Abstract
Hepatic stellate cell (HSC) activation is a key phenomenon in development of liver fibrosis. Recently, Akkermansia muciniphila has been introduced as a next-generation microbe residing in the mucosal layer of the human gut. Due to the probable risks associated with the use of live probiotics, the tendency to use heat-killed bacteria has been raised. Herein, we investigated the potential anti-fibrotic effects of heat-killed A. muciniphila MucT on activation of HSCs. The human LX-2 cells were stimulated by various concentrations of LPS to evaluate the optimal concentration for HSC activation. Cell viability of LX-2 cells treated with LPS and heat-killed A. muciniphila MucT was measured by MTT assay. Scanning electron microscopy was used to analyze the morphology of heat-killed bacteria. Quiescent and LPS-stimulated LX-2 cells were coinfected with heat-killed A. muciniphila MucT. The gene expression of α-SMA, TIMP, Col1, TGF-β, TLR4, and PPARγ was analyzed using quantitative real-time PCR. Our results showed that LPS treatment led to a significant increase in fibrosis markers in a concentration-independent manner (P < 0.0001), and significantly downregulated the expression of PPARγ (P < 0.0001). The heat-killed A. muciniphila MucT could significantly modulate the expression of fibrosis markers particularly in MOI 10 (P < 0.0001), and reversed the HSC activation in LPS-stimulated LX-2 cells. In conclusion, we demonstrated that heat-killed A. muciniphila MucT was safe and capable to ameliorate LPS-induced HSC activation through modulation of fibrosis markers. Further in vivo studies are required to validate the anti-fibrotic properties of heat-killed A. muciniphila MucT.
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Acknowledgments
The authors wish to thank all laboratory staff of Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran, and Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran. This article has been also extracted from a PhD thesis (Registration No: B-9428) from Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran, Tehran, Iran.
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The study was funded by a research grant (Project No: RIGLD 1017) from 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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SKAR performed the microbiological experiments, cell culture, molecular tests, data analysis, and wrote the manuscript draft; SA participated in molecular testing; MA contributed to microbiological experiments and cell culture experiments; AY, SDS, and MRZ contributed to study design, methodology, conceptualization, and project administration; AY critically revised the manuscript; FV and AM participated in manuscript revision. All authors approved the final version of the manuscript.
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This work does not contain any studies related with human participants or animals. The study was approved by the Institutional Ethical Review Committee of Research Institute for Gastroenterology and Liver Diseases at Shahid Beheshti University of Medical Sciences (Project No. IR.SBMU.RIGLD.REC.1395.211).
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Keshavarz Azizi Raftar, S., Abdollahiyan, S., Azimirad, M. et al. The Anti-fibrotic Effects of Heat-Killed Akkermansia muciniphila MucT on Liver Fibrosis Markers and Activation of Hepatic Stellate Cells. Probiotics & Antimicro. Prot. 13, 776–787 (2021). https://doi.org/10.1007/s12602-020-09733-9
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DOI: https://doi.org/10.1007/s12602-020-09733-9