Metabolomics reveals pro-inflammatory effects of 12R-HETE and ALOX12B in maternal allergic asthma

Abstract

Approximately 10% of pregnant women worldwide suffer from asthma, significantly increasing risks like preterm birth, low birth weight, and gestational hypertension. Current diagnostic methods are limited in early detection, underscoring the need for more effective diagnostic tools and treatment strategies to improve maternal and fetal health. In this study, following house dust mites (HDM) stimulation, significant changes were observed in lung tissue morphology, lung function, and inflammation in both pregnant rats and their offspring. Hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) analysis revealed substantial differences in metabolite levels between the parent groups in both positive and negative ion modes. Additionally, significant variations in metabolite levels were observed between the offspring groups. Notably, targeted metabolomic analysis showed elevated levels of 12R-hydroxy-5,8,10,14-eicosatetraenoic acid (12R-HETE) in plasma and lung tissue of both the parental and offspring HDM model groups. Concurrently, arachidonate 12-lipoxygenase, 12R type (ALOX12B) expression was significantly increased in the lung tissue, particularly in bronchial epithelium, as indicated by immunohistochemistry (IHC). In bronchial epithelial cells (16HBE), overexpression of ALOX12B raised mRNA and protein levels of IL-33, IL-6, MUC5AC, and GM-CSF, while ALOX12B knockdown reduced these inflammatory factors. This indicates that the ALOX12B pathway plays a crucial role in the type 2 inflammatory response in asthma. In summary, HDM induces the accumulation of 12R-HETE by promoting ALOX12B expression, thereby exacerbating type 2 inflammatory reactions and contributing to the development of allergic asthma. Consequently, 12R-HETE or ALOX12B may serve as potential biomarkers for diagnosing pregnancy-related allergic asthma or as novel therapeutic targets.

HDM induces the accumulation of 12R-HETE by promoting ALOX12B expression, thereby exacerbating TH2-type inflammatory reactions and contributing to the development of allergic asthma. Consequently, 12R-HETE or ALOX12B may serve as potential biomarkers for diagnosing pregnancy-related allergic asthma or as novel

Introduction

Asthma is a chronic inflammatory disease characterized by airway inflammation, airway remodeling, and airway hyperresponsiveness (AHR). Clinical manifestations include wheezing, shortness of breath, chest tightness, and cough¹. Allergic asthma is the most common form of asthma, which can be triggered by allergens such as HDM^2,3. In recent years, the prevalence of allergic asthma has been on the rise globally, especially notably in children and pregnant women^4–6.

Clinical reports have shown that acute exacerbation of bronchial asthma in pregnant women during pregnancy can lead to an increased rate of adverse pregnancy outcomes and an increased rate of various complications⁷. Studies have indicated that compared to non-asthmatic mothers, uncontrolled asthma increases the risk of early-onset asthma in offspring, which is more pronounced if asthma is not controlled early in pregnancy⁸. Currently, some traditional clinical trials for asthma, such as quantitative sputum cell count and lung function tests, are not easily performed in specific populations⁹. Therefore, identifying early diagnostic biomarkers and tools to improve monitoring of airway dysfunction and inflammation through non-invasive methods is a key objective for successful management of pregnancy asthma.

Metabolomics, as a powerful tool, can characterize a large number of low-molecular-weight and low-concentration compounds, and infer the function of these metabolites and their related metabolic pathways. As a valuable tool for analyzing metabolites in complex substrates, metabolomics has demonstrated important value in metabolite screening and research¹⁰. HILIC is an emerging liquid chromatography (LC) separation mode that retains polar and ionic metabolites using a highly hydrophilic stationary phase, combined with a highly organic mobile phase compatible with mass spectrometry (MS), making HILIC widely used in metabolomics research¹¹. Several studies have shown that metabolomics has important applications in asthma research, with numerous biomarkers related to asthma immunity and inflammation identified through metabolomics technology, including tryptophan, arginine, oleic acid, and sphingosine-1-phosphate (S1P)^12–14. By analyzing changes in metabolites in asthma patients, metabolomics can reveal the pathogenic mechanisms of asthma, identify potential biomarkers, and provide a theoretical basis for the early diagnosis and risk prediction of asthma¹⁵.

