Pitfalls in AR42J-model of cerulein-induced acute pancreatitis.

ABSTRACT:

Background

AR42J are immortalized pancreatic adenocarcinoma cells that share similarities with pancreatic acinar cells. AR42J are often used as a cell-culture model of cerulein (CN)-induced acute pancreatitis (AP). Nevertheless, it is controversial how to treat AR42J for reliable induction of AP-like processes. Gene knockout and/or overexpression often remain challenging, as well. In this study, we demonstrate conditions for a reliable induction of proinflammatory markers upon CN treatment in AR42J and high transfection efficacy using Glyoxalase-I (Glo-I) as a target of interest.

Methods

Effects of dexamethasone (dexa) and CN on cell morphology and amylase secretion were analyzed via ELISA of supernatant. IL-6, TNF-? and NF-?B-p65 were measured via qRT-PCR, ELISA and Western Blot (WB). Transfection efficacy was determined by WB, qRT-PCR and immune fluorescence of pEGFP-N1-Glo-I-Vector and Glo-I-siRNA.

Results

Treatment of AR42J with 100 nm dexa is mandatory for differentiation to an acinar-cell-like phenotype and amylase production. CN resulted in secretion of amylase but did not influence amylase production. High levels of CN-induced amylase secretion were detected between 3 and 24 hours of incubation. Treatment with LPS alone or in combination with CN did not influence amylase release compared to control or CN. CN treatment resulted in increased TNF-? production but not secretion and did not influence IL-6 mRNA. CN-induced stimulation of NF-?B was found to be highest on protein levels after 6h of incubation. Transient transfection was able to induce overexpression on protein and mRNA levels, with highest effect after 12 to 24 hours. Gene-knockdown was achieved by using 30 pmol of siRNA leading to effective reduction of protein levels after 72 hours. CN did not induce amylase secretion in AR42J cell passages beyond 35.

Conclusion

AR42J cells demonstrate a reliable in-vitro model of CN-induced AP but specific conditions are mandatory to obtain reproducible data.

SUBMITTER: Hollenbach M 

PROVIDER: S-EPMC7833168 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature