Gastric nNOS reduction accompanied by natriuretic peptides signaling pathway upregulation in diabetic mice

AIM:To investigate the relationship between neuronal nitric oxide synthase(nNOS)expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin(STZ)-induced diabetic mice.METHODS:Diabetic mice were induced by injection of STZ solution.Immunofluorescence labeling of HuC/D,nNOS and natriuretic peptide receptor-A,B,C(NPRs)in the gastric fundus(GF)was used to observe nNOS expression and whether NPRs exist on enteric neurons.The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting.An isometric force transducer recorded the electric field stimulation(EFS)-induced relaxation and contraction in the diabetic GF.An intracellular recording method assessed EFSinduced inhibitory junction potentials(IJP)on the GF.GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide(CNP)for 24 h,after which their nNOS expressions were detected by western blotting.RESULTS:Eight weeks after injection,43 diabetic mice were obtained from mouse models injected with STZ.Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF.The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced,while the contraction was enhanced in the diabetic GF.EFSinduced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice.However,both NPR-A and NPR-B were detected on enteric neurons,and their expression levels were upregulated in the diabetic GF.The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP.CONCLUSION:These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.

Pseudotargeted metabolomics for exploring the changes of neurotransmitters profile in aging rat brain and the potential neuroprotective effect of alkaloids from Uncaria rhynchophylla

Background:Aging is an essential risk factor for most neurodegenerative diseases,including Alzheimer’s disease and Parkinson’s disease.However,changes in the levels of neurotransmitters that are associated with aging are not well understood.Methods:Methods such as liquid-liquid extraction,protein precipitation,and solid-phase extraction,using 20 different extraction solvents,were evaluated to optimize the extraction of neurotransmitters.A pseudotargeted metabolomics approach was developed to detect neurotransmitters in brain tissues using ultra-high-performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry.Alkaloids that crossed into the brain were used to evaluate the effect of glutamic acid-induced excitatory neurotoxicity in SH-SY5Y cells.Results:The overall extraction efficiency using protein precipitation was high.The changes in neurotransmitters’levels in the brain exhibited changes during the different growth cycles.The levels of seven neurotransmitters(aspartic acid,tyrosine,isoleucine,leucine,tryptophan,valine,andγ-aminobutyric acid)were significantly different.Meanwhile,alkaloids could reduce the excitatory neurotoxicity of glutamic acid-induced SH-SY5Y cells via suppression of oxidative stress.Conclusion:Significant differences were observed in neurotransmitter profiling between 1-and 8-month-old rats,and the discrepant neurotransmitters were associated with aging.Seven indole alkaloids from Uncaria rhynchophylla,which could cross the blood-brain barrier,were screened and used to explore their protective effects against aging.Uncaria rhynchophylla alkaloids exhibited a neuroprotective effect by inhibiting oxidative stress,indicating that the alkaloid could be a potential therapeutic candidate for neurological disorders caused by glutamic acid toxicity.