Although progress has been made in metabolomics research in asthma, research on early biomarkers for maternal asthma during pregnancy and offspring maternal asthma is still relatively limited. In our previous studies, we established metabolomics and lipidomics technologies for samples such as maternal rat plasma and amniotic fluid during pregnancy, and found that exogenous compounds could lead to dysregulation of amniotic fluid lipid metabolism, affecting fetal growth and development¹⁶; subsequently, we established an allergic pregnancy asthma rat model and found that dysregulation of amniotic fluid lipids during pregnancy asthma could lead to abnormal fetal lung development and fetal growth restriction¹⁷.

The aim of this study is to identify common potential metabolic biomarkers in pregnant women with allergic asthma and their offspring with allergic diseases through metabolomics analysis, providing theoretical basis and foundation for the management of asthma during pregnancy, monitoring of allergic diseases in offspring, and the development of targeted drugs.

Results

Exposure of mother rats to HDM can induce airway hyperresponsiveness and airway inflammation

To detect the effect of HDM-induced asthma, several indicators such as pathological changes, lung function, and inflammatory factors were analyzed. Lung function results showed (Fig. 1B) that the HDM group of rats exhibited respiratory obstruction, characterized by a decrease in mid-respiratory rate and an increase in airway resistance. Hematoxylin and Eosin (HE) and Periodic Acid-Schiff (PAS) staining (Fig. 1C) revealed that, compared to the PBS group, the HDM group of rats had increased airway inflammation cell infiltration and mucin secretion. In addition, The results from qPCR (Fig. 1D) indicated an increase in the expression of IL-4, IL-5, IL-13, and MUC5AC mRNA in the lung tissue of the HDM group (p < 0.05). Furthermore, ELISA analysis (Fig. 1E) demonstrated elevated protein levels of IL-5 in the bronchoalveolar lavage fluid and increased IgE levels in the serum (p < 0.05). These results suggest that HDM can induce airway hyperresponsiveness and trigger type 2 inflammation in rats.

Fig. 1. Exposure of maternal rats to HDM can induce airway hyperresponsiveness and airway inflammation.

A Schematic diagram of parental rats modeling. B HDM-induced parental rats lung function changes (n = 6). C Lung inflammation in asthmatic rats induced by HDM HE, PAS staining (200×, scale bar: 100 μm) and inflammation score. D Expression of IL-4, IL-5, IL-13, MUC5AC mRNA in parental rat lung tissue (n = 6). E Levels of IL-5 in parental rat BALF and IgE in serum (n = 6). Mean ± SEM were used to describe the central tendency of each group data and estimate the precision of each mean. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Pregnancy exposure to HDM in rats increases the risk of allergic diseases in offspring

Exposure to environmental stimuli during the perinatal period can affect the offspring’s asthma, with HDM being an important factor in inducing allergic asthma. We also analyzed pathological changes, lung function, inflammatory factors and other indicators in young rats. Interestingly, similar results were observed in young rats: decreased breathing rate, increased airway resistance (Fig. 2B), increased inflammatory cell infiltration, and increased mucus secretion (Fig. 2C). The results from qPCR showed (Fig. 2E) that in the HDM+HDM group, the expression levels of IL-4, IL-5, IL-13 and MUC5AC mRNA increased (p < 0.05). The results of ELISA showed that the protein levels of IL-5 in bronchoalveolar lavage fluid and IgE in serum increased (p < 0.05) (Fig. 2D). The severity increased in the four groups: PBS+PBS, HDM+PBS, PBS+HDM, and HDM+HDM. These results indicate that exposure of pregnant rats to HDM increases the risk of their offspring developing allergic diseases.

Fig. 2. Increased risk of allergic disease in offspring of pregnant rats exposed to HDM.

A Schematic diagram of HDM-induced offspring rats. B Lung function changes in HDM-induced offspring rats (n = 6). C Lung inflammation in asthmatic rats induced by HDM HE, PAS staining (200×, scale bar: 100 μm) and inflammation score. D Expression of IL-4, IL-5, IL-13, MUC5AC mRNA in offspring rat lung tissue (n = 6). E Levels of IL-5 in offspring rat BALF and IgE in serum (n = 7). mean ± SEM were used to describe the central tendency of each group’s data and the precision of each mean estimate. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Metabolic dysregulation in asthmatic rats characterized by significantly Elevated 12R-HETE Levels

Non-targeted metabolomics analysis was employed to investigate the changes in plasma metabolites in asthmatic rats. All QC samples clustered at the center of the PCA score plot (Fig. S1), indicating that the analytical system was reliable, and therefore, the differences in metabolic profiles could reflect the compositional differences between the groups. An unsupervised PCA model was constructed, and the results demonstrated (Fig. 3A) that in both positive and negative ion modes, there was a clear separation between the PBS group and the HDM group, as well as between the PBS+PBS group and the HDM+HDM, HDM+PBS, and PBS+HDM groups.

