| [Background]Nesfatin-1,product of the precursor NEFA/nucleobindin2(NUCB2),was initially identified as anorectic hypothalamic neuropeptide,acting in a leptin-independent manner.In central,nesfatin-1 immunoreactive cells are present in a number of discrete neuronal populations or nuclei,which are implicated in the feeding and metabolic regulation,including the hypothalamic arcuate nucleus(ARC),paraventricular nucleus(PVN),supraoptic nucleus(SON),lateral hypothalamic area(LHA),and dorsal motor nucleus of the vagus(DMV),nucleus tractus solitarius(NTS)in the brain stem.In peripheral,nesfatin-1 immunoreactive cells are localized in the gastric mucosal gland,pancreatic islets,the duodenum and adipocyte.Nesfatin-1 is co-expressed with thyrotropin releasing hormone(TRH),corticotropin releasing hormone(CRH)and somatostatin in the paraventricular nucleus(PVN).These regulatory peptides are involved in the regulation of gastric acid secretion,suggesting that central nesfatin-1 may be involved in the regulation of gastric acid secretion.Our previous studies have found that lateral ventricle injection of nesfatin-1 can inhibit gastric acid secretion and downregulate histidine decarboxylase,but the concrete approach is not yet clear.Studies have found that injected nesfatin-1 into the fourth intracerebral ventricle inhibit 2-deoxy-D-glucosestimulated gastric acid production and activated the expression of c-Fos in DMV.Nesfatin-1 also activate T-type calcium channel in primary cultured DMV neurons and activates vagal afferent neurons by stimulating Ca2+ influx through N-type channels.We postulate that vagus nerve may participate in the process of nesfatin-1-induced inhibition of gastric acid secretion.Therefore,we examined in the present study the hypothesis that i.c.v injection of different concentrations of nesfatin-1 may affect gastric acid secretion and the level and activity of H~+/K~+-ATPase,and we also investigated whether vagal system signal pathway is involved in the above effect of nesfatin-1.[Aim]To observe the effects of vagotomy on intracerebroventricular(ICV)injection of nesfatin-l-induced decreases in gastric acid secretion.[Methods]1.Male Sprague-Dawley rats weighing 200-250g were maintained under controlled temperature(21-23 ℃)and light(light on 06:00-18:00h)with ad libitum access to chow and water.The rats were maintained in individual cages after ICV cannula implanted.2.Intracerebroventricular cannulation:Animals were anesthetized with 5%chloral hydrate(0.8ml/100g),cannulae were placed unilaterally into the lateral ventricles,A-0.8mm behind the bregma,L +1.5 mm from the midline,and D 3.6 mm below the skull surface.The cannula was maintained in place by glass ionomer cement between two screw spikes.After cannula implanted,animals were housed single in a cage,and given a 5-7 days recovery.3.Normal adult rats were randomly divided into 4 groups,which were administered by nesfatin-1(1,5,50pmol)and equal sterile water(5ul)into the lateral ventricle.Two hours after the treatment,the volume of gastric secretion was measured and the gastric tissue was preserved at-80 ℃.4.The expression of protein and mRNA level of H~+/K~+-ATPase in control and nesfatin-1-treated groups were examined by RT-PCR.And the activity of H~+/K~+-ATPase was measured.5.Subdiaphragamatic vagotomy:under pentobarbital sodium anesthesia ’(50 mg/kg),a 2-cm ventral midline abdominal incision was made.The dorsal and ventral branches of the vagus nerve were exposed carefully and dissected away from the esophagus.Each branch of the vagus nerve was tied with surgical sutures at 2 points separated by 1 cm and severed between the sutures.All rats allowed to recuperate for 5-7 days.6.In another group,rats were divided into vagotomy and sham group.Nesfatin-1(50pmol)and sterile water were respectively injected and the secretion of gastric acid was measured 2h after treatment and the gastric tissue was preserved by-80 ℃.7.The expression of protein and mRNA level of H~+/K~+-ATPase in vagotomy and sham group were examined by western-blotting and RT-PCR.And the activity of H~+/K~+-ATPase was measured.[Results]1.Nesfatin-1(5 and 50pmol/rat)induced a significant decrease in gastric acid secretion and increase of pH 2 hour after i.c.v injection compared to vehicle(P<0.05),while nesfatin-1(1 pmol/rat)not.Furthermore,nesfatin-1 in 50pmol/rat showed more significant suppression in gastric acid secretion compared to 5 pmol/rat(P<0.05 both in gastric acid content and pH).2.Compared to control group,nesfatin-1(5 and 50pmol/rat)down-regulated H~+/K~+-ATPase expression and activity 2 hour after i.c.v injection(P<0.05).50pmol/rat showed more significant suppression in H~+/K~+-ATPase expression and activity compared to 5 pmol/rat(P<0.05).3.Intracerebroventricular injection nesfatin-1(50pