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ORIGINAL ARTICLE

Red ginseng polysaccharide attenuates sepsis-induced acute lung injury via suppressing oxidative stress-mediated ER stress through activation of Nrf2/AMPK pathways

Qiuhong Liu1, Jinqiao Zhou2*, Jingrui Yang3, Xiaoqin Zhang3, Yuna Li1, Li Zhang1

1Department of Respiratory, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China;

2Department of Neurosurgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China;

3School of Laboratory Medicine, Xinxiang Medical University, Zhengzhou, Henan, China

Abstract

The present study elucidates the protective efficacy of red ginseng polysaccharide (RGP) against septic-associated acute lung injury (ALI), a critical condition exacerbated by oxidative stress and endoplasmic reticulum (ER) stress. Utilizing lipopolysaccharide (LPS)-induced mouse and cellular models, we assessed lung pathology through histological staining, injury scoring, and lung wet-to-dry ratios along with inflammatory cytokine levels. Cell viability and apoptosis were quantified using cell counting kit-8 and TUNEL assays, respectively, while fluorescence labeling was employed to gauge reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Our findings indicated that RGP markedly attenuated LPS-induced lung damage, inflammation, oxidative injury, ER stress, and apoptotic processes. Mechanistic insights revealed that RGP exerted its protective effects by activating the nuclear factor erythroid-2-related factor 2 (Nrf2)/adenosine monophosphate-activated protein kinase (AMPK) pathway in LPS-stressed lung tissues. Inhibition of Nrf2 or AMPK abrogated RGP’s benefits on apoptosis, ROS production, and MMP preservation in murine lung type II epithelial (MLE-12) cells, underscoring the Nrf2/AMPK-mediated mechanism of RGP’s action against LPS-induced ALI. These results underscore the potential of RGP as a novel therapeutic agent for sepsis-associated ALI.

Key words: acute lung injury, endoplasmic reticulum stress, lipopolysaccharide, oxidative stress, red ginseng polysaccharide

*Corresponding Author: Jinqiao Zhou, Department of Neurosurgery, the First Affiliated Hospital of Zhengzhou University, No. 1, Jianshe East Road, Erqi District, Henan, 450000, China. Email: [email protected]

Academic Editor: Prof. Tommaso Beccari – Università degli Studi di Perugia, Italy

Received: 21 August 2024; Accepted: 13 September 2024; Published: 1 January 2025

DOI: 10.15586/ijfs.v37i1.2760

© 2025 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

Sepsis, a condition characterized by dysregulation of host defense mechanisms, is intricately associated with organ dysfunction (Liu et al., 2022a). The lungs, as the most sensitive and critical organs in sepsis, frequently succumb to acute lung injury (ALI) (Hwang et al., 2019). Sepsis-induced ALI is typified by inflammatory cell infiltration, alveolar epithelium damage, pulmonary edema, and compromised gas exchange (Zhou and Liao, 2021). Emerging evidence underscores the pivotal role of excessive oxidative stress and endoplasmic reticulum (ER) stress in the pathogenesis of ALI (Guo et al., 2021; Wang et al., 2022a). Reactive oxygen species (ROS) are known to elicit oxidative stress within lung epithelial cells, thereby inducing ER stress and subsequent mitochondrial damage (Huang et al., 2020; Yang et al., 2020). Consequently, targeting the inhibition of inflammation and/or oxidative stress-mediated ER stress emerges as a promising avenue for the prevention and treatment of sepsis-induced ALI, offering new therapeutic prospects in the management of this critical condition.

