Lipoxin A4 Ameliorates Lung Development in Neonatal Rats with Hyperoxia-induced Bronchopulmonary Dysplasia by Reducing Mitochondrial Fission

doi:10.21203/rs.3.rs-960380/v1

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Lipoxin A4 Ameliorates Lung Development in Neonatal Rats with Hyperoxia-induced Bronchopulmonary Dysplasia by Reducing Mitochondrial Fission

https://doi.org/10.21203/rs.3.rs-960380/v1

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Bronchopulmonary dysplasia (BPD) is a common chronic pulmonary disease in premature infants. Hyper oxygen exposure and mechanical ventilation are common causes. The pulmonary pathological changes of BPD were mainly micro alveolar simplification and pulmonary vascular development disorder. Lipoxin A4(LXA4) is an important endogenous lipid and anti-inflammatory mediator that promotes the regression of inflammation and is referred to as the brake signal of inflammatory response. The purpose of this study was to evaluate the intervention of LXA4 on pulmonary inflammation and oxidative stress in neonates with hyperoxia lung injury and to explore the mechanism of Drp1 and NLRP3 inflammasome. A BPD model was established 7 days after neonatal rats were exposed to 85% oxygen. Rats were divided in four groups: control, hyperoxia, hyperoxia + LXA4 and hyperoxia+LXA4+Mdivi-1. Lung injury was evaluated via histological examination; lung development was assessed using morphometric analyses. Pulmonary micro vessels were tested by vWF-positive staining and Evans Blue. Expression of Drp1, NF-κB, and NLRP3 was analyzed via western blot or immunohistochemistry. Dihydroethidium (DHE) levels was examined to determine oxidative stress in vitro. We found that LXA4 significantly reduced lung injury of rats, simplified alveoli, and promoted weight gain. In addition, we found that LXA4 can reduce the protein expression of pDrp1, pNF-κB and NLRP3. Therefore, we concluded that LXA4 intervention can reduce the inflammatory response in the lungs and reduce the degree of lung injury, and through the role of NLRP3 inflammatory chrome in the hypertoxic rat BPD model.

Health Economics & Outcomes Research

Health Policy

Pulmonology

Bronchopulmonary dysplasia

Lipoxin A4

Mitochondrial fission

Drp1

Oxidative stress

Bronchopulmonary dysplasia (BPD) is a common complication in premature infants, which seriously affects their survival rate and quality of life. Although in recent years, with continuous improvements in neonatal intensive care unit (NICU) care, very low birth weight (lower than 1500 g) and ultra-low birth weight (lower than 1000 g) infants have shown an improvement in survival rates, the incidence of BPD has also increased significantly [1]. Lung injury is caused by various factors during the perinatal period. In the process of lung injury and repair, lung development inhibition and pulmonary fibrosis may be the main causes of BPD[2]. The main pathological condition of BPD is pulmonary stagnation, including alveolar structure simplification and pulmonary microvasculature dysplasia, which seriously affect the physiological functions of the lung [3, 4]. Oxidative stress, inflammation, and other factors cause pulmonary development disorders, which manifest as alveolar simplification and pulmonary microvasculature reduction, leading to the emergence of a “new BPD” [5, 6]. Abnormal mitochondria and mitochondrial dysfunction are the basis of the pathological mechanism underlying several lung diseases, including, but not limited to chronic obstructive pulmonary disease (COPD), asthma, and lung cancer [7]. High levels of oxygen cause oxidative stress through generation of reactive oxygen species (ROS) in lung tissue, which activates dynein-associated protein 1 (Drp1) mediated mitochondrial fission [8]. Change in mitochondrial dynamics results in abnormal mitochondrial function and structure, leading to mitochondrial apoptosis. Therefore, we aimed to analyze abnormal mitochondrial structure and dysfunction in BPD, its occurrence, and development.

Drp1 is a factor that mediates mitochondrial fission[9]. Mitochondria are in dynamic equilibrium, constantly connecting (mitochondrial fusion) and separating themselves (mitochondrial fission). Fusion, fission, and motility are non-classical functions of mitochondria and are collectively termed mitochondrial dynamics [10, 11, 12]. Studies have shown that abnormal changes in mitochondrial dynamics may lead to disrupted mitochondrial structure and dysfunction, which are closely related to the occurrence and development of lung disease like COPD[13] and pulmonary arterial hypertension [14].

