Gastric ICC And NOS Neuron Damages And Its Mechanism In Diabetic Murine

Gastroparesis is a chronic disorder that is defined as the delayed gastric emptying of solids and liquids in the absence of physical obstruction Gastroparesis occurs in type 1 and type 2 diabetes mellitus patients?25%–55%?or can be idiopathic Diabetic gastroparesis affects nutritional state,glycemic control and patient quality of life.Gastric motility requires interactions between smooth muscle,enteric and extrinsic autonomic nerves,and interstitial cells of Cajal?ICC?.Several factors contribute to gastroparesis,including acute hyperglycemia,dysregulation of gastrointestinal hormones,oxidative stress which induces the loss of neural nitric oxide synthase?nNOS?expression in the myenteric plexus and ICC network disturbances.ICC are the pacemaker cells in gastrointestinal tract that trigger the generation of slow waves in the tunica muscularis,and the damage to the ICC network is likely to contribute to gastropathy and gastroparesis.Stem cell factor?SCF?-Kit signaling is important for the maintenance of ICC phenotypes,proliferation,and differentiation.Membrane-bound SCF?mSCF?expressed by gastric smooth muscle cells?GSMCs?has been linked to smooth muscle myopathy and ICC depletion in murine diabetic gastroparesis.Exogenous SCF partially reverses the pathological changes in ICC in diabetic mice.With the exception of classical RAS,local RAS exists in various tissues.It is well known that angiotensin ??Ang??is associated with increased gene expression and the production of growth factors,such as endothelial growth factor?EGF?,platelet activating factor?PAF?,platelet-derived growth factor?PDGF?,and insulin-like growth factor-1?IGF-1?.Therefore,we hypothesized that Ang? might be involved in the production of mSCF.It is generally considered that enteric neuropathy is one of the causative factors of diabetic gastroparesis.Our previous study demonstrated that there is a loss of NOS neurons in diabetic mice.However,the underlying mechanism which nNOS expression was down-regulated and inhibitory neuron function impaired remain unclear.Although oxidative stress,formation of advanced glycation end products,and protein kinase C upregulation may be taken as important factors.In fact,multiple lethal pathways may be activated during chronic hyperglycemia.It has been reported that chronically activated neuronal P2X₇R,leading to preferential loss of nitrergic neurons in Crohn's disease and inflammatory bowel diseases.The effect of P2X₇R on diabetes-induced gastric NOS neurons damage and its mechanism were investigated by using quantitative RT-RCR,immunofluorescence,western blot,isometric muscle recording,[Ca2+]i measurement and whole-cell patch clamp.Results as follows:Local RAS may compensate for ICC loss in diabetic stomach and its mechanism1.The blood glucose levels of the diabetic mice were approximately3 times higher than those of control mice and body weight were decreased in diabetic mice.2.The immunohistochemistry showed that a significant decrease in c-Kit immunoreactivity in diabetic mice compared with control group.Including a near-complete loss of intramuscular ICC?ICC-IM?and a quantitative reduction of myenteric ICC?ICC-MY?.Additionally,western blot results indicated c-Kit and mSCF protein expression levels were down-regulated in diabetic mice.3.The realtime fluorescence quantitative PCR results showed that the mRNA level of AT1aR and AT1bR were increased in diabetic mice.ACE expression in gastric mucosa and Ang? receptor 1?AT1?in gastric smooth muscle layers were upregulated in diabetic mice.4.The isometric tension measurement showed Ang? significantly increased both amplitude and basal tension in normal and diabetic mice,and compared with normal mice,the excitatory response to Ang? was more sensitive in diabetic mice at the same concentration?10^-88 mol/L?.5.Ang? levels in plasma were measured by competitive quantitative enzyme-linked immunosorbent assay,the results showed plasma Ang? levels were significantly elevated in diabetic mice compared with controls.6.An assay for CCk-8 showed that the OD values in Ang?-treated GSMCs were markedly increased compared to those of the control group in a dose-dependent manner.Ang? might induce GSMC proliferation via AT1R but not AT2R.PI3K inhibitor LY294002significantly inhibited Ang?-induced GSMC proliferation.7.Western blot analysis determined Ang? significantly increased the mSCF expression level in GSMCs.AT1R inhibitor ZD7155 and LY294002?a PI3K inhibitor?significantly inhibited Ang?-induced mSCF expression.8.Ang? treatment led to significant increases in the phosphorylation of Akt-Ser473 in GSMCs.Akt-Ser473 phosphorylation was antagonized by AT1R inhibitor ZD7155 and LY294002?a PI3K inhibitor?.CONCLUSIONS:we observed that diabetes-induced gastric ICC loss was accompanied by the up-regulation of the ACE/Ang?/AT1receptor signal pathway.We also found that Ang? promoted cell proliferation and mSCF expression in cultured GSMCs via the PI3K/Akt signaling pathway.Our results suggest that a systemic or local ACE/Ang?/AT1 receptor signal pathway could play an important role in GSMC proliferation and could maintain the gastric ICC phenotype.Diabetes-induced gastric nitric oxide neurons damage mediated by P2X₇R in diabetic mice1.The immunohistochemistry and western blot results showed that nNOS expression was significantly down-regulated in diabetic mice,meanwhile,electric field stimulation-induced NO sensitive relaxation was significantlydecreased.2.The immunohistochemistry and western blot results showed myenteric neurons expressed P2X₇R and pannexin-1,and the mRNA and protein level of P2X₇R and pannexin-1 were up-regulated in diabetic mice.3.The calcium fluorescent showed that BzATP,a P2X7R activator,evoked intracellular calcium?[Ca2+]i?increase in Hek293 cells with heterologous expression of P2X₇R?Hek293-P2X₇R cells?and the same dose of ATP increased intracellular calcium([Ca²⁺]i)are more obviously in Hek293-P2X₇R cells than in Hek293 cells.4.The whole-cell patch-clamp recording showed that application of BzATP activated an inward current in Hek293-P2X₇R in a concentration dependent manner and showing no desensitization.5.Hek293-P2X₇R cells could take up of YO-PRO-1,but not in untransfected Hek293 cells.In addition,the uptake of YO-PRO-1 by Hek293-P2X₇R cells was blocked by oxATP,a P2X₇R antagonist and CBX?a pannexin-1 inhibitor?.CONCLUSIONS:?1?P2X₇R expressed in enteric neurons may be involved in diabetes-induced NOS neurons damage.?2?In diabetic mice,chronically activated neuronal P2X₇R,P2X₇R and pannexin-1 may form the pore to induce macromolecular substances and calcium into the cell then cause NOS neurons death.SUMMARYThe above results can be summarized as follows:1.The loss of nNOS expression in the myenteric plexus and ICC network disturbances may contribute to gastrointestinal disorders.2.Ang? promoted cell proliferation and mSCF expression in cultured GSMCs.Diabetes-induced gastric ICC loss was accompanied by the up-regulation of the ACE/Ang?/AT1 receptor signal pathway.The up-regulation of ACE and AT1R in the stomach may help compensate for ICC loss in STZ-induced diabetic mice.3.The P2X₇R of enteric neurons may be involved in diabetes-induced NOS neuron damage via activating P2X₇R and pannexin-1?forming the pore?which induce macromolecular substances and calcium into the cell.