An extensive body of literature has concluded that infections caused by Gram-negative bacteria are a primary contributor to the development of ALI, with lipopolysaccharide (LPS), a key component of the bacterial outer membrane, playing a pivotal role in initiating lung injury and inflammatory responses (Fukatsu et al., 2022; Li et al., 2021). Exposure to LPS results in a vigorous activation of macrophages and the infiltration of inflammatory cells, notably neutrophils, into the lung (Hong et al., 2022; Wang et al., 2022b). This influx of neutrophils leads to the uncontrolled release of inflammatory cytokines and the generation of ROS, thereby precipitating oxidative stress (Dhlamini et al., 2022; Zhou et al., 2021). It is particularly intriguing that the activation or inhibition of multiple signaling pathways is closely associated with the regulation of inflammatory responses and oxidative stress in the context of LPS-induced ALI. The transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) is identified as a critical antioxidant regulator, and its activation is shown to prevent oxidative stress-induced cellular or tissue damage (He et al., 2020). Adenosine monophosphate-activated protein kinase (AMPK), a cellular energy sensor present in various eukaryotes, also plays a role in alleviating a range of inflammatory and oxidative stress-induced diseases, including ALI (Sang et al., 2022; Trefts and Shaw, 2021). Evidence suggests that activated Nrf2 suppresses the overproduction of mitochondrial ROS and subsequently activates the AMPK signaling pathway (Chu et al., 2022). Collectively, these findings underscore the potential of the Nrf2/AMPK signaling pathway to serve as a therapeutic target for attenuating ALI through its anti-inflammatory and antioxidant properties.

Medicinal herbs and their derivatives are increasingly recognized for their critical role in preventing diseases, especially those resulting from inflammation and oxidative stress, such as ALI (Yang et al., 2021; Yimam et al., 2023). Among these, red ginseng (RG), owing to its ‘warming effect’, has seen a rise in its incorporation into herbal formulas, including the famous Shenfu injection (Xu et al., 2020; Zhang and Li, 2023). Previously, more research was conducted on ginsenosides and less on red ginseng polysaccharide (RGP), which is one of the active ingredients of RG (Chen et al., 2019). The pharmacological potential of RGP, particularly its antitumor and antioxidant properties, has recently garnered attention (Wang et al., 2023; Zhai et al., 2022). Notably, RGP is shown to activate the Nrf2 pathway, enhancing antioxidant defenses and mitigating inflammatory responses, as evidenced by its efficacy in improving myocardial infarction outcomes (Lian et al., 2022). However, the protective effects of RGP against septic ALI, both in vitro and in vivo, remain largely uncharted.

In this study, we aimed to elucidate the protective mechanisms of RGP on LPS-induced ALI, with a particular focus on its modulation of the Nrf2/AMPK signaling axis. Our findings provide novel insights into the therapeutic potential of RGP, offering a promising avenue for the development of innovative treatments targeting sepsis-associated ALI.

Materials and Methods

Construction of ALI model and animal grouping

Ethical approval for animal experiments was obtained from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (approval No.: 2022-03-B111). Male wild-type C57BL/6 mice, aged 6–8 weeks were randomly divided into five groups, namely, Control, LPS, LPS+dextromethorphan (DXM), LPS+RGP (200 mg/kg) and LPS+RGP (400 mg/kg). In brief, phosphate-buffered saline (PBS) solution was used to treat control animals. Mice in the LPS group were given 10 mg/kg LPS intraperitoneally (i.p.). Then, control drug dextromethorphan (DXM; D4902; Sigma-Aldrich, Shanghai, China) at a dose of 5 mg/kg or RGP (30% purity; Fufeng Sinoute Biotech Co. Ltd, Shanxi, China) at a dose of 200 mg/kg or 400 mg/kg (Lian et al., 2022) was administrated intragastrically 2 h before, and 0 h and 6 h after LPS injection. Each group’s 48-h survival rate was determined (10 mice per group). At 12 h after LPS challenge, the plasma and lungs were collected from the surviving mice in each group (randomly selected, 6 mice in each group). The lung tissue sections were prepared for histological analysis, fluorescence staining, Western blotting analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) apoptosis assay. The plasma samples were applied for enzyme-linked immunosorbent serological assay (ELISA).

