18β-Glycyrrhetinic Acid Protectes Neonatal Rats with Hyperoxia Exposure Through Inhibiting ROS/NF-κB/NLRP3 Inammasome

Bronchopulmonary dysplasia (BPD) is a common devastating pulmonary complication in preterm infants. Oxygen supplementation is a lifesaving therapeutic measure used for premature infants with pulmonary insuciency. However, oxygen toxicity is a signicant trigger for BPD, and oxidative stress-induced inammatory responses, in turn, worsens the oxidative toxicity resulting in lung injury and arresting of lung development. Glycyrrhiza radix is commonly used in the medicine and food industries. 18β-Glycyrrhetinic acid (18β-GA), a primary active ingredient of Glycyrrhiza radix, has a powerful anti-oxidative and anti-inammatory effects. This study aimed to determine whether 18β-GA has protective effects on neonatal rats with hyperoxia exposure. Newborn Sprague-Dawley rats were kept in either 21% (normoxia) or 80% O 2 (hyperoxia) continuously from postnatal day (PN) 1 to 14. 18β-GA was injected intragastrically at 50 or 100 mg/kg body weight once a day from PN 1 to 14. We examined the body weights and alveolar development, and measured ROS level and the markers of pulmonary inammation. Mature-IL-1β and NF-κB pathway proteins, and the NLRP3 inammasome, were assessed; concurrently, caspase-1 activity was measured. Our results indicated that hyperoxia resulted in alveolar simplication and decreased bodyweight of neonatal rats. Hyperoxia exposure increased ROS level and pulmonary inammation, and activated NF-κB and the NLRP3 inammasome. 18β-GA treatment decreased ROS level, inhibited the activation of NF-κB and the NLRP3 inammasome, decreased pulmonary inammation, improved alveolar development, and increased the bodyweight of neonatal rats with hyperoxia exposure. Our study demonstrates that 18β-GA protects neonatal rats with hyperoxia exposure through inhibiting ROS/NF-κB/NLRP3 inammasome.