The regulation of mitochondrial ROS (mtROS) is a double-edged sword [15]. Studies have shown that moderate levels of mtROS can protect cells from various cellular stresses, but the continuous production of mtROS can lead to mitochondrial dysfunction and the oxidation of DNA, proteins, and lipids [16]. Mitochondrial dysfunction resulting because of defects in dynamics leads to increased ROS production, which may further activate the apoptotic pathway in mitochondria [17, 18]. In addition, ROS may be a modulator of mitochondrial dynamics. Studies have found that increased levels of mtROS in Alzheimer's disease models may result in mitochondrial shortening and Drp1 activation [19].

Lipoxin A4 (LXA4) is an important endogenous anti-inflammatory lipid mediator, which regulates the function of a variety of inflammatory cells and the expression of inflammation-related genes, and promotes the timely resolution of the inflammatory response [20]. In the anti-inflammatory process, LXA4 helps catalyze the synthesis of arachidonic acid by different lipoxygenases (lipoxygenase LO) through a transcellular pathway[21] and binds to the corresponding cell membrane receptors to attenuate inflammation [22]. However, research related to LXA4 intervention in BPD is still in its nascent stages.

Based on the above-mentioned research, we aimed to study the role and signaling pathways associated with LXA4 and the relationship between hyperoxia-induced inflammation and mitochondrial dynamics. We observed that LXA4 significantly inhibits the translocation of Drp1 to mitochondria, thereby regulating changes in mitochondrial dynamics and mtROS production, and acts as a regulator of NLRP3 inflammasome activation and NF-κB signaling. We further analyzed whether LXA4 can reduce hyperoxia-induced lung injury in neonatal rats by reducing the expression of Drp1.

Animals and BPD model

All animals were provided by the Animal Center of the Chinese Academy of Science (Shanghai, China) and were kept in the laboratory animal room of Wenzhou Medical University. All animal studies were in accordance with the guidelines of the animal experiment manual of Wenzhou Medical University (ethic number: wydw 2014-0058). Sprague-Dawley (SD) rats were maintained in the experimental room at a constant temperature and humidity with 12 h light-dark cycles, and were allowed food and water ad libitum. After mating and 21 days of pregnancy, the rats naturally gave birth to pups. These pups were combined and randomly assigned to the nursing rats for 6 hours. Pups were then randomly divided into four groups: control, hyperoxia, hyperoxia+LXA4(H+LXA4), and hyperoxia+LXA4+Mdivi-1(H+LXA4+Mdivi-1). LXA4 (2 ng/g) (sigma-Aldrich, USA) was administered intraperitoneally (i.p.) to the pups for a total of four days on postnatal days 0, 2, 4, 6 and Mdivi-1(25mg/kg)(MCE, USA) was also intraperitoneally to the pups every day from days 0-7. LXA4 and Mdivi-1 dosages were chosen according to previous studies and our previous experiments. Every pup was given the same volume of saline.

For the BPD model, it was based on the study of hyperoxia groups were placed in airtight plexiglass boxes, given 80-85% oxygen from postnatal day 0 to 7. Rats in the control group were placed in room air. An airtight organic glass box with an inlet pipe and air flow, and placed in the box at the top of the oxygen analyzer to detect oxygen concentration inside the box. The box was placed in the room at 22-27 °C. The box was opened every 24 h and used to replace the feeding mother rats from the hyperoxia group to the control group to prevent oxygen poisoning, and to change the bedding and add water and feed.

Lung tissue and histological examination

Five pups were randomly selected from each group on postnatal days 7. The rats were anesthetized with 1% pentobarbital by intraperitoneal injection. The left lung was perfused and fixed with 4% paraformaldehyde (PFA). A soft indwelling needle was used to infuse the PFA at 20 cm H2O pressure for 1 min to ensure infusion into each alveolus. The tissues were then stored in PFA at 4 °C for 48 h, embedded in paraffin, sliced into 4 μm-thick sections, and prepared for hematoxylin and eosin (HE) (Solarbio, Beijing, China). The radical alveolar counts (RAC), mean alveolar diameter (MAD) and mean linear intercept (MLI) were evaluated lung development. Five sections were taken from each group at P7 by HE staining, and 10 fields in each group were randomly selected for photographing under a 100-fold microscope (Ni-U; Nikon, Tokyo, Japan); All images were acquired at 100× magnification and were assessed by investigators blinded to the experimental groups.

