expression of inflammatory markers, including IL-1β and MCP-1. In summary, these findings suggest that Akt signaling plays a critical role in the LPS-induced inflammatory and oxidative stress responses in RAW 264.7 cells.
KeywordsAkt, inflammation, lipopolysaccharide, Nrf2, NF-κB
Macrophages are important innate immune system immune cells required to regulate inflammation though antigen presentation, polarization, and phagocytosis (Eshghjoo et al. 2022; Chen et al. 2024). Lipopolysaccharides (LPS) is a gram-negative bacterium used to induce inflammation-mediated damage models in liver, kidney, lung, endothelial cells, and macrophage (Huynh et al. 2020; Jin et al. 2022; Jin et al. 2023a; Jin et al. 2023b; Nguyen et al. 2024). LPS-stimulated macrophages activate signaling pathways, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to trigger inflammatory mechanisms (Luyendyk et al. 2008; Hyam et al. 2013; Nguyen et al. 2024). In unstimulated macrophages, IκB binds to NF-κB and maintains it in a stable state. However, when stimulated with substances that cause inflammatory damage, such as LPS, IκB is degraded though ubiquitination, allowing NF-κB to translocate to the nucleus (Park et al. 2016). The subsequent activation of NF-κB results in the secretion of various inflammatory mediators, including IL-1β and monocyte chemoattractant protein-1 (MCP-1), which collectively contribute to the inflammatory response (Huynh et al. 2020).
Reactive oxygen species (ROS) can lead to immune dysfunction and impaired cell proliferation when deficient in the cell. However, the excessive release of superoxide radicals, generated by macrophages activated due to cellular oxidative stress, can lead to apoptosis, aging, and inflammatory diseases (Jin et al. 2022). Therefore, antioxidants are used to protect cells and tissues from high concentrations of ROS caused by oxidative stress. Nuclear factor erythoid 2-related factor 2 (Nrf2), a transcriptional regulator of intracellular oxidative stress, undergoes increased translocation into the nucleus in response to inflammation-stimulating factors. This translocation leads to the release of antioxidants, which can inhibit ROS and suppress inflammation (Jeon et al. 2021). However, the relationship between inflammation-mediated signals and antioxidant mechanisms though Nrf2 activity remains unclear. This study investigated the role of Akt signaling on LPS-induced inflammatory and oxidative stress responses in RAW 264.7 cells.
Rabbit antibodies against phosphorylated NF-κB (#8242), phosphorylated Akt (Ser473) (#4058), Akt (#2685), phosphorylated ERK1/2 (Th202/Tyr204) (#9101), ERK1/2 (#4695), and Nrf2 (#12721) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The mouse antibody against β-tubulin (SC-166729) was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). LPS (from Escherichia coli O111, EL2630) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LY294002 (S1105), an Akt inhibitor, was purchased from Selleckchem (Houston, TX, USA). Phosphate-buffered saline (PBS, #EBA-1105) was purchased from Elpisbio (Daejeon, Korea).
The murine macrophage cell line, RAW 264.7 (#40071), was procured from the Korean Cell Line Bank (Seoul, Korea). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (100 U/ml, Gibco, Waltham, MA, USA), and incubated at 37°C in a 5% CO2 humidified environment.
RAW 264.7 cells were plated in a 96-well plate and then incubated at 37°C with 5% CO2 for 24 h. Subsequently, the cells were treated with varying concentrations of LY294002 or LPS for 20 h. Cell viability was assessed using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT, Sigma-Aldrich, St. Louis, MO, USA) with absorbance readings taken at 570 nm using a microplate reader (SpectraMax iD3, Molecular Devices, LLC, San Jose, CA). The cells were incubated with MTT solution in Opti-MEM Reduced Serum Medium (Gibco, Waltham, MA, USA) at 37°C for 4 h.
RAW 264.7 cells were collected using PBS and centrifuged at 13,000 rpm for 1 min at 4°C. Nuclear extraction was conducted following a previously reported method with minor adjustments (Chang et al. 2022). Cells were incubated with a hypotonic buffer containing 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.9), 1 M KCl, 100 mM DTT, and 1X protease inhibitor cocktail for 5 min. Subsequently, 10% NP-40 was added, and the mixture was incubated on ice for 15 min. The samples were then centrifuged at 5,000 rpm for 5 min, and the resulting supernatant contained cytoplasmic proteins. The pellet was washed with PBS, resuspended in a hypertonic buffer comprising 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.9), 2.5 M NaCl, 100 mM DTT, and 1X protease inhibitor cocktail, and incubated on ice for 15 min. After centrifugation at 13,000 rpm for 10 min, the supernatant was collected as nuclear protein. Finally, 40 µl of the samples were loaded onto SDS-PAGE gels.