Cell treatment

ALI cellular model was constructed by continuous stimulation of murine lung type II epithelial (MLE-12) cell line (CRL-2110™; ATCC, Manassas, VA, USA), a mouse lung epithelial cell line, for 24 h with LPS (L4516; Sigma-Aldrich) at a final concentration of 500 ng/mL (Liu et al., 2022b; Xiao et al., 2020). In certain groups, MLE-12 cells were pretreated with Nrf2 inhibitor ML385 (20 μM; SML1833; Sigma-Aldrich) for 2 h or AMPK inhibitor Compound C (10 μM; 171260; Sigma-Aldrich) for 1 h, and then co-incubated with 40-μg/mL RGP for 24 h prior to LPS stimulation.

Histological analysis

Lung tissues were fixed in 10% formalin for 24 h, embedded in paraffin, and cut into 5-μm thick sections for hematoxylin and eosin (H&E) staining. Histopathological changes were observed under a light microscope (Olympus Corporation, Tokyo, Japan), and the severity of lung injury was assessed using a semi-quantitative scale (He et al., 2022; Li et al., 2015).

Analysis of wet-to-dry ratio of lungs

The collected lung tissues were blotted and weighed to obtain wet weight and then dried in an oven at 80°C for 48 h to obtain dry weight. The wet-to-dry ratio of the lungs was calculated to assess the degree of pulmonary edema.

ELISA assays

Using mouse ELISA kits (Beyotime Biotechnology, Shanghai, China), the protein levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were determined in plasma samples, according to the manufacturer’s instructions.

Measurement of reactive oxygen species generation

The production of ROS in tissues and cells was measured with the fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCF-DA) using ROS assay kit (S0033S; Beyotime Biotechnology). Lung tissue homogenate supernatants and rinsed MLE-12 cells were incubated with 10-μM DCF-DA for 20 min at 37°C in the dark. Samples were photographed using a fluorescent microscope (Zeiss, New York, NY, USA).

Quantitative real time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from tissues using the TRIzolTM reagent (Invitrogen, Waltham, MA, USA). PrimeScriptTM RealTime (RT) reagent kit (Takara, Japan) was used to create complementary DNA (cDNA). qRT-PCR was performed on the Light Cycler 480 II RT-PCR system (Roche, Basel, Switzerland) using SYBR Premix Ex Taq II (TaKaRa, Shiga, Japan). The 2–ΔΔCt method was used to calculate relative RNA abundance. The expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was taken as a negative control. The following primers were used in this study: Bcl-2-associated X-protein (Bax): 5’-AGACAGGGGCCTTTTTGCTAC-3’ (forward), 5’-AATTCGCCGGAGACACTCG-3’ (reverse); B-cell lymphoma 2 (Bcl-2): 5’-GTCGCTACCGTCGTGACTTC-3’ (forward), 5’-CAGACATGCACCTACCCAGC-3’ (reverse); and GAPDH: 5’-AGGTCGGTGTGAACGGATTTG-3’ (forward), 5’-GGGGTCGTTGATGGCAACA-3’ (reverse).

Western blot analysis

Lung tissues were homogenized and lysed using radioimmunoprecipitation assay (RIPA; Thermo Scientific, Shanghai, China), and the lysate was centrifuged at 12,000 ×g for 5 min at 4°C. The supernatant was stored at –20°C. The contents of target proteins were determined according to the bicinchoninic acid assay (BCA), and separated by electrophoresis. After transferring to nitrocellulose membranes (Millipore, Bedford, MA, USA), proteins were hybridized with primary antibodies overnight at 4°C and secondary antibodies (1:2,000 dilution; #7074; Cell Signaling Technology, Boston, MA, USA) for 2 h at room temperature. Immunodetection was performed using the enhanced chemiluminescence reagent (Beckman Coulter, Brea, CA, USA).

TUNEL assays

Apoptosis in lung tissues was analyzed by TUNEL staining using One Step TUNEL apoptosis assay kit (C1086; Beyotime Biotechnology). Images were obtained using a microscope.