Fig. 3. The levels of metabolites in the blood plasma of asthmatic rats induced by HDM in parent and offspring changed.

A PCA of plasma metabolome of parent and offspring rats (ESI+, ESI-), each symbol represents data from one sample (n = 6 or 9). B Volcano plot of metabolites in negative ion mode of parent rats: red represents upregulated differential metabolites, purple represents downregulated differential metabolites, metabolites with no difference are labeled in gray. C Analysis of 12R-HETE content and correlation in parent plasma and lung tissue (n = 6). D Analysis of 12R-HETE content and correlation in offspring plasma and lung tissue (n = 6). E Correlation analysis of 12R-HETE and IL-5/IgE in parent lung tissue (n = 7 or 10). F Correlation analysis of 12R-HETE and IL-5/IgE in offspring lung tissue (n = 7 or 10). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Based on the screening criteria of p < 0.05 and Fold Change≥1.5 or ≤0.667, we found 52 and 75 significantly changed metabolites in the positive ion mode in parent and offspring, respectively, and 42 and 57 significantly changed metabolites in parent and offspring in the negative ion mode (Tables S1–S4). It is worth noting that a significant change of 12R-HETE in the plasma of pregnant rats was found in the negative ion mode (Fig. 3B).

Subsequently, through targeted quantitative analysis of 12R-HETE levels in plasma and lung tissue, we found that the levels of 12R-HETE in the plasma and lung tissue of HDM-exposed parent rats significantly increased, with a clear positive correlation (Fig. 3C). Similarly, the levels of 12R-HETE in the plasma and lung tissue of HDM-exposed offspring rats also significantly increased, with a clear positive correlation (Fig. 3D). Figure 3E and Fig. S2 show a clear positive correlation between the changes in 12R-HETE in parent and offspring rats and type2 inflammation. These results indicate that exposure of pregnant mothers to HDM significantly alters the metabolites of both the mothers and their offspring, particularly the significant increase in 12R-HETE, which is closely related to type 2 inflammation.

Increased expression of ALOX12B in HDM-induced asthmatic rats and their offspring

The key enzyme in the 12R-HETE metabolic pathway is ALOX12B enzyme (12R-LOX)¹⁸. To further confirm that HDM increases the level of 12R-HETE by promoting ALOX12B enzyme expression in asthmatic rats, we determined the content of 12R-LOX in the lung tissues of parental and offspring rats. The results from qPCR indicated a notable increase in ALOX12B mRNA expression in the parental HDM group as well as in the offspring HDM+HDM group. (p < 0.05), while there was no significant change in mRNA in the HDM+PBS group and PBS+HDM group (Figs. 4A, and S3). WB analysis indicated that the protein expression of the ALOX12B enzyme correlated with its mRNA expression levels (Figs. 4B, and S3). In addition, IHC staining of lung tissue sections revealed that ALOX12B enzyme expression increased most significantly in the parental HDM group and offspring HDM+HDM group, particularly in bronchial epithelial cells (Fig. 4C). These results indicate that HDM stimulation promotes the expression of ALOX12B enzyme in both parental and offspring rats, especially in bronchial epithelial cells.

Fig. 4. Increased expression of ALOX12B in asthma rats induced by HDM.

A Changes in protein levels and quantitative analysis of 12R-LOX (ALOX12B enzyme) in parent (n = 3). B Changes in protein levels and quantitative analysis of 12R-LOX (ALOX12B enzyme) in offspring (n = 4). C Lung tissue section stained with ALOX12B enzyme antibody IHC (200×, scale bar: 100 μm). ^*p < 0.05, ^**p < 0.01.

Overexpression of ALOX12B promotes the accumulation of 12R-HETE and exacerbates type 2 inflammatory responses

To further investigate the role of ALOX12B enzyme and its metabolic product 12R-HETE in regulating TH2 inflammation in allergic asthma, we first quantitatively analyzed the levels of 12R-HETE in HDM-stimulated human bronchial epithelial cells (16HBE) and found a significant increase in 12R-HETE levels in 16HBE cells after HDM induction (p < 0.05) (Fig. 5A); Simultaneously, qPCR analysis revealed a significant upregulation of ALOX12B mRNA levels in the HDM group (p < 0.05) (Fig. 5B), aligning with the trends observed in in vivo experiments.

Fig. 5. Overexpression of ALOX12B promotes the accumulation of 12R-HETE and exacerbates type 2 inflammatory responses.

