Omeprazole activates aryl hydrocarbon receptor to reduce hyperoxia-induced oxidative stress in the peripheral blood mononuclear cells from premature infants

Abstract Objective To investigate the correlation between the aryl hydrocarbon receptor (AhR) and reactive oxygen species (ROS) in peripheral blood mononuclear cells (PBMCs) of premature infants, to demonstrate the protective role of AhR against hyperoxia-induced oxidative stress in premature infants and to provide a rational basis for the use of omeprazole (OM) as a new treatment for bronchopulmonary dysplasia (BPD). Methods From January 2021 to June 2021, 1–3 ml of discarded peripheral blood was collected from premature infants of gestational age less than 32 weeks who were not taking inhaled oxygen and were admitted to the Department of Neonatology of the Affiliated Hospital of Southwest Medical University. Using a random number table, the PBMCs were randomly assigned to each of the following groups: the control group, air + OM group, hyperoxia group, and hyperoxia + OM group. After 48 h of in vitro modeling and culture, PBMCs and the culture medium of each group were collected. Immunofluorescence analysis was used to examine ROS levels in PBMCs. A full-spectrum spectrophotometer was used to examine malondialdehyde (MDA) levels in the culture medium. Enzyme-linked immunosorbent assay (ELISA) was used to examine monocyte chemotactic protein 1 (MCP-1) levels in culture medium. Immunofluorescence analysis was used to examine the intracellular localization of AhR. Western blotting was used to examine the expression level of AhR in PBMCs. Results Compared with those in the control group, the levels of ROS, MDA, and MCP-1 and the cytoplasm-nuclear translocation rate of AhR in the air + OM group did not change significantly (p > 0.05), but the expression level of AhR increased significantly (p < 0.05). The levels of ROS, MDA, and MCP-1 and the cytoplasm-nuclear translocation rate of AhR significantly increased in the hyperoxia group (p < 0.05), and the expression level of AhR was significantly reduced (p < 0.05). Compared with those in the hyperoxia group, the levels of ROS, MDA, and MCP-1 in the hyperoxia + OM group were significantly reduced (p < 0.05), and the cytoplasm-nuclear translocation rate of AhR and the expression level of AhR were significantly increased (p < 0.05), but did not reach the level of the control group (p < 0.05). Conclusion OM can activate AhR to inhibit hyperoxia-induced oxidative stress in the PBMCs from premature infants.


Background
With the development and promotion of neonatal life support technology and the widespread use of new medical equipment, such as ventilators, the survival rates of preterm infants have continued to increase, as has the risks of oxygen exposure to preterm infants [1].Exposure to high oxygen levels can induce the mitochondrial respiratory chain to generate a large amount of oxygen free radicals through the leakage mechanism, which are then transformed into reactive oxygen species (ROS) and lead to lipid peroxidation damage and the release of inflammatory factors, thereby inducing cell damage.Premature infants lack antioxidant response kinases, so they cannot effectively eliminate excessive ROS accumulation in the body.ROS can hinder alveolar development and pulmonary angiogenesis and eventually develop into bronchopulmonary dysplasia (BPD), which seriously affects the quality of life of premature infants [2][3][4].Because the pathogenesis of BPD is still unclear, there is a lack of specific effective treatment methods in clinical practice.
Aryl hydrocarbon receptor (AhR) is a basic helixloop-helix (bHLH) Per-Arnt-Sim homology domain (PAS) protein that belongs to the bHLH superfamily of transcription factors.AhR is highly expressed in human lung, thymus, kidney and liver tissue.AhR mainly regulates downstream genes, such as CYP1A1, CYP1A2, GST-a, NQO1, and ALDH, and participates in cell energy metabolism [5][6][7][8].Studies have shown that AhR plays an important role in maintaining the stability of body's environment [9]; AhR has an anti-inflammatory effect on smoking-induced lung inflammation [10].AhR has endogenous and exogenous ligands.Due to the different structures and properties of these ligands, different biological effects can occur after they bind with AhR [11].
Omeprazole (OM) is a proton pump inhibitor that is mainly used in the clinic to treat gastric ulcers, duodenal ulcers and other diseases [12].Its mechanism of action is to inhibit the function of H þ /K þ -ATP, selectively regulate parietal cell enzymes to inhibit gastric acid secretion, and reduce ROS, thereby alleviating oxidative stress damage in epithelial cells and reducing the inflammatory response induced by NF-jB and STAT3/6 [13][14][15][16].Studies have shown that the AhR agonist OM can inhibit hyperoxia-induced oxidative stress damage by activating AhR and upregulating AhR expression [17].
This study aims to investigate the correlation between AhR and ROS in the PBMCs from premature infants, to demonstrate the protective role of AhR with respect to hyperoxia-induced oxidative stress in premature infants and to provide a rational basis for the use of OM as a new treatment for BPD.