Cell viability assay

Cell viability was determined using Cell Counting Kit-8 assay (CCK-8) (C0037; Beyotime Biotechnology). Following different treatments, MLE-12 cells with a molecular mass of 100 µL were incubated in 96-well plates at a density of 2,000/well. Absorbance was detected at the wavelength of 450 nm after the addition of 10-μL CCK-8 solution.

Flow cytometry (FCM)

The Annexin V-FITC apoptosis detection kit (C1062S; Beyotime Biotechnology) was used to evaluate apoptotic cells according to the manufacturer’s instructions. MLE-12 cells were washed with PBS, resuspended in 1× binding buffer, and stained with 5-µL Annexin V-FITC for 10 min. The cells were analyzed using a flow cytometer (BD Biosciences, San Diego, CA, USA) after counterstained with 10-µL propidium iodide (PI) for 10 min.

Detection of mitochondrial membrane potential (MMP)

The MMP was detected using MMP assay kit with JC-1 staining working solution (C2006; Beyotime Biotechnology). Briefly, MLE-12 cells (1×105 cells resuspended in 500-μL cell culture medium) were incubated for 20 min at 37°C with 0.5-mL JC-1. After centrifugation at 600 ×g for 3 min at 4°C, the supernatant was discarded. After washing twice with JC-1 staining buffer, cells were resuspended in JC-1 staining buffer and observed with fluorescence microscopy.

Statistical analysis

Data were presented as mean ± standard deviation (SD). GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) was used for data analysis using ANOVA, followed by Tukey’s test; p < 0.05 was considered as statistically significant.

Results

Red ginseng polysaccharide ameliorates LPS-induced lung injury and inflammation in mice

To ascertain the protective efficacy of RGP against sepsis-associated ALI, we established a mouse model of LPS-induced ALI. Initially, we monitored the survival rate over 48 h post-LPS challenge, revealing that treatment with 400-mg/kg RGP conferred a protective effect on mouse survival against LPS-induced effects, akin to the positive control drug dextromethorphan (DXM; Figure 1A). Histopathological alterations in each group of mice were further examined via H&E staining. Inflammatory cell infiltration, interstitial edema, alveolar wall thickening, and tissue damage were commonly observed in LPS-induced mice; both DXM intervention and 400-mg/kg RGP markedly alleviated the pulmonary injury induced by LPS (Figure 1B), which was also quantified using lung injury scores (Figure 1C). Subsequently, we assessed the impact of RGP on LPS-induced pulmonary edema. As depicted in Figure 1D, the lung wet-to-dry weight ratio was significantly elevated in the LPS group, compared to the control group, an increase that was mitigated by treatment with either DXM or RGP. To investigate the effect of RGP on LPS-induced inflammatory responses, we measured the expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in plasma via ELISA. As illustrated in Figure 1E, LPS administration resulted in substantial elevations in the plasma levels of these cytokines, which were reversed by either DXM or RGP. The anti-inflammatory action of RGP underscored its potential as a functional food ingredient, offering promise in mitigating inflammation-related diseases, including sepsis-induced ALI.

Figure 1. RGP ameliorates LPS-induced lung injury and inflammation in mice. LPS (10 mg/kg) was intraperitoneally administrated to induce ALI; 2 h before and 0 h and 6 h after LPS challenge, DXM (5 mg/kg) or RGP (200 mg/kg or 400 mg/kg) was given to each administration group. (A) Survival curves of five groups (n = 10). (B) H & E staining of lung tissues from each group of mice (scale bar: 200 µm; n = 6). (C) The lung injury score was calculated by a semi-quantitative scale in different groups (n = 6). (D) The lung wet-to-dry weight ratio (n = 6). (E) Levels of TNF-α, IL-1β, and IL-6 in plasma were determined by ELISA (n = 6). Results are presented as mean ± SD obtained from six independent experiments; *p < 0.05; ***p < 0.001.