A 12R-HETE levels in 16HBE cells (n = 6). B ALOX12B levels in 16HBE cells (n = 3). C mRNA expression levels of ALOX12B upon overexpression (n = 3). D Protein expression levels of ALOX12B upon overexpression (n = 3). E mRNA expression levels of inflammatory factors following ALOX12B overexpression (n = 3). F Protein expression levels of inflammatory factors following ALOX12B overexpression (n = 7). G Cell viability after exogenous 12R-HETE treatment at various concentrations (n = 3). H Inflammatory cytokine RNA levels at various 12R-HETE concentrations (n = 3). I Changes in 12R-HETE levels after ALOX12B overexpression and HDM treatment (n = 5). Mean ± SD was used to describe the central tendency of each group’s data and the estimated precision of each mean. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Next, we conducted an overexpression experiment of ALOX12B in 16HBE cells, and the qPCR and WB analysis showed that both the mRNA and protein levels of ALOX12B exhibited an increasing trend (p < 0.05) (Figs. 5C, D, and S4); Targeted quantitative analysis of 16HBE cells overexpressing ALOX12B showed that the overexpression of ALOX12B significantly increased the level of 12R-HETE in the cells (p < 0.05) (Fig. 5G). This phenomenon also mimicked the significant accumulation of 12R-HETE when ALOX12B expression was elevated after HDM stimulation in vitro and in vivo experiments.

In addition, the overexpression of ALOX12B in the 16HBE cells also led to a significant increase in the levels of inflammatory factors GMCSF, IL-6, and IL-33 mRNA (p < 0.05) (Fig. 5E), with the protein levels showing a consistent trend with mRNA levels (p < 0.05) (Fig. 5F). These results indicate that following HDM stimulation, the upregulation of ALOX12B in airway epithelial cells leads to the accumulation of 12R-HETE, thereby promoting type 2 inflammatory response and subsequently facilitating the development of allergic asthma.

ALOX12B knockdown alleviates HDM-induced type 2 inflammatory response

To further validate the role of ALOX12B enzyme in regulating TH2-type inflammation in allergic asthma, we performed a gene knockdown experiment to inhibit the expression of ALOX12B in 16HBE cells for reverse verification. The results showed that after knocking down ALOX12B, the mRNA and protein levels of ALOX12B in 16HBE cells did exhibit a decreasing trend (p < 0.05) (Figs. 6A, B, and S5); The mRNA levels of inflammation-related factors GMCSF, IL-6, IL-33, and MUC5AC (Fig. 6C) as well as their protein levels (Fig. 6D) were significantly decreased (p < 0.05). These results suggest that ALOX12B plays a role in TH2 type inflammation in cells, and HDM can regulate the expression of Th2 inflammatory factors GMCSF, IL-6, IL-33, and the mucin molecule MUC5AC through the ALOX12B pathway.

Fig. 6. ALOX12B knockdown alleviates HDM-induced type 2 inflammatory response.

A Knockdown of ALOX12B mRNA expression (n = 6). B Knockdown of ALOX12B protein expression (n = 3). C Decreased mRNA expression levels of inflammatory factors after ALOX12B knockdown (n = 3). D Increased protein expression levels of inflammatory factors after overexpression of ALOX12B (n = 5). Data are presented as mean ± SEM to describe the central tendency of each group and the estimated precision of the mean. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Discussion

Based on a HDM-induced asthma model during pregnancy, to the best of our knowledge, this study systematically reveals for the first time the critical role of the ALOX12B/12R-HETE pathway in the pathogenesis of asthma in both maternal and offspring groups, driving a significant type 2 inflammatory response. Notably, consistent upregulation of the arachidonic acid metabolite 12R-HETE was observed in the plasma and lung tissues of HDM-exposed dams and their offspring, along with markedly increased ALOX12B expression, particularly in bronchial epithelial cells. These findings demonstrate that 12R-HETE and ALOX12B not only play key roles in amplifying allergic inflammation but also exhibit strong potential as novel molecular biomarkers for monitoring asthma during pregnancy. Their detectable presence in the systemic circulation further supports the feasibility of non-invasive assessment, offering a promising strategy for early diagnosis and intervention in gestational asthma.