The cells were lysed with a 2X SDS buffer consisting of 100 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 200 mM β-mercaptoethanol, and 0.02% bromophenol blue. The protein extracts were then separated by SDS-PAGE and analyzed by western blotting, following previously established protocols (Jin et al. 2023a).
The quantitative RT-PCR (qRT-PCR) assay was performed according to previous report by minor modification (Jang et al. 2022). After treatment, total RNA was isolated using a Tri-RNA reagent (Favorgen, Pingtung, China) and subsequently synthesized to complementary DNA by the reverse transcription 5X master mix (Eplisbio, Daejeon, South Korea). The primer IL-1β and MCP-1 were synthesized by BIONNER (Daejeon, South Korea). Primer sequences are listed in Table 1. The relative gene expressions were calculated using the 2-ΔΔct method, normalized by the housekeeping gene β-actin.
Table 1 List of primer sequences used for qRT-PCR
Name | Forward primer sequence (5’-3’) | Reverse primer sequence (3’-5’) |
---|---|---|
IL-1β | AACCTGCTGGTGTGTGACGTTC | CAGCACGAGGCTTTTTTGTTGT |
MCP-1 | CCACTCACCTGCTGCTACTCAT | TGGTGATCCTCTTGTAGCTCTCC |
β-actin | CGTGCGTGACATCAAAGAGAA | TGGATGCCACAGGATTCCAT |
Immunofluorescence (IF) analysis was performed by the following previous report with minor modifications (Meng et al. 2023). RAW 264.7 cells were seeded onto glass coverslips and treated with LPS or Akt inhibitor in a 12-well plate. Cells were rinsed with PBS twice and fixed 4% paraformaldehyde for 10 min. Then, 0.2% Triton X-100 (JUNSEI) was added for 10 min for permeabilization at room temperature (RT). After blocking, RAW 264.7 cells were incubated with primary antibody against Nrf2 or NF-κB (1:200 dilution) overnight at 4°C and incubated with Alexa Fluor 488-conjugated secondary antibody (1:250 dilution) for 1 h at RT. Finally, all cells were mounted with 4’,6-diamidino-2-phenylindole (DAPI) solution, and images were obtained using a K1-Fluo laser scanning confocal microscope (Nanoscope Systems, Daejeon, Korea). Fluorescence values were detected and merged using ImageJ.
Statistical analyses were performed using GraphPad Prism 5 (version 5.02, GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons was performed. All data are expressed as the mean ± standard error of the mean (SEM) and all experiments were performed independently at least 3 times. p < 0.05 was considered statistically significant.
To investigate the viability of RAW 264.7 cells treated with LY294002 or LPS, the cells were treated with 2.5, 5, 10, and 20 µM of LY294002 and 50, 100, 250, 500, and 1000 ng/ml of LPS for 20 h, and cell viability was measured by MTT assay. Neither treatment showed any cell toxicity compared to the control (Fig. 1A, 1B). To determine whether Akt and ERK1/2 act in crosstalk, the cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml of LPS for 1 h. The phosphorylation of ERK1/2 was not reduced when treated with the Akt inhibitor, while the phosphorylation of Akt was reduced compared to the control (Fig. 1C).
To investigate the role of the Akt signaling pathway in antioxidative mechanisms, we examined Nrf2 nuclear translocation. As shown in Fig. 2A, the expression of Nrf2 in the nucleus is decreased in the LPS-stimulated group, whereas blocking the Akt signaling pathway with the inhibitor LY294002 reversed the effects of LPS on Nrf2 nuclear translocation. Additionally, we investigated Nrf2 translocation though immunofluorescence analysis and observed that the Akt signaling pathway is involved in inhibiting Nrf2 nuclear translocation (Fig. 2B, 2C).
To examine whether Akt inhibition regulates the nuclear translocation of NF-κB, a known downstream regulator, we evaluated NF-κB translocation by immunofluorescence analysis. The cells were pre-treated with 10 µM LY294002 for 1 h, followed by treatment with 1 µg/ml of LPS for 1 h. As shown in Fig. 3A and 3B, nuclear NF-κB expression was significantly reduced by the Akt inhibitor compared to the LPS-treated group.
