A Study Of The Effect Of Nesfatin-1on Gastric Acid Secretion And It’s Mechanism In Rat Gastric Mucosa Cells

Nesfatin-1is a recently identified82-amino-acid peptide by Oh-I et al. as the amino-terminal fragment of nucleobindin2（NUCB2）. Nesfatin-1/NUCB2is not onlyexpressed in appetite control hypothalamic nuclei and brainstem, but also in theperiphery, including the pituitary, stomach, pancreas, testis, and adipose tissue,especially in the gastrointestinal tract. It is known that the function of nesfatin-1involved in the regulation of food intake, gastric emptying, gastroduodenal motility,stress responses, glucose/insulin regulation when administered peripherally orcentrally. It was demonstrated that the expression of NUCB2mRNA in the rat gastricoxyntic mucosa was at least10-fold higher compared with the rat brain. At thecellular level, immunoreactivity of nesfatin-1was detected in endocrine cells of themid and basal portion of the gastric glands of the oxyntic mucosa, and coexpressedwith ghrelin, somatostatin or histidine decarboxylase. These results suggest thatnesfatin-1may play an important role in gastric acid secretion. In this study, weaimed to study the effects of nesfatin-1on basal and histamine-stimulated gastric acidin rat gastric mucosa cells in vitro and the mechanism.Aim1. To study the effects of nesfatin-1on basal and histamine-induced gastric acid secretion in rat gastric mucosa cells in vitro;2. To characterize the effects of nesfatin-1on the expression of the H⁺/K⁺-ATPasemRNA and protein;3. To characterize the effect of nesfatin-1on parietal cell ultrastructural changes.Methods1. The gastric mucosa cells were isolated from SD rats by enzymolysis, DMEM/F12which including10%fetal bovine serum was used to culture the cell and the gastricmucosa cells were identified by immunofluorescence staining;2. The effects of nesfatin-1on gastric acid secretion in rat gastric mucosa cells wasinvestigated by accumulation14C-aminopyrine（14C-AP）;3. The H⁺/K⁺-ATPase alpha（α） and beta（β） subunit mRNA and protein expressionswere examined by Real-Time PCR and Western Blot;4. Ultrastructural analysis was performed using transmission electron microscopy.Results1. Isolation and identification of gastric mucosa cell in primary culture （1） The ratgastric mucosa cells were attached to the culture vessels by24h.（2）Immunofluorescence staining shows that parietal cells stained with an antibody to theH⁺/K⁺-ATPase β subunit, ECL cells were visualized by immunostaining for histamine,D cells were visualized by immunostaining for somatostatin, G cells were werevisualized by immunostaining for gastrin.The cytoplasm were stained by FITC（green）, the nuclear were stained by DAPI （blue）.2. The effects of nesfatin-1on basal and histamine induced gastric acid secretion inrat gastric mucosa cells in vitro （1） Nesfatin-1（10-1and10-2μmol/l） inhibited gastric acid secretion at2h and3h;（2） Nesfatin-1(10^-1μmol/l) inhibited histamine（10-4mol/l）-stimulated gastric acid secretion, the action of nesfatin-1reach a maximalinhibitory effect at1h;（3）Nesfatin-1(10^-1and10-2μmol/l) inhibitedhistamine（10-4mol/l）-stimulated gastric acid secretion.3. The effects of nesfatin-1on the expression of the H⁺/K⁺-ATPase mRNA andprotein （1） Nesfatin-1(10^-1μmol/L) inhibited the expression of H⁺/K⁺-ATPase αsubunit mRNA at1,2,3h and inhibited the expression of H⁺/K⁺-ATPase β subunitmRNA at1,2h; Nesfatin-1at the dose range from10-4to10^-1μmol/L dosedependently inhibited the expression of H⁺/K⁺-ATPase α subunit and β subunitmRNA at2h. Nesfatin-1at dose of10^-1μmol/l inhibited the H⁺/K⁺-ATPase α subunitprotein expression at1,2,3h and inhibited the H⁺/K⁺-ATPase β subunit proteinexpression at2,3h; Nesfatin-1at dose of10-3,10-2,10^-1μmol/L dose dependentlyinhibited H⁺/K⁺-ATPase α subunit and β subunit protein expression when pretreatedwith nesfatin-1for2h.（2） Nesfatin-1(10^-1μmol/l) inhibited the elevation ofH⁺/K⁺-ATPase α, β subunit mRNA and protein expression induced by histamine （10-4mol/l） for1h.4. The effect of nesfatin-1on the change of parietal cell ultrastructureNesfatin-1(10^-1μmol/l) changed the histamine-induced（10-4mol/l） parietal cellsconformational rearrangements.ConclusionsThese data suggest that nesfatin-1can inhibits basal and histamine-induced gastricacid secretion, and this effect is possibly due to by down-regulation of thetranscription and the translation of H⁺/K⁺-ATPase and change the cytological arrangements of parietal cell.