12R-HETE is an important metabolite in the arachidonic acid (AA) metabolic pathway¹⁹. Currently, it is widely believed that AA can be metabolized by three different enzyme systems, namely cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYP) enzymes²⁰. Mammalian LOXs are traditionally divided into 5-, 8-, 12-, or 15-LOX based on the position of oxygen insertion in AA. In humans, there are two different 12-LOXs, platelet-type, also known as 12S-LOX (expressed by the ALOX12 gene); epidermal-type, known as 12R-LOX (expressed by the ALOX12B gene). Although both LOXs produce 12-HETE, the 12S-LOX enzyme produces 12S-HETE enantiomer, while the 12R-LOX enzyme almost exclusively produces the 12R-HETE enantiomer²¹.

AA and its metabolites have attracted extensive attention in cardiovascular diseases, carcinogenesis, and many inflammatory diseases such as asthma and arthritis, especially in the context of inflammatory diseases^22–24. In the ovalbumin-induced asthma model, ALOX5-deficient mice showed an inhibitory response to acetylcholine-induced airway hyperresponsiveness and impaired lung eosinophilic inflammation²⁵. Similarly, inhibition of 5-LOX production in macrophages improved monosodium urate crystal-induced gouty arthritis²⁶. Studies using knockout mice have shown that ALOX15 gene deletion results in inhibition of acetylcholine-induced airway hyperresponsiveness and prolonged survival time in a fungal-induced allergic asthma model²⁷. However, research on 12R-HETE and 12R-LOX (ALOX12B enzyme) is limited, mainly in some skin diseases such as ichthyosis and psoriasis^28,29. The research on 12R-HETE and 12R-LOX in allergic asthma and inflammation is scarce, with only one paper directly indicating the crucial role of 12R-LOX in the expression of mucin MUC5AC; the metabolism product of 12R-HETE can induce the expression of MUC5AC, activate the ERK signaling pathway, and promote the translocation of transcription factor Sp1¹⁸. In our study, we found that the expression of ALOX12B is significantly increased in HDM-sensitized pregnant asthmatic rats, and its metabolite 12R-HETE accumulates significantly in both the sensitized parent and offspring rats exposed to allergens. Further in vitro and in vivo experiments demonstrate that HDM promotes the generation of 12R-HETE through the ALOX12B pathway, enhancing the expression of Th2 inflammatory factors GMCSF, IL-6, IL-33, and mucin molecule MUC5AC.

Asthma is closely related to Th2 cytokines, which play an important role in the pathogenesis of asthma, especially in allergic asthma³⁰. Th2 cells, as helper T cells, secrete cytokines IL-4, IL-5, and IL-13 that can drive and maintain chronic airway inflammation³¹. In addition, factors secreted by epithelial cells also play important roles in the pathogenesis of asthma. For example, IL-33 can activate Th2 cells and other inflammatory cells³², IL-6 has various pro-inflammatory effects in airway inflammation³³, MUC5AC is a mucin associated with excessive mucus secretion in the airways³⁴, and GM-CSF can induce the recruitment, accumulation, activation, and migration of eosinophils³⁵. These factors act together to exacerbate airway inflammation and remodeling. Our in vitro and in vivo studies suggest that ALOX12B and its metabolite 12R-HETE play pro-inflammatory and airway remodeling-promoting roles in the development of house dust mite-induced allergic asthma.

In conclusion, through multi-dimensional metabolomics analysis of the mother and offspring, we found that 12R-HETE may be an important potential metabolic biomarker in HDM-induced maternal allergic asthma. Further research revealed that HDM increases the level of 12R-HETE by regulating the expression of the key enzyme 12R-LOX, thereby exacerbating the type 2 inflammatory response in allergic asthma. In the future, we can further verify the role of ALOX12B or 12R-HETE in maternal allergic asthma by conducting more in-depth animal experiments, such as using ALOX12B gene knockout animals or supplementing animals with extra 12R-HETE, as well as collecting clinical samples for testing and analysis to explore the association of 12R-HETE with other related metabolic pathways in asthma. In conclusion, this study provides a new perspective on the pathogenesis of allergic asthma and offers potential new targets for early clinical diagnosis and intervention.

Materials and methods

Materials and reagents

The main reagents used are: House Dust Mite (Greer, USA), Acetyl-β-methylcholine chloride (Sigma, USA), Methanol, Acetonitrile (Merck, Germany), Formic acid, Isopropanol, Ammonium formate and Ammonium acetate, all of which are of 99.8% mass spectrometry purity (ROE, USA), Methyl Tert-Butyl Ether (MTBE, ROE, USA), 12R-Hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12R-HETE, MedBio, China).

