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. 2024 Sep 25;15(1):171–179. doi: 10.4103/mgr.MEDGASRES-D-24-00044

Comparative study on the anti-inflammatory and protective effects of different oxygen therapy regimens on lipopolysaccharide-induced acute lung injury in mice

Xinhe Wu 1,#, Yanan Shao 2,#, Yongmei Chen 3, Wei Zhang 3, Shirong Dai 3, Yajun Wu 1, Xiaoge Jiang 2, Xinjian Song 2,*, Hao Shen 2,*
PMCID: PMC11515059  PMID: 39324894

Abstract

Oxygen therapy after acute lung injury can regulate the inflammatory response and reduce lung tissue injury. However, the optimal exposure pressure, duration, and frequency of oxygen therapy for acute lung injury remain unclear. In the present study, after intraperitoneal injection of lipopolysaccharide in ICR mice, 1.0 atmosphere absolute (ATA) pure oxygen and 2.0 ATA hyperbaric oxygen treatment for 1 hour decreased the levels of proinflammatory factors (interleukin-1beta and interleukin-6) in peripheral blood and lung tissues. However, only 2.0 ATA hyperbaric oxygen increased the mRNA levels of anti-inflammatory factors (interleukin-10 and arginase-1) in lung tissue; 3.0 ATA hyperbaric oxygen treatment had no significant effect. We also observed that at 2.0 ATA, the anti-inflammatory effect of a single exposure to hyperbaric oxygen for 3 hours was greater than that of a single exposure to hyperbaric oxygen for 1 hour. The protective effect of two exposures for 1.5 hours was similar to that of a single exposure for 3 hours. These results suggest that hyperbaric oxygen alleviates lipopolysaccharide-induced acute lung injury by regulating the expression of inflammatory factors in an acute lung injury model and that appropriately increasing the duration and frequency of hyperbaric oxygen exposure has a better tissue-protective effect on lipopolysaccharide-induced acute lung injury. These results could guide the development of more effective oxygen therapy regimens for acute lung injury patients.

Keywords: acute lung injury, cytokines, hyperbaric oxygen, inflammation, lipopolysaccharide, normobaric oxygen, oxygen therapy

Introduction

Acute lung injury (ALI) is a common severe clinical disease characterized by an excessive inflammatory response, alveolar epithelial and pulmonary capillary endothelial injury, diffuse alveolar and interstitial edema, and progressive hypoxemia.1,2,3 Currently, the main clinical treatment strategies for ALI are respiratory support, anti-infection therapy, fluid replacement, and symptomatic supportive treatment.4 Due to the lack of specific drugs, the treatment efficacy for this disease is poor, and the mortality rate is as high as 40%.5 Consequently, it is essential to identify a protective treatment for ALI.

Numerous studies have shown that oxygen therapy inhibits inflammatory reactions and reduces inflammatory injuries.6,7,8,9,10 Currently, there are various common clinical oxygen therapy methods, among which hyperbaric oxygen (HBO) therapy is a treatment method based on exposure to pure oxygen under enhanced atmospheric pressure, with 14 approved indications by the Undersea and Hyperbaric Medical Society, involving various types of diseases such as carbon monoxide poisoning, intracranial abscesses, necrotizing soft tissue infections, and acute traumatic ischemia.8,11,12,13 Recently, with the ongoing global coronavirus disease 2019 (COVID-19) pandemic, the clinical application of HBO in patients with COVID-19 or post-COVID-19 syndrome has received increasing attention.12,14 Clinical studies have revealed that HBO can suppress chronic inflammation and improve patients’ respiratory function and hypoxia, alleviating long-term COVID-19-related symptoms.15,16,17,18,19 An animal study has shown that 2.5 atmosphere absolute (ATA) HBO can alleviate lipopolysaccharide (LPS)-induced ALI in rats by downregulating tumor necrosis factor-alpha (TNF-α) expression.20 There are also reports that 2.0 ATA HBO pretreatment can alleviate lung inflammation and oxidative stress in a nitric oxide-dependent manner to protect against lung injury induced by acute pancreatitis.21 However, in various lung injury animal models, HBO can significantly alleviate the inflammatory response, improve tissue oxygenation capacity, downregulate the levels of inflammatory factors, alleviate tissue organ damage, and reduce animal mortality22,23,24,25,26,27; however, some studies have suggested that HBO therapy does not significantly improve the inflammatory response or even has harmful effects.28,29,30 Therefore, more research is required to support the selection of oxygen therapy regimens for ALI patients.

