Nrf2 attenuates hyperglycemia-induced nNOS impairment in adult mouse primary enteric neuronal crest cells and normalizes stomach function

Abstract

Enteric neuronal cells play a vital role in gut motility in humans and experimental rodent models. Patients with diabetes are more vulnerable to gastrointestinal dysfunction due to enteric neuronal degeneration. In this study, we examined the mechanistic role and regulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) in hyperglycemia-induced enteric neuronal cell apoptosis in vitro by using adult mouse primary enteric neuronal crest cells (pENCs). Our data show that hyperglycemia (HG) or inhibition of Nrf2 induces apoptosis by elevating proinflammatory cytokines, reactive oxygen species (ROS) and suppresses neuronal nitric oxide synthase (nNOS-α) via PI3K/Nrf2-mediated signaling. Conversely, treating pENCs with cinnamaldehyde (CNM), a naturally occurring Nrf2 activator, prevented HG-induced apoptosis. These novel data reveal a negative feedback mechanism for GSK-3 activation. To further demonstrate that loss of Nrf2 leads to inflammation, oxidative stress, and reduces nNOS-mediated gastric function, we have used streptozotocin (STZ)-induced diabetic and Nrf2 null female mice. In vivo activation of Nrf2 with CNM (50 mg/kg, 3 days a week, ip) attenuated impaired nitrergic relaxation and delayed gastric emptying (GE) in conventional type 1 diabetic but not in Nrf2 null female mice. Supplementation of CNM normalized diabetes-induced altered gastric antrum protein expression of 1) p-AKT/p-p38MAPK/p-GSK-3β, 2) BH4 (cofactor of nNOS) biosynthesis enzyme GCH-1, 3) nNOSα, 4) TLR4, NF-κB, and 5) inflammatory cytokines (TNF-α, IL-1β, IL-6). We conclude that activation of Nrf2 prevents hyperglycemia-induced apoptosis in pENCs and restores nitrergic-mediated gastric motility and GE in STZ-induced diabetes female mice.NEW & NOTEWORTHY Primary neuronal cell crust (pENCs) in the intestine habitats nNOS and Nrf2, which was suppressed in diabetic gastroparesis. Activation of Nrf2 restored nNOS by suppressing inflammatory markers in pENCs cells. Inhibition of Nrf2 reveals a negative feedback mechanism for the activation of GSK-3. Activation of Nrf2 alleviates STZ-induced delayed gastric emptying and nitrergic relaxation in female mice. Activation of Nrf2 restored impaired gastric BH₄ biosynthesis enzyme GCH-1, nNOSα expression thus regulating nitric oxide levels.

Keywords: Nrf2; enteric neuronal cells; inflammation; nNOS; siRNA-Nrf2.