Pulmonary capillary permeability test

Five pups from different groups were selected for the Evans blue experiment to analyze the permeability of lung capillaries. On the 7th day, all pups were anesthetized and 3% Evans blue dye(Solarbio, Beijing, China) was injected into the femoral vein. The mice were allowed to rest on a warm pad for 1 h to allow the dye to flow through the circulatory system and reach the whole body. Next, the mice were anesthetized and the thoracic cavity was opened to infuse pre-cooled saline into the left heart to cleanly infuse excess dye in the circulatory system. The lung was carefully removed to analyze the lung tissue and take images for comparison. The lung tissue was homogenized in solution according to the manufacturer’s instructions and the OD values were obtained at 610nm. The value of residual dye was measured by comparison with standard curves.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

An apoptosis kit(Roche, USA) was used and manufacturer instructions were followed. After embedding in paraffin, sections of lung tissues were obtained. After deparaffinization and 20 µg/ml DNase-free proteinase K (10 mg/ml)( ST533, Beyotime, Beijing, China) treatment, 0.5% Triton-X in PBS was added to each tissue, the sections were washed three times with PBS; filter paper was used to carefully absorb excess water around the tissue, 50 µl of TUNEL reaction mixture was dripped on each lung tissue and they were incubated at 37°C in the dark for 1 h. Samples were then washed thrice with PBS, and were stained by DAPI. The sections were mounted and analyzed using a fluorescence microscope(Ni-U; Nikon, Tokyo, Japan).

Immunohistochemical and immunofluorescence staining

Seven days after hyperoxia, five rats from each group were sacrificed for sections. The sections were selected for immunohistochemical staining. The lung sections were deparaffinized in citrate buffer for 10 min at 96℃, then were treated with 3% H2O2 for 30 min and BSA for 1 h at 37℃. The sections were incubated with the following antibodies overnight at 4℃: NLRP3 (1:1000, DF7438; Affinity Biosciences, Cincinnati, OH,USA) They were then treated with horseradish peroxidase-conjugated donkey-goat secondary antibody (1:1000, Sc-2020, Santa Cruz Biotechnology) at 37 °C for 45 min. Finally, the sections were treated with 3,3-diaminobenzidine. Images were captured using a microscope (Ni-U; Nikon, Tokyo, Japan).

Five sections in each group were chosen for the immunofluorescence staining. The lung sections were deparaffinized in citrate buffer for 10 min at 96℃, then were treated with BSA for 1 h at 37℃. The sections were incubated with the following antibodies overnight at 4℃: pDrp-1(1:200, AF8470; Affinity Biosciences, Cincinnati, OH,USA), vWF(1:200, AF3000; Affinity Biosciences, Cincinnati, OH,USA). the next were then treated with secondary antibody while for negative controls PBS was used in the place of the antibody. The sections were then incubated with secondary antibody, Alexa Fluor-594donkey anti-rabbit IgG (1:500, ab150076; Abcam) for 4 h at room temperature, followed by DAPI incubation for nuclear staining. Images were captured using a microscope (Ni-U; Nikon, Tokyo, Japan).

Western blot

The BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to measure protein levels. Forty micrograms of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V for 1 h and transferred to polyvinylidene difluoride (PVDF) membranes at 200 A for 1 h. Membranes were then blocked in 5% skimmed milk dissolved in Tris-buffered saline and polysorbate 20 (TBST) for 3 h at room temperature, with shaking. Then, they were incubated with primary antibodies such as Bax (1:500, 50599–2-Ig; Proteintech), B-cell lymphoma-2 (Bcl-2; 1:1000, #2876; Cell Signaling Technology), cleaved-caspase-3 (1:1000, #9664; Cell Signaling Technology, Danvers, MA,USA), NLRP3(1:1000, DF7438; Affinity Biosciences, Cincinnati, OH,USA), ASC(PYCARD,1:1000, DF6304; Affinity Biosciences, Cincinnati, OH,USA), Cleaved-Caspase 1(1:1000, 22915-1-AP; Proteintech), Drp1(1:1000, DF7037; Affinity Biosciences, Cincinnati, OH,USA), pDrp1(se616) (1:1000, AF8470; Affinity Biosciences, Cincinnati, OH,USA), NF-κB(1:1000, AF5006; Affinity Biosciences, Cincinnati, OH,USA), pNF-κB(1:1000, AF2006; Affinity Biosciences, Cincinnati, OH,USA) ,and β-actin(1:1000, AF7018; Affinity Biosciences, Cincinnati, OH,USA) overnight at 4 °C. The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody for 2 h after washing three times with TBST, and developed using enhanced chemiluminescence reagents (Thermo Scientific Pierce; Thermo Fisher Scientific, Waltham, MA, USA). Densitometry values were detected for all bands and standardized relative to β-actin for each sample. The protein bands were detected using Pierce™ ECL western blotting substrate (Thermo Fisher Scientific, Logan, USA) and visualized using the ChemiDoc XRS+ Imaging System (Bio-Rad, Berkeley, USA).