To explore the best oxygen therapy regimen after ALI, we used a mouse model of ALI induced by LPS and simulated different oxygen treatment regimens. Different pressure treatment groups (1.0/2.0/3.0 ATA), different exposure duration treatment groups (1/3 hours), and different exposure frequency treatment groups (once/twice a day) were used for the intervention. The expression of inflammatory factors in peripheral blood and lung tissues and organs and lung tissue injury scores were determined by analyzing the expression of inflammatory factors in peripheral blood and lung tissues and organs. Our study aimed to compare the therapeutic effects of different oxygen regimens on lung tissue injury in ALI mice from the perspective of inhibiting inflammation and to provide a preliminary experimental basis for selecting oxygen therapy regimens for ALI patients.

Materials and Methods

Animal models and different oxygen therapy protocols

Male ICR mice weighing 25–30 g were purchased from Nantong University Laboratory Animal Center (license No. SYXK (Su)-2012-0030). All animals were placed in plastic cages, allowed free access to standard mouse chow and water, and were maintained on a light/dark cycle for 12 hours at a controlled temperature (24 ± 2°C) for 1 week prior to the experiment. The animal experiments were approved by the Animal Ethics Committee of Nantong University, Jiangsu Province, China (approval No. S20190920-303). All experiments were designed and reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.31 ICR mice were injected intraperitoneally with LPS (Cat# L2630; Sigma Aldrich, St. Louis, MO, USA) at 5 mg/kg body mass dissolved in normal saline; the control group was injected with the same volume of normal saline. Mice were subjected to different oxygen therapy protocols immediately after LPS injection (Figure 1).

Figure 1.

Figure 1

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Experimental flow chart of different oxygen therapy regimens.

Protocol 1: Experimental flow chart of different exposure pressures; gray represents the control group, the same dose of normal saline was injected; green represents the LPS (5 mg/kg) group; orange from light to dark represents the 1.0, 2.0, and 3.0 ATA exposure pressure groups, respectively. Protocol 2: Experimental flow chart of different exposure durations and continuous exposure for 1 or 3 hours under 2.0 ATA. Protocol 3: Experimental flow chart of different exposure frequencies under 2.0 ATA, a single exposure for 3 hours or two exposures for 1.5 hours. ATA: Atmosphere absolute; Con: control; Exp.: experiment; ip.: intraperitoneal injection; LPS: lipopolysaccharide.

Protocol 1

The mice were randomly assigned to five groups (n = 4–7 per group): the control group, the LPS group, the LPS + 1.0 ATA group, the LPS + 2.0 ATA group, and the LPS + 3.0 ATA group. The control and LPS groups were treated with normobaric air at 1.0 ATA in 21% oxygen. The LPS + 1.0 ATA group received 100% oxygen at 1.0 ATA for 60 minutes. The mice were placed in a gas intervention box with a total volume of approximately 3.5 L (Wuhu Diving Equipment Factory, Wuhu, Anhui, China). The LPS + 2.0 ATA HBO group received 100% oxygen at 2.0 ATA for 60 minutes, and the LPS + 3.0 ATA HBO group received 100% oxygen at 3.0 ATA for 60 minutes. The mice were placed in a 100-liter chamber (Wuhu Diving Equipment Factory) with a compression/decompression time of 5 minutes. After 24 hours of LPS induction, the surviving mice in each group were euthanized by intraperitoneal injection of 2.5% avertin (0.15 mL/10 g, 2,2,2-tribromoethanol, Cat#T48402; 2-methyl-2-butanol, Cat# 152463, Sigma Aldrich) compound anesthetics, and the samples were collected for subsequent experiments.

Protocol 2

Based on the results of protocol 1, the effects of different durations of exposure to 2.0 ATA HBO on inflammatory factors in ALI model mice were observed. The mice were randomly allocated to the following four groups (n = 4–8 per group): control group, LPS group, LPS + 2.0 ATA HBO + 1 h group, and LPS + 2.0 ATA HBO + 3 h group. The control and LPS groups were treated with normobaric air at 1.0 ATA in 21% oxygen. The LPS + 2.0 ATA HBO + 1 h group received 100% oxygen at 2.0 ATA for 1 hour, and the LPS + 2.0 ATA HBO + 3 h group received 100% oxygen at 2.0 ATA for 3 hours. After 24 hours of LPS induction, the surviving mice in each group were euthanized by intraperitoneal injection of 2.5% Avertin compound anesthetics, and samples were collected for subsequent experiments.

