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Original Research Article

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.

Akt Signaling Pathway Inhibits Nrf2 to Enhance Oxidative Stress and Induces NF-κB Activation to Promote Inflammation in RAW 264.7 Cells

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.

Received: June 17, 2024; Revised: August 7, 2024; Accepted: August 14, 2024

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.

Reagents and antibodies

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).

Cell culture

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.

Cell viability

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.

Nuclear and cytoplasmic extraction

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.

Western blot analysis

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).

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

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

NameForward primer sequence (5’-3’)Reverse primer sequence (3’-5’)
IL-1βAACCTGCTGGTGTGTGACGTTCCAGCACGAGGCTTTTTTGTTGT
MCP-1CCACTCACCTGCTGCTACTCATTGGTGATCCTCTTGTAGCTCTCC
β-actinCGTGCGTGACATCAAAGAGAATGGATGCCACAGGATTCCAT

Immunofluorescence analysis

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 analysis

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.

LPS-induces Akt activation in RAW 264.7 cells

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).

Figure 1.LPS-induces Akt activation in RAW 264.7 cells. To determine the optimal concentration of LY294002 or LPS for our experiments, we assessed toxicity in RAW 264.7 cells via MTT assay. (A) RAW 264.7 cells were exposed to 50, 100, 250, 500, and 1000 ng/ml LPS for 20 h. (B) RAW 264.7 cells were treated with 2.5, 5, 10, and 20 µM of LY294002 for 20 h. (C) The RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 1 h. Total cell lysates were subjected to western blot analysis using Akt phosphorylation, total Akt, ERK1/2 phosphorylation, total ERK1/2, and β-tubulin. The results are presented as mean ± SEM, with n = 3 in each group.

Akt signaling pathway regulates Nrf2 nuclear translocation in LPS-induced RAW 264.7 cells

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).

Figure 2.Akt signaling pathway regulates Nrf2 nuclear translocation in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 24 h. (A) Cells were subjected to nucleus fractionation to determine the expression levels of Nrf2 and Lamin B1 in the nucleus, and then, protein expression was detected by western blot. (B) Nrf2 translocation was analyzed by immunostaining with Nrf2 antibody. (C) Quantitively bar graph indicates relative nuclear Nrf2 expression. For quantification, we counted the number of DAPI-positive nuclei to determine the total cell count and then identified the number of cells with high nuclear Nrf2 expression. The bar graph reflects this count as a proportion of the total cell count in each image. The scale bar represents 30 µm. **p < 0.05 compared to the control group, #p < 0.05 compared to the LPS-treated group.

Akt signaling pathway is associated with NF-κB activation in LPS-induced RAW 264.7 cells

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.

Figure 3.Akt signaling pathway is associated with NF-κB activation in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 1 h. (A) NF-κB translocation was analyzed by immunostaining with NF-κB antibody. The quantification was the number of NF-κB in the nucleus was divided by the total number of DAPI-positive nuclei. The scale bar represents 30 µm. **p < 0.01 compared to the control group, #p < 0.05 compared to the LPS-treated group.

LY294002 suppressed LPS-induced IL-1β and MCP-1 expression in RAW 264.7 cells

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).

Figure 4.LY294002 suppressed LPS-induced IL-1β and MCP-1 expression in RAW 264.7 cells. (A, B) RAW 264.7 cells were treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 12 h. Total RNA samples were subjected to qRT-PCR using IL-1β and MCP-1 primer. (C) Schematic illustration created using Biorender.com. Data are expressed as the mean ± SEM (n = 3). ***p < 0.001 compared with the control group, ###p < 0.001 compared with the LPS-treated group.

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).

