Oleic acid attenuates asthma pathogenesis via Th1/Th2 immune cell modulation, TLR3/4-NF-κB-related inflammation suppression, and intrinsic apoptotic pathway induction

Abstract

WHO reported that asthma was responsible for 455,000 deaths in 2019 and asthma patients was evaluated 262 million in May 2023. The incidence is expected to increase as the average life expectancy increases, highlighting asthma as a significant health challenge in an aging society. The etiology of asthma is linked to an imbalance of Th1 and Th2 cells, respiratory inflammation, and pulmonary cell proliferation. The purpose of this study is to investigate the anti-asthmatic effect and potential mechanism of oleic acid. The anti-inflammatory effect of oleic acid was evaluated in an LPS-induced RAW 264.7 cell model, and immune modulation and the anti-apoptotic effect were measured in an ovalbumin-induced BALB/c mouse model. A variety of analytical procedures, such as MTT, qPCR, ELISA, Western blotting, immunofluorescence, gene transfection, immunohistochemistry, and several staining methods (Diff Quik, H&E, PAS), were used to evaluate the effectiveness and mechanisms of these methods. The results from in vitro experiments showed that oleic acid could reduce the levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β), and molecular docking studies suggested that oleic acid could interact with TLR3 and TLR4 proteins to form ligand-protein complexes, showing good binding affinity. Additionally, oleic acid attenuated the expression of MAPK pathway components (JNK, p38 MAPK) and NF-κB pathway constituents (IκB, NF-κB, COX-2, PGE₂). In vivo results indicated that oleic acid reduced the levels of inflammatory cells (WBCs and eosinophils) and IgE activity, reduced the expression of the Th2 cell transcription factor GATA-3, and decreased the levels of Th2/Th17-related cytokines (IL-4, TNF-α, and IL-6). Oleic acid also alleviated OVA-induced pathological changes in the lung, such as epithelial cell proliferation, inflammatory cell infiltration, and mucus hypersecretion. OVA restored apoptosis in lung epithelial cells by modulating the expression of Bcl-2 and Bax. In summary, oleic acid has potential as a novel candidate for asthma treatment through its ability to regulate immune cells, exert anti-inflammatory effects, and promote apoptosis, thereby ameliorating asthma manifestations.

Keywords: apoptosis; asthma; immune balance; inflammation; oleic acid.

Figure 1

Safety of oleic acid in RAW 264.7 cells and effect of oleic acid on inflammatory cytokines. (A) Cell viability was reduced in a dose-dependent manner by oleic acid treatment up to 100 μM; notably, 50 μM oleic acid treatment maintained approximately 80% cell viability. ^* p < 0.05 vs. 0 μM oleic acid treatment; ^** p < 0.001 vs. 0 μM oleic acid treatment; ^$ p < 0.05 vs. 1 μM oleic acid treatment; ^$$ p < 0.001 vs. 1 μM oleic acid treatment; ^# p < 0.05 vs. 5 μM oleic acid treatment. (B) Compared with the control treatment, LPS treatment increased cell viability to 130%, whereas oleic acid treatment decreased viability in a dose-dependent manner (n = 6). ^* p < 0.05 vs. 0 μM oleic acid; ^** p < 0.001 vs. 0 μM oleic acid; ^$ p < 0.05 vs. 10 ng/mL LPS; ^$$ p < 0.001 vs. 10 ng/mL LPS; ^# p < 0.05 vs. 1 μM oleic acid; ^## p < 0.001 vs. 1 μM oleic acid; ^@ p < 0.05 vs. 5 μM oleic acid; ^@@ p < 0.001 vs. 5 μM oleic acid; ^& p < 0.05 vs. 10 μM oleic acid. Oleic acid decreased the expression level of LPS-induced cytokines in RAW 264.7 cells in a concentration-dependent manner (n = 5). Cells were treated with 0-50 μM oleic acid for 2 h and then exposed to 10 ng/mL LPS for 24 h. (C) TNF-α levels were measured by ELISA. (D) TNF-α quantified by RT−PCR. (E) IL-6 levels were assessed by ELISA. (F) IL-6 levels were determined by RT−PCR. (G) IL-1β levels were analyzed by ELISA. (H) In RAW 264.7 cells, treatment with 25 μM or 50 μM oleic acid reduced NO production. ^** p < 0.001 vs. 0 μM oleic acid treatment; ^$ p < 0.05 vs. 10 ng/mL LPS treatment; ^$$ p < 0.001 vs. 10 ng/mL LPS treatment; ^# p < 0.05 vs. 5 μM oleic acid treatment; ^## p < 0.001 vs. 5 μM oleic acid treatment; ^@ p < 0.05 vs. 10 μM oleic acid treatment; ^@@ p < 0.001 vs. 10 μM oleic acid treatment; ^& p < 0.05 vs. 25 μM oleic acid treatment. The results are expressed as the mean ± standard deviation.