Protocol 3

Mice were randomly assigned to different groups (n = 4–7 per group): the control group, the LPS group, the LPS + 2.0 ATA HBO + 3 h group, and the LPS +2.0 ATA HBO + 1.5 h × 2 group. The control and LPS groups were exposed to normobaric air at 1.0 ATA in 21% oxygen. The LPS + 2.0 ATA HBO + 3 h group received 100% oxygen at 2.0 ATA for 3 hours, while the LPS + 2.0 ATA HBO + 1.5 h × 2 group received 100% oxygen at 2.0 ATA for 1.5 hours twice daily at 12-hour intervals. After 24 hours of LPS induction, the surviving mice in each group were euthanized by intraperitoneal injection of 2.5% Avertin compound anesthetics, and samples were collected for subsequent experiments.

Enzyme-linked immunosorbent assay for cytokines

ICR mice were anesthetized, and blood was collected from the heart. Serum samples were obtained from blood samples by centrifugation (2000 × g, 20 minutes, 4°C). The levels of interleukin (IL)-1β, IL-6 and TNF-α in the serum were detected using enzyme-linked immunosorbent assay (ELISA) kits (Cat-EK201B/3-96, Cat-EK282/4-96, Cat-EK206/3-96, Multisciences, Hangzhou, China) following the manufacturer’s instructions.

Quantitative polymerase chain reaction

Total RNA was isolated from lung tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and quantified using a One Drop OD-1000+ Spectrophotometer (One Drop, Shanghai, China). Reverse transcription (+gDNA wiper) was performed using the HiScript II RT SuperMix software package for quantitative polymerase chain reaction (qPCR) (Vazyme, Nanjing, Jiangsu, China). For qPCR assays, Universal SYBR qPCR Master Mix software (Vazyme) was used. The relative transcript levels of the target genes were normalized to that of β-actin (Actb in mice). The sequences of the primers used are listed in Table 1.

Table 1.

Polymerase chain reaction primer sequence

Gene name Primer sequence (5'–3')
Mouse illb Forward: TCC AGG ATG AGG ACA TGA GCA C
Reverse: GAA CGT CAC ACA CCA GCA GGT TA
Mouse II6 Forward: CAC GGC CTT CCC TAC TTC AC
Mouse Tnf Reverse: TGC AAG TGC ATC ATC GTT GT
Mouse II10 Forward: GTT CTA TGG CCC AGA CCC TCA C
Reverse: GGC ACC ACT AGT TGG TTG TCTTTG
Forward: TGC TAA CCG ACT CCTTAA TGC A
Reverse: TCA TGG CCT TGA GAC ACC TTG
Mouse Arg1 Forward: CAG GAA GAA TGG AAG AGT CAG
Reverse: GGT GAC TCC CTG CAT ATC TG
Mouse CD206 Forward: TGA TTA CGA GCA GTG GAA GC
Mouse Actb Reverse: GCT ACG ACG TGG GCT ACA G
Forward: ACA CCC GCC ACC AGT TC
Reverse: TAC AGC CCG GGG AGC AT
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Actb: Beta-actin; Arg1: arginase-1; CD206: mannose receptor, C, type 1; II1-10: interleukin-10; II1b: interleukin-lbeta; II6: interleukin-6; Tnf: tumor necrosis factor-α.

Histological evaluation

Formalin-fixed lung tissues embedded in paraffin wax were cut into 4 μm sections and morphologically examined via hematoxylin and eosin staining (Leica RM2125 RTS, Shanghai, China). The pathological scoring of tissue was performed by a professional histologist. Lung injury scores ranged from 0–4 points (0, minimal harm or very mild harm; 1, 2, 3, and 4 represented mild, moderate, severe, and maximum damage, respectively).32

Statistical analysis

The data were statistically analyzed using GraphPad Prism 8.0 software (GraphPad Software, Boston, MA, USA, www.graphpad.com) and are expressed as the mean ± standard error of the mean (SEM). The Shapiro–Wilk test was used to test the normality of the distribution of the data. Normally distributed data among more than two groups were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test. Normally distributed data with heterogeneous variance were compared between two or more groups using Brown-Forsythe and Welch analysis of variance tests. P < 0.05 was considered to indicate statistical significance.

