The effect of bifidobacterium bifidum on tlr 2,9 genes expression in caco-2 cell line as intestinal epithelial cell line model

Maryam Ahmady,1 Seyed davar siadat,2,* Mahsa alebrahim,3

1. Department of Molecular and Cellular Sciences,Faculty of Advanced sciences and Technologies,Tehran Medical Sciences,Islamic Azad University,Tehran,Iran
2. Microbiology Research Center Mycobacteriology,Pasteur Institute of Iran
3. Department of Physiology,Faculty of Advanced sciences and Technologies,Tehran Medical Sciences,Islamic Azad University,Tehran,Iran

Abstract

Introduction

The human gastrointestinal tract consists of more than 100 trillion microbial cells which is called as gut microbiota.gut microbiota has essential roles in human functions specially metabolic and immune system through their symbiotic interactions with the host.recognition pamps(pathogen-associated molecular pattern )of microorganisms by pattern-recognition receptors (prrs) is the primary component of innate immunity that is responsible for the maintenance of host-microbial interactions in intestinal mucosa.tlrs are subset of prrs that regulate inherent immune responses.gut microbiota affect host immunity due to have various pamps that constantly interact with tlrs.bifidobacterium bifidum is an important member of gut microbiota,they are anaerobic,rod shaped,gram positive bacteria that localization in anaerobic flora and belongs to the actinobacteria phylum and member of the bifidobacteriaceae family.according to the importance of bifidobacterium bifidum in gut ,and tlr as a kind of prrs,in this study we focused on the effect of b.bifidum ontlr2,9 genes expression in caco-2 cell line as intestinal epithelial cell line.

Methods

In this study b.bifidum was cultured in de man, rogosa and sharpe agar(mrs agar)at anaerobic atmosphere including 80%co2,10%h2,10%n2.caco-2 cell line(ibrc3010094)was prepared in dmem supplemented fbs and penicillin and streptomycin high glucose,caco-2 cells was treated with b.bifidum in multiplicity of infection(moi) = 74 for overnight.total rnas were extracted by thiazol solution(rnx) and reverse transcripted to cdna using cdna fermentaz synthesis kit.revers transcription-guantitative pcr( rtq-pcr)was performed to assay tlr2 and tlr 9 gene expression through syber green method. gene expression were measured based on delta delta ct (∆∆ct) method and gapdh as house keeping gene.finally, the significancy of the results is determined by independent sample t-test by spss and prism.

Results

Our rt-qpcr showed that the tlr2 and tlr9 mrna levels were change by b.bifidum in caco-2 cell line. tlr2 and tlr9 gene expression were decreased by b.bifidum at moi 74.this down regulatory effects of b.bifidum on tlr2 and tlr9 gene expression was significant based on p<0.05,p<0.01 respectively.

Conclusion

In conclusion we demonstrated that b.bifidum was able to down regulate the expression of tlr2 and tlr9 genes.according to the regulation of tlrs signaling in inflammatory response,the understanding of the important gut microbiota members could be effective in the improvement of gut microbiota interactions.

Keywords

Bifidobacterium bifidum,gut microbiota,toll like receptors,caco-2 cell line,inflammatory response