Figure 2

Effect of oleic acid on the MAPK signaling pathway. (A) Oleic acid reduced LPS-induced inflammation by inhibiting the activation of the JNK and p38 MAPK signaling pathways in RAW 264.7 cells (n = 4). Cells were treated with 0–50 μM oleic acid for 2 h and then exposed to 10 ng/mL LPS for 30 min. Phosphorylated JNK (B) and p-p38 MAPK (C) protein levels, which were normalized to those of GAPDH, decreased. ^* p < 0.05 vs. 0 μM oleic acid treatment; ^** p < 0.001 vs. 0 μM oleic acid treatment; ^$ p < 0.05 vs. 10 ng/mL LPS treatment; ^# p < 0.05 vs. 5 μM oleic acid treatment; ^@ p < 0.05 vs. 10 μM oleic acid treatment; ^& p < 0.05 vs. 25 μM oleic acid treatment. The results are expressed as the mean ± standard deviation.

Figure 3

Effect of oleic acid on the NF-κB signaling pathway. (A) Oleic acid attenuated LPS-induced inflammation by suppressing the NF-κB/COX-2/PGE₂ pathway in RAW 264.7 cells (n = 4). After treatment with 0–50 μM oleic acid for 2 h, the cells were treated with 10 ng/mL LPS for either 30 min or 24 h. (B–E) Phosphorylated protein levels were normalized to those of GAPDH. (F) Immunofluorescence revealed that LPS treatment enhanced the translocation of NF-κB p65 (green fluorescence) to the nucleus and upregulated COX-2 (red fluorescence) in the cytoplasm, whereas oleic acid dose-dependently reduced both COX-2 expression and NF-κB translocation. ^* p < 0.05 vs. 0 μM oleic acid treatment; ^** p < 0.001 vs. 0 μM oleic acid treatment; ^$ p < 0.05 vs. 10 ng/mL LPS treatment; ^# p < 0.05 vs. 5 μM oleic acid treatment; ^@ p < 0.05 vs. 10 μM oleic acid treatment; ^& p < 0.05 vs. 25 μM oleic acid treatment. The results are expressed as the mean ± standard deviation. Scale bar, 50 μm; magnification, 1000×.

Figure 4

In silico study of oleic acid (yellow) docked into the TLR3 (TLR3 PDB ID: 1ZIW) or TLR4 (TLR4/MD-2 PDB ID: 5IJD) protein complex and the effect of oleic acid on inflammatory cytokines by TLR4 knockdown. (A) Oleic acid docked into the LRR 9-12 region and into the C-terminal region of TLR3. (B) Oleic acid docked into the TLR4/MD-2 protein complex. TLR4/MD-2 PDB ID: 5IJD. Extension of ligand and binding pose region were expressed with magenda and cyan lines border, respectively. Twenty-four hours after transfection with siTLR4, RAW 264.7 cells were incubated with oleic acid across a concentration range of 0–50 μM for 2 h, followed by LPS treatment for 2 h (n = 4). The protein and RNA levels of TNF-α (C, D), IL-6 (E, F), and IL-1β (G), which were elevated by 10 ng/mL LPS induction, were dampened by siTLR4 transfection and similarly decreased in the oleic acid treatment group. Notably, IL-6 expression was significantly lower in the oleic acid treatment group than in the siTLR4 group. ^* p < 0.05 vs. 0 μM; ^** p < 0.001 vs. 0 μM; ^$ p < 0.05 vs. 0 μM+LPS; ^$$ p < 0.001 vs. 0 μM+LPS; ^# p < 0.05 vs. 0 μM+LPS+siTLR4; ^## p < 0.001 vs. 0 μM+LPS+siTLR4; ^@ p < 0.05 vs. 5 μM+LPS+siTLR4; ^@@ p < 0.001 vs. 5 μM+LPS+siTLR4. The results are expressed as the mean ± standard deviation.