Results

Effects of different hyperbaric oxygen exposure pressures on inflammatory factors in the peripheral blood and lung tissue of the lipopolysaccharide-induced acute lung injury mouse model

To assess the impact of various exposure pressures on inflammatory factors in the peripheral blood and lung tissue of mice with LPS-induced ALI, we used ELISA and qPCR to analyze the levels of inflammatory factors in the peripheral blood and lung tissue of the mice in each group. Compared with those in the control group, the levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in the peripheral blood significantly increased after 24 hours of LPS induction (P < 0.001, Figure 2A). Compared with those in the LPS group, the levels of IL-1β and IL-6 were increased after treatment with 1.0 ATA pure oxygen or 2.0 ATA HBO for 1 hour (P < 0.05 or P < 0.01, Figure 2A), and 2.0 ATA HBO also decreased the level of TNF-α (P < 0.01, Figure 2A). However, 3.0 ATA HBO therapy had no significant effect on the levels of the inflammatory cytokines mentioned above. In lung tissue, the mRNA levels of the proinflammatory cytokines Il1b, Il6, and Tnf were significantly increased after LPS induction (P < 0.01 or P < 0.001, Figure 2B). Compared with those in the LPS group, Il1b, Il6, and Tnf expressions in the 1.0 and 2.0 ATA HBO treatment groups were inhibited after 1 hour (P < 0.05 or P < 0.01, Figure 2B). However, 3.0 ATA HBO therapy had no significant effect on the levels of the inflammatory factors mentioned above, which was consistent with the results of the peripheral blood test.

Figure 2.

Figure 2

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Effects of different exposure pressures on inflammatory factors in the peripheral blood and lung tissues of LPS-induced ALI model mice.

Note: (A) IL-1β. IL-6. and TNF-α levels in serum detected by ELISA. (B) Relative mRNA levels of the proinflammatory factors Il1b, Il6, and Tnfa in lung tissue. (C) Relative mRNA levels of the anti-inflammatory factors Il10, Arg1 and CD206 in lung tissue. The data are expressed as the mean ± SEM (n = 4–7 samples for each group). *P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). ALI: Acute lung injury; ATA: atmosphere absolute; Con: control; IL: interleukin; LPS: lipopolysaccharide; TNF: tumor necrosis factor.

In addition to measuring the levels of proinflammatory factors, we measured the mRNA levels of the anti-inflammatory factors Il10, Arg1, and CD206 in lung tissue. Treatment with 2.0 ATA HBO for 1 hour significantly increased the mRNA levels of Il10 and Arg1 in lung tissue (P < 0.01, Figure 2C), while 1.0 and 3.0 ATA HBO treatment for 1 hour had no significant effect on the levels of these anti-inflammatory factors. In addition, any of the oxygen treatments had no significant effect on the mRNA level of CD206 in lung tissue. In conclusion, in a mouse model of ALI induced by LPS, 1.0 and 2.0 ATA HBO treatments for 1 hour inhibited the expression of proinflammatory factors to varying degrees. Only 2.0 ATA HBO therapy increased the mRNA levels of Il10 and Arg1. These results indicate that 2.0 ATA HBO exhibited the greatest anti-inflammatory effect in our study.

Exposure to different pressures of hyperbaric oxygen results in different degrees of reduction in pathological injury to lung tissue in the mouse model of lipopolysaccharide-induced acute lung injury

To investigate the effects of different exposure pressures on LPS-induced lung tissue injury, we compared the histomorphology of lung organs in mice subjected to different exposure pressures by hematoxylin and eosin staining of tissue sections. Compared with the control group, the LPS group exhibited severe lung injury, including alveolar wall telangiectasia and congestion, interstitial edema, neutrophil infiltration, and leaky red blood cells in the alveolar space (Figure 3A). After 1 hour of pure oxygen treatment at 1.0 ATA, telangiectasis, congestion, and neutrophil infiltration of the alveolar wall began to improve, but the differences were not significant. When the pressure increased to 2.0 ATA, the above pathological changes in the lung tissue were significantly reduced (P < 0.05; Figure 3B). However, no significant improvement was observed when the pressure was increased to 3.0 ATA. The above results indicate that the improvement effect of oxygen therapy on tissue pathological injury increases with increasing oxygen partial pressure within a certain range. However, beyond a certain threshold, the improvement effect disappears, consistent with the detection of tissue inflammatory factor expression levels. Given the above results, we focused on the effect of 2.0 ATA HBO on inflammatory factors and pathological injury to lung tissue in ALI model mice.

