Protective Effects of Atractylodis lancea Rhizoma on Lipopolysaccharide-Induced Acute Lung Injury via TLR4/NF-κB and Keap1/Nrf2 Signaling Pathways In Vitro and In Vivo

Acute lung injury (ALI) is a syndrome caused by an excessive inflammatory response characterized by intractable hypoxemia both inside and outside the lung, for which effective therapeutic drugs are lacking. Atractylodis rhizoma, a traditional Chinese medicine, has excellent anti-inflammatory and antiviral properties in addition to protecting the integrity of the cellular barrier. However, few studies of Atractylodis rhizoma for the treatment of ALI have been published, and its mechanism of action remains unclear. In the present study, the chemical composition of the ethanolic extract of Atractylodis rhizoma (EEAR) was initially clarified by high performance liquid chromatography (HPLC), after which it was studied in vivo using a lipopolysaccharide (LPS)-induced ALI rat model. Treatment with EEAR significantly reduced the lung wet/dry (W/D) ratio, neutrophil infiltration, and malondialdehyde (MDA) and myeloperoxidase (MPO) formation, and enhanced superoxide dismutase (SOD) and glutathione (GSH) depletion in rats with ALI, thereby improving lung barrier function and effectively reducing lung injury. In addition, EEAR significantly reduced histopathological changes, decreased the expression of inflammatory factors (such as tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), inducible nitric oxide synthase (INOS), and cyclooxygenase-2 (COX-2)), and inhibited the activation of the NF-κB signaling pathway, thus reducing inflammation. In addition, EEAR was found to also reduce oxidative stress in ALI by upregulating the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins heme oxygenase-1 (HO-1) and NADPH quinone acceptor oxidoreductase 1 (NQO-1). EEAR also reduced LPS-induced inflammatory factor expression in THP-1 cells in vitro by inhibition of the NF-κB signaling pathway, and reduced damage from lipopolysaccharide (LPS)-induced oxidative stress in THP-1 cells by promoting the expression of Nrf2 and its downstream targets HO-1 and NQO-1, the molecular mechanism of which was consistent with in vivo observations. Therefore, we conclude that EEAR attenuates oxidative stress and inflammatory responses via TLR4/NF-κB and Keap1/Nrf2 signaling pathways to alleviate LPS-induced ALI, suggesting that Atractylodis rhizoma is a potential drug candidate for the treatment of ALI.


Introduction
Acute lung injury (ALI) is a clinical syndrome characterized by intractable hypoxemia due to various intra-and extra-pulmonary pathogenic factors, causing acute diffuse lung injury leading to acute respiratory failure, the pathogenesis of which has not yet been fully elucidated. The pathology of ALI is characterized by alveolar inflammatory exudate and increased pulmonary vascular permeability causing pulmonary edema, leading to impaired

Effects of EEAR on Wet-Dry (W/D) Specific Gravity and the Inflammatory R Lungs of Rats with ALI
Since an inflammatory response is involved in the development and p ALI, inflammation in the lungs of rats with ALI was examined. As displayed the W/D ratio of the lung tissue was significantly higher in the model grou control group (p < 0.01). Compared with the model group, the W/D ratio of the high-dose EEAR (EEARH) and dexamethasone (Dex) groups was signi (p < 0.05). Compared with the control group, the levels of TNF-α, IL-1β, a bronchoalveolar lavage fluid (BALF) of rats in the model group were signif (p < 0.01), and were significantly lower (p < 0.05) in the EEARH and Dex gro EEAR and Dex treatments ( Figure 2C-E). In addition, measurement by R flammatory factor mRNA expression in the lung tissue of rats with ALI dem the mRNA expression levels of TNF-α, IL-1β, IL-6, cyclooxygenase-2 (COXible nitric oxide synthase (INOS) were significantly higher in the model gro control group (p < 0.01), while the mRNA expression levels of TNF-α, IL-1β and INOS were significantly lower in the EEARH and Dex groups after the a of the drug treatments (p < 0.05) ( Figure 2F-J).