Cell culture and cell counting assays

Pulmonary microvascular endothelial cells (PMVECs) were purchased from Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in MCE supplemented with 10% FBS (both from Gibco, Grand Island, NY, USA), in a humidified incubator(5% CO2, 37°C). The LXA4 drug concentrations used were 50 and 100 ng/ml. The cells in the hyperoxia group were placed in an incubator with hyperoxia conditions and an oxygen concentration of 95%.

To assess whether LXA4 can attenuate hyperoxia-induced cell death, we evaluated the viability of PMVECs using Cell Counting Kit-8 assays (CCK8, Beyotime, China), as per the manufacturer's instructions. Cells were plated in 96-well plates at a density of 1 x 10⁵ cells/well. After 24 h, 10 μl of CCK-8 solution was added to each well, and the cells were incubated for 4 h at 37°C in the spectrophotometer. OD values were measured at 490 nm, and the results were expressed as the percentage of live cells relative to the number observed in the control group.

Assessment of oxidative stress

The level of cellular oxidative stress was analyzed using the DHE staining kit(Beyotime, China). PMVECs were seeded in a 6-well plate, hyperoxia conditions were applied, and drugs were administered. Next, the cells were treated with the DHE dye for 15 min, and the cells were observed using a fluorescence microscope.

Immunofluorescence staining of cells

PMVECs were seeded in a 12-well plate; a circular slide was placed in the well, and after subjecting cells to hyperoxia and drug intervention, they were washed thrice with PBS. Paraformaldehyde (PFA; 10%) was added for fixation. Cells were again washed thrice with PBS, 10% goat serum was added, and the samples were blocked for 1 h. Tweezers were used to carefully remove the cell climbing slide, which was placed on a glass slide, and cells were incubated in a refrigerator at 4°C with pDrp1(se616) (1:200, AF8470; Affinity Biosciences, Cincinnati, OH,USA),and NLRP3(1:1000, DF7438; Affinity Biosciences, Cincinnati, OH,USA) overnight. The next day, the slide was washed with PBS and incubated with a fluorescent secondary antibody in the dark for 1 h. After washing with PBS, an anti-fluorescence quencher containing DAPI was added dropwise, the slide was covered with a cover glass, and observed using a fluorescence microscope.

Statistical analysis

The experiments were performed in triplicate and repeated at least three times. The data are presented as the mean ± SD or SEM and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (equal variance) or Dunnett T3's post-hoc test (unequal variance) for multiple comparisons. Correlation analyses were performed using Spearman's rank correlation. Statistical analysis was carried out using SPSS Statistics 19.0 (SPSS Inc., Chicago, USA) or GraphPad 6.0 (GraphPad Software, San Diego, USA). Values of P<0.05 were considered statistically significant.

LXA4 improved alveolar development in rats

From the results shown in Figure 1, the alveoli of the normal group showed clear secondary separation on the 7th day, with complete alveolar structure, uniform size, and thin alveolar spacing. In the hyperoxia-treated group, the number of alveoli and secondary separation was significantly reduced. The number of alveoli in the H+LXA4 and H+LXA4+Mdivi-1 groups were higher than that in the hyperoxia group, and the compartments were not significantly thickened (Fig1 A). The RAC value of the H+LXA4 and the H+LXA4+Mdivi-1 groups was lower than that of the control group and higher than that of the hyperoxia group, indicating that LXA4 may improve lung development induced by hyperoxia (Fig1 C). Comparison of the various groups (Fig1 D) showed that the MLI of the H group was significantly higher than that of the other three groups, indicating that the inner alveolar diameter increased after hyperoxia stimulation, and the structure was simplified. The alveolar simplification was improved after LXA4 intervention.

