Impairment of gastric nitrergic and NRF2 system in apolipoprotein E knockout mice

Abstract

Background and aim: Gastric motility dysfunction is most commonly seen in diabetic and idiopathic gastroparesis patients. Recently we reported that impaired nitrergic relaxation and a reduced NO (nitric oxide) bioavailability were responsible for gastric motility dysfunction in diabetic female rats. One of the main factors involved in the inactivation of the nitrergic system is oxidative stress commonly seen in diabetic patients. Hyperlipidemia may also be one of the detrimental causes for impaired gastric motility associated with diabetes. In the current study, we investigated whether apolipoprotein E knockout mice (ApoE-KO), an oxidative stress animal model with a hyperlipidemia burden, also displays an impaired nitrergic system. To test this, nitrergic relaxation (AUC/mg tissue) was measured at 2 Hz through electric field stimulation using gastric pyloric strips prepared from C57BL WT or ApoE-KO female mice. Protein expression was determined by Western blots.

Results: Nitrergic relaxation was reduced in gastric strips from ApoE-KO versus WT mice. Protein levels of nNOS (neuronal nitric oxide synthase), GCH-1 (GTP cyclohydrolase 1), Nrf2 (nuclear factor E-2 related factor 2) and GCSc (glutamate-cysteine ligase catalytic) were also reduced in ApoE-KO compared to controls, with no significant change in GCSm (glutamate-cysteine ligase modifier) and HO-1 (heme oxygenase 1). The activities of DHFR (dihydrofolate reductase) and antioxidant enzymes were also reduced in ApoE-KO mice.

Conclusions: This novel study is the first to reveal that a deficiency in ApoE impairs gastric motility functions, and that hyperlipidemia and the suppression of selective antioxidants may be an underlying mechanism for this pathological change.

Fig. 1

a Nitrergic relaxation in WT and apolipoprotein E knockout mice (ApoE-KO) gastric muscular pyloric tissues in vivo. Nitrergic relaxation was measured in WT and ApoE-KO pyloric gastric tissue. Nitrergic relaxation is reduced in ApoE-KO mice when compared to WT mice. b Expression of nNOSα in WT and ApoE-KO female mice gastric muscular tissue. Representative immunoblot and densitometric analysis data for nNOSα protein in female mice gastric fundus muscular tissue. There was a significant decrease in nNOSα in female ApoE-KO mice compared to WT mice. c Representative immunoblot and densitometric analysis data for nNOSα protein in female mice gastric antrum muscular tissue. A significant decrease was seen in nNOSα in female ApoE-KO mice compared to WT mice. Values are mean ± SE (N = 4). Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group

Fig. 2

Expression of GCH-1, dihydrofolate reductase (DHFR) in WT and apolipoprotein E knockout (ApoE-KO) female mice gastric antrum muscular tissue. a Representative immunoblot and densitometric analysis data for GCH-1 protein in female mice gastric antrum. A significant decrease was seen in GCH-1 in female ApoE-KO mice compared to WT mice (0.49 ± 0.04 vs 0.68 ± 0.04). Values are mean ± SE (N = 4). Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group. b Representative immunoblot and densitometric analysis data for DHFR protein in female mice gastric antrum. Values are mean ± SE (N = 4)

Fig. 3

Expression of Nrf2, GCSc, GCSm and HO-1 in WT and apolipoprotein E knockout (ApoE-KO) female mice gastric antrum muscular tissue. Representative immunoblot and densitometric analysis data for Nrf2 protein in female mice gastric antrum. a A significant decrease in Nrf2 in female ApoE-KO mice compared to WT mice. Values are mean ± SE (N = 4). Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group. b Representative immunoblot and densitometric analysis data for GCSc protein in female mice gastric antrum. c Representative immunoblot and densitometric analysis data for GCSm protein in female mice gastric antrum. d Representative immunoblot and densitometric analysis data for HO-1 protein in female mice gastric antrum. Values are mean ± SE (N = 4)