Effects of EEAR on Wet-Dry (W/D) Specific Gravity and the Inflammatory Response in the Lungs of Rats with ALI
Since an inflammatory response is involved in the development and progression of ALI, inflammation in the lungs of rats with ALI was examined. As displayed in Figure 2B, the W/D ratio of the lung tissue was significantly higher in the model group than in the control group (p < 0.01). Compared with the model group, the W/D ratio of lung tissue in the high-dose EEAR (EEARH) and dexamethasone (Dex) groups was significantly lower (p < 0.05). Compared with the control group, the levels of TNF-α, IL-1β, and IL-6 in the bronchoalveolar lavage fluid (BALF) of rats in the model group were significantly higher (p < 0.01), and were significantly lower (p < 0.05) in the EEARH and Dex groups following EEAR and Dex treatments ( Figure 2C-E). In addition, measurement by RT-qPCR of inflammatory factor mRNA expression in the lung tissue of rats with ALI demonstrated that the mRNA expression levels of TNF-α, IL-1β, IL-6, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (INOS) were significantly higher in the model group than in the control group (p < 0.01), while the mRNA expression levels of TNF-α, IL-1β, IL-6, COX-2, and INOS were significantly lower in the EEARH and Dex groups after the administration of the drug treatments (p < 0.05) ( Figure 2F-J).

Effects of EEAR on Neutrophils and Oxidative Stress Levels in the Lungs of Rats with ALI
ALI represents a pathological change due to pulmonary and systemic inflammatory responses. It has been demonstrated [26][27][28] that the production of lung inflammation in ALI is associated with excessive activation and aggregation of neutrophils in the lungs. Furthermore, excessive aggregation and activation of neutrophils can lead to the release of large quantities of reactive oxygen species (ROS) [29,30] which can further activate neutrophils and cause oxidative cellular damage, which aggravates the lung injury. Therefore, we examined neutrophil and oxidative stress levels in the lungs of rats with ALI. The results indicated that the level of myeloperoxidase (MPO) was significantly higher in the model group compared with the control group (p < 0.001), with a significant decrease (p < 0.05) in the EEARH and Dex groups after the administration of the respective drug treatments ( Figure 3A). In terms of oxidative stress, the expression of malondialdehyde (MDA) was significantly higher, and the expression of superoxide dismutase (SOD) and glutathione (GSH) significantly lower, in the model group compared with the control group (p < 0.05), with a significant recovery in the EEARH and the Dex groups following the administration of the respective drug treatments (p < 0.05) ( Figure 3B-D). In addition, immunohistochemistry demonstrated that the numbers of neutrophils and the concentrations of their expression product, MPO, were significantly elevated in the model group, with both decreasing significantly after administration of the drug treatments ( Figure 3E,F).

Effects of EEAR on Neutrophils and Oxidative Stress Levels in the Lungs of Rats with ALI
ALI represents a pathological change due to pulmonary and systemic inflammatory responses. It has been demonstrated [26][27][28] that the production of lung inflammation in ALI is associated with excessive activation and aggregation of neutrophils in the lungs. Furthermore, excessive aggregation and activation of neutrophils can lead to the release of large quantities of reactive oxygen species (ROS) [29,30] which can further activate neutrophils and cause oxidative cellular damage, which aggravates the lung injury. Therefore, we examined neutrophil and oxidative stress levels in the lungs of rats with ALI. The results indicated that the level of myeloperoxidase (MPO) was significantly higher in the model group compared with the control group (p < 0.001), with a significant decrease (p < 0.05) in the EEARH and Dex groups after the administration of the respective drug treatments ( Figure 3A). In terms of oxidative stress, the expression of malondialdehyde (MDA) was significantly higher, and the expression of superoxide dismutase (SOD) and glutathione (GSH) significantly lower, in the model group compared with the control group (p < 0.05), with a significant recovery in the EEARH and the Dex groups following the administration of the respective drug treatments (p < 0.05) ( Figure 3B-D). In addition, immunohistochemistry demonstrated that the numbers of neutrophils and the concentrations of their expression product, MPO, were significantly elevated in the model group, with both decreasing significantly after administration of the drug treatments ( Figure 3E,F).

Effect of EEAR on Lung Barrier Function in Rats with ALI
ALI is usually accompanied by damage to the alveolar-capillary barrier [31][32][33], and improving pulmonary epithelial barrier function is a principal means of treating ALI. Therefore, the barrier function of the lung epithelial cells was examined in the present study. The results demonstrated that the expression of the tight junction proteins zonula occluden-1 (ZO-1) and occludin were significantly reduced in the model group, with the lung barrier structure broken and significantly abnormal. There was significant recovery after the administration of EEAR, in which the effect of EEARH was superior to that of low-dose EEAR (EEARL) ( Figure 4A-D). This indicates that EEAR reduced barrier dysfunction in the rat lungs and alleviated ALI.