Introduction
Bronchopulmonary dysplasia (BPD) is described as the multiple external injurious stimuli (oxygen toxicity, baro-and volu-trauma, and infection/in ammation) damage the immature, developing lung, and arrest lung development ( Baraldi et al., 2007;Nakanishi et al., 2016). Among 10-year-old children born extremely preterm, those who had BPD were at increased risk of cognitive and academic achievement (Sriram et al., 2018). Lowering the high incidence of BPD may be vital to improving long-term outcomes after extremely or very preterm birth. Unfortunately, current prevention strategies such as corticosteroids (Doyle et al., 2017), surfactant (Isayama et al., 2016), less invasive respiratory strategies (Sweet et al., 2019), careful oxygen usage (Saugstad Ola et al., 2018),and antioxidants (Poggi et al., 2014) have not been able to diminish the BPD incidence.
For the developing lung, oxygen toxicity is a signi cant trigger for BPD (Morty et al., 2018;Bhandari et al., 2010;Wang et al., 2018). Lung development progresses in ve distinct stages: embryonic, pseudo glandular, canalicular, saccular, and alveolar (Kotecha et al., 2000). Human preterm infants who develop BPD are primarily born in the saccular stage of lung development (human 24-38 weeks of gestation age). In rodent models, the saccular stage of lung development begins at embryonic day 18 and continues through postnatal (PN) day 5 (Joshi et al., 2007). The rodent pups from birth exposed to high concentration oxygen have similar histological changes with hyperoxia-exposed human preterm infants (Nardiello et Buczynski et al., 2013). Therefore, hyperoxia-exposure neonatal rat models are essential for better understanding the molecular mechanisms of BPD and testing the e cacy of potential therapeutic agents.
The molecular mechanisms of BPD are complex. The newborns pass from the hypoxic environment of the womb to the relatively hyperoxic extrauterine environment, and they experience increased oxidative stress. Preterm neonates are more susceptible to oxidative stress because of developmental de cits in antioxidant defenses (Kinsella et al., 2006;Negi et al., 2012). In addition, an additive oxygen therapy for the treatment of respiratory instabilities can increase oxidative stress (Perrone et al., 2016). Although premature infants are usually exposed to only the least required amount of supplemental oxygen, studies show considerable evidence of oxidant stress (Ozsurekci et al., 2016;Perrone et al., 2016). The excessive reactive oxygen species (ROS) trigger the oxidative stress response, and damage the developing lung.
And oxidative stress can activate in ammatory responses, representing increased pro-in ammatory cytokines and chemokines, and in ammatory cell in ltration in lung tissue. The in ammatory cells produce more ROS to worsens the oxidative toxicity, which in turn recruits more in ammatory cells to the lung (Rosanna et al., 2012). These complex and cross-talk processes between oxidative stress and in ammation ultimately lead to lung injury and impairment of lung development, which contribute to BPD (Rosanna et al., 2012;Balany et al., 2015). However, the mechanism of the interaction between oxidative stress and in ammation has not yet been conclusively clari ed.
The NLRP3 (NOD-, LRR-, and pyrin-domain containing protein 3) in ammasome is the most extensively studied in ammasome complex (Agostini et al., 2004), and NLRP3 in ammasome can be activated during oxidative stress and systemic infections (Groslambert et al., 2018;Yang et al., 2019). NLRP3 recruits ASC (apoptosis-associated Specklike protein containing CARD) and caspase-1, and format the NLRP3 in ammasome. The NLRP3 in ammasome mediates the post-translational processing of IL-1β by active caspase-1, which cleaves pro-IL-1β to produce mature IL-1β. Mature IL-1β interacts with its receptor, IL1R, to signal in ammatory pathways. Activation of the NLRP3 in ammasome is considered to be a two-step process that requires two signals: the rst priming signal triggers nuclear factor-κB (NF-κB)dependent upregulation of NLRP3 and pro-IL-1β expression (Bauernfeind et al., 2009); the second signal is to induce NLRP3 activation (Swanson et al., 2019;Yang et al., 2019). ROS has been considered as a trigger of NLRP3 in ammasome activation (Teng et al., 2020; Wei et al., 2020), and many chemical compounds are reported to activate NLRP3 in ammasome via increasing intracellular ROS levels (Zhong et al., 2013). In addition, ROS can activate the NF-κB signaling pathway, resulting in the ampli cation of in ammatory response (Zha et al., 2014). ROS-mediated NF-κB signaling plays an essential role in activating the NLRP3 in ammasome (Teng et al., 2020;Peng et al., 2020). Therefore, ROS/NF-κB/NLRP3 in ammasome signaling is closely associated with the overlapping or feedback pathways between oxidative stress and in ammatory response.
Caffeine is a kind of plant alkaloid, and exhibits practical bene t in the treatment or prevention of BPD in several clinical studies (Coulter et  Based on these considerations, we hypothesized that 18β-GA had a protective effect in neonatal rats with hyperoxia exposure. In this study, we investigated the role of ROS/NF-κB/NLRP3 in ammasome in this process.

Animals
Pregnant Sprague-Dawley (SD) rats were purchased from the Department of Animals, Experimental Center, Shengjing Hospital of China Medical University (Shenyang, China). All newborn SD rats were born on days 21-23 of gestation. Both male and female neonatal rats were used in all studies. Each maternal rat was adjusted to feed 6-8 pups to minimize nutritional differences on lung development. Maternal rats were rotated between room air and oxygen-exposed litters daily to prevent oxygen toxicity. The rat's living environment was as follows: 12 h alternating light/dark cycle, the temperature at 25°C-26°C, and humidity at 60 %-70 %. The chamber was opened once per day for 0.5 h to replace the food and water. All animal procedures were approved by the Laboratory Animal Ethics Committee of Shengjing Hospital of China Medical University (Shenyang, China).

Bodyweight measurement and lung sample collection
The bodyweights of all neonatal rats were measured at PN 14, after which they were sacri ced by an intraperitoneal injection of pentobarbital (50 mg/kg body weight). BALF was centrifuged at 500 g for 10 min immediately after extraction. The BALF supernatant was frozen at -80°C until further analysis. The pulmonary artery was perfused with PBS to remove all blood from the lungs. The lungs for the molecular analysis were immediately frozen in liquid nitrogen and stored at -80°C. For immunohistochemical analysis, the lungs were in ated to 25 cm H 2 O with 4% paraformaldehyde (PFA), then fully xed in 4% PFA for 48 h and embedded in para n.