The comparison of the MAD for the three groups showed that the development of the pulmonary artery in the H group was blocked, and the pulmonary artery diameter in the LXA group was significantly improved relative to the H group. Lung damage improved after using an inhibitor (Fig1 E). These results indicate that LXA4 can improve hypertoxic lung injury.

LXA4 could improve hyperoxia-induced pulmonary vascular reduction and pulmonary capillary permeability

The results shown in Figure 2 indicate that hyperoxia leads to a decrease in the number of small pulmonary arteries, which can be rescued using LXA4 and an inhibitor. The vWF-positive small vessels were stained using immunofluorescence staining and counted (Fig2 A, C). Hyperoxia conditions increased the permeability of pulmonary capillaries. Evans blue staining showed that darker color was associated with greater permeability abrogation. After the use of LXA4 and inhibitor, the vascular permeability was improved under high oxygen conditions (Fig2 B, D).

LXA4 attenuates hyperoxia-induced lung tissue apoptosis

We used the TUNEL assay to analyze apoptosis in lung cells. The results showed that hyperoxia caused a significant increase in lung tissue apoptosis, and LXA4 intervention effectively alleviated the apoptosis induced by hyperoxia. The use of inhibitors had the same effect (Fig3 A). We also used western blotting to analyze the expression of three apoptosis-related proteins, Bcl-2, Bax, and cleaved-caspase3, and found that hyperoxia induced significant changes in apoptosis-related proteins of lung tissue, but these proteins reduced lung apoptosis after intervention with LXA4 and an inhibitor (Fig 3 B-H).

LXA4 improved mitochondrial fission caused by hyperoxia and alleviated the activation of the NLRP3 inflammasome

We analyzed mitochondrial fission using the phosphorylation level of the Drp-1 protein as a marker. As shown in Figures 4 and 5, the ratio of DRP-1/pDrp-1 increased after hyperoxia stimulation, and the phenotype improved after LXA4 intervention (Fig 4D). We analyzed the activation of NLRP3 inflammasome and NF-KB under hyperoxia-inducing conditions and after administering LXA4 intervention using western blotting (Fig 4 A-C and E). The results showed that hyperoxia stimulation could activate NLRP3 bodies and increased the expression of the ASC and Casp-1 proteins. In addition, hyperoxia could induce a combination of inflammasomes. Further, we used fluorescence and immunohistochemical analysis for evaluating the expression changes of pDRP1 and NLRP3 bodies in lung tissues (Fig 5 A, B).

LXA4 alleviated hyperoxia-induced vascular endothelial cell activity and oxidative stress levels

We used the CCK8 assay to identify changes in activity under different conditions. We induced hyperoxia for varying durations (Fig 6A) and used different LXA4 concentrations to explore its protective effect on hyperoxia-damaged cells (Fig 6B). Based on the results, we selected two concentrations for further experiments. We used DHE dyes to analyze the oxidative stress level of different groups. Greater fluorescence levels were observed with increased oxidation levels in hyperoxia groups. We observed that LXA4 reduced the oxidative stress level induced by hyperoxia in vascular endothelial cells (Fig 6 C, D).

LXA4 could reduce the activation of mitochondrial fission and NLRP3 inflammasome

We used cellular immunofluorescence analysis to observe the expression of pDRP1 and NLRP3 after exposure to different hyperoxia conditions and LXA4 intervention (Fig7 A, B). Hyperoxia induced the phosphorylation of Drp1 and increased the expression of NLRP3. The expression of these two proteins was also increased after LXA4 intervention. We concluded that LXA4 may reduce mitochondrial fission in pulmonary vascular endothelial cells and the activation of NLRP3 bodies induced by hyperoxia.