Effect of EEAR on Lung Barrier Function in Rats with ALI
ALI is usually accompanied by damage to the alveolar-capillary barrier [31][32][33], and improving pulmonary epithelial barrier function is a principal means of treating ALI. Therefore, the barrier function of the lung epithelial cells was examined in the present study. The results demonstrated that the expression of the tight junction proteins zonula occluden-1 (ZO-1) and occludin were significantly reduced in the model group, with the lung barrier structure broken and significantly abnormal. There was significant recovery after the administration of EEAR, in which the effect of EEARH was superior to that of lowdose EEAR (EEARL) ( Figure 4A-D). This indicates that EEAR reduced barrier dysfunction in the rat lungs and alleviated ALI.

Effects of EEAR on the TLR4/NF-κB Signaling Pathway
LPS is a major component of the cell wall of Gram-negative bacteria and an important causative agent of inflammation [34]. It has been shown [35,36] that LPS-induced ALI is associated with the NF-κB signaling pathway, and in ALI, LPS is specifically recognized by TLR4, which activates the NF-κB signaling pathway to induce the release of multiple inflammatory factors, including TNF-α, IL-1β, and IL-6. We observed a significant increase in the expression of TLR4, Myeloid differentiation factor 88 (Myd88), phospho-IκB alpha (p-IκBα), and phospho-p65 (p-p65) in the model group using Western blot analysis of ALI rat lung tissue. Each sample demonstrated a different extent of decrease in expression after the administration of the EEAR treatment, with the greatest effect observed in the EEARH samples ( Figure 5A-E).   by TLR4, which activates the NF-κB signaling pathway to induce the release of multiple inflammatory factors, including TNF-α, IL-1β, and IL-6. We observed a significant increase in the expression of TLR4, Myeloid differentiation factor 88 (Myd88), phospho-IκB alpha (p-IκBα), and phospho-p65 (p-p65) in the model group using Western blot analysis of ALI rat lung tissue. Each sample demonstrated a different extent of decrease in expression after the administration of the EEAR treatment, with the greatest effect observed in the EEARH samples ( Figure 5A-E).

Effects of EEAR on the Keap1/Nrf2 Signaling Pathway
Oxidative damage plays an important role in the development and pathology of ALI [37]. The relationship between antioxidants and ALI has received increasing attention over recent years, including study of the Keap1/Nrf2 signaling pathway. It has been shown [38,39] that this pathway is able to mitigate the extent of ALI by inhibiting the activation of NF-κB and affecting the release of inflammatory factors in vivo. In addition, the Keap1/Nrf2 signaling pathway, when activated during periods of oxidative stress, promotes the expression of the downstream protein HO-1, thereby alleviating the level of oxidative stress in the host [40]. By examining the lung tissue in rats with ALI, we found that the Nrf2 mRNA expression levels and those of its downstream targets, NQO-1 and HO-1, were significantly lower in the model group (p < 0.05), while EEAR promoted Nrf2 mRNA expression levels and those of its downstream targets (p < 0.05) ( Figure 6A-C). The immunofluorescence plots also indicated a significant decrease in Nrf2 expression in the model group and a significant increase following the administration of EEAR ( Figure 6D). Finally, the Keap1/Nrf2 signaling pathway was examined by Western blot analysis. The results indicate that the expression of Keap1 was significantly higher in the model group, while the expression of Nrf2, HO-1, and NQO-1 was significantly lower, an effect reversed by the administration of EEAR ( Figure 6E-I).

Effects of EEAR on the Keap1/Nrf2 Signaling Pathway
Oxidative damage plays an important role in the development and pathology of ALI [37]. The relationship between antioxidants and ALI has received increasing attention over recent years, including study of the Keap1/Nrf2 signaling pathway. It has been shown [38,39] that this pathway is able to mitigate the extent of ALI by inhibiting the activation of NF-κB and affecting the release of inflammatory factors in vivo. In addition, the Keap1/Nrf2 signaling pathway, when activated during periods of oxidative stress, promotes the expression of the downstream protein HO-1, thereby alleviating the level of oxidative stress in the host [40]. By examining the lung tissue in rats with ALI, we found that the Nrf2 mRNA expression levels and those of its downstream targets, NQO-1 and HO-1, were significantly lower in the model group (p < 0.05), while EEAR promoted Nrf2 mRNA expression levels and those of its downstream targets (p < 0.05) ( Figure 6A-C). The immunofluorescence plots also indicated a significant decrease in Nrf2 expression in the model group and a significant increase following the administration of EEAR ( Figure 6D). Finally, the Keap1/Nrf2 signaling pathway was examined by Western blot analysis. The results indicate that the expression of Keap1 was significantly higher in the model group, while the expression of Nrf2, HO-1, and NQO-1 was significantly lower, an effect reversed by the administration of EEAR ( Figure 6E-I).