The mRNA expression levels of IL-1β and MCP-1, downstream molecules of NF-κB, were analyzed by qRT-PCR. In Fig. 4A-4C, LPS stimulation significantly increased the expression of IL-1β and MCP-1 in macrophages. Furthermore, the data show that treatment with LY294002 significantly suppressed LPS-induced IL-1β and MCP-1 expression (Fig. 4D).
In the innate immune response to inflammation, the regulation of monocyte-to-macrophage differentiation by inflammatory responses represents immune cell activation and plays a central role in inflammation. LPS is known to activate macrophage polarization, induce the release of inflammatory cytokines such as tumor necrosis factor-α and IL-1β, and induce ROS production (Bobryshev et al. 2016; Wang et al. 2020). Previous studies in our group have shown that LPS stimulation induces the mRNA expression of pro-inflammatory cytokines such as IL-1β and MCP-1 though MAPK and NF-κB signaling pathways (Hyunh et al. 2020; Van Nguyen et al. 2022; Jin et al. 2023a; Nguyen et al. 2024). In addition, LPS activates several signaling pathways to increase transcriptional activity and induce a mediated inflammatory response (Li et al. 2020).
Our study is significant as it elucidates the mechanistic relationship between LPS-induced inflammatory signaling pathways and oxidative stress responses in macrophages, a crucial aspect of understanding inflammatory diseases. The research highlights that while LPS stimulates inflammatory pathways and oxidative stress, the precise mechanisms linking these processes have remained unclear. By focusing on the role of Akt signaling in these interactions, the study provides new insights into how LPS-induced inflammation can modulate oxidative stress responses through Akt and Nrf2 pathways.
Our findings demonstrate that inhibiting Akt, an upstream mechanism of the inflammatory response, restores Nrf2 nuclear translocation (Fig. 2). It has been reported that Nrf2 binds to antioxidant response element sequences within the promoters of various antioxidant and detoxification genes, such as Heme Oxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase 1 (NQO-1) in the nucleus (Park et al. 2024). Therefore, our data suggests that Akt signaling may be interfering with the normal regulatory mechanism of Nrf2. One possible mechanism is that Akt activation could lead to increased phosphorylation of proteins involved in Nrf2 degradation or prevent Nrf2 from translocating to the nucleus effectively. To fully understand how Akt inhibition restores Nrf2 activity, additional studies are needed to explore the specific molecular interactions and post-translational modifications involved. Investigating how Akt affects Nrf2 stability, localization, and interaction with other regulatory proteins will provide deeper insights into this regulatory pathway and we design future experiments to elucidate these molecular mechanisms further.
Furthermore, we observed that Akt inhibition affects the LPS-stimulated nuclear translocation of NF-κB and its transcriptional targets, such as IL-1β and MCP-1. (Fig. 3 and 4). These findings offer new perspectives on the molecular mechanisms underlying inflammatory diseases and could lead to the development of targeted therapeutic strategies that modulate Akt signaling to mitigate inflammation and oxidative stress.
Consequently, drugs targeting Akt inhibition are expected to reduce macrophage inflammation and enhance antioxidant responses, thereby improving inflammatory diseases.
The authors declare that they have no conflict of interest.
This work was supported by National Research Foundation of Korea (KNRF-2022R1A2C40017761231482092640102).
DTT 2024; 3(2): 105-110
Published online September 30, 2024 https://doi.org/10.58502/DTT.24.0009
Copyright © The Pharmaceutical Society of Korea.
Hami Yu* , Lan Phuong Phan*, Kyung-Sun Heo
College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon, Korea
Correspondence to:Kyung-Sun Heo, kheo@cnu.ac.kr
*The authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Macrophage plays a critical role in inducing inflammatory response, activated though TLR4 receptor by endotoxin lipopolysaccharide (LPS). It has been reported that cell inflammation is related to the oxidative stress response. However, it is not clear how the LPS-induced inflammatory signaling pathway is mechanistically related to oxidative stress responses. Here, we examined the impact of LPS-induced inflammation and oxidative stress using RAW 264.7 cells. LPS treatment up to 1 µg/ml did not show any cell toxicity. LPS stimulation causes Akt and ERK1/2 activation. However, treatment with LY294002, an Akt signaling inhibitor, completely inhibited Akt activation but not ERK1/2 activation. Interestingly, We found that LPS induces down-regulation of Nrf2 nuclear expression and translocation, whereas inhibition of Akt activation with LY294002 reverses the effects of LPS on Nrf2, as judged by Western blotting with nuclear fraction protein and immunofluorescence analysis. In addition, to investigate the role of Akt signaling in NF-κB activation-induced inflammatory response, NF-κB nuclear translocation and its target gene expression were analyzed by immunofluorescence assay and qRT-PCR analysis. LPS-induced NF-κB nuclear translocation was suppressed by LY294002 treatment. Furthermore, LY294002 inhibited LPS-induced mRNA expression of inflammatory markers, including IL-1β and MCP-1. In summary, these findings suggest that Akt signaling plays a critical role in the LPS-induced inflammatory and oxidative stress responses in RAW 264.7 cells.