AG RNAex Pro Reagent, SteadyPure RNA Extraction Kit (Accurate Biotechnology, China), Hifaire lll 1st Strand cDNA Synthesis, SYBR Green (Yeasen, China). RPMI 1640, Pancreatic enzyme, fetal calf serum (BasalMedia, China). Antibody ALOX12B (Cat:PK08394S, 1:1000, Abmart, China), Antibody β-actin (Cat:3700S, 1:1000, CST, America), Rat Interleukin-5 (IL-5) ELISA Kits, Rat Immunoglobulin E (IgE) ELISA Kits, Human Interleukin-33, Human MUC5AC (Elabscience, China), pcDNA3.1-ALOX12B (GENEWIZ, China), shRNA-ALOX12B (Abmart, China), Lipofectamine 3000 (Thermofisher, USA).

16HBE cells were donated to the Key Laboratory of Children’s Health and Traditional Chinese Medicine in Jiangsu Province.

Preparation of sensitizing solution and nasal drops

Dissolve the whole bottle of HDM (17.4 mg) in PBS to prepare a 10 mg/ml stock solution, and store it at 4 °C. Prepare the sensitizing solution for intraperitoneal injection at a concentration of 1 mg/ml, and the nasal drop solution at a concentration of 2 mg/ml.

Animals and HDM-induced bronchial asthma

We have complied with all relevant ethical regulations for animal use. 6-week-old female SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, with the batch number No.110011230102593818. All animal experiments were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (Ethics No. 202302A050). The animals were housed in cages at a temperature of 24–26 °C and a relative humidity of 55%, with free access to food and water. After 1 week of adaptation feeding, the rats were randomly divided into two groups, with 12 rats in each group, namely the control group (PBS group) and the sensitization group (HDM group). The sensitization group was intraperitoneally injected with sensitizing solution HDM (1 mg/ml) on days 0, 3, and 6. The control group was administered an equal volume of PBS injections. Mating was initiated 1 week after the final sensitization injection. The date of first observation of active sperm was designated as Gestation Day (GD) 0. On GD0, GD3, GD6, GD9, and GD12 of pregnancy, intranasal stimulation with HDM (2 μg/μl) was performed. Eight pregnant rats were assessed for lung function on GD16, On GD18, for in vitro experiments and prior to tissue collection, rats were administered pentobarbital as a deep anesthetic (80 mg/kg, i.p.). Subsequently, after anesthesia, blood, bronchoalveolar lavage fluid, and lung tissue samples were collected for analysis. The pathology tissues were fixed in 4% paraformaldehyde for histopathological evaluation. The blank control group received the same procedures (Fig. 1A).

Each group of 4 remaining pregnant rats gave natural birth. Each group retained 20 fetal rats, divided into offspring control group and offspring stimulation group. From the 21st day after birth, offspring were given HDM nasal drops for 6 consecutive days, 50 μl per day, while the offspring control group was switched to PBS nasal drops. After a 10-h fast, all fetal rats were tested for lung function on the 26th day after birth, and 28 days later, for in vitro experiments and prior to tissue collection, rats were administered pentobarbital as a deep anesthetic (80 mg/kg, i.p.). Subsequently, blood, lung lavage fluid, and lung tissue samples were collected (Fig. 2A).

Respiratory function tests

Through the use of a pulmonary function testing apparatus to monitor the airway reactivity of various groups of pregnant rats and young rats, aerosolized acetylcholine solutions of different mass concentrations (0, 12.50, 25.00, 50.00 mg/ml) were used to induce asthma. The enhanced pause (Penh) values of the various groups of rats and the maximum expiratory flow at 50% of the vital capacity (EF₅₀) under the stimulation of acetylcholine solutions of different mass concentrations were recorded, indicating an increase in airway reactivity. During the measurements, the rats were placed in a closed container, and their breathing caused changes in the pressure inside the container. The respiratory curves were obtained by monitoring the changes in pressure.

Real-time quantitative PCR (RT-qPCR)

Using RNA extraction kit to isolate total RNA from lung tissues of pregnant rats and young rats, the concentration and purity were determined. Subsequently, RNA was reverse transcribed into cDNA according to the manufacturer’s instructions. The RNA expression was detected using the QuantStudio 7 PCR system with SYBR Green Master Mix. Then, the relative expression level of the target gene was calculated using the comparative 2^-ΔΔCT method. The primers were obtained from Sangon Biotech, and their sequences are shown in the Table 1.

Table 1.