Effect of EEAR on LPS-Induced Inflammatory Factor Expression in THP-1 Cells In Vitro
To further confirm the anti-inflammatory effect of EEAR, LPS-stimulated THP-1 cells were used to simulate an in vitro model of ALI. Firstly, the required EEAR dose was confirmed by CCK-8 assay, with 12.5 µg/mL utilized as the low-dose group and 25 µg/mL as the high-dose group ( Figure 7A). Secondly, inflammatory factor expression was measured in the cell supernatants. The expression of TNF-α, IL-1β, IL-6, and Monocyte chemotactic protein-1 (MCP-1) were each significantly higher in the model group, and significantly lower after treatment with EEAR ( Figure 7B

Effect of EEAR on LPS-Induced Inflammatory Factor Expression in THP-1 Cells In Vitro
To further confirm the anti-inflammatory effect of EEAR, LPS-stimulated THP-1 cells were used to simulate an in vitro model of ALI. Firstly, the required EEAR dose was confirmed by CCK-8 assay, with 12.5 μg/mL utilized as the low-dose group and 25 μg/mL as the high-dose group ( Figure 7A). Secondly, inflammatory factor expression was measured in the cell supernatants. The expression of TNF-α, IL-1β, IL-6, and Monocyte chemotactic protein-1 (MCP-1) were each significantly higher in the model group, and significantly lower after treatment with EEAR ( Figure 7B-E). In addition, these observations were reflected in the RT-qPCR results ( Figure 7F-H).

Effects of EEAR on the TLR4/NF-κB Signaling Pathway In Vitro
The effects of EEAR on the TLR4/NF-κB signaling pathway were confirmed using additional in vitro experiments. The results suggest that the expression of TLR4, Myd88, and p-IκBα were significantly higher in the model group than in the control group. It was also found that the expression of p65 in the nucleus was significantly increased. The expression of each protein was lower to varying degrees following treatment with EEAR ( Figure 8A-E). These results suggest that EEAR also inhibits activation of the TLR4/NF-κB signaling pathway in vitro, consistent with the results of the in vivo experiments.

Effects of EEAR on the TLR4/NF-κB Signaling Pathway In Vitro
The effects of EEAR on the TLR4/NF-κB signaling pathway were confirmed using additional in vitro experiments. The results suggest that the expression of TLR4, Myd88, and p-IκBα were significantly higher in the model group than in the control group. It was also found that the expression of p65 in the nucleus was significantly increased. The expression of each protein was lower to varying degrees following treatment with EEAR ( Figure 8A-E). These results suggest that EEAR also inhibits activation of the TLR4/NF-κB signaling pathway in vitro, consistent with the results of the in vivo experiments.

Effects of EEAR on the TLR4/NF-κB Signaling Pathway In Vitro
The effects of EEAR on the TLR4/NF-κB signaling pathway were confirmed using additional in vitro experiments. The results suggest that the expression of TLR4, Myd88, and p-IκBα were significantly higher in the model group than in the control group. It was also found that the expression of p65 in the nucleus was significantly increased. The expression of each protein was lower to varying degrees following treatment with EEAR ( Figure 8A-E). These results suggest that EEAR also inhibits activation of the TLR4/NF-κB signaling pathway in vitro, consistent with the results of the in vivo experiments.

Effects of EEAR on the Keap1/Nrf2 Signaling Pathway In Vitro
Whether EEAR was able to reduce LPS-induced oxidative stress in THP-1 cells via the Keap1/Nrf2 signaling pathway was further clarified in in vitro experiments. RT-qPCR results indicate that the expression of Nrf2, HO-1, and NQO-1 was significantly lower in the model group than in the control group (p < 0.05), but significantly higher following administration of EEAR (p < 0.01) ( Figure 9A-C). In addition, Western blot analysis demonstrated that in the model group, the expression of Keap1 protein was significantly higher, while the expression of the antioxidant protein Nrf2 and its downstream targets, NQO-1 and HO-1, were significantly lower. These observations were reversed after treatment with EEAR. This indicates that EEAR also alleviated LPS-induced oxidative stress in THP-1 cells in vitro via the Keap1/Nrf2 signaling pathway, consistent with the results of the in vivo experiments.