Red ginseng polysaccharide suppresses oxidative stress and ER stress in LPS-induced mouse lung tissues

The assessment of DCF-DA fluorescence intensity revealed that pretreatment with DXM or RGP markedly attenuated the LPS-induced overproduction of ROS, as evidenced in Figure 2A. LPS administration led to the up-regulation of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), as well as the activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) in mouse lung tissues, accompanied by increased phosphorylation levels of eukaryotic initiation factor 2 alpha (eIF2α), PERK, and inositol requiring protein-1α (IRE1α). Notably, these LPS-induced alterations were significantly mitigated by pretreatment with DXM or RGP, as depicted in Figures 2B and 2C. Collectively, these findings suggested that RGP has the potential to prevent oxidative stress and ER stress, thereby alleviating LPS-induced ALI.

Figure 2. RGP suppresses LPS-induced oxidative stress and ER stress in mouse lung tissues. (A) The fluorescence images of ROS production stained by DCF-DA (scale bar: 200 µm). (B) The protein levels of GRP78, CHOP, p-eIF2α, and total eIF2α were determined in lung tissues of different groups. (C) The phosphorylation and total levels of PERK and IRE1α were determined in lung tissues of different groups. Results are presented as mean ± SD (n = 6) obtained from six independent experiments; **p < 0.01; ***p < 0.001.

Red ginseng polysaccharide inhibits LPS-induced lung cell apoptosis in LPS-challenged mice

The level of apoptosis was subsequently evaluated using TUNEL labeling to delineate the impact of RGP on LPS-induced apoptosis within the pulmonary tissues of mice. Quantitative analysis of apoptotic cells indicated a notably higher count in the LPS-treated group whereas a pronounced decrease in apoptotic cells was observed following treatment with DXM or RGP (Figure 3A). The results of qRT-PCR and Western blot analyses demonstrated that both DXM and RGP effectively mitigated the LPS-induced elevation of Bax messenger RNA (mRNA) and protein levels, along with the corresponding decrease in Bcl-2 expression (Figures 3B and 3C). Collectively, these results suggested that RGP intervention could efficaciously inhibit LPS-induced apoptosis in lung tissue cells, underscoring its potential therapeutic implications in mitigating lung injury.

Figure 3. RGP inhibits LPS-induced lung cell apoptosis in LPS-challenged mice. (A) The apoptosis of lung tissue cells was detected by TUNEL assay (scale bar: 200 µm). (B) The mRNA expressions of Bax and Bcl-2 in lung tissues of different groups were determined by qRT-PCR. (C) The protein levels of Bax and Bcl-2 in lung tissues of different groups were determined by Western blot analysis. Results are presented as mean ± SD (n = 6) obtained from six independent experiments; ***p < 0.001.

Red ginseng polysaccharide activates Nrf2/AMPK signaling pathway in LPS-induced ALI mice

Further analysis was conducted to evaluate the activities of Nrf2 and AMPK, molecular pathways known to mitigate lung injury (Qiu et al., 2018; Yang et al., 2022) using Western blot analysis (Figures 4A and 4B). Our findings indicated that LPS exposure resulted in a slight, albeit nonsignificant, increase in Nrf2 expression and a significant decrease in AMPK phosphorylation within lung tissues. However, pretreatment with DXM or RGP led to the up-regulation of Nrf2 and a marked enhancement of AMPK phosphorylation in LPS-challenged mice. These results suggested that both DXM and RGP are capable of effectively activating the Nrf2/AMPK signaling pathway, which plays a pivotal role in conferring protection against LPS-induced ALI.

Figure 4. RGP activates Nrf2/AMPK signaling pathway in LPS-induced ALI mice. (A) The protein levels of Nrf2 were determined in lung tissues of different groups. (B) The protein levels of p-AMPK and total AMPK were determined in lung tissues of different groups. Results are presented as mean ± SD (n = 6) obtained from six independent experiments; ***p < 0.001.