Lung histological and morphometric analyses
In ation-xed lungs were processed to obtain 3 µm thick para n sections, stained with hematoxylin and eosin for examination of the lung architecture. Six images were randomly selected for each sample. Alveolarization was evaluated by the radial alveolar count (RAC) value (Cooney et al., 1982) which was obtained by drawing a line from the center of terminal bronchioles to the nearest connective tissue septum and counting the number of the alveoli on the line. These assessments were carried out independently by two pathologists who were blinded to the grouping.

In ammatory cells in BALF and Giemsa staining
BALF supernatant cell smears were examined using Giemsa staining (D010-1-1, Nanjing Jiancheng Research Institute of Biotechnology, Jiangsu, China) according to the manufacturer's instructions. The in ammatory cells were counted and classi ed under a light microscope.

ROS Measurement
We centrifugated the homogenates of lung tissues at 10,000 g for 15 min at 4°C and collected the supernatant for subsequent testing. ROS level was assayed by a ROS assay kit (WanLeiBio, China), which used a stable non-uorescent dichlorodihydro uorescein diacetate (DCFH-DA) as a probe. DCFH-DA freely entered cells and was then hydrolyzed by esterases to create non-uorescent DCFH. However, DCFH was rapidly oxidized by ROS in the cells to generate strong uorescent DCF. Hence, ROS levels were assayed indirectly via measuring DCF uorescence.

Caspase-1 activity assay in lung tissues
Caspase-1 activity in lung homogenates was analyzed with a caspase-1 activity assay kit (C1101, Beyotime, China) according to the manufacturer's instructions.

Statistical analysis
Data analysis was performed using GraphPad Prism version 8.0 (GraphPad Software). Experimental data were presented as the mean ± standard deviation. Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test (equal variance) or Dunnett T3's post hoc test (unequal variance). P < 0.05 was considered statistically signi cant.

Effect of 18β-GA on bodyweight of neonatal rats with hyperoxia exposure
The bodyweights of neonatal rats in the different groups were measured at PN14. Exposure to 80% O 2 resulted in a decrease in the bodyweights of neonatal rats (P < 0.01; Fig. 1B). However, treatment with 18β-GA increased the bodyweights of neonatal rats exposed to 80% O 2 (P < 0.01; Fig. 1B).

18β-GA increased alveolarization of neonatal rats with hyperoxia exposure
The neonatal rats exposed to 21% O 2 had a standard distal lung architecture with well-formed alveoli and a standard RAC value (Fig. 2). However, distal lung histology in neonatal rats exposed to 80% O 2 showed simpli cation of the distal lung architecture, with fewer, larger alveoli and a lower RAC value (P < 0.01; Fig. 2). The hyperoxia exposed and 18β-GA-treated rats had increased alveolarization and a higher RAC value than the rats with hyperoxia exposure alone (P < 0.01; Fig. 2). These results demonstrated that hyperoxia exposure resulted in alveolar simpli cation; however, 18β-GA treatment protected neonatal rats with hyperoxia exposure from alveolar simpli cation.