Effects of EEAR on the Keap1/Nrf2 Signaling Pathway In Vitro
Whether EEAR was able to reduce LPS-induced oxidative stress in THP-1 cells via the Keap1/Nrf2 signaling pathway was further clarified in in vitro experiments. RT-qPCR results indicate that the expression of Nrf2, HO-1, and NQO-1 was significantly lower in the model group than in the control group (p < 0.05), but significantly higher following administration of EEAR (p < 0.01) ( Figure 9A-C). In addition, Western blot analysis demonstrated that in the model group, the expression of Keap1 protein was significantly higher, while the expression of the antioxidant protein Nrf2 and its downstream targets, NQO-1 and HO-1, were significantly lower. These observations were reversed after treatment with EEAR. This indicates that EEAR also alleviated LPS-induced oxidative stress in THP-1 cells in vitro via the Keap1/Nrf2 signaling pathway, consistent with the results of the in vivo experiments.

Discussion
ALI is a syndrome characterized by an excessive inflammatory response causing dyspnea and hypoxemia due to damage in the alveolar epithelial cells and capillary endothelial cells caused by a variety of injury-causing factors, both inside and outside the lung. It is a critical condition causing high morbidity and mortality [41,42]. The pathogenesis of ALI can be divided into three principal phases: increased alveolar-capillary membrane permeability, imbalance in the inflammatory and anti-inflammatory responses, and changes in the pulmonary and bronchial circulation [43,44]. Currently, clinical treatments for ALI commonly include pharmacotherapy and mechanical ventilation [45][46][47]. Although ventilator treatment strategies can provide a degree of relief to patients, they are not effective in reducing the morbidity and mortality rate, while conventional Western