Figure 3.

Figure 3

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Exposure to different pressures of HBO results in different degrees of pathological injury to the lung tissue in an LPS-induced ALI mouse model.

Note: (A) Histopathological changes in lung tissue. In the control group, lung tissue morphology was normal; in the LPS group, interstitial lung edema, neutrophil infiltration, and leakage of erythrocytes from the alveolar lumen were observed; in the LPS + 2.0 ATA HBO group, lung histopathological injuries were significantly attenuated; and in the LPS +1.0 ATA HBO group and LPS +3.0 ATA HBO group, no significant improvement was observed. Scale bars: 50 μm (upper), 5 μm (lower). (B) Lung injury scores. The data are expressed as the mean ± SEM (n = 3–5 in each group). *P < 0.05, ***P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). ALI: Acute lung injury; ATA: atmosphere absolute; Con: control; HBO: hyperbaric oxygen; LPS: lipopolysaccharide.

Effects of different hyperbaric oxygen exposure times on inflammatory factor levels in the peripheral blood and lung tissue of the mouse model of lipopolysaccharide-induced acute lung injury

To further assess the impact of treatment duration on the anti-inflammatory protective effect in LPS-induced ALI model mice, we compared the levels of inflammatory factors in the peripheral blood and lung tissues of mice exposed to 2.0 ATA HBO for 1 or 3 hours. ELISA detection of inflammatory factors in peripheral blood revealed that compared with LPS treatment, 2.0 ATA HBO for 1 and 3 hours inhibited the levels of IL-1β, IL-6, and TNF-α (P < 0.01, or P < 0.05; Figure 4A), with the increase in IL-6 being more significantly inhibited at 3 hours (P < 0.05; Figure 4A). qPCR analysis of lung tissues revealed that treatment with 2.0 ATA HBO for 1 and 3 hours inhibited the LPS-induced increases in Il1b, Il6, and Tnfα (P < 0.05, or P < 0.01, Figure 4B), with a more pronounced trend toward the inhibition of Il1b and Il6 after exposure for 3 hours. Additionally, we detected the mRNA levels of the anti-inflammatory factors Il10, Arg1, and CD206 in lung tissue. Compared with those in the LPS group, the mRNA levels of Il10 and Arg1 in the 2.0 ATA HBO group significantly increased after 1 and 3 hours (P < 0.05, P < 0.01, or P < 0.001; Figure 4C), and the CD206 mRNA level was upregulated only in the 3 h group (P < 0.01; Figure 4C). The mRNA levels of Il10, Arg1, and CD206 in the LPS + 2.0 ATA HBO + 3 h group were significantly higher than those in the LPS + 2.0 ATA HBO + 1 h group (P < 0.05 or P < 0.01, Figure 4C). These results suggest that 2.0 ATA HBO for 3 hours resulted in a better anti-inflammatory effect in the LPS-induced ALI mouse model.

Figure 4.

Figure 4

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Effects of different durations of HBO exposure on inflammatory factor levels in the peripheral blood and lung tissues of LPS-induced ALI model mice.

Note: (A) IL-1β. IL-6. and TNF-α levels in serum detected by ELISA. (B) Relative mRNA levels of the proinflammatory factors Il1b, Il6, and Tnfa in lung tissue. (C) Relative mRNA levels of the anti-inflammatory factors Il10, Arg1 and CD206 in lung tissue. The data are expressed as the mean ± SEM (n = 4–8 samples for each group). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). ALI: Acute lung injury; ATA: atmosphere absolute; Con: control; HBO: hyperbaric oxygen; IL: interleukin; LPS: lipopolysaccharide; TNF: tumor necrosis factor.