Research object
Forty premature infants of gestational age less than 32 weeks and without oxygen treatment were admitted to the Department of Neonatology of the Affiliated Hospital of Southwest Medical University from January 2021 to June 2021.The inclusion criteria were as follows: premature babies with gestational age <32 weeks and no oxygen; the exclusion criteria were as follows: congenital heart disease and other congenital diseases [18].There were no statistically significant differences in sex, gestational age, or birth weight among the premature infants in each group (Table 1).This study was approved by the Ethics Committee of the Affiliated Hospital of Southwest Medical University (KY2021026), and the family members agreed and signed an informed consent form.

Separation of PBMCs by Ficoll density gradient centrifugation
One to three milliliters of discarded peripheral blood was collected with the anticoagulant ethylenediaminetetraacetic acid (EDTA).The collected peripheral blood was thoroughly mixed with Hanks' balanced salt solution (HBSS) in equal proportions, and PBMCs were collected by Ficoll density gradient centrifugation.

Establishment of cell cultures and the hyperoxia model
PBMCs were collected and randomly assigned into one of the following four groups by the random number table method: control group, air þ OM group, hyperoxia group, and hyperoxia þ OM group.The control group was placed in a 5% CO 2 incubator at 37 � C for 48 h.In the air þ OM group, 5 lmol/L OM was added, and the cells were placed in a 5% CO 2 incubator for 48 h at 37 � C. In the hyperoxia group, the air mixer was fed CO 2 and O 2 mixed at a ratio of 5:95, and the cells were placed in a 5% CO 2 incubator for 48 h at 37 � C after 10 min of hyperoxia.After 48 h, an ML-IICB digital intelligent oxygen meter was used to examine the culture flask and discard samples with an oxygen concentration of <90%.In the hyperoxia þ OM group, 5 lmol/L OM was added and the modeling method was the same as that in the hyperoxia group.After the cells in each group were cultured in vitro for 48 h, the PBMCs and culture medium from each group were collected by centrifugation [18].

Examination of ROS levels in PBMCs by fluorescence microscopy
PBMCs were collected from each group, and the cell density was adjusted to 2 � 10 6 /ml.A total of 200 ll of the cell suspension was evenly spread on an adhered glass slide and fixed with 4% paraformaldehyde for 20 min at room temperature.In a dark environment, relevant solutions were prepared in strict accordance with the MitoSOX TM Red instructions.The cells were evenly coated with MitoSOX TM Red (400 ll), incubated at 37 � C for 30 min, incubated with DAPI at room temperature for 3 min, and mounted with 20 ll of antifluorescence quencher.A fluorescence microscope was used to examine the red fluorescence, and Image J analysis software was used to analyze and process the image, determine the average absorbance value and perform statistical analysis.

Examination of malondialdehyde (MDA) levels in culture medium by thiobarbituric acid colorimetry
As the final product of lipid peroxidation, MDA can condense with thiobarbituric acid and form a red product.The culture medium of each group was collected, and the level of MDA in the culture medium was examined by thiobarbituric acid colorimetry.The relevant reagents were prepared in strict accordance with the MDA kit instructions.The samples in each group were placed in a constant-temperature water bath at 95 � C for 80 min, the centrifuge tube was cooled with running water, the supernatant of each group was collected and transferred to a 96-well plate, and 200 ll of sample was added to each well.A fullspectrum spectrophotometer was used to measure the absorbance value of each well at 532 nm.The MDA level in plasma was calculated according to the MDA calculation formula.

Examination of monocyte chemotactic protein 1 (MCP-1) levels in culture medium by enzymelinked immunosorbent assay (ELISA)
After the MCP-1 kit was rewarmed at room temperature for 30 min, the ELISA plate was removed, 50 ll of standard solution was added to the blank wells in the order of the standards, 50 ll of distilled water was added to the blank control wells, and 40 ll of sample was added to the remaining wells.Then, 10 ll of biotin-labeled antibody was added.After sealing the plate with a sealing film, the plate was incubated at 37 � C for 30 min.After the plate was washed 5 times with the washing solution, 50 ll of enzyme label solution was added to the standard group and sample group, and the plate was sealed with sealing film, placed in a humid box and incubated at 37 � C for 1 h.After being washed 5 times with the washing solution, the color developing solution was added and incubated at 37 � C for 10 min, and 50 ll of stop solution was added.A full-spectrum spectrophotometer was used to measure the absorbance value of each well at 450 nm, and the MCP-1 level in the medium was calculated according to the standard curve.