Discussion
ALI is a syndrome characterized by an excessive inflammatory response causing dyspnea and hypoxemia due to damage in the alveolar epithelial cells and capillary endothelial cells caused by a variety of injury-causing factors, both inside and outside the lung. It is a critical condition causing high morbidity and mortality [41,42]. The pathogenesis of ALI can be divided into three principal phases: increased alveolar-capillary membrane permeability, imbalance in the inflammatory and anti-inflammatory responses, and changes in the pulmonary and bronchial circulation [43,44]. Currently, clinical treatments for ALI commonly include pharmacotherapy and mechanical ventilation [45][46][47]. Although ventilator treatment strategies can provide a degree of relief to patients, they are not effective in reducing the morbidity and mortality rate, while conventional Western drugs such as dexamethasone can cause adverse side effects, including coagulation dysfunction, gastric ulcers, and osteoporosis. Chinese herbal medicines are generally considered clinically effective in preventing and treating ALI, with a number of experimental studies of Chinese herbal medicines demonstrating beneficial therapeutic effects. This provides the rationale for researching and developing other herbal medicines for the prevention and treatment of ALI.
Atractylodis rhizoma is a traditional Chinese medicine which has previously been shown [48] to exhibit excellent anti-inflammatory properties and to provide protection of epithelial cell barrier function, both aspects lacking in the pathogenesis of acute lung injury. Therefore, one could speculate that Atractylodis rhizoma may represent a potential candidate for the treatment of ALI.
LPS is a pathogenic endotoxin of the outer membrane of Gram-negative bacteria and is widely used to establish animal models of ALI [49]. In the present study, a rat model of ALI was established by tracheal drip injection of LPS. Lung tissue examination of the model group indicated that the lung tissue W/D ratio was significantly higher than in other groups, with correspondingly higher levels of the inflammatory factors TNF-α, IL-6, and IL-1β in BALF, with increased numbers of neutrophils, indicating that the model had been successfully replicated. EEAR significantly reduced the level of inflammatory factors and neutrophilia in rat BALF, decreased the lung W/D ratio, reduced pulmonary edema, decreased levels of MDA in rat lung tissue, and significantly increased GSH and SOD activity. The results demonstrate that EEAR significantly protected against LPS-induced lung inflammatory injury.
A number of herbal medicines have been found to be effective for treating ALI [50,51], with their mechanisms of action occurring through three possible pathways: an antiinflammatory response, anti-oxidative stress, and protection of airway epithelial barrier function. Numerous studies have concluded [52][53][54] that the TLR4/NF-κB pathway is capable of rapidly initiating intracellular inflammatory signaling pathways in ALI in response to LPS. The binding of TLR4 to LPS can activate multiple signaling pathways, the principal pathway being the MyD88 pathway, which activates IκB kinase (IKK) after a cascade of signals, followed by NF-κB inhibitor (IκB) phosphorylation, and the shedding of IκB from NF-κB which is subsequently ubiquitinated. NF-κB thus becomes activated from its inhibited state and then rapidly translocates to the nucleus, binding to its target protein and swiftly upregulating the expression of pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-8. In the present study, EEAR was demonstrated to reduce the expression of inflammatory factors in the lungs of rats with ALI in both in vivo and ex vivo experiments via inhibition of the TLR4/NF-κB signaling pathway, thereby alleviating the inflammatory response to ALI.
LPS induces the aggregation and hyperactivation of inflammatory cells, which generates a large degree of ROS-induced oxidative stress. Antioxidation is a key strategy for treating ALI [55]. The Keap1/Nrf2 signaling pathway is an important pathway that inhibits oxidative stress. Keap1 and Nrf2 are normally bound and in an inactive state. When stimulated in some way, their binding becomes unstable, releasing Nrf2 which binds to the antioxidant responsive element (ARE), which regulates the transcription of a variety of downstream antioxidant proteins and metabolic enzyme genes, thereby inhibiting the oxidative stress response in vivo. NQO-1 and HO-1, downstream target genes of the Keap1/Nrf2 signaling pathway, have anti-inflammatory, antioxidant, and anti-apoptotic properties [56,57]. In the present study, we found that EEAR inhibited the expression of Keap1 and promoted the expression of Nrf2 and its downstream proteins NQO-1 and HO-1, thus reducing the level of oxidative stress in acute lung injury.
Over recent years, with the rapid development of molecular biology and cell biology research techniques, experimental investigation of the prevention and treatment of ALI using Chinese medicines has made significant progress. However, research on their mechanisms of action is limited, and mostly at the level of identification of inflammatory factors, oxidative stress, and inflammatory cell infiltration. In-depth research and exploration remain somewhat uncommon. In the present study, we have only provided a preliminary exploration of the efficacy and mechanism of action of EEAR against ALI. It is clear that EEAR reduces the symptoms of ALI via the TLR4/NF-κB and Keap1/Nrf2 signaling pathways ( Figure 10), but additional study of the mechanisms of action is required, including using inhibitors and knockout rats. In addition, Atractylodis rhizoma has multiple chemical components and identification of the factor or factors that assist in the prevention and treatment of ALI requires additional research. exploration of the efficacy and mechanism of action of EEAR against ALI. It is clear that EEAR reduces the symptoms of ALI via the TLR4/NF-κB and Keap1/Nrf2 signaling pathways ( Figure 10), but additional study of the mechanisms of action is required, including using inhibitors and knockout rats. In addition, Atractylodis Rhizoma has multiple chemical components and identification of the factor or factors that assist in the prevention and treatment of ALI requires additional research.

Extract of Atractylodis rhizoma
Atractylodis rhizoma was obtained from the Hubei Tianji Chinese Herbal Pieces Company (Wuhan, China). The authenticity of the medicinal materials was confirmed by Kun Yu, an associate Professor from the College of Pharmacy of Hubei University of Chinese Medicine. A total of 200 g of Atractylodis rhizoma was crushed and then passed through a No. 2 sieve, soaked overnight in a 10-fold greater volume of 80% ethanol, then extracted 3 times in an ultrasonic water bath for 2 h each time, then filtered, after which the extracts were combined and concentrated in a rotary evaporator. The residue was dried under vacuum and weighed, from which the yield of volatile alcoholic extract was calculated.

Conversion of Dosage
The Chinese Pharmacopoeia (2020 Edition) lists the clinical dosage of Rhizoma atractylodis as 3-9 g, with 9 g representing the dose for an adult having a standard weight of 60 kg. Thus, the daily adult dose is 150 mg/kg. Using an accepted dose conversion between humans and rats, the dosage given to rats should be 925.5 mg/kg. Thus, a low dose was defined as 925.5 mg/kg and a high dose as 7404.0 mg/kg [25]. Based on the yield in an 80% ethanol extract of Rhizoma atractylodis (34%), the dosage of the ethanol extract of Rhizoma atractylodis in rats ranged from 314.7 mg/kg to 2517.4 mg/kg.

