Sera with anti-enteric neuronal antibodies from patients with irritable bowel syndrome promote apoptosis in myenteric neurons of guinea pigs and human SH-Sy5Y cells.

BACKGROUND

Sera anti-enteric neuronal antibodies (AENA), neuronal inflammation, and degeneration in myenteric plexus in patients with irritable bowel syndrome (IBS) were reported. Effects of sera AENA in patients with IBS are unclear.

METHODS

Patients with IBS met Rome III criteria were enrolled. Controls included healthy subjects (HS) and patients with slow transit functional constipation, inflammatory bowel disease, chronic intestinal pseudo-obstruction, and autoimmune diseases. Indirect immunofluorescence was used to detect AENA. Anti-enteric neuronal antibodies intensities were termed as "1" = weak fluorescence (mild positive); "2" = moderate fluorescence (moderate positive); "3" = very high fluorescence (intensive positive). Intensities of ≥1 were defined as positive and ≥2 were defined as obvious positive. Cultured myenteric neurons of small intestine from guinea pigs and human SH-Sy5Y cells were incubated with fetal bovine serum (FBS), HS sera, or IBS sera with or without AENA. Indirect immunofluorescence with anti-PGP9.5/DAPI/anti-active caspase-3 or TUNEL, Western blot, and flow cytometry were used to detect apoptosis.

KEY RESULTS

Overall, 293 patients with IBS were enrolled (41.7 ± 11.5 years). AENA-positive and obvious positive rates in IBS were higher than HS (76.8% vs 33%; 43.7% vs 7%; all P < 0.001). Myenteric neurons incubated with AENA moderate or intensive positive IBS sera showed higher rates of anti-active caspase-3 and TUNEL-positive cells than HS or FBS (20% ± 7.3% and 35% ± 13.3% vs 4.3% ± 1.5% and 0.9% ± 0.4%, respectively; 6.2% ± 2.0% and 10.2% ± 4.6% vs 1.3% ± 1.9% and 0.5%±0.5%, respectively; all P < 0.05). Human SH-Sy5Y cells incubated with AENA moderate or intensive positive IBS sera showed increased cleaved caspase-3 and Bax expression and decreased Bcl-2 expression. Flow cytometry showed apoptosis rates of these two groups were higher than that of AENA mild positive, negative, HS, and FBS (7.6%±0.8% and 10.7%±1.3% vs 5.0%±0.8%, 3.8%±0.3%, 3.4%±0.2% and, 2.8%±0.2%, P < 0.05).

CONCLUSIONS AND INFERENCES

The AENA obvious positive rate in patients with IBS was higher than HS, and sera with higher levels of AENA promoted neuronal apoptosis. AENA-mediated neuropathy might exist in a subset of patients with IBS.