Keywords: Akt, inflammation, lipopolysaccharide, Nrf2, NF-&kappa,B
Macrophages are important innate immune system immune cells required to regulate inflammation though antigen presentation, polarization, and phagocytosis (Eshghjoo et al. 2022; Chen et al. 2024). Lipopolysaccharides (LPS) is a gram-negative bacterium used to induce inflammation-mediated damage models in liver, kidney, lung, endothelial cells, and macrophage (Huynh et al. 2020; Jin et al. 2022; Jin et al. 2023a; Jin et al. 2023b; Nguyen et al. 2024). LPS-stimulated macrophages activate signaling pathways, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to trigger inflammatory mechanisms (Luyendyk et al. 2008; Hyam et al. 2013; Nguyen et al. 2024). In unstimulated macrophages, IκB binds to NF-κB and maintains it in a stable state. However, when stimulated with substances that cause inflammatory damage, such as LPS, IκB is degraded though ubiquitination, allowing NF-κB to translocate to the nucleus (Park et al. 2016). The subsequent activation of NF-κB results in the secretion of various inflammatory mediators, including IL-1β and monocyte chemoattractant protein-1 (MCP-1), which collectively contribute to the inflammatory response (Huynh et al. 2020).
Reactive oxygen species (ROS) can lead to immune dysfunction and impaired cell proliferation when deficient in the cell. However, the excessive release of superoxide radicals, generated by macrophages activated due to cellular oxidative stress, can lead to apoptosis, aging, and inflammatory diseases (Jin et al. 2022). Therefore, antioxidants are used to protect cells and tissues from high concentrations of ROS caused by oxidative stress. Nuclear factor erythoid 2-related factor 2 (Nrf2), a transcriptional regulator of intracellular oxidative stress, undergoes increased translocation into the nucleus in response to inflammation-stimulating factors. This translocation leads to the release of antioxidants, which can inhibit ROS and suppress inflammation (Jeon et al. 2021). However, the relationship between inflammation-mediated signals and antioxidant mechanisms though Nrf2 activity remains unclear. This study investigated the role of Akt signaling on LPS-induced inflammatory and oxidative stress responses in RAW 264.7 cells.
Rabbit antibodies against phosphorylated NF-κB (#8242), phosphorylated Akt (Ser473) (#4058), Akt (#2685), phosphorylated ERK1/2 (Th202/Tyr204) (#9101), ERK1/2 (#4695), and Nrf2 (#12721) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The mouse antibody against β-tubulin (SC-166729) was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). LPS (from Escherichia coli O111, EL2630) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LY294002 (S1105), an Akt inhibitor, was purchased from Selleckchem (Houston, TX, USA). Phosphate-buffered saline (PBS, #EBA-1105) was purchased from Elpisbio (Daejeon, Korea).
The murine macrophage cell line, RAW 264.7 (#40071), was procured from the Korean Cell Line Bank (Seoul, Korea). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (100 U/ml, Gibco, Waltham, MA, USA), and incubated at 37°C in a 5% CO2 humidified environment.
RAW 264.7 cells were plated in a 96-well plate and then incubated at 37°C with 5% CO2 for 24 h. Subsequently, the cells were treated with varying concentrations of LY294002 or LPS for 20 h. Cell viability was assessed using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT, Sigma-Aldrich, St. Louis, MO, USA) with absorbance readings taken at 570 nm using a microplate reader (SpectraMax iD3, Molecular Devices, LLC, San Jose, CA). The cells were incubated with MTT solution in Opti-MEM Reduced Serum Medium (Gibco, Waltham, MA, USA) at 37°C for 4 h.