Sequences of the primers used

Human GAPDH	forward	5′-GGAGTCCACTGGCGTCTTCAC-3′
	reverse	5′-GCTGATGATCTTGAGGCTGTTGTC-3′
Human IL-33	forward	5′-TCAGGTGACGGTGTTGATGGTAAG-3′
	reverse	5′-CACAGAGTGTTCCTTGTTGTTGGC-3′
Human IL-6	forward	5′-GGTGTTGCCTGCTGCCTTCC-3′
	reverse	5′-GTTCTGAAGAGGTGAGTGGCTGTC-3′
Human MUC5AC	forward	5′-CCCGCCCACCTCTTCTACCC-3′
	reverse	5′-TGACCACCACGAGCCCATCC-3′
Human CCL20	forward	5′-GTTTGCTCCTGGCTGCTTTGATG-3′
	reverse	5′-AAGTTGCTTGCTGCTTCTGATTCG-3′
Human GMCSF	forward	5′-ACCTGAGTAGAGACACTGCTGCTG-3′
	reverse	5′-GCTCCAGGCGGGTCTGTAGG-3′
Human ALOX12B	forward	5′-AGTGGATGGATGGCTACGAGACC-3′
	reverse	5′-TCCTGCTTGGCTCTGATCTCCTC-3′
RAT β-actin	forward	5′-CACGATGGAGGGGCCGGACTCATC-3′
	reverse	5′-TAAAGACCTCTATGCCAACACAGT-3′
RAT IL-4	forward	5′-ACAAGGAACACCACGGAGAACG-3′
	reverse	5′-TCTTCAAGCACGGAGGTACATCAC-3′
RAT IL-5	forward	5′-GAGGATGCTTCTGTGCTTGAACG-3′
	reverse	5′-CATCGTCTCATTGCTCGTCAACAG-3′
RAT IL-13	forward	5′-TCGCTTGCCTTGGTGGTCTTG-3′
	reverse	5′-CTTCTGGTCTTGTGTGATGTTGCTC-3′
RAT MUC5AC	forward	5′-GGAGTGCCGTGCTGAGGATAAC-3′
	reverse	5′-CAGTGGAGGTTGGACAGTTGATGG-3′
RAT ALOX12B	forward	5′-GCTCCCAACTGTCGTGTCTACC-3′
	reverse	5′-TGGCTCTGATCTCTTCTTGTCTGTG-3′

Enzyme-linked immunosorbent assay (ELISA)

According to the manufacturer’s instructions, measure the expression levels of IgE in serum and IL-5 in bronchoalveolar lavage fluid using ELISA kits.

Non-targeted metabolomics analysis

Sample preparation: In brief, collect 20 mg of mouse lung tissue into a 2 mL centrifuge tube, add 200 μl of double distilled water, and then homogenize for 10 min using a ball mill. Take 20 μl of lung tissue homogenate or 20 μl of plasma, mix with 225 μl of cold methanol solution. After vortexing for 10 s, add 750 μl of MTBE, vortex again for 10 s, then shake at 4 °C for 10 min, followed by adding 188 μl of ultrapure water and vortexing for 20 s. After centrifugation at 18,000 rpm for 2 min at 4 °C, dry the lower layer solution (220 μl) in a vacuum centrifuge and reconstitute in 110 μl of acetonitrile water solvent (4:1, v/v) including internal standard CUDA at 1 μg/ml. Then vortex the samples for 10 min and proceed with sonication. Finally, take the supernatant for metabolomics analysis. Obtain QC samples by taking 15 μl from each sample and process them following the same method as the samples.

Chromatographic conditions: The untargeted metabolomics was performed on Dionex UItiMate 3000 UPLC system (Santa Clara, CA, USA) and Q Exactive HF MS instrument (Thermo Fisher Scientific, USA). Chromatographic separation was carried out using Waters ACQUITY UPLC BEH Amide (2.1 × 150 mm, 1.7 μm) at 45 °C. The mobile phase in both positive and negative ion modes consisted of the same mobile phases, A (H2O) and B (ACN: H2O, 95:5, v/v), with the addition of 10 mM AmF and 0.125% FA. The flow rate was set at 0.4 mL/min. The elution gradient was as follows: 0–2 min, 100% B; 2–7.7 min, linear decrease to 70% B; 7.7–9.5 min, linear decrease to 40% B; 9.5–10.25 min, linear decrease to 30% B; return to initial 100% B at 12.75 min and held until 17 min.