Figure 5

Anti-asthmatic effect of oleic acid according to BALF and serum IgE analysis and histopathological observation. The populations of (A) WBCs and (B) eosinophils (Eos) in the bronchoalveolar lavage fluid (BALF) were elevated due to OVA exposure and subsequently reduced by oleic acid treatment. Notably, oleic acid reduced the number of white blood cells and eosinophils in a concentration-dependent manner (n = 4). (C) Observation of inflammatory cells in BALF through Diff Quik staining showed a dose-dependent decrease with oleic acid treatment. Scale bar, 100 μm; magnification, 400×. (D) Serum IgE levels, which were elevated in the OVA-induced group, decreased in a dose-dependent manner with oleic acid treatment at various concentrations (n = 8). (E) Lung morphology, characterized by hypersecretion of mucus, proliferation of epithelial cells, and infiltration of inflammatory cells around the bronchoalveolar ducts and blood vessels, which were altered due to OVA, improved with oleic acid treatment in a dose-dependent manner. Scale bar, 100 μm; magnification, × 200. (F) Bronchoalveolar mucus secretion induced by OVA was reduced by treatment with varying concentrations of oleic acid (in a dose-dependent manner). Scale bar, 100 μm; magnification, × 200. a, CON; b, OVA; c, 1 mg/kg DEX; d, 50 mg/kg oleic acid; e, 125 mg/kg oleic acid; f, 250 mg/kg oleic acid. The results are expressed as the mean ± standard deviation. ^* p < 0.05 vs. CON; ^** p < 0.001 vs. CON; ^$ p < 0.05 vs. OVA; ^$$ p < 0.001 vs. OVA; ^# p < 0.05 vs. 1 mg/kg DEX; ^## p < 0.001 vs. 1 mg/kg DEX; ^@ p < 0.05 vs. 50 mg/kg oleic acid; ^@@ p < 0.001 vs. 50 mg/kg oleic acid; ^& p < 0.05 vs. 125 mg/kg oleic acid.

Figure 6

Regulation of Th2 cell and Th17 cell–related cytokines and inflammatory mechanism using oleic acid. (A) ELISA analysis demonstrated that oleic acid similarly reduced the protein levels of IL-4, TNF-α, and IL-6 in a dose-dependent fashion (n = 4). At 250 mg/kg, the levels were reduced to a degree akin to that of the dexamethasone treatment group. (B) RT−PCR analysis revealed that oleic acid reduced the cDNA levels of the Th2 cell-related cytokine IL-4 and the Th17 cell-related cytokines TNF-α and IL-6 in a dose-dependent manner (n = 4). Notably, at 250 mg/kg oleic acid, the reduction was comparable to that of the control group. (C) When observed by fluorescence immunostaining, compared to the merged photo in the OVA-treated group, the expression (translocation) of GATA-3 (red fluorescence) in the 250 mg/kg oleic acid-treated group was altered, similar to that in the DEX-treated group. Scale bar, 50 μm; magnification, 1000×. (D) Immunofluorescence indicated that oleic acid suppressed the expression of p-NF-κB (green fluorescence) and COX-2 (red fluorescence), which were upregulated by OVA treatment in the nucleus and cytoplasm. This suppression was similar to that observed in the DEX treatment group. Scale bar, 50 μm; magnification, ×1000. (E) Immunohistochemistry for PGE₂ expression revealed that oleic acid treatment decreased the levels of PGE₂ (brown staining), which were elevated by OVA treatment. a, CON; b, OVA; c, 1 mg/kg DEX; d, 250 mg/kg oleic acid. Scale bar, 100 μm; magnification, × 200. The results are presented as the mean ± standard deviation. ^* p < 0.05 vs. CON; ^** p < 0.001 vs. CON; ^$ p < 0.05 vs. OVA; ^$$ p < 0.001 vs. OVA; ^# p < 0.05 vs. 1 mg/kg DEX; ^@ p < 0.05 vs. 50 mg/kg oleic acid.

Figure 7

Induction of apoptosis in lung epithelial cells by oleic acid by regulating the Bcl-2 family. (A) TUNEL staining showed that apoptosis (green fluorescence) in lung epithelial cells, which was inhibited by OVA treatment, was significantly induced by oleic acid treatment. (B) Oleic acid induces apoptosis in lung epithelial cells by regulating the Bcl-2 family. Treatment with oleic acid decreased the expression of the antiapoptotic protein Bcl-2 (green fluorescence) and increased the expression of the proapoptotic protein Bax (red fluorescence). Scale bar, 50 μm; magnification, 1000×.