RAW 264.7 cells were collected using PBS and centrifuged at 13,000 rpm for 1 min at 4°C. Nuclear extraction was conducted following a previously reported method with minor adjustments (Chang et al. 2022). Cells were incubated with a hypotonic buffer containing 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.9), 1 M KCl, 100 mM DTT, and 1X protease inhibitor cocktail for 5 min. Subsequently, 10% NP-40 was added, and the mixture was incubated on ice for 15 min. The samples were then centrifuged at 5,000 rpm for 5 min, and the resulting supernatant contained cytoplasmic proteins. The pellet was washed with PBS, resuspended in a hypertonic buffer comprising 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.9), 2.5 M NaCl, 100 mM DTT, and 1X protease inhibitor cocktail, and incubated on ice for 15 min. After centrifugation at 13,000 rpm for 10 min, the supernatant was collected as nuclear protein. Finally, 40 µl of the samples were loaded onto SDS-PAGE gels.
The cells were lysed with a 2X SDS buffer consisting of 100 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 200 mM β-mercaptoethanol, and 0.02% bromophenol blue. The protein extracts were then separated by SDS-PAGE and analyzed by western blotting, following previously established protocols (Jin et al. 2023a).
The quantitative RT-PCR (qRT-PCR) assay was performed according to previous report by minor modification (Jang et al. 2022). After treatment, total RNA was isolated using a Tri-RNA reagent (Favorgen, Pingtung, China) and subsequently synthesized to complementary DNA by the reverse transcription 5X master mix (Eplisbio, Daejeon, South Korea). The primer IL-1β and MCP-1 were synthesized by BIONNER (Daejeon, South Korea). Primer sequences are listed in Table 1. The relative gene expressions were calculated using the 2-ΔΔct method, normalized by the housekeeping gene β-actin.
Table 1 . List of primer sequences used for qRT-PCR.
Name | Forward primer sequence (5’-3’) | Reverse primer sequence (3’-5’) |
---|---|---|
IL-1β | AACCTGCTGGTGTGTGACGTTC | CAGCACGAGGCTTTTTTGTTGT |
MCP-1 | CCACTCACCTGCTGCTACTCAT | TGGTGATCCTCTTGTAGCTCTCC |
β-actin | CGTGCGTGACATCAAAGAGAA | TGGATGCCACAGGATTCCAT |
Immunofluorescence (IF) analysis was performed by the following previous report with minor modifications (Meng et al. 2023). RAW 264.7 cells were seeded onto glass coverslips and treated with LPS or Akt inhibitor in a 12-well plate. Cells were rinsed with PBS twice and fixed 4% paraformaldehyde for 10 min. Then, 0.2% Triton X-100 (JUNSEI) was added for 10 min for permeabilization at room temperature (RT). After blocking, RAW 264.7 cells were incubated with primary antibody against Nrf2 or NF-κB (1:200 dilution) overnight at 4°C and incubated with Alexa Fluor 488-conjugated secondary antibody (1:250 dilution) for 1 h at RT. Finally, all cells were mounted with 4’,6-diamidino-2-phenylindole (DAPI) solution, and images were obtained using a K1-Fluo laser scanning confocal microscope (Nanoscope Systems, Daejeon, Korea). Fluorescence values were detected and merged using ImageJ.
Statistical analyses were performed using GraphPad Prism 5 (version 5.02, GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons was performed. All data are expressed as the mean ± standard error of the mean (SEM) and all experiments were performed independently at least 3 times. p < 0.05 was considered statistically significant.
To investigate the viability of RAW 264.7 cells treated with LY294002 or LPS, the cells were treated with 2.5, 5, 10, and 20 µM of LY294002 and 50, 100, 250, 500, and 1000 ng/ml of LPS for 20 h, and cell viability was measured by MTT assay. Neither treatment showed any cell toxicity compared to the control (Fig. 1A, 1B). To determine whether Akt and ERK1/2 act in crosstalk, the cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml of LPS for 1 h. The phosphorylation of ERK1/2 was not reduced when treated with the Akt inhibitor, while the phosphorylation of Akt was reduced compared to the control (Fig. 1C).
To investigate the role of the Akt signaling pathway in antioxidative mechanisms, we examined Nrf2 nuclear translocation. As shown in Fig. 2A, the expression of Nrf2 in the nucleus is decreased in the LPS-stimulated group, whereas blocking the Akt signaling pathway with the inhibitor LY294002 reversed the effects of LPS on Nrf2 nuclear translocation. Additionally, we investigated Nrf2 translocation though immunofluorescence analysis and observed that the Akt signaling pathway is involved in inhibiting Nrf2 nuclear translocation (Fig. 2B, 2C).