  1. Bobryshev YV, Ivanova EA, Chistiakov DA, Nikiforov NG, Orekhov AN (2016) Macrophages and their role in atherosclerosis: pathophysiology and transcriptome analysis. Biomed Res Int 2016:9582430. doi: 10.1155/2016/9582430
    Pubmed KoreaMed CrossRef
  2. Chang JW, Kim S, Lee EY, Leem CH, Kim SH, Park CS (2022) Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells. Korean J Physiol Pharmacol 26:479-499. doi: 10.4196/kjpp.2022.26.6.479
    Pubmed KoreaMed CrossRef
  3. Chen R, Zhang H, Tang B, Luo Y, Yang Y, Zhong X, Chen S, Xu X, Huang S, Liu C (2024) Macrophages in cardiovascular diseases: molecular mechanisms and therapeutic targets. Signal Transduct Target Ther 9:130. doi: 10.1038/s41392-024-01840-1
    Pubmed KoreaMed CrossRef
  4. Eshghjoo S, Kim DM, Jayaraman A, Sun Y, Alaniz RC (2022) Macrophage polarization in atherosclerosis. Genes (Basel) 13:756. doi: 10.3390/genes13050756
    Pubmed KoreaMed CrossRef
  5. Huynh DTN, Baek N, Sim S, Myung CS, Heo KS (2020) Minor ginsenoside Rg2 and Rh1 attenuates LPS-induced acute liver and kidney damages via downregulating activation of TLR4-STAT1 and inflammatory cytokine production in macrophages. Int J Mol Sci 21:6656. doi: 10.3390/ijms21186656
    Pubmed KoreaMed CrossRef
  6. Hyam SR, Lee IA, Gu W, Kim KA, Jeong JJ, Jang SE, Han MJ, Kim DH (2013) Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages. Eur J Pharmacol 708:21-29. doi: 10.1016/j.ejphar.2013.01.014
    Pubmed CrossRef
  7. Jang EJ, Kim H, Baek SE, Jeon EY, Kim JW, Kim JY, Kim CD (2022) HMGB1 increases RAGE expression in vascular smooth muscle cells via ERK and p-38 MAPK-dependent pathways. Korean J Physiol Pharmacol 26:389-396. doi: 10.4196/kjpp.2022.26.5.389
    Pubmed KoreaMed CrossRef
  8. Jeon H, Jin Y, Myung CS, Heo KS (2021) Ginsenoside-Rg2 exerts anti-cancer effects through ROS-mediated AMPK activation associated mitochondrial damage and oxidation in MCF-7 cells. Arch Pharm Res 44:702-712. doi: 10.1007/s12272-021-01345-3
    Pubmed CrossRef
  9. Jin Y, Jeon H, Le Lam Nguyen T, Kim L, Heo KS (2023a) Human milk oligosaccharides 3'-sialyllactose and 6'-sialyllactose attenuate LPS-induced lung injury by inhibiting STAT1 and NF-κB signaling pathways. Arch Pharm Res 46:897-906. doi: 10.1007/s12272-023-01470-1
    Pubmed CrossRef
  10. Jin Y, Nguyen TLL, Myung CS, Heo KS (2022) Ginsenoside Rh1 protects human endothelial cells against lipopolysaccharide-induced inflammatory injury through inhibiting TLR2/4-mediated STAT3, NF-κB, and ER stress signaling pathways. Life Sci 309:120973. doi: 10.1016/j.lfs.2022.120973
    Pubmed CrossRef
  11. Jin Y, Tangchang W, Kwon OS, Lee JY, Heo KS, Son HY (2023b) Ginsenoside Rh1 ameliorates the asthma and allergic inflammation via inhibiting Akt, MAPK, and NF-κB signaling pathways in vitro and in vivo. Life Sci 321:121607. doi: 10.1016/j.lfs.2023.121607
    Pubmed CrossRef
  12. Li J, Qin Y, Chen Y, Zhao P, Liu X, Dong H, Zheng W, Feng S, Mao X, Li C (2020) Mechanisms of the lipopolysaccharide-induced inflammatory response in alveolar epithelial cell/macrophage co-culture. Exp Ther Med 20:76. doi: 10.3892/etm.2020.9204
    Pubmed KoreaMed CrossRef
  13. Luyendyk JP, Schabbauer GA, Tencati M, Holscher T, Pawlinski R, Mackman N (2008) Genetic analysis of the role of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine and tissue factor gene expression in monocytes/macrophages. J Immunol 180:4218-4226. doi: 10.4049/jimmunol.180.6.4218
    Pubmed KoreaMed CrossRef
  14. Meng RY, Li CS, Hu D, Kwon SG, Jin H, Chai OH, Lee JS, Kim SM (2023) Inhibition of the interaction between Hippo/YAP and Akt signaling with ursolic acid and 3'3-diindolylmethane suppresses esophageal cancer tumorigenesis. Korean J Physiol Pharmacol 27:493-511. doi: 10.4196/kjpp.2023.27.5.493
    Pubmed KoreaMed CrossRef
  15. Nguyen DV, Jin Y, Nguyen TLL, Kim L, Heo KS (2024) 3'-Sialyllactose protects against LPS-induced endothelial dysfunction by inhibiting superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis. Life Sci 338:122410. doi: 10.1016/j.lfs.2023.122410
    Pubmed CrossRef
  16. Park JM, Park JE, Park JS, Leem YH, Kim DY, Hyun JW, Kim HS (2024) Anti-inflammatory and antioxidant mechanisms of coniferaldehyde in lipopolysaccharide-induced neuroinflammation: involvement of AMPK/Nrf2 and TAK1/MAPK/NF-κB signaling pathways. Eur J Pharmacol 979:176850. doi: 10.1016/j.ejphar.2024.176850
    Pubmed CrossRef
  17. Park JS, Park MY, Cho YJ, Lee JH, Yoo CG, Lee CT, Lee SM (2016) Anti-inflammatory effect of erdosteine in lipopolysaccharide-stimulated RAW 264.7 cells. Inflammation 39:1573-1581. doi: 10.1007/s10753-016-0393-4
    Pubmed CrossRef
  18. Van Nguyen D, Nguyen TLL, Jin Y, Kim L, Myung CS, Heo KS (2022) 6'-Sialylactose abolished lipopolysaccharide-induced inflammation and hyper-permeability in endothelial cells. Arch Pharm Res 45:836-848. doi: 10.1007/s12272-022-01415-0
    Pubmed CrossRef
  19. Wang Y, Liu H, Zhao J (2020) Macrophage polarization induced by probiotic bacteria: a concise review. Probiotics Antimicrob Proteins 12:798-808. doi: 10.1007/s12602-019-09612-y
    Pubmed CrossRef