Chronic inflammation and oxidative stress are common pathological changes associated with BPD[23], and abnormal lung inflammation is an important factor affecting alveolar development [24]. In this study, we analyzed the protective mechanism of LXA4 in alleviating oxidative stress in lung tissues. The protective effect of LXA4 on hypertoxic lung injury and its effects on lung inflammation, oxidative stress, and alveolar development were evaluated in rats. The results showed that abnormal alveolar development in rats exposed to hyperoxia conditions was associated with mitochondrial activity. Abnormal changes in mitochondrial dynamics are prevalent in a variety of pulmonary inflammatory diseases, and excessive mitochondrial proliferation has been proved to be related to hyperoxia injury [25, 26]. LXA4 is an endogenous anti-inflammatory compound with anti-inflammatory and antioxidant effects. Studies have shown that LXA4 can inhibit hyperoxia-induced lung inflammation [27, 28]. Previously, our group has shown that LXA4 can alleviate mitochondrial autophagy and inhibit ROS [29]. Therefore, we studied the mechanisms associated with LXA4 in combating the inflammatory response induced by hyperoxia, the activation of inflammasomes, and the division of mitochondria.

The neonatal rat hypertoxic lung injury model is widely used. Hyperoxia exposure can cause specific pathological changes, such as alveolar simplification, vascular malformations, and pulmonary fibrosis [30, 31, 32]. In our study, we showed that exposure to a hyperoxic environment delayed weight gain in newborn rats. H&E staining of lung tissue showed simplified alveolar development, indicating the establishment of a hyperoxic lung injury model. We evaluated the concentration of LXA4, and our results showed that 7 days after exposure to hyperoxia, body weight gain in rats was delayed and the alveoli were significantly simplified. The intervention using LXA4 reduced oxidative stress and inflammation, thereby improving alveolar development. RAC, MDA, and MLI are important indicators for evaluating alveolar growth. After intervention using LXA4, the number of and diameter of alveoli increased, indicating that LXA4 promotes the development of lungs and reduces growth disorders caused by hyperoxia. These data indicate that lipoxinA4 can reduce the severity of hyperoxic lung injury. Mdivi-1 is an inhibitor of Drp1, and Mdivi-1 intervention showed results similar to that of LXA4 intervention against hyperoxia lung injury.

In a previous study, we showed that LXA4 can reduce the phosphorylation level of IκB-α, inhibit the activation of NF-κB, and reduce the degree of the neuroinflammatory response [22]. NF-κB is associated with a variety of pathophysiological responses such as inflammation, oxidative stress, cell proliferation, and immunity. Excessive activation of these pathways may lead to the production of various pro-inflammatory cytokines and further aggravate tissue damage [33, 34, 35]. Our results indicate that hyperoxia causes NF-κB activation, whereas LXA4 intervention attenuates this activation. Studies have shown that hyperoxia induces NLRP3 activation. The NLRP3 inflammasome is extensively studied and is involved in the occurrence of a variety of chronic inflammations [36, 37]. NLRP3 protein polymerizes and combines with the ASC adaptor to induce the translocation and activation of caspase-1[38, 39]. Activated caspase-1 triggers the secretion of mature pro-inflammatory cytokines such as the IL-1 precursor, which is converted to active IL-1 and released to the extracellular environment to recruit inflammatory cells and activate the inflammatory response [40, 41, 42]. NLRP3 inflammasome activation can be classified into two stages. The first stage is induced by the activation of the NF-κB pathway, which initiates the transcription and translation of IL1β, IL-18, and NLRP3[43]. The second stage is related to the assembly of the NLRP3 inflammasome[44]. The activation of the NLRP3 inflammasome leads to the occurrence of BPD, mainly through the regulation of inflammation, pyrolysis, apoptosis, and fibrosis. Therefore, inhibition of NLRP3 inflammasome activation may serve as a new strategy for BPD treatment. Hyperoxia is a pro-inflammatory cytokine that promotes the activation of NLRP3 inflammasomes, such as IL-1β and IL-18, which is consistent with our experimental results. Our results suggest that LXA4 attenuates hyperoxia and induces inflammation by inhibiting the activation of the following substances. NF-κB and NLRP3 inflammasome are expressed in lung tissues.

