Impact of Nano-Pomegranate Seed Oil on the Expression of TLR2 and TLR4 Genes in A549 Cells Sensitized with Alternaria alternata Cellular Extract

Abstract

Background: Allergies impact nearly 30% of the world's population, with fungi being remarkable contributors to allergic sensitivity. Exposure to fungi allergens can trigger allergic reactions and severe asthma has been linked to hypersensitivity to fungi such as Aspergillus and Alternaria spp. Alternaria alternata, a common allergen in respiratory allergic diseases, releases proteases that initiate Th2 responses, causing inflammatory cytokine production. Objectives: Given the anti-inflammatory properties of Pomegranate Seed Oil (PSO) and the potential of Nano-emulsions (NEs) to enhance drug delivery, this study investigates the impact of PSO-loaded NEs on TLR2 and TLR4 gene expression in A549 cells sensitized with A. alternata extract (ALT). Methods: A. alternata (ATCC 6663) was cultured and processed to obtain a cytosolic extract, with protein content measured using the Bradford method. Using a Soxhlet apparatus, PSO was extracted from cleaned, dried seeds and analyzed by gas chromatography. Alginate nanospheres containing PSO were prepared through a modified water-in-oil emulsification method and characterized for particle size, polydispersity index, and zeta potential. A549 cells were cultured and treated with various ALT, PSO, and PSO-loaded NEs combinations. Following treatment, RNA was extracted, and real-time RT-PCR was conducted to analyze TLR2 and TLR4 gene expression. Results: All treatment groups showed an increase in TLR2 gene expression compared to the control, with the ALT combined with PSO (P+ALT) causing the highest increase at 4.82-fold. Free PSO (P) and free NEs (NP) resulted in 3.92-fold and 2.93-fold increases, respectively, while the ALT and PSO-loaded NEs (NP+ALT) led to 2.26-fold and 2.50-fold increases. For TLR4 gene expression, the ALT treatment increased expression by 2.29-fold, but treatments containing PSO (P, P+ALT, NP+ALT) reduced TLR4 expression, with P+ALT and NP+ALT causing 0.45-fold and 0.61-fold decreases. Conclusion: The study confirms that herbal extracts like PSO selectively upregulate TLR2 and downregulate TLR4, suggesting targeted therapeutic potential in inflammation and immune modulation. PSO-loaded NEs demonstrated superior anti-inflammatory effects, supporting their development for treating inflammatory diseases and warranting further research into their molecular mechanisms and therapeutic applications.

Keywords

• Alternaria alternata
• Pomegranate seed oil
• Nano-emulsions
• asthma
• Anti-inflammatory.