Article

Original Research Article

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.

Akt Signaling Pathway Inhibits Nrf2 to Enhance Oxidative Stress and Induces NF-κB Activation to Promote Inflammation in RAW 264.7 Cells

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.

Received: June 17, 2024; Revised: August 7, 2024; Accepted: August 14, 2024

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.

Abstract

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

Introduction

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.

Materials|Methods

Reagents and antibodies

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).

Cell culture

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.

Cell viability

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.

Nuclear and cytoplasmic extraction

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.

Western blot analysis

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).

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

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.

NameForward primer sequence (5’-3’)Reverse primer sequence (3’-5’)
IL-1βAACCTGCTGGTGTGTGACGTTCCAGCACGAGGCTTTTTTGTTGT
MCP-1CCACTCACCTGCTGCTACTCATTGGTGATCCTCTTGTAGCTCTCC
β-actinCGTGCGTGACATCAAAGAGAATGGATGCCACAGGATTCCAT


Immunofluorescence analysis

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 analysis

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.

Results

LPS-induces Akt activation in RAW 264.7 cells

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).

Figure 1. LPS-induces Akt activation in RAW 264.7 cells. To determine the optimal concentration of LY294002 or LPS for our experiments, we assessed toxicity in RAW 264.7 cells via MTT assay. (A) RAW 264.7 cells were exposed to 50, 100, 250, 500, and 1000 ng/ml LPS for 20 h. (B) RAW 264.7 cells were treated with 2.5, 5, 10, and 20 µM of LY294002 for 20 h. (C) The RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 1 h. Total cell lysates were subjected to western blot analysis using Akt phosphorylation, total Akt, ERK1/2 phosphorylation, total ERK1/2, and β-tubulin. The results are presented as mean ± SEM, with n = 3 in each group.