Mass spectrometry conditions: The mass spectrometer operates under both positive and negative ion modes with the same operating conditions. The mass spectrometry parameters are as follows: the scan range (m/z) is 60–900; the sheath gas flow rate is 60 arb; the auxiliary gas flow rate is 25 arb; the spray voltage is 3.6 kV in positive ion mode and 4.0 kV in negative ion mode; the capillary temperature is 300 °C; the auxiliary gas heater temperature is 370 °C. The resolution of MS2 spectra is 70,000, and the acquisition of MS-data is dependent on data-dependent scan mode with a resolution of 17,500. Fragmentation information of the top 10 precursor ions is used for data acquisition. Normalized collision energy (NCE) is set at 20, 40, and 60. Equally pooled samples are collected as quality control (QC) samples, and every 6 samples are analyzed together with one QC sample.

Targeted quantification of 12R-HETE

The preparation method of samples and control compounds is the same as non-target sample processing.

A Dionex UltiMate 3000 ultra-high performance liquid chromatography system (Santa Clara, CA, USA) coupled online via an electrospray ionization source (ESI) with a TSQ Vantage™ MS/MS instrument (Thermo Fisher Scientific, Waltham, MA, USA) was applied for targeted metabolism in plasma and lung.

The Mass spectrometry parameters for detection in negative ion mode were optimized as the follows: spray voltage 3200 V, vaporizer temperature, sheath gas pressure 45 bar, auxiliary gas pressure 25 bar, capillary temperature 350 °C, S-LensAmplitude, collision energy (CE). flow rate is 0.3 (mL/min), injection volume is 2 μL, and the run time is 3 min.

Cell culture

16HBE cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin, and cultured in a 5% CO₂ incubator at 37 °C. When the cells reached 70–80% confluence, passaging was performed using trypsin digestion.

Cell transfection

Seed 16HBE cells in a 6-well plate. Proceed with subsequent experiments when the cell confluence reaches 70% ~ 80%. According to the manufacturer’s instructions, transfect the plasmids pcDNA3.1 and ALOX12B overexpression plasmid into the cells using Lipofectamine 3000. The experiments were divided into a control group (Vector group) and an overexpression group (OE-ALOX12B group). Additionally, transfect cells with small interfering RNA targeting ALOX12B (shALOX12B). Cells infected with HDM (150 μg/ml) were subsequently transfected with shRNA, dividing into the control group (NC-shRNA group) and the knockdown group (ALOX12B-shRNA group). Gene expression was tested at the mRNA and protein levels 24 and 48 h after transfection, respectively.

Immunohistochemistry

The tissue embedded in paraffin was cut into 5 μm sections for IHC analysis. After deparaffinization and antigen retrieval, the tissue was blocked with 10% goat serum, then incubated overnight at 4 °C with rat ALOX12B antibody (1:1000), followed by incubation at room temperature for 1 h with goat anti-mouse antibody. The tissue sections were then detected with 3,3′-diaminobenzidine as the chromogen and counterstained with hematoxylin. Images were captured using a Zeiss Axio Examiner microscope.

Statistics and reproducibility

The original mass spectrometry files were compared for spectral information in the MS-DAIL 4.24 software to complete metabolite identification. The data was uploaded to the MetaboAnalyst 5.0 website (https://www.metaboanalyst.ca/) for data visualization analysis.

Using GraphPad Prism 8.0 software for statistical analysis, quantitative data were expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Differences between multiple groups were evaluated by one-way analysis of variance (One-Way ANOVA) test, with P < 0.05 considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, with * representing the experimental group compared to the control group.

Author contributions

L.W.: Validation, Software, Data curation, Writing – original draft. X.W.: Validation, Data curation, Visualization. W.W.: Validation, Visualization. Z.S.: Investigation. H.F.: Data curation. C.Q.: Validation. W.L.: Supervision. L.L.: Conceptualization. T.X.: Formal analysis. C.S.: Investigation. C.S.: Conceptualization. J.X.: Supervision, Funding acquisition, Writing – review & editing. F.G.: Funding acquisition. J.S.: Funding acquisition, Supervision, Writing – review & editing.

Peer review

Peer review information

Communications Biology thanks Shuhai Lin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Zheng-Jiang Zhu and Joao Valente.

Data availability

All source data used to produce the graphs in this work are provided in Supplementary Data. Uncropped blots/gels are provided in Supplementary Figs. 3-4. The sequence of the newly generated plasmid can be accessed via Addgene under the accession ID:247174. All other data are available upon reasonable request to the corresponding author.

Competing interests

The authors declare no competing interests.

Declaration of using tool

During the preparation of this work, the authors used ChatGPT4.0 in order to improve language and readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-025-08979-9.