To examine whether Akt inhibition regulates the nuclear translocation of NF-κB, a known downstream regulator, we evaluated NF-κB translocation by immunofluorescence analysis. The cells were pre-treated with 10 µM LY294002 for 1 h, followed by treatment with 1 µg/ml of LPS for 1 h. As shown in Fig. 3A and 3B, nuclear NF-κB expression was significantly reduced by the Akt inhibitor compared to the LPS-treated group.
The mRNA expression levels of IL-1β and MCP-1, downstream molecules of NF-κB, were analyzed by qRT-PCR. In Fig. 4A-4C, LPS stimulation significantly increased the expression of IL-1β and MCP-1 in macrophages. Furthermore, the data show that treatment with LY294002 significantly suppressed LPS-induced IL-1β and MCP-1 expression (Fig. 4D).
In the innate immune response to inflammation, the regulation of monocyte-to-macrophage differentiation by inflammatory responses represents immune cell activation and plays a central role in inflammation. LPS is known to activate macrophage polarization, induce the release of inflammatory cytokines such as tumor necrosis factor-α and IL-1β, and induce ROS production (Bobryshev et al. 2016; Wang et al. 2020). Previous studies in our group have shown that LPS stimulation induces the mRNA expression of pro-inflammatory cytokines such as IL-1β and MCP-1 though MAPK and NF-κB signaling pathways (Hyunh et al. 2020; Van Nguyen et al. 2022; Jin et al. 2023a; Nguyen et al. 2024). In addition, LPS activates several signaling pathways to increase transcriptional activity and induce a mediated inflammatory response (Li et al. 2020).
Our study is significant as it elucidates the mechanistic relationship between LPS-induced inflammatory signaling pathways and oxidative stress responses in macrophages, a crucial aspect of understanding inflammatory diseases. The research highlights that while LPS stimulates inflammatory pathways and oxidative stress, the precise mechanisms linking these processes have remained unclear. By focusing on the role of Akt signaling in these interactions, the study provides new insights into how LPS-induced inflammation can modulate oxidative stress responses through Akt and Nrf2 pathways.
Our findings demonstrate that inhibiting Akt, an upstream mechanism of the inflammatory response, restores Nrf2 nuclear translocation (Fig. 2). It has been reported that Nrf2 binds to antioxidant response element sequences within the promoters of various antioxidant and detoxification genes, such as Heme Oxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase 1 (NQO-1) in the nucleus (Park et al. 2024). Therefore, our data suggests that Akt signaling may be interfering with the normal regulatory mechanism of Nrf2. One possible mechanism is that Akt activation could lead to increased phosphorylation of proteins involved in Nrf2 degradation or prevent Nrf2 from translocating to the nucleus effectively. To fully understand how Akt inhibition restores Nrf2 activity, additional studies are needed to explore the specific molecular interactions and post-translational modifications involved. Investigating how Akt affects Nrf2 stability, localization, and interaction with other regulatory proteins will provide deeper insights into this regulatory pathway and we design future experiments to elucidate these molecular mechanisms further.
Furthermore, we observed that Akt inhibition affects the LPS-stimulated nuclear translocation of NF-κB and its transcriptional targets, such as IL-1β and MCP-1. (Fig. 3 and 4). These findings offer new perspectives on the molecular mechanisms underlying inflammatory diseases and could lead to the development of targeted therapeutic strategies that modulate Akt signaling to mitigate inflammation and oxidative stress.
Consequently, drugs targeting Akt inhibition are expected to reduce macrophage inflammation and enhance antioxidant responses, thereby improving inflammatory diseases.
The authors declare that they have no conflict of interest.
This work was supported by National Research Foundation of Korea (KNRF-2022R1A2C40017761231482092640102).
Table 1 List of primer sequences used for qRT-PCR
Name | Forward primer sequence (5’-3’) | Reverse primer sequence (3’-5’) |
---|---|---|
IL-1β | AACCTGCTGGTGTGTGACGTTC | CAGCACGAGGCTTTTTTGTTGT |
MCP-1 | CCACTCACCTGCTGCTACTCAT | TGGTGATCCTCTTGTAGCTCTCC |
β-actin | CGTGCGTGACATCAAAGAGAA | TGGATGCCACAGGATTCCAT |