Akt signaling pathway regulates Nrf2 nuclear translocation in LPS-induced RAW 264.7 cells

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).

Figure 2. Akt signaling pathway regulates Nrf2 nuclear translocation in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 24 h. (A) Cells were subjected to nucleus fractionation to determine the expression levels of Nrf2 and Lamin B1 in the nucleus, and then, protein expression was detected by western blot. (B) Nrf2 translocation was analyzed by immunostaining with Nrf2 antibody. (C) Quantitively bar graph indicates relative nuclear Nrf2 expression. For quantification, we counted the number of DAPI-positive nuclei to determine the total cell count and then identified the number of cells with high nuclear Nrf2 expression. The bar graph reflects this count as a proportion of the total cell count in each image. The scale bar represents 30 µm. **p < 0.05 compared to the control group, #p < 0.05 compared to the LPS-treated group.

Akt signaling pathway is associated with NF-κB activation in LPS-induced RAW 264.7 cells

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.

Figure 3. Akt signaling pathway is associated with NF-κB activation in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 1 h. (A) NF-κB translocation was analyzed by immunostaining with NF-κB antibody. The quantification was the number of NF-κB in the nucleus was divided by the total number of DAPI-positive nuclei. The scale bar represents 30 µm. **p < 0.01 compared to the control group, #p < 0.05 compared to the LPS-treated group.

LY294002 suppressed LPS-induced IL-1β and MCP-1 expression in RAW 264.7 cells

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).

Figure 4. LY294002 suppressed LPS-induced IL-1β and MCP-1 expression in RAW 264.7 cells. (A, B) RAW 264.7 cells were treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 12 h. Total RNA samples were subjected to qRT-PCR using IL-1β and MCP-1 primer. (C) Schematic illustration created using Biorender.com. Data are expressed as the mean ± SEM (n = 3). ***p < 0.001 compared with the control group, ###p < 0.001 compared with the LPS-treated group.

Discussion

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.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by National Research Foundation of Korea (KNRF-2022R1A2C40017761231482092640102).

Fig 1.

Figure 1.LPS-induces Akt activation in RAW 264.7 cells. To determine the optimal concentration of LY294002 or LPS for our experiments, we assessed toxicity in RAW 264.7 cells via MTT assay. (A) RAW 264.7 cells were exposed to 50, 100, 250, 500, and 1000 ng/ml LPS for 20 h. (B) RAW 264.7 cells were treated with 2.5, 5, 10, and 20 µM of LY294002 for 20 h. (C) The RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 1 h. Total cell lysates were subjected to western blot analysis using Akt phosphorylation, total Akt, ERK1/2 phosphorylation, total ERK1/2, and β-tubulin. The results are presented as mean ± SEM, with n = 3 in each group.
Drug Targets and Therapeutics 2024; 3: 105-110https://doi.org/10.58502/DTT.24.0009

Fig 2.

Figure 2.Akt signaling pathway regulates Nrf2 nuclear translocation in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 24 h. (A) Cells were subjected to nucleus fractionation to determine the expression levels of Nrf2 and Lamin B1 in the nucleus, and then, protein expression was detected by western blot. (B) Nrf2 translocation was analyzed by immunostaining with Nrf2 antibody. (C) Quantitively bar graph indicates relative nuclear Nrf2 expression. For quantification, we counted the number of DAPI-positive nuclei to determine the total cell count and then identified the number of cells with high nuclear Nrf2 expression. The bar graph reflects this count as a proportion of the total cell count in each image. The scale bar represents 30 µm. **p < 0.05 compared to the control group, #p < 0.05 compared to the LPS-treated group.
Drug Targets and Therapeutics 2024; 3: 105-110https://doi.org/10.58502/DTT.24.0009

Fig 3.

Figure 3.Akt signaling pathway is associated with NF-κB activation in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pre-treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 1 h. (A) NF-κB translocation was analyzed by immunostaining with NF-κB antibody. The quantification was the number of NF-κB in the nucleus was divided by the total number of DAPI-positive nuclei. The scale bar represents 30 µm. **p < 0.01 compared to the control group, #p < 0.05 compared to the LPS-treated group.
Drug Targets and Therapeutics 2024; 3: 105-110https://doi.org/10.58502/DTT.24.0009

Fig 4.