18β-GA decreased pulmonary in ammation of neonatal rats with hyperoxia exposure
We detected in ammatory cells in the BALF of neonatal rats in different groups. Neonatal rats exposed to 80% O 2 had a signi cantly elevated in ammatory cell count in their BALF, especially neutrophils and macrophages (P < 0.05; Table 1). Treatment with 18β-GA decreased neutrophil and macrophage count in the BALF of neonatal rats exposed to 80% O 2 (P < 0.05; Table 1). Further, we investigated neutrophils and macrophages in ltration in lung tissues, by measuring the speci c enzyme activities of MPO and NAG in lung homogenates. MPO and NAG activities increased in rats exposed to 80% O 2 (P < 0.01; Fig. 3A-B). The increased MPO and NAG activities were reversed by 18β-GA treatment (P < 0.01; Fig. 3A-B). These results suggest that 18β-GA treatment halts the in ammatory cell in ltration caused by hyperoxia exposure. Chemokines play an essential role in mediating in ammatory cell recruitment to the lung during hyperoxic exposure (Endesfelder et al., 2020). As shown in Fig. 4A-C, we observed a robust increase in the expression of the cytokine-induced neutrophil chemoattractant-1 (CINC-1) (P < 0.01; Fig. 4A), macrophage in ammatory protein-2 (MIP-2) (P < 0.01; Fig. 4B), and macrophage migration inhibitory factor (MIF) (P < 0.01; Fig. 4C) in the hyperoxia group compared to the normoxic group, which was alleviated by 18β-GA treatment.
Next, we measured the pro-in ammatory cytokines levels in the BALF. Rats exposed to 80% O 2 had increased expression of IL-1β, IL-6, and TNF-α in their BALF (P < 0.01; Fig. 5A-C). 18β-GA treatment reversed the increase in pro-in ammatory cytokines expression under hyperoxic conditions (P < 0.01; Fig. 5A-C). These results suggest that 18β-GA treatment reduces the in ammatory response caused by hyperoxia exposure.
3.4 18β-GA decreased pulmonary ROS level of neonatal rats with hyperoxia exposure As depicted in Fig. 6 showed that rats exposed to 80% O 2 had a signi cantly elevated ROS level in the lung tissues. Treatment with 18β-GA reversed the increase in ROS level under hyperoxic conditions (P < 0.01).

Treatment with 18β-GA inhibited the activity of NF-κB and the NLRP3 in ammasome and decreased mature-IL-1β levels
We investigated whether 18β-GA treatment inhibited the activity of the NF-κB pathway using western blot analysis. As shown in Fig. 7, phosphor-NF-κB/NF-κB (P < 0.01) and phosphor-IκBα/IκBα protein relative expression (P < 0.01) were signi cantly increased in rats exposed to 80% O 2 , and 18β-GA treatment reversed these changes (P < 0.01). These results indicated that 18β-GA treatment inhibited the activation of the NF-κB pathway in rats exposed to hyperoxia.
Next, we tested the effect of 18β-GA on the NLRP3 in ammasome. As shown in Fig. 8A-C, rats exposed to 80% O 2 had increased expression of NLRP3, ASC, and cleaved caspase-1 (P < 0.01), reversed by 18β-GA treatment. Furthermore, we found that rats exposed to 80% O 2 had increased caspase-1 activity which was abolished by treatment with 18β-GA (P < 0.01; Fig. 8D). These results indicated that 18β-GA treatment inhibited the increased activity of NLRP3 in ammasome induced by hyperoxia exposure.
Since hyperoxia exposure increased the expression of cleaved caspase-1, we further tested whether hyperoxia exposure could increase the level of mature IL-1β. Similarly, with cleaved caspase-1, rats exposed to 80% O 2 had increased expression of mature IL-1β, and 18β-GA-treated rats had a lower protein expression of mature IL-1β (P < 0.01; Fig. 9). Based on the above ndings, 18β-GA treatment downregulated the increased expression of mature-IL-1β induced by hyperoxia exposure, through inhibiting the activity of the NLRP3 in ammasome.

