Reuterin Isolated from Lactobacillus reuteri Indonesian Strain Affected Interleukin-8 and Human Beta Defensin-2 on Pathogens Induced-HaCat Cells

Abstract

Probiotic Lactobacillus reuteri has positive effects on health through inhibiting pathogenic bacteria and the ability to reduce inflammation. This study investigates the ability of reuterin isolated from L. reuteri Indonesian strain for increasing mRNA expression of interleukin (IL)-8 and human beta-defensin (hBD)-2 gene by epithelial cells, after exposure to oral bacteria. L. reuteri isolated from Indonesian's saliva, and species was confirmed by PCR, using 16S rRNA specific gene. To produce reuterin, the isolate was mixed in glycerol-containing MRS broth. Reuterin molecule's weight was counted by SDS-PAGE. Streptococcus mutans ATCC-25175 and Porphyromonas gingivalis ATCC-33277 were put in water (80°C) for 30 min, and each killed bacterial (107 CFU/mL) was inoculated into HaCat cell line (105 cell/mL). Reuterin was added in different concentrations (100%, 50%, 25%, 12,5%) and different incubation time at 37°C, 5% CO2. RNA was extracted, and a reverse transcription procedure was performed to obtain cDNA. Subsequently, a quantitative PCR method was performed to analyse the transcription level of IL-8 and HBD-2 mRNA expressed by inflamed HaCat cells. All results were statistically analysed by ANOVA test. PCR assays showed that clinical isolates were L. reuteri. Quantitative PCR results showed reuterin decreased the expression of IL-8 and increased the expression of hBD-2 in all concentrations and time periods set in this study (p < 0.05). Reuterin isolated from L. reuteri Indonesian strain increased expression of human beta defensin-2 as antimicrobial peptide and may be useful in combating inflammation.

Keywords: Anti-Inflammatory; Human Beta Defensin-2; Interleukin-8 Reuterin; Lactobacillus reuteri.