Effects of different hyperbaric oxygen exposure times on pathological injury to lung tissue in the mouse model of lipopolysaccharide-induced acute lung injury

To further compare the anti-inflammatory and protective effects of different treatment durations of 2.0 ATA HBO on lung tissue, we analyzed the effects of 1 and 3 hours of treatment with 2.0 ATA HBO on lung tissue morphology. Hematoxylin and eosin staining of lung tissue sections revealed that LPS-induced pathological damage, such as telangiectasia and congestion, interstitial edema, neutrophil infiltration, and leakage of red blood cells in the alveolar cavity, was significantly reduced after 1 hour of 2.0 ATA HBO. Furthermore, lung tissue damage improved significantly with a 3-hour duration of 2.0 ATA HBO. This was superior to a single exposure of 1 hour, as indicated by measurements of tissue inflammatory factor expression levels (P < 0.05, Figure 5A and B). These results suggest that 2.0 ATA HBO for 3 hours provides better anti-inflammatory protection in lung tissue.

Figure 5.

Figure 5

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Effects of different HBO exposure times on pathological injury to lung tissue in an LPS-induced ALI mouse model.

(A) Histopathological changes in lung tissue. The LPS group exhibited pathological damage, such as capillary dilatation and congestion, interstitial edema, neutrophil infiltration, and alveolar luminal erythrocyte leakage, which were attenuated by 1 and 3 hours of 2.0 ATA HBO treatment. Scale bars: 50 μm (upper), 5 μm (lower). (B) Lung injury scores. The data are expressed as the mean ± SEM (n = 3–5 in each group). * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). ALI: Acute lung injury; ATA: atmosphere absolute; Con: control; HBO: hyperbaric oxygen; LPS: lipopolysaccharide.

Effects of 2.0 ATA hyperbaric oxygen exposure on inflammatory factors in the peripheral blood and lung tissues of the mouse model of lipopolysaccharide-induced acute lung injury

In clinical HBO treatment, the duration of conventional single-agent treatment ranges from 1 to 1.5 hours.33 Our research revealed that a single 3-hour exposure exhibited a more significant effect on suppressing inflammation. Considering the typical single HBO treatment time in clinical practice, we compared the effects of dividing 3 hours into two exposures and the inhibitory effect of a single 3-hour exposure on inflammation. The ELISA results for peripheral blood showed that compared with the LPS group, both single 3-hour HBO exposure and two 1.5-hour HBO exposures inhibited the levels of IL-1β, IL-6, and TNF-α (P < 0.01 or P < 0.05, Figure 6A). qPCR analysis of lung tissue revealed that compared with those in the LPS group, the LPS + 2.0 ATA HBO + 3 h group and the LPS +2.0 ATA HBO + 1.5 h × 2 group exhibited decreased mRNA levels of Il1b, Il6, and Tnfα (P < 0.01, Figure 6B) and significantly increased mRNA levels of Il10 and Arg1 (P < 0.05 or P < 0.01, Figure 6C). There was no significant difference in the effect. However, only the 2.0 ATA HBO + 3 h group exhibited upregulated CD206 mRNA in lung tissue. The above results indicate that a single exposure to 2.0 ATA HBO for 3 hours and two exposures had similar effects on the levels of inflammatory factors.

Figure 6.

Figure 6

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Effects of different frequencies of HBO exposure on inflammatory factor levels in the peripheral blood and lung tissues of LPS-induced ALI model mice.

IL-1β. IL-6. and TNF-α levels in serum detected by ELISA. (B) Relative mRNA levels of the proinflammatory factors Il1b, Il6, and Tnfa in lung tissue. (C) Relative mRNA levels of the anti-inflammatory factors Il10, Arg1 and CD206 in lung tissue. The data are expressed as the mean ± SEM (n = 4–7 samples for each group). *P < 0.05, **P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). ALI: Acute lung injury; ATA: atmosphere absolute; Con: control; HBO: hyperbaric oxygen; IL: interleukin; LPS: lipopolysaccharide; TNF: tumor necrosis factor.

Figure 7.

Figure 7

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Exposure to different frequencies of HBO similarly alleviates pathological lung tissue injury in an LPS-induced ALI mouse model.

