Understanding the Effect of Der p 2 on Tight Junctional Protein ZO-1 Expression in Human Bronchial Epithelial Cells

Suardi, Siti Sarah Batrisyia (2020) Understanding the Effect of Der p 2 on Tight Junctional Protein ZO-1 Expression in Human Bronchial Epithelial Cells. [Project Paper] (Submitted)

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Abstract

Human airway epithelium represents a physical barrier against airborne foreign molecules including house dust mite (HDM) allergens. Junctional system, made up of junctional proteins such as tight junctions and adherens junctions, play key roles in the epithelial barrier integrity. HDM allergens have been reported to increase airway epithelial permeability via junctional protein disruption, which eventually initiate the development of allergic airway inflammation leading to asthma. Over the years, it was believed that the cleavage of tight junctional proteins is largely dependent on the serine/cysteine protease activity of HDM proteolytic allergens (mite allergen groups 1, 3, 6 and 9). However, recent studies showed that the HDM-induced modulation of epithelial immune and barrier responses can be independent of proteolytic activities. Interestingly, the HDM allergen, Der p 2, a fatty acid/lipid binding protein was shown to upregulate the expression of tight junctional molecule claudin-2 in airway epithelial cells. Hence, we hypothesized that HDM allergens without proteolytic activities could influence tight junctional protein expression. Objective: This study aims to examine the effects of non-proteolytic HDM allergens, namely Der p 2 on the expression of tight junctional protein zonula occludens-1 (ZO-1) in human bronchial epithelial cells (16HBE14o-). Methodology: The human bronchial epithelial cells 16HBE14o- was propagated and maintained in growth medium of 10% EMEM until reaching 80-90% confluency. The highest non-cytotoxic concentration of Der p 2 was determined by cytotoxicity assay (MTT) and lactate dehydrogenase (LDH) activity assay. Then, the effect of the Der p 2 on the expression of tight junctional protein ZO-1 in 16HBE14o- was examined by using Western Blot. Results: Der p 2 at all tested concentrations (5, 10, 20, 40, 80 µg/mL) were shown to be non-cytotoxic to the growth of 16HBE-14o cells. This was further confirmed by LDH activity assay which proved that 20 µg/mL and 80 µg/mL Der p 2 were not cytotoxic to the cells. However, the effect of Der p 2 on tight junctional protein expression ZO-1 in 16HBE14o- was inconclusive due to the failure in detecting ZO-1 at appropriate molecular weight. Conclusion: Der p 2 at concentrations of 80 µg/mL and below were not cytotoxic to the growth of 16HBE14o- cells. Further studies have to be conducted to examine the effect of Der p 2 on ZO-1 expression through appropriate detection of ZO-1 at accurate molecular weight.

Item Type:	Project Paper
Faculty:	Faculty of Medicine and Health Science
Depositing User:	Ms. Nor Safa'aton Saidin
Date Deposited:	22 Aug 2023 07:57
Last Modified:	22 Aug 2023 07:57
URI:	http://psaspb.upm.edu.my/id/eprint/1341

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