Extract of Atractylodis rhizoma
Atractylodis rhizoma was obtained from the Hubei Tianji Chinese Herbal Pieces Company (Wuhan, China). The authenticity of the medicinal materials was confirmed by Kun Yu, an associate Professor from the College of Pharmacy of Hubei University of Chinese Medicine. A total of 200 g of Atractylodis rhizoma was crushed and then passed through a No. 2 sieve, soaked overnight in a 10-fold greater volume of 80% ethanol, then extracted 3 times in an ultrasonic water bath for 2 h each time, then filtered, after which the extracts were combined and concentrated in a rotary evaporator. The residue was dried under vacuum and weighed, from which the yield of volatile alcoholic extract was calculated.

Conversion of Dosage
The Chinese Pharmacopoeia (2020 Edition) lists the clinical dosage of Rhizoma atractylodis as 3-9 g, with 9 g representing the dose for an adult having a standard weight of 60 kg. Thus, the daily adult dose is 150 mg/kg. Using an accepted dose conversion between humans and rats, the dosage given to rats should be 925.5 mg/kg. Thus, a low dose was defined as 925.5 mg/kg and a high dose as 7404.0 mg/kg [25]. Based on the yield in an 80% ethanol extract of Rhizoma atractylodis (34%), the dosage of the ethanol extract of Rhizoma atractylodis in rats ranged from 314.7 mg/kg to 2517.4 mg/kg.

Sample Preparation and HPLC Chromatographic Conditions
A 0.5 g quantity of EEAR powder was placed into a 100 mL conical flask with a stopper, to which methanol was added and extracted in an ultrasonic water bath, then cooled to 25 • C. The samples were shaken well and allowed to stand, after which methanol was added to adjust the weight, then centrifuged at 4 • C at 5000× g for 10 min. The supernatant was filtered through a 0.22 µm membrane and stored until required for further use.

In Vivo Experimental Design
Sterile-pathogen-free (SPF) male Sprague Dawley (SD) rats (140 ± 20 g) were purchased from the experimental animal center of Three Gorges University (Yichang, China; animal license No. SCXK (E) 2022-0012). The rats were maintained in an animal room at a temperature of 24 ± 1 • C and humidity of 50-70% with a 12 h/12 h light/dark cycle and adaptive feeding for a week. The husbandry and all surgical procedures on the experimental animals met all ethical requirements and were approved by the animal ethics committee of the Hubei University of Chinese Medicine (approval code: NO. 00273292, 10 November 2020).
The SD rats (n = 60) were randomly divided into the following five groups, 12 rats per group: normal group, model group, EEARL group, EEARH group, and Dex group. The ethanol extract of Atractylodis Rhizome was diluted with normal saline to prepare 31.47 mg/mL and 251.74 mg/mL solutions. Dexamethasone was dissolved in normal saline to prepare a 0.5 mg/mL solution. On days 1 to 7, a daily volume of normal saline (1 mL/100 g) was administered to the normal and model groups by gavage, and the corresponding drug concentrations to the other groups by gavage (1 mL/100 g). LPS (5 mg/kg) was administered to all groups other than the normal group two hours after gavage on the seventh day by intratracheal instillation. After 6 h, the animals were anesthetized using pentobarbital sodium. Subsequently, lung tissue samples and BALF were collected and evaluated using immunohistochemistry (IHC), immunofluorescence, Western blot analysis, and enzyme-linked immunosorbent assays (ELISAs).

Lung Wet/Dry Ratios
The whole lungs were removed, washed with PBS solution three times, and then the water on the surface of the lungs was dried with absorbent paper and weighed immediately, giving the wet weight. Then they were put into the incubator, baked at 80 • C for 48 h, and then weighed again, giving the dry weight, and then the ratio of wet weight to dry weight (W/D) was calculated.

Immunohistochemical Staining
For IHC, lung tissues were fixed in a 4% (w/v) solution of paraformaldehyde in PBS overnight, rinsed with PBS, and stored in 70% (v/v) ethanol. The samples were embedded in paraffin and 5 µm sections were obtained. Once deparaffinized, the sections were processed for antigen retrieval by incubation in 10 mM sodium citrate buffer (pH 6.0) containing 0.05% (v/v) Tween 20 at 95 • C for 10 min, washed twice with 0.1% (v/v) Triton X-100 in PBS, blocked for 45 min in 2% (v/v) donkey serum in 0.1% (v/v) Triton X-100 in M PBS, and incubated overnight at 4 • C with primary antibody (1:200) for MPO (Abcam, Cambridge, UK, ab208670), Ly-6 g (Abcam, Cambridge, UK, ab238132). The sections were washed in PBS and incubated for 2 h at room temperature with secondary antibody (1:500, Servicebio Technology Co., Ltd. Wuhan, China).