Examination of the intracellular localization of AhR in PBMCs by fluorescence microscopy
PBMCs were collected from each group, and the cell density was adjusted to 2 � 10 6 /ml.Then, 200 ll of the cell suspension was evenly spread on the adhered glass slide, fixed with 4% paraformaldehyde at room temperature for 20 min, and incubated at room temperature with 0.3% Triton X-100 for 10 min.The cells were blocked with 3% bovine serum albumin at room temperature for 60 min.The cells were evenly covered with the primary antibody (50 ll of rabbit anti-human AhR antibody; 1:50) and incubated overnight at 4 � C in a humid chamber.Then, the attached glass slide was removed, and 50 ll rhodamine-labeled goat anti-rabbit IgG secondary antibody (1:50) was added.The cell layer was evenly covered and incubated at 37 � C in the dark for 60 min.DAPI was added and incubated at room temperature for 3 min, and 20 ll of anti-fluorescence quencher was used to seal the cover glass.Fluorescence microscopy was used to examine the intracellular localization of AhR.

Examination of the AhR protein expression levels in PBMCs by Western blotting
PBMCs from each group were collected and counted in parallel, and 0.1 ml of RIPA buffer was added per 10 6 cells.The samples were placed on ice for 10 min and then they were sonicated to lyse the cells.The supernatant was collected after centrifugation at 4 � C and 12,000 rpm for 5 min.The protein concentration was determined by the BCA method.The proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked with a blocking solution (5% skim milk powder in TBST) for 1 h.Rabbit anti-human AhR (1:1000) and rabbit anti-human GAPDH antibodies (1:10000) were added and incubated overnight at 4 � C. HRP-labeled goat anti-rabbit IgG (1:2000) was added, incubated at 37 � C for 1 h, and then washed 3 times with TBST for 10 min.The dark room was entered, excess luminescent agent was wiped off, and the film was exposed.Image J analysis software was used to analyze the gray value of the target band, and the results are expressed as AhR/GAPDH and were statistically analyzed.

Statistical analysis
SPSS 21.0 statistical software was used to perform statistical analysis of the data.Measurement data are expressed as the mean ± standard deviation (x±s).Oneway analysis of variance was used for comparisons between multiple groups, and the LSD-t test was used for pairwise comparisons between groups.p < 0.05 was considered statistically significant.Enumeration data are expressed as percentages (%).The chi-square test was used for comparisons among multiple groups, and p < 0.05 was considered statistically significant; pairwise comparisons between groups were performed using the chi-square test, and p < 0.009 was considered statistically significant.

OM inhibits hyperoxia-induced ROS generation in the PBMCs from premature infants
Immunofluorescence analysis was used to examine the amount of ROS produced by PBMCs, red fluorescence labeled ROS, and blue fluorescence labeled nuclei.The stronger the red fluorescence was, the more ROS were generated (Figure 1 1(B)).These results suggest that OM can inhibit hyperoxia-induced ROS generation in the PBMCs from premature infants.

OM inhibits hyperoxia-induced MDA production in the PBMCs from premature infants
Compared with the control group (0.99 ± 0.08 nmol/ml), the air þ OM group (1.06 ± 0.13 nmol/ml) had no significant changes in MDA levels (p > 0.05).Compared with those in the control group, MDA levels in the hyperoxia group increased significantly (2.19 ± 0.49 nmol/ml) (p < 0.05).Compared with those in the hyperoxia group, MDA levels in the hyperoxia þ OM group (1.52 ± 0.29 nmol/ml) were significantly reduced (p < 0.05), but the levels did not reach those of the control group (p < 0.05).These results suggest that OM can inhibit hyperoxia-induced MDA production in the PBMCs from premature infants (Figure 2).

OM inhibits hyperoxia-induced MCP-1 production in the PBMCs from premature infants
Compared with the control group (88.13 ± 3.81 pg/ml), the air þ OM group (98.18 ± 10.34 pg/ml) had no significant changes in MCP-1 levels (p > 0.05).Compared with those in the control group, MCP-1 levels in the hyperoxia group were significantly increased (133.52 ± 11.85 pg/ml) (p < 0.05).Compared with those in the hyperoxia group, MCP-1 levels in the hyperoxia þ OM group (114.84 ± 3.82 pg/ml) were significantly reduced (p < 0.05), but the levels did not reach those of the control group  (p < 0.05).OM inhibited hyperoxia-induced MCP-1 production in the PBMCs from premature infants (Figure 3).

