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Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: B. coagulans and B. subtilis (natto)

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Abstract

Probiotics are used as a treatment for different intestinal disorders. They confer health benefits by different ways. This study was aimed to investigate immunomodulatory effect of Bacillus probiotic spores on the production of lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) in HT-29 intestinal epithelial cells. Differentiated intestinal epithelial cell line was used as a model for the study of colonization of purified spores (Bacillus subtilis (natto) and B. coagulans) and their anti-inflammatory effects. MTT assay and trypan blue staining were used for the detection of optimal concentration of the purified spores and LPS. Pre-treatment assay was done by treatment of the cells with the purified spores for 2 h, followed by challenges with LPS for 3 and 18 h. Post-treatment assay was done by initial treatment of the cells with LPS for 18 h, followed by the spores for 3 and 6 h. Levels of IL-8 secretion and its mRNA expression were measured by ELISA and relative Q real-time PCR. Our results showed similar rates of adherence to intestinal epithelial cells by the spore probiotics, while displaying no cytotoxic effect. In the pre-treatment assay, a significant decrease in IL-8, at both protein and mRNA levels, was measured for B. coagulans spores after the addition of LPS, which was higher than those observed for Bacillus subtilis (natto) spores. In the post-treatment assay, while Bacillus subtilis (but not B. coagulans) diminished the LPS-stimulated IL-8 levels after 3 h of incubation, the inhibitory effect was not constant. In conclusion, ability of Bacillus spore probiotics for adherence to intestinal epithelial cell and their anti-inflammatory effects, through interference with LPS/IL-8 signaling, was shown in this study. Further studies are needed to characterize responsible bacterial compounds associated with these effects.

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Acknowledgments

We are grateful to Dr. Mojtaba Jafari, for the preparation of Bacillus probiotics strains. This study was a part of MSc thesis that was supported by Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

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Authors and Affiliations

  1. Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Masoumeh Azimirad & Masoud Alebouyeh

  2. Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Masoud Alebouyeh

  3. Department of Cell and Molecular Biology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran

    Masoumeh Azimirad & Tahereh Naji

Authors
  1. Masoumeh Azimirad
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  2. Masoud Alebouyeh
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  3. Tahereh Naji
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Correspondence to Masoud Alebouyeh.

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Azimirad Masoumeh, Alebouyeh Masoud, and Naji Tahereh declare that they have no conflict of interest.

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Supplementary Fig. 1

RT-PCR for quality control of RNA extraction and cDNA synthesis. M: DNA Ladder mix; Lanes 1–10: PCR products for the prepared cDNAs from the pre- and post-treated cells. Lane 11: negative control (DOCX 67 kb)

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Azimirad, M., Alebouyeh, M. & Naji, T. Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: B. coagulans and B. subtilis (natto). Probiotics & Antimicro. Prot. 9, 56–63 (2017). https://doi.org/10.1007/s12602-016-9234-x

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  • DOI: https://doi.org/10.1007/s12602-016-9234-x

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