Red ginseng polysaccharide ameliorates LPS-induced injury in MLE-12 cells by activating the Nrf2/AMPK pathway

Based on the above in vivo findings, we proceeded to explore the potential of RGP to mitigate LPS-induced cellular injury in MLE-12 cells. CCK-8 assays revealed that RGP at concentrations of 10, 20, and 40 μg/mL exhibited no cytotoxic effects, with significant cytotoxicity only observed at 80 μg/mL (Figure 5A). Following LPS stimulation, cell viability was notably diminished; however, treatment with RGP at 20 μg/mL or 40 μg/mL significantly attenuated the LPS-induced decline in cell viability (Figure 5B). Consequently, we selected a concentration of 40 μg/mL RGP for subsequent experiments. To elucidate the involvement of the Nrf2/AMPK signaling pathway in RGP’s mechanism of action, cells were co-treated with ML385 (an inhibitor of Nrf2) or Compound C (an inhibitor of AMPK). In alignment with the in vivo findings, RGP demonstrated inhibition of LPS-induced cell death in MLE-12 cells whereas co-administration with ML385 or Compound C notably counteracted the anti-apoptotic effects of RGP (Figure 5C). Mitochondria are widely considered the primary site of ROS formation, and MMP is an important parameter reflecting mitochondrial function (Correia-Álvarez et al., 2020; Rehfeldt et al., 2021). In this study, LPS markedly induced ROS production and a reduction in MMP within MLE-12 cells, indicative of oxidative stress injury and mitochondrial dysfunction. On the contrary, RGP protected MLE-12 cells from LPS-induced ROS overproduction (Figure 5D) and MMP reduction (Figures 5E and 5F), and its protective effects were restored by the inhibitors of Nrf2 and AMPK.

Figure 5. RGP ameliorates LPS-induced injury in MLE-12 cells by activating the Nrf2/AMPK pathway. MLE-12 cells were treated with different concentrations of RGP (0, 10, 20, 40, and 80 μg/mL), and the cell viability was evaluated by CCK-8 assay. (A) MLE-12 cells were treated with different concentrations of RGP (0, 10, 20, and 40 μg/mL) in the presence of LPS (500 ng/mL) for 24 h, and the cell viability was evaluated by CCK-8 assay. (B) MLE-12 cells were treated with RGP (40 μg/mL) or co-treated with ML385 or Compound C in the presence of LPS (500 ng/mL) for 24 h; then cells were collected and used for apoptosis, oxidative stress, and ER stress detection. (C) Apoptosis rate was analyzed by FCM. (D) The intracellular ROS production was assessed by DCF-DA fluorescence staining. (E) JC-1 staining results: green represents MMP monomers and red represents MMP aggregate. (F) Representative histograms depicting MMP levels. Results are presented as mean ± SD (n = 3) obtained from three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001.

Discussion

Sepsis-induced ALI is a complex hypoxic condition characterized by multifaceted pathological mechanisms, including apoptosis, inflammation, oxidative stress, and ER stress (Chen et al., 2018; Qian et al., 2020; Zeng et al., 2017). RGP, a principal bioactive component of RG, has demonstrated tumor-suppressive (Zhai et al., 2022) and antioxidant properties (Kang et al., 2023; Lian et al., 2022). The current study identified RGP as a promising therapeutic agent for alleviating sepsis-associated ALI, suggesting that its protective effects against LPS-induced ALI may be mediated through the activation of the Nrf2/AMPK signaling pathway.

Extensive evidence is observed that LPS could act as a cytotoxic agent capable of inducing ALI both in vivo and in vitro, leading to pathological damage, apoptosis, and inflammation (Li et al., 2023a, 2023b). In this study, RGP exhibited therapeutic effects comparable to the positive control drug DXM in LPS-induced ALI mice. This was evidenced by enhanced survival proportions, diminished pulmonary pathological lesions, lower lung injury scores and wet-to-dry ratios, and reduced plasma pro-inflammatory cytokine levels. In addition, RGP demonstrated an inhibitory effect on apoptosis within the lung tissues of mice with sepsis-induced ALI.