OM increases the hyperoxia-induced cytoplasm-nuclear translocation of AhR in the PBMCs from premature infants
AhR is labeled with red fluorescence, and the nucleus is labeled with blue fluorescence, which combined forms purple.In the control group, a separated red halo was observed around the nucleus, suggesting that AhR did not undergo cytoplasm-nuclear translocation.In the hyperoxia group, there was no separated red aperture around the nuclei of PBMCs, and uniform red fluorescence was observed in the nucleus, indicating that AhR had undergone cytoplasm-nuclear translocation (Figure 4(A)).Compared with the control group (18.3%), the air þ OM group (17.2%) had no significant change in the AhR cytoplasm-nucleus translocation rate (p > 0.05).Compared with that in the control group, the AhR cytoplasm-nuclear translocation rate in the hyperoxia group (38.5%) was

OM promotes AhR protein expression in the PBMCs from premature infants
Compared with that in the control group (0.63 ± 0.02), the expression of AhR protein in the air þ OM group (0.83 ± 0.02) did not change significantly (p > 0.05).Compared with that in the control group, the protein expression of AhR in the hyperoxia group was significantly reduced (0.23 ± 0.05) (p < 0.05).Compared with that in the hyperoxia group, the protein expression of AhR in the hyperoxia þ OM group (0.47 ± 0.05) was significantly increased (p < 0.05), but the level did not reach the level of the control group (p < 0.05).OM upregulated hyperoxia-induced AhR expression in the PBMCs from premature infants (Figure 5(A,B)).

Discussion
BPD is a common type of chronic lung disease that is mainly characterized by alveolar simplification and pulmonary angiogenesis.BPD is characterized by reduced alveolar numbers, enlarged alveoli, simplified alveolar structure, abnormal pulmonary microvascular morphology and sustained inflammation [19][20][21][22][23][24].Oxidative stress is an important factor in the pathogenesis of BPD.Hyperoxia induces the production of large amounts of ROS, which in turn upregulate the expression of proapoptotic proteins, promote the opening of mitochondrial permeability transition pores, lead to lipid peroxidation damage of cell membranes, and even induce apoptosis in alveolar epithelial cells, resulting in long-term chronic lung disease (CLD).Inflammation plays an important role in the pathogenesis of BPD.Hyperoxia can induce the activation of NF-jB, proinflammatory cytokines, and chemotactic proteins, thereby inducing severe lung inflammation and further acute/chronic lung injury [25][26][27][28].Studies have shown that OM can inhibit oxidative stress and has anti-inflammatory effects by activating AhR, which is mainly related to the inhibition of ROS and MCP-1 production, in H441 cells [17、29-31].
In this study, our data show that hyperoxia induced ROS, MDA and MCP-1 generation in the PBMCs from premature infants.ROS, MDA and MCP-1 showed the same trend, which is consistent with previous research by our group.Interestingly, after OM administration, the levels of ROS, MDA and MCP-1 decreased significantly.These findings further demonstrate that OM can reduce the generation of oxygen free radicals in oxidative stress and that OM may reduce hyperoxiainduced oxidative stress damage and inflammatory damage in the PBMCs of preterm infants.AhR is located in the cytoplasm under normal conditions and binds to the heat shock protein 90 (HSP90) dimer and hepatitis B virus X protein-associated protein 2 (XAP2) to form a complex.When AhR is activated, AhR shuttles from the cytoplasm to the nucleus, forming a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT) and initiating the transcription of downstream genes, which are involved in cell energy metabolism [32][33][34].Interestingly, our data show that OM promotes the expression of AHR protein, but there is no obvious change in ROS, MDA, MCP-1 and cytoplasm-nuclear shuttling of AhR under normal conditions.However, OM can increase the cytoplasm-nuclear shuttling of AhR and activate AhR activity, thereby inhibiting the hyperoxia-induced generation of ROS, MDA and MCP-1, inhibiting oxidative stress and inducing antiinflammatory effects.In summary, OM can activate AhR to inhibit hyperoxia-induced oxidative stress in the PBMCs from premature infants.OM may become a therapeutic agent for the prevention and treatment of BPD in preterm infants.However, OM-mediated inhibition of oxidative stress and the associated specific anti-inflammatory signaling pathways and mechanisms need further experimental and clinical study in premature infants.

Figure 4 .
Figure 4. OM increases the cytoplasm-nuclear translocation of AhR induced by hyperoxia in PBMCs of premature infants.�P > 0.009vs control group; �p < 0.009 vs control group; $p < 0.009 vs hyperoxia group.
(A)).Compared with the control group (26.25 ± 2.86), the air þ OM group (22.33 ± 0.84) had no significant changes in ROS (p > 0.05).Compared with those in the control group, ROS levels in the hyperoxia group increased significantly (46.16 ± 6.59) (p < 0.05).Compared with those in the hyperoxia group, ROS levels in the hyperoxia þ OM group (32.54 ± 10.03) were significantly reduced (p < 0.05); however, the levels did not reach those in the control group (p < 0.05) (Figure