Figure 4.LY294002 suppressed LPS-induced IL-1β and MCP-1 expression in RAW 264.7 cells. (A, B) RAW 264.7 cells were treated with 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 12 h. Total RNA samples were subjected to qRT-PCR using IL-1β and MCP-1 primer. (C) Schematic illustration created using Biorender.com. Data are expressed as the mean ± SEM (n = 3). ***p < 0.001 compared with the control group, ###p < 0.001 compared with the LPS-treated group.
Drug Targets and Therapeutics 2024; 3: 105-110https://doi.org/10.58502/DTT.24.0009

Table 1 List of primer sequences used for qRT-PCR

NameForward primer sequence (5’-3’)Reverse primer sequence (3’-5’)
IL-1βAACCTGCTGGTGTGTGACGTTCCAGCACGAGGCTTTTTTGTTGT
MCP-1CCACTCACCTGCTGCTACTCATTGGTGATCCTCTTGTAGCTCTCC
β-actinCGTGCGTGACATCAAAGAGAATGGATGCCACAGGATTCCAT

References

  1. Bobryshev YV, Ivanova EA, Chistiakov DA, Nikiforov NG, Orekhov AN (2016) Macrophages and their role in atherosclerosis: pathophysiology and transcriptome analysis. Biomed Res Int 2016:9582430. doi: 10.1155/2016/9582430
    Pubmed KoreaMed CrossRef
  2. Chang JW, Kim S, Lee EY, Leem CH, Kim SH, Park CS (2022) Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells. Korean J Physiol Pharmacol 26:479-499. doi: 10.4196/kjpp.2022.26.6.479
    Pubmed KoreaMed CrossRef
  3. Chen R, Zhang H, Tang B, Luo Y, Yang Y, Zhong X, Chen S, Xu X, Huang S, Liu C (2024) Macrophages in cardiovascular diseases: molecular mechanisms and therapeutic targets. Signal Transduct Target Ther 9:130. doi: 10.1038/s41392-024-01840-1
    Pubmed KoreaMed CrossRef
  4. Eshghjoo S, Kim DM, Jayaraman A, Sun Y, Alaniz RC (2022) Macrophage polarization in atherosclerosis. Genes (Basel) 13:756. doi: 10.3390/genes13050756
    Pubmed KoreaMed CrossRef
  5. Huynh DTN, Baek N, Sim S, Myung CS, Heo KS (2020) Minor ginsenoside Rg2 and Rh1 attenuates LPS-induced acute liver and kidney damages via downregulating activation of TLR4-STAT1 and inflammatory cytokine production in macrophages. Int J Mol Sci 21:6656. doi: 10.3390/ijms21186656
    Pubmed KoreaMed CrossRef
  6. Hyam SR, Lee IA, Gu W, Kim KA, Jeong JJ, Jang SE, Han MJ, Kim DH (2013) Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages. Eur J Pharmacol 708:21-29. doi: 10.1016/j.ejphar.2013.01.014
    Pubmed CrossRef
  7. Jang EJ, Kim H, Baek SE, Jeon EY, Kim JW, Kim JY, Kim CD (2022) HMGB1 increases RAGE expression in vascular smooth muscle cells via ERK and p-38 MAPK-dependent pathways. Korean J Physiol Pharmacol 26:389-396. doi: 10.4196/kjpp.2022.26.5.389
    Pubmed KoreaMed CrossRef
  8. Jeon H, Jin Y, Myung CS, Heo KS (2021) Ginsenoside-Rg2 exerts anti-cancer effects through ROS-mediated AMPK activation associated mitochondrial damage and oxidation in MCF-7 cells. Arch Pharm Res 44:702-712. doi: 10.1007/s12272-021-01345-3
    Pubmed CrossRef
  9. Jin Y, Jeon H, Le Lam Nguyen T, Kim L, Heo KS (2023a) Human milk oligosaccharides 3'-sialyllactose and 6'-sialyllactose attenuate LPS-induced lung injury by inhibiting STAT1 and NF-κB signaling pathways. Arch Pharm Res 46:897-906. doi: 10.1007/s12272-023-01470-1