RNA Extraction and RT-qPCR
Total RNA in THP-1 cells or lung tissue was extracted using Trizol reagent (Vazyme Biotech Co., Ltd., Nanjing, China), in accordance with the manufacturer's instructions. RNA purity was determined from the A 260nm /A 280nm absorption ratio and was considered acceptable at a value between 1.8 and 2.0. The RNA was reverse-transcribed into cDNA using a commercial kit (Vazyme Biotech Co., Ltd., Nanjing, China). Primers were synthesized by the Tsingke Biotechnology Co., Ltd. RT-qPCR was performed using a ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) in a Roche Light Cycler 96 system (Roche, Basel, Switzerland). The PCR reaction was conducted as follows: 95 • C for 30 s, then 40 cycles at 95 • C for 10 s, and 60 • C for 30 s. Melt curve analysis was conducted after exposure of the samples to 95 • C for 15 s, 60 • C for 60 s, and 95 • C for 15 s. All gene expression values were normalized to β-actin expression and calculated using the 2 −∆∆CT method [59]. The specific primer sequences for all genes quantified are listed in Table 1.

Cell Culture and CCK-8 Assay
THP-1 cells (China Cell Line Bank, Beijing, China) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), and 100 U/mL penicillin and 100 U/mL streptomycin at 37 • C in a humidified atmosphere containing 5% CO 2 . In all experiments, the cells were allowed to acclimate for 24 h prior to any treatment. Analysis was conducted in accordance with a previous study [60]. The viability of THP-1 macrophages following the various treatments was measured using a CCK-8 cell proliferation and cytotoxicity assay kit (CA1210; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Briefly, THP-1 cells were seeded in 10% FBS-RPMI at a density of 5 × 10 4 cells/100 µL in 96-well plates, after which the cells were treated with PMA (100 ng/mL) and EEAR (0, 12.5, 25, 50, 100, 200 µg/mL) for 24 h. A 10 µL CCK-8 solution was added to each well and incubated at 37 • C for 1.5 h, in accordance with the manufacturer's instructions, after which the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Western Blot Analysis
Lung tissue samples or cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors, for 30 min. Total protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Elabscience Biotechnology Co., Ltd. Wuhan, China). A total of 20 µg of protein solution was electrophoretically transferred onto a PVDF membrane following separation on a 10% SDS-polyacrylamide gel. The membrane was blocked using 5% (w/v) nonfat dry milk blocking solution for 1 h, followed by overnight incubation at 4 • C with the respective primary antibody (1:1000). The membrane was then incubated for an additional hour with the appropriate HRP-conjugated secondary antibody (1:5000 dilution) at room temperature after thoroughly washing three times with phosphate buffered saline containing Tween (PBST). Bands were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA) and band intensities quantified using Image J software [61]

Isolation of Nuclear and Cytosolic Fractions
Cytoplasmic and nuclear extracts were prepared using an NE-PER nuclear and cytoplasmic extraction reagent kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), in accordance with the manufacturer's instructions. All steps were performed on ice, or at 4 • C.

Statistical Analysis
The results are presented as means ± S.E.M. All data were analyzed using GraphPad Prism version 8.0 software (San Diego, CA, USA). A Student's t-test or one-way ANOVA followed by Bonferroni test was used to compare two independent variables: # p < 0.05, ## p < 0.01, * p < 0.05, ** p < 0.01.

Conclusions
The results indicate that EEAR was effective in reducing the pathological symptoms of ALI, alleviating pulmonary edema, and improving the lung barrier. EEAR not only reduced the secretion of inflammatory factors in the lung by inhibition of TLR4/NF-κB signaling pathway activation, but also reduced the oxidative stress response in the lung via the Keap1/Nrf2 signaling pathway, thus alleviating ALI. The results help to clarify the efficacy of EEAR in preventing and treating ALI, as well as to clarify its mechanism of action. They provide the basis for the further development of therapeutic drugs for ALI.