(A) Histopathological changes in lung tissue. Lung tissues in the LPS group exhibited pathological injuries, such as gross edema, neutrophil infiltration, and alveolar erythrocyte leakage, which were alleviated after two 1.5-hour and one 3-hour HBO treatments. Scale bars: 50 μm (upper), 5 μm (lower). (B) Lung injury scores. The data are expressed as the mean ± SEM (n = 3–5 in each group). ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). ALI: Acute lung injury; ATA: atmosphere absolute; Con: control; HBO: hyperbaric oxygen; LPS: lipopolysaccharide.

Exposure to different frequencies of hyperbaric oxygen similarly alleviated pathological lung tissue injury in the mouse model of lipopolysaccharide-induced acute lung injury

To further investigate the effects of 2.0 ATA HBO exposure frequency in treating LPS-induced ALI, we compared the effects of two 1.5-hour exposures and a single 3-hour exposure on lung tissue morphology. Both exposure frequencies significantly reduced lung tissue pathological changes compared to those in the LPS group (P < 0.01, Figure 6A and B). There was no significant difference between the two groups, indicating that different frequencies of HBO have similar effects on the ALI.

Discussion

This study aimed to investigate the protective effects of different oxygen regimens on lung tissue inflammation in a mouse model of LPS-induced ALI. The results showed that 2.0 ATA HBO treatment improved LPS-induced inflammatory injury in lung tissue by regulating the expression of inflammatory factors in serum and lung tissue. With an exposure duration of 3 hours, the anti-inflammatory effect of HBO increased, and the effect on improving lung tissue injury improved. At different exposure frequencies, a single exposure for 3 hours exhibited the same anti-inflammatory effect as two exposures for 1.5 hours. These results suggest that oxygen therapy for ALI can appropriately prolong the exposure duration or increase the exposure frequency according to the actual situation of patients to achieve better tissue anti-inflammatory protective effects.

We found that different oxygen regimens exhibit varying effects on improving lung tissue injury, possibly related to their effects on inflammatory factors. Assessing inflammatory factors can help clinicians accurately diagnose the disease as early as possible, monitor the effectiveness of treatment regimens, and adjust treatment if necessary.34 In patients with ALI, elevated IL-6 upon admission is closely related to disease severity. The proinflammatory cytokines IL-8, TNF-α, IL-1β, and IL-18 are the most promising molecular biomarkers for predicting morbidity and mortality.35,36 Our study revealed that 2.0 ATA HBO treatment significantly inhibited the levels of these proinflammatory factors to a greater extent than did 1.0 ATA pure oxygen exposure, which is consistent with the results of Halbach et al.37 and Hauser et al.38 During the inflammatory response, anti-inflammatory factors are key to inhibiting excessive inflammatory responses.39 Anti-inflammatory factors (such as Il4, Il10, and Arg1) can activate anti-inflammatory signals, terminate the proinflammatory response, and ultimately promote the recovery of lung injury by eliminating apoptotic neutrophils at inflammatory sites.40,41 Previous research has shown that by increasing the expression of IL-10, HBO inhibits the secretion of IL-6 and reduces the mortality of ALI model mice.40,42,43 In our study, treatment with 2.0 ATA HBO for 3 hours significantly upregulated the mRNA levels of the anti-inflammatory factors Il10, Arg1, and CD206 compared with exposure for 1 hour, and the effect on improving lung tissue injury was greater. Interestingly, we found that under 2.0 ATA HBO conditions, two 1.5-hour exposures were also effective in reducing lung tissue damage, with a similar effect to that of a single 3-hour exposure. Among the anti-inflammatory factors, we found that a single 3-hour exposure significantly upregulated the mRNA level of CD206 in lung tissue, while two 1.5-hour exposures only showed an upregulation trend. This may be linked to each cell’s different peak times of inflammatory factors. Therefore, it is necessary to monitor the expression of inflammatory factors at different time points to help guide clinical diagnosis and treatment.