    Pubmed CrossRef
  10. Jin Y, Nguyen TLL, Myung CS, Heo KS (2022) Ginsenoside Rh1 protects human endothelial cells against lipopolysaccharide-induced inflammatory injury through inhibiting TLR2/4-mediated STAT3, NF-κB, and ER stress signaling pathways. Life Sci 309:120973. doi: 10.1016/j.lfs.2022.120973
    Pubmed CrossRef
  11. Jin Y, Tangchang W, Kwon OS, Lee JY, Heo KS, Son HY (2023b) Ginsenoside Rh1 ameliorates the asthma and allergic inflammation via inhibiting Akt, MAPK, and NF-κB signaling pathways in vitro and in vivo. Life Sci 321:121607. doi: 10.1016/j.lfs.2023.121607
    Pubmed CrossRef
  12. Li J, Qin Y, Chen Y, Zhao P, Liu X, Dong H, Zheng W, Feng S, Mao X, Li C (2020) Mechanisms of the lipopolysaccharide-induced inflammatory response in alveolar epithelial cell/macrophage co-culture. Exp Ther Med 20:76. doi: 10.3892/etm.2020.9204
    Pubmed KoreaMed CrossRef
  13. Luyendyk JP, Schabbauer GA, Tencati M, Holscher T, Pawlinski R, Mackman N (2008) Genetic analysis of the role of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine and tissue factor gene expression in monocytes/macrophages. J Immunol 180:4218-4226. doi: 10.4049/jimmunol.180.6.4218
    Pubmed KoreaMed CrossRef
  14. Meng RY, Li CS, Hu D, Kwon SG, Jin H, Chai OH, Lee JS, Kim SM (2023) Inhibition of the interaction between Hippo/YAP and Akt signaling with ursolic acid and 3'3-diindolylmethane suppresses esophageal cancer tumorigenesis. Korean J Physiol Pharmacol 27:493-511. doi: 10.4196/kjpp.2023.27.5.493
    Pubmed KoreaMed CrossRef
  15. Nguyen DV, Jin Y, Nguyen TLL, Kim L, Heo KS (2024) 3'-Sialyllactose protects against LPS-induced endothelial dysfunction by inhibiting superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis. Life Sci 338:122410. doi: 10.1016/j.lfs.2023.122410
    Pubmed CrossRef
  16. Park JM, Park JE, Park JS, Leem YH, Kim DY, Hyun JW, Kim HS (2024) Anti-inflammatory and antioxidant mechanisms of coniferaldehyde in lipopolysaccharide-induced neuroinflammation: involvement of AMPK/Nrf2 and TAK1/MAPK/NF-κB signaling pathways. Eur J Pharmacol 979:176850. doi: 10.1016/j.ejphar.2024.176850
    Pubmed CrossRef
  17. Park JS, Park MY, Cho YJ, Lee JH, Yoo CG, Lee CT, Lee SM (2016) Anti-inflammatory effect of erdosteine in lipopolysaccharide-stimulated RAW 264.7 cells. Inflammation 39:1573-1581. doi: 10.1007/s10753-016-0393-4
    Pubmed CrossRef
  18. Van Nguyen D, Nguyen TLL, Jin Y, Kim L, Myung CS, Heo KS (2022) 6'-Sialylactose abolished lipopolysaccharide-induced inflammation and hyper-permeability in endothelial cells. Arch Pharm Res 45:836-848. doi: 10.1007/s12272-022-01415-0
    Pubmed CrossRef
  19. Wang Y, Liu H, Zhao J (2020) Macrophage polarization induced by probiotic bacteria: a concise review. Probiotics Antimicrob Proteins 12:798-808. doi: 10.1007/s12602-019-09612-y
    Pubmed CrossRef

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