Discussion
In this study, we exposed neonatal rats in 80% O 2 from birth to PN 14, and 18β-GA treatment was concurrently applied. Our results show that 18β-GA treatment increased bodyweight and improved alveolar simpli cation of neonatal rats with hyperoxia exposure. Under hyperoxia conditions, 18β-GA treatment decreased ROS level and in ammation response, and inhibited the activity of NF-κB and the NLRP3 in ammasome. Our results indicated that 18β-GA treatment protected neonatal rats undergoing hyperoxia exposure from alveolar simpli cation by inhibiting ROS/NF-κB/NLRP3 in ammasome.
Advances in the management of preterm infants over the past 50 years, the survival rate of extremely and very premature infants has improved. In contrast, the incidence of BPD is almost unchanged (Stoll et al., 2015). The overall incidence of BPD in infants born at < 28 weeks of gestational age is estimated to be 48%-68% (Stoll et al., 2010). To date, no adequate or preventive therapy of BPD is available. 18β-GA, as an anti-oxidative and anti-in ammatory drug, has been proved to play a protective role in pulmonary diseases ( ). We exposed the neonatal rats to 80% O 2 from birth to PN 14, which represented the saccular and alveolar stage in rats lung development. In our study, we demonstrated that compared with rats exposed to 21% O 2 , rats exposed to 80% O 2 had decreased bodyweight and alveolar simpli cation with a lower RAC value. And these results were similar to that in Chen's study (Chen et al., 2020). Our results indicated that 18β-GA treatment in hyperoxia exposed rats increased weight gain, and improved alveolarization, as demonstrated by a higher RAC value. The above data showed that 18β-GA treatment had a protective effect in hyperoxia-exposed neonatal rats.
Exposure to high levels of oxygen inevitably leads to the production and accumulation of excessive ROS and activation of in ammatory response, and the in ammatory reaction triggered by hyperoxia, in turn, worsens the oxidative toxicity. Pulmonary in ammation of BPD is characterized by chemotactic factors, Our study found that ROS level was signi cantly increased in neonatal rats with hyperoxic exposure. Treatment with 18β-GA decreased the ROS level of rats under hyperoxic conditions. And we con rmed that chemotactic factors CINC-1, MIP-2, and MIF were signi cantly increased in the rats with hyperoxia exposure; a large number of neutrophils and macrophages and pro-in ammatory cytokines (IL-1β, IL−6, and TNF-α) moved into the BALF of rats exposed to hyperoxia. We also found that hyperoxia exposure increased MPO and NAG activities in lung tissues, indicating that hyperoxia causes a large in ux of neutrophils and macrophages into the lung tissues of rats. However, the in ux of chemotactic factors, in ammatory cells, and pro-in ammatory cytokines was reversed by 18β-GA treatment. Based on the above ndings, our results indicated that 18β-GA, as a kind of free radical scavenger, inhibited the in ammatory response induced by hyperoxia.
ROS/NF-κB/NLRP3 in ammasome signaling may be a link between oxidative stress and in ammatory response. ROS acts as the priming signal to activate the NLRP3 in ammasome and then promote the secretion of mature IL-1β (Teng et al., 2020;Wei et al., 2020). Another study showed that hyperoxia exposure impaired alveolarization of neonatal mice by activiting NLRP3 in ammasome (Liao et al., 2015). In the present study, we con rmed that the hyperoxia exposure increased ROS level, activated NF-κB and the NLRP3 in ammasome, and increased the expression level of mature IL-1β. However, 18β-GA treatment reversed these effects. Therefore, our results indicated that 18β-GA protected neonatal rats with hyperoxia exposure through inhibiting ROS/NF-κB/NLRP3 in ammasome.

Limitations
BPD is a complex multifactorial disease, including oxygen toxicity, perinatal in ammation, perinatal hypoxia, and mechanical ventilation (Nardiello et  ). This study exposed neonatal rats with 80% O 2 for 14 days, and future studies are needed to investigate the protective role of 18β-GA in neonatal rats exposed to different oxygen concentrations and, or duration of oxygen exposure, even other risk factors of BPD.

Conclusion
In summary, our study suggested that 18β-GA treatment protected neonatal rats with hyperoxia exposure. The possible mechanism is that 18β-GA, as a kind of free radical scavenger, decreased the hyperoxiainduced in ammatory response through inhibiting NF-κB/NLRP3 in ammasome signaling.    Effects of 18β-GA on the chemokines in lung tissues. 18β-GA decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage in ammatory protein-2 (MIP-2), and macrophage migration inhibitory factor (MIF) in lung homogenates of rats exposed to hyperoxia conditions. The values are the mean ± standard deviation. NS not signi cant; ** P < 0.01 vs. N group; ## P < 0.01 vs. H group.

Figure 7
Effects of 18β-GA on the NF-κB pathway in rat lungs. 18β-GA reduced NF-κB pathway activation in rats

Figure 9
Effects of 18β-GA on the mature IL-1β. 18β-GA decreased the protein expression of mature IL-1β in rats exposed to hyperoxia conditions. Representative image and semiquantitative analysis of mature IL-1β protein expression in lung tissue homogenates. The values are the mean ± standard deviation. NS not signi cant ** P < 0.01 vs. N group; ## P < 0.01 vs. H group.