The variations in the extent of improvement in tissue hypoxia and the levels of reactive oxygen species (ROS) generated by different HBO treatments may account for the impact of various oxygen regimens on inflammatory factors. An excessive inflammatory response results in tissue inflammatory hypoxia,44,45 activating inflammatory cells and releasing numerous proinflammatory factors, such as TNF-α, IL-1β, IL-6, and IL-8, consequently worsening tissue damage.46,47 Our research revealed that treatment with 1.0 ATA pure oxygen can suppress certain proinflammatory factors but does not significantly impact lung tissue injury; only treatment with 2.0 ATA HBO can effectively reduce lung tissue injury. Furthermore, an improvement in lung tissue injury was more pronounced when the duration of 2.0 ATA HBO treatment was extended to 3 hours or when 2.0 ATA HBO was administered 2 times for 1.5 hours each; this improvement may be linked to enhanced tissue hypoxia efficiency. It is widely recognized that HBO is more effective than normobaric oxygen in ameliorating tissue hypoxia by elevating the dissolved oxygen in plasma and extending the oxygen diffusion distance,11,44 a fact that has been confirmed in cases of focal brain tissue ischemia.48,49,50,51 One study reported that in a skeletal muscle injury model, a single exposure to HBO for 2 hours improved the hypoxic environment and oxygenated the injured muscle tissue for 30 hours.52 Meng et al.53 demonstrated that intermittent HBO (100% oxygen, 2.0 ATA, 1.5 hours, twice a day) enhanced the recovery of neurological function after spinal cord injury by inhibiting the expression of inflammatory factors and promoting the expression of brain-derived neurotrophic factors. Clinical studies have indicated that HBO can aid in the return of inflammatory factors to steady-state levels by regulating IL-6 and TNF-α, thus improving oxygen levels and increasing blood oxygen saturation in patients with COVID-19,15,54,55 which are similar to the results of our study. Furthermore, hyperoxia during HBO therapy induces ROS production, which exhibits antibacterial effects and has been shown in patients with severe and refractory infectious diseases.14,56 Our study also revealed a reduction in inflammatory cell infiltration in lung tissues after treatment with 2.0 ATA HBO. Notably, when the pressure was increased to 3.0 ATA, we found no improvement in LPS-induced ALI, which may be associated with ROS levels and tissue recovery damage caused by high-pressure exposure. A growing body of literature has indicated that low levels of ROS act as oxidation–reduction signaling molecules to maintain cellular homeostasis, participate in defense against pathogens, and inhibit inflammatory responses. However, high levels of ROS can not only lead to gene mutations but also promote inflammatory responses and induce cell death and disease.57,58,59 Oter et al.60 have demonstrated that 3.0 ATA HBO therapy can lead to oxidative stress damage in the lungs and brain of rats, similar to our study’s results. It has also been reported in the literature30,60,61 that the lung is one of the most sensitive tissues to oxygen toxicity. Compared with 3.0 ATA, the oxidation parameters are lower when the HBO pressure ranges from 2 to 2.5 ATA, and clinical treatment is relatively safe. Given that oxidative stress is proportional to the exposure pressure of HBO, clinicians are encouraged to use relatively low exposure pressures whenever possible to ensure safe and effective patient treatment. In conclusion, these data indicate that oxygen therapy has a protective effect on inflammatory injury in lung tissue, and further experiments are needed to verify and elucidate the underlying mechanisms involved.

Several limitations of our study deserve attention. First, the results of this study showed that although HBO treatment at 2.0 ATA significantly improved inflammatory injury to the lung tissue, a long-term effect was not observed. Therefore, future studies will utilize biochemical and pathological indicators to detect lung injury in mice over a longer period and compare the effects of different treatment courses. Second, we observed that 1.0 ATA pure oxygen started inhibiting the expression of some proinflammatory factors. Recently, many studies have shown that micropressure can also benefit patients.60,61,62,63,64,65 Consequently, our future research will include the pressure gradient between micro pressures. Finally, this study examined only exposure stress, duration, and frequency. We did not explore the specific mechanisms involved in this change, which will be the focus and direction of our future research.

In conclusion, our results suggest that appropriately prolonging the duration or increasing the frequency of HBO exposure has a better anti-inflammatory protective effect on the lung tissue of ALI patients. In the future, more experimental data are needed to optimize the clinical application of oxygen therapy and provide more guidance for clinicians.

Acknowledgments:

We thank the Home for Researchers editorial team (www.home-for-researchers.com) for the language editing service.

Funding Statement

Funding: This study was supported by the Senile Health Scientific Research Project of Jiangsu Province (No. LR2021026) and the Scientific Research Project of the Nantong Municipal Health Commission (Nos. QN2022034 and QA2021034).

Footnotes

Conflicts of interest

The authors declare that they have no conflicts of interest.

Data availability statement:

All relevant data are within the paper.

References

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