ROS is a well-known factor that causes cell death and is related to mitochondrial division. Mitochondria are the main site for cellular aerobic respiration and play a key role in regulating pattern recognition and receptor signaling pathways, including the regulation of NF-κB and NLRP3 inflammasome activation [45]. Mitochondria are the main source of ROS. External stimulation promotes the production of ROS via mitochondria, which in turn stimulates signaling pathways and alerts the immune system. Mitochondrial function shows a decline, and the production of ROS increases, causing tissue and organ damage. In addition, mitochondria are also the main targets of ROS damage. Excessive ROS causes mitochondrial lysis and disrupts the dynamic homeostasis of mitochondria. Mitochondrial dynamics are related to ROS [46, 47]. Drp1 is an important mitochondrial fission-related factor and a key regulator of mitochondrial fission [48]. The expression of Drp1 in lung tissues in our study was significantly increased under hyperoxia conditions and decreased after intervention with LXA4. Studies have shown that mitochondrial fission mediated by Drp1 is an early phenomenon related to mitochondrial dysfunction and apoptosis. After mitochondrial breakage, ROS/mtROS production is enhanced [49, 50]. As a result of fission, cytochrome c leakage occurs through the mitochondrial permeability transition pore (mPTP), resulting in mitochondrial-dependent initiation of cardiomyocyte apoptosis [51]. However, the association between mitochondrial dynamics (especially fission) and lung epithelial cells undergoing apoptosis is unclear. Drp1 is also an important mediator of apoptosis. Inhibition of Drp1 via RNAi or using a dominant-negative mutant (DRP1K38A) may delay the occurrence of mitochondrial breakdown, cytochrome c release, caspase activation, and cell death [52]. We also studied the effect of LXA4 on primary rat lung microvascular endothelial cells under hyperoxic conditions. We showed that the expression of the Drp1 protein in primary rat lung microvascular endothelial cells decreased after hyperoxia exposure, and increased after LXA4 intervention.

Hyperoxia induces mitochondrial fission and mitochondrial oxidative stress, which in turn activates the NF-kB pathway and NLRP3 inflammasomes. Our results show that LXA4 can reduce mitochondrial fission to relieve inflammation caused by oxidative stress in the lungs. Thus, LXA4 alleviates hyperoxia-induced lung injury in rats by reducing mitochondrial fission. However, our study has limitation that we did not observe mitochondrial structure by electron microscopy. Our next study will focus on the mitochondrial structure

In summary, our study provides comprehensive evidence supporting that LXA4 had potential beneficial on BPD. And the potential protective mechanisms by reducing mitochondrial fission. LXA4 as could reduced the pulmonary inflammatory, lung cell apoptosis and attenuating cellar oxidative stress damage after hyperoxia. Our study provided a new mechanism with LXA4 against mitochondrial fission, providing potential evidence for LXA4 treatment for BPD

BPD, bronchopulmonary dysplasia; LXA4, Lipoxin A4; Drp1, dynamin-related protein 1; HE, hematoxylin-eosin; Mdivi-1, mitochondrial division inhibitor-1; vWF, von Willebrand Factor; PMVECs, Pulmonary microvascular endothelial cells

Authors’ contributions

Yu BY and Liang ZJ drafted the manuscript and designed this study. Zhu JJ and Pan BT helped create the figures and write parts of the manuscript. Wang XY and Huo R contributed to analyze the data. Chen SQ contributed to critical revisions of the manuscript.

Ethics approval and consent to participate

All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health and were approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University (approval code number: wydw2014–0058).

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This study was funded by the Natural Science Foundation of Zhejiang Province (no. LY21H040007).

Competing interests

The authors declare that they have no conflicts of interest.

Acknowledgements

Not applicable

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Lipoxin A4 Ameliorates Lung Development in Neonatal Rats with Hyperoxia-induced Bronchopulmonary Dysplasia by Reducing Mitochondrial Fission

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Abstract

Figures

Introduction

Materials And Methods

Animals and BPD model

Lung tissue and histological examination

Pulmonary capillary permeability test

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

Immunohistochemical and immunofluorescence staining

Western blot

Cell culture and cell counting assays

Assessment of oxidative stress

Immunofluorescence staining of cells

Statistical analysis

Result

LXA4 improved alveolar development in rats

LXA4 could improve hyperoxia-induced pulmonary vascular reduction and pulmonary capillary permeability

LXA4 attenuates hyperoxia-induced lung tissue apoptosis

LXA4 improved mitochondrial fission caused by hyperoxia and alleviated the activation of the NLRP3 inflammasome

LXA4 alleviated hyperoxia-induced vascular endothelial cell activity and oxidative stress levels

LXA4 could reduce the activation of mitochondrial fission and NLRP3 inflammasome

Discussion

Conclusion

Abbreviations

Declarations

Authors’ contributions

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Funding

Competing interests

Acknowledgements

References

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