Effect of montelukast on leukotriene B4 metabolism in asthma

doi:10.3969/j.issn.1674-8115.2023.05.008

Abstract

Abstract:

Objective ·To observe the effect of montelukast on the expressions of key genes in LTB4 (leukotriene B4) metabolic pathway in treating asthma and investigate the candidate intervene targets of asthma. Methods ·The acute, subacute, and chronic asthmatic mouse models characterizing by allergic airway disease (AAD) were set up by ovalbumin (OVA) and Al(OH)₃ sensitization and challenge and intervened by intragastric administration of montelukast and finally challenged by OVA for chronic asthma model. The pulmonary functions of mice were tested by unconstrained whole body plethysmograph, to quest the change patterns of airway hyperresponsiveness (AHR). The eosinophil (EOS) infiltration and goblet cell (GCL) hyperplasia in mouse lungs were detected by hematoxylin-eosin (HE) staining, to quest the pathologic features of airway allergic inflammation. The levels of immunoglobulin E (IgE), interferon γ (IFN-γ), and interleukin (IL) in bronchoalveolar lavage fluid (BALF) and serum were detected by ELISA and Milliplex kits, to quest the helper T cell type 2 (Th2) inflammation status. The transcription and protein levels of 5-lipoxygenase activating protein (ALOX5AP), leukotriene A4 hydrolase (LTA4H), and leukotriene B4 receptor 1 (BLT1) genes, which encoded the rate-limiting enzymes in LTB4 synthesis pathway, were detected by RT-qPCR, Western blotting and immunohistochemistry (IHC). Results ·The asthmatic mouse model could be set up by OVA and Al (OH)₃ and was presented as AHR characterized by increasing enhanced pause (Penh) value, eosinophilic inflammation and high mucous secretion pathologically characterized by airway EOS infiltration and GCL hyperplasia, Th2 inflammation immunologically characterized by the increasing levels of IgE, IL-4, and IL-13 as well as decreasing levels of IFN-γ, IL-2, and IL-12 in BALF and serum. Montelukast could alleviate AAD effectively. The transcription and protein levels of ALOX5AP, LTA4H, and BLT1 genes increased in asthma. Montelukast can inhibit the expression of ALOX5AP gene and promote the expressions of LTB4 and BLT1 genes in asthmatic chronic phase. When challenged by OVA once again, montelukast can induce the significantly high expressions of LTB4 and BLT1 genes. Conclusion ·Montelukast has the effect of relieving allergic inflammation in asthma mice, but it can stimulate the production and accumulation of LTB4 and is significant in chronic phase. When challenged by OVA a second time, LTB4 could be promoted to combine with BLT1 and attend in the pathogenesis of asthma. The results suggested that there was a potential risk of activation of LTB4 by montelukast. The rate-limiting enzyme LTA4H and its receptor BLT1 metabolism may be potential targets for asthma treatment.

Key words: asthma, 5-lipoxygenase activating protein (ALOX5AP), leukotriene A4 hydrolase (LTA4H), leukotriene B4 receptor, montelukast

CLC Number:

R562

HU Yu, XIE Liang, ZOU Dan, FU Hongling, LOU Lili, XIE Keqi, LIU Hanmin. Effect of montelukast on leukotriene B4 metabolism in asthma[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(5): 580-591.

Figures/Tables 9

Fig 1 Setup of mice model with asthma

Tab 1 List of primers

Primer Name	Direction	Sequence (5′→3′)
ALOX5AP	Forward	TGTCGGCTATCTGGGAGAGAGA
ALOX5AP	Reverse	ATCCGCTTGCCGAAGATGTA
LTA4H	Forward	TTGATTGGAACACCTGGCTCTA
LTA4H	Reverse	TGTCAGAGTCACGTCGTAATTGG
BLT1	Forward	GGCACTAAGACAGATTCAAGGATT
BLT1	Reverse	ACATGCCACCAGGAGAAGAAG
Actin	Forward	TGGCTCCTAGCACCATGAAGA
Actin	Reverse	GCCACCGATCCACACAGAGT

Fig 2 Penh values of different mice groups

Fig 3 HE staining and scores of mice lung samples

Fig 4 Expression levels of IgE and cytokines in BALF and serum of mice

Fig 5 Transcription and protein levels of ALOX5AP gene

Fig 6 Transcription and protein levels of LTA4H gene

Fig 7 Transcription and protein levels of BLT1 gene

Fig 8 Transcription and protein levels of ALOX5AP, LTA4H and BLT1 genes after re-challenged by OVA in asthma mice models

TrendMD

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