(1598) The Protease-Activated Receptor 2 as an Effector Pathway for Zonulin-Mediated Duodenal Dysmotility

Introduction: Pre-haptoglobin 2, also known as zonulin, has been associated with gastrointestinal (GI) dysfunction in models of inflammation. We have shown lipopolysaccharide (LPS)-injected Zonulin transgenic mice (Ztm) have attenuated duodenal dysmotility, when zonulin mRNA expression is not increased. We hypothesized that inhibiting zonulin would also result in attenuated duodenal dysmotility.

Methods: Adult (8-12 weeks old), male and female, Ztm, Ztm-protease-activated receptor 2 (PAR2) knockout (Ztm-P) and wildtype (WT) mice on a C57Bl/6 background were included in experiments. Inflammation was triggered by a 10mg/kg intraperitoneal LPS injection. Control mice were injected with vehicle (V). Zonulin inhibition included, knocking out PAR2, a zonulin-signaling pathway, or exposure to a zonulin inhibitor, AT1001 (A), for 1-week pre-injection. GI transit was examined by fluorescein-isothiocyanate-dextran (FITC), 6 hours after injection. Duodenal zonulin and PAR2 mRNA expression, corrected for the 18S housekeeping gene (dCq), were examined.

Results: LPS-injected Ztm-P mice had 30% less FITC in the duodenum than WT mice (WT 56.9%, Ztm-P 26.1%, p=0.003), representing faster duodenal transit. Zonulin mRNA expression was increased in Ztm-P, LPS-injected mice compared to V (LPS 16.6 SD 2 dCq, V 20.8 SD 1 dCq, p=0.001). LPS-injected Ztm had no increase in PAR2 mRNA expression (LPS 13.2 SD 1.7 dCq, V 12.5 SD 1.4 dCq, p=0.45). LPS-injected Ztm-A had similar duodenal dysmotility as WT mice (WT 56.9%, Ztm-A 45.8%, p=0.07). Ztm-A had an increase in the mRNA expression of zonulin (LPS 13.9 SD 2.6 dCq, V 17.5 SD 2.5 dCq, p=0.02) and PAR2 (LPS 11.9 SD 1 dCq, V 15.3 SD 2.3 dCq, p=0.003).

Conclusions: LPS-injected Ztm-P mice had attenuated duodenal dysmotility despite an increase in zonulin mRNA expression, and LPS-injected Ztm had a similar motility phenotype without an increase in PAR2 mRNA expression, whereas Ztm-A mice had no attenuation in duodenal dysmotility with an increase in zonulin and PAR2 mRNA expression. These findings suggest a primary role for PAR2 in zonulin-mediated GI function during acute inflammation. The unexpected phenotype of Ztm-A may be due to downregulation of zonulin and PAR2 pre-injection resulting in an over compensatory response after LPS injection and subsequent GI dysfunction.