Abstract
Purpose :
A previous study using immortalized colonic adenocarcinoma cells (CaCo-2) demonstrated upregulation of the endogenous antimicrobial peptide (AMP) human beta-defensin 3 (hBD3, gene: DEFB103) mRNA expression when the cells were treated with one of three Chinese herbal extracts (andrographolide, oridonin, isoliquiritigenin). The current study determined the ability of corneal epithelial cells to upregulate DEFB103 expression when treated with these herbal compounds.
Methods :
Immortalized human corneal epithelial cells (hTCEpi, passage: 42-55) were treated with increasing concentrations of each herbal compound (0.5 – 1000 μM) and cell viability was assessed using an MTT assay. The dosing levels were further refined around this maximal treatment level to measure the inducible AMP expression. Near confluent monolayers of hTCEpi cells were treated with each of the herbal compounds individually for 24 hours, followed by RNA isolation and quantitative PCR (qPCR) targeting hBD3 mRNA expression. The reference transcript GAPDH was also measured and all data was normalized to the vehicle control (andrographolide: 0.1% DMSO, oridonin: 0.15% DMSO, isoliquiritigenin: 1.0% ethanol). Statistical significance was determined based on an ANOVA followed by a Dunnett’s multiple comparisons.
Results :
The treatment doses of the different herbal compounds resulted in differential cell viability with the highest dosing in andrographolide (121.5 μM) followed by isoliquiritigenin (13.5 μM) and oridonin (4.5 μM). Subsequently, a more focused range of treatments around the highest dose was performed. All treatments resulted in a dose-dependent increase in DEFB103 mRNA expression with the most dramatic upregulation seen with andrographolide (360-fold, 75 μM, P < 0.0001) followed by oridonin (4-fold, 5 μM, P = 0.02) and isoliquiritigenin (3-fold, 15 μM, P < 0.0001).
Conclusions :
With the rise of antibiotic resistant infections at the ocular surface, treatment focusing on upregulation of endogenous AMPs may be considered for patients to combat these resistant bacterial infections. Additional ex vivo and in vivo studies are planned to assess the tolerability and utility of these herbal compounds to induce DEFB103 expression in native corneal epithelium.
This is a 2020 ARVO Annual Meeting abstract.