Endoplasmic reticulum stress arises from the accumulation of unfolded or misfolded proteins, leading to a disruption in ER’s structural and functional equilibrium (Dandekar et al., 2015; Schwarz and Blower, 2016). This stress is a critical pathogenic mechanism in sepsis-associated ALI and holds potential as a biomarker and therapeutic target for sepsis (Ahmad et al., 2022; Zeng et al., 2023). In a mouse model of LPS-induced ALI, pretreatment with RGP ameliorated lung oxidative damage by reducing ROS production and mitigated ER stress by down-regulating ER stress markers GRP78 and CHOP (Arab et al., 2023) and decreasing phosphorylation levels of the unfolded protein response (UPR) sensors eIF2α, PERK, and IRE1α (Men et al., 2023). In LPS-stimulated MLE-12 cells, RGP exhibited anti-apoptotic effects and enhanced mitochondrial function, evidenced by decreased ROS release and increased MMP, suggesting a protective role against LPS-induced ALI through anti-apoptotic, anti-inflammatory, and anti-stress mechanisms. Further studies are warranted to fully elucidate this hypothesis.

To further elucidate the protective mechanism of RGP, we examined its influence on the Nrf2/AMPK pathway. Nrf2, a redox-sensitive transcriptional regulator, orchestrates the expression of antioxidant genes to maintain cellular redox balance and mitigate ER stress (Huang et al., 2020). AMPK plays a pivotal role in modulating energy metabolism and mitochondrial biogenesis in response to energy deficits (Sang et al., 2022; Tang et al., 2022).

Accumulating evidence suggests that various herbal bioactive components confer protection against ALI by activating the Nrf2/AMPK signaling pathway. For instance, Lv et al. (2017) reported that xanthohumol effectively protected LPS-induced ALI against oxidative stress and inflammation damage, which are largely dependent upon up-regulation of the Nrf2 pathway via activation of AMPK/GSK3β. Lycium barbarum polysaccharide (LBP) ameliorated lung inflammation and edema in a mouse model of hyperoxia-induced ALI through an Nrf2/AMPK-dependent mechanism (Zheng et al., 2019). Sophoricoside exhibited anti-inflammatory effects in LPS-induced ALI mice and macrophages via Nrf2/AMPK signaling (Wu et al., 2021). Kinsenoside prevented LPS-induced ALI by activating Nrf2/AMPK and regulating mitochondrial function (Yang et al., 2022). In our study, RGP up-regulated Nrf2 expression and induced AMPK phosphorylation in LPS-induced ALI mice. Furthermore, the inhibition of Nrf2/AMPK signaling in MLE-12 cells with ML385 and compound C partially negated RGP’s anti-apoptotic, antioxidant, and anti-ER stress effects in LPS-treated cells.

The current study, however, has limitations. First, the genomic differences between mice and humans (Lewis et al., 2016) in response to sepsis necessitate validation of these findings in murine models of cecal ligation and puncture (CLP). In addition, the complex pathophysiology of ALI involves multiple signaling pathways, warranting further research to achieve a comprehensive understanding of the underlying mechanisms. Further studies are required to delineate the regulatory role of RGP on the Nrf2/AMPK signaling pathway.

Conclusions

The present study demonstrates that RGP, a key component of RG, protects against sepsis-induced ALI by modulating the Nrf2/AMPK pathway. These findings highlight the potential of RGP as a functional food ingredient with therapeutic applications in managing sepsis-related complications, thereby contributing to the growing field of nutraceuticals to enhance immune and respiratory health through diet.

Availability of Data and Materials

All data generated or analyzed in this study are included in this published article. The datasets used and/or analyzed in the present study are available from the corresponding author on reasonable request.

Author Contributions

Qiuhong Liu and Jinqiao Zhou designed the study, completed the experiment, and supervised data collection. Jingrui Yang analyzed and interpreted the data. Xiaoqin Zhang, Yuna Li, and Li Zhang prepared the manuscript for publication and reviewed draft of the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

None.

Funding and Competing Interests

This work was supported by the Henan Province Medical Science and Technology Key Project, jointly constructed by the Provincial and Ministerial Departments (Grant No. SBGJ202102172). The authors stated that there was no conflict of interest to disclose.

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