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Updated: Mar 5, 2026
Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids
Published on: June 2, 2023
Establishment of immortalized mouse intestinal epithelial cells line and study of effects of Arg-Arg on inflammatory
Kang Zhan1, Maocheng Jiang1, Yannan Sui1
1College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, People's Republic of China.
Abstract:
Primary mouse intestinal epithelial cells (MIEs) are not ideal models for long-term culture in vitro and a limited amount of approximate three generations. In addition, the mechanism that arginine-arginine dipeptide (Arg-Arg) regulates mouse intestinal inflammatory response remains unknown. Therefore, the aim of this study was to establish immortal MIEs and study the effects of Arg-Arg on inflammatory response after challenging the MIEs with lipopolysaccharide (LPS) or staphylococcal enterotoxin C (rSEC). Our data showed that immortalized MIEs could be cultured over 100 generations. The immortalized MIEs showed positive reaction against cytokeratine 18 antigen, E-cadherin, and peptide transporters (Pept1) using indirect immunofluorescence. Cytokeratine 18 and Pept1 can be expressed in immortalized MIEs by immunoblotting. Fatty acid-binding proteins (FABPs) and villin known as intestinal epithelial cell functional protein were constitutively expressed in immortalized MIEs. For inflammatory response, these results showed that Arg-Arg can decrease the LPS-induced expression of IL-1β and the rSEC-induced expression of TNF-α; however, it can upregulate the LPS-induced expression of IL-6 and TNF-α and the rSEC-induced expression level of IL-1β. In addition, in the MAPK signaling pathway, pSAPK/JNK and p-Erk1/2 in LPS with Arg-Arg treatment were upregulated than that in LPS treatment. p-p38 in LPS with Arg-Arg treatment was attenuated than that in LPS treatment. pSAPK/JNK and p-p38 in rSEC with Arg-Arg treatment were enhanced than that in rSEC treatment. Conversely, p-Erk1/2 in rSEC with Arg-Arg treatment was attenuated than that in rSEC treatment. These novel findings suggest that Arg-Arg dipeptide plays an important role for regulation of the immunologic balance in mouse intestinal inflammatory response.
Insights
This study established immortalized mouse intestinal epithelial cells (MIEs) for long-term culture. The arginine-arginine (Arg-Arg) dipeptide was found to modulate inflammatory responses in MIEs challenged with LPS or staphylococcal enterotoxin C.
Area of Science:
- Gastroenterology
- Immunology
- Cell Biology
Background:
- Primary mouse intestinal epithelial cells (MIEs) have limited in vitro culture lifespan.
- The role of arginine-arginine (Arg-Arg) dipeptide in regulating intestinal inflammation is not well understood.
Purpose of the Study:
- To establish immortalized MIEs for extended in vitro studies.
- To investigate the effects of Arg-Arg dipeptide on inflammatory responses in MIEs stimulated with lipopolysaccharide (LPS) or staphylococcal enterotoxin C (rSEC).
Main Methods:
- Establishment of immortalized MIEs.
- Characterization of immortalized MIEs using indirect immunofluorescence and immunoblotting for epithelial markers (cytokeratine 18, E-cadherin, peptide transporters (Pept1), fatty acid-binding proteins (FABPs), and villin).
- Assessment of inflammatory cytokine expression (IL-1β, TNF-α, IL-6) and MAPK signaling pathway activation (pSAPK/JNK, p-Erk1/2, p-p38) in response to LPS or rSEC challenge with and without Arg-Arg treatment.
Main Results:
- Immortalized MIEs were successfully cultured for over 100 generations and expressed key intestinal epithelial markers.
- Arg-Arg modulated the expression of IL-1β, TNF-α, and IL-6 in response to LPS and rSEC.
- Arg-Arg influenced the activation of MAPK signaling pathways (JNK, Erk1/2, p38) differently depending on the inflammatory stimulus.
Conclusions:
- The established immortalized MIEs serve as a robust model for studying intestinal epithelial cell function and inflammatory responses.
- The arginine-arginine dipeptide plays a significant role in regulating the immunologic balance within the mouse intestinal inflammatory response.
- These findings provide novel insights into the mechanisms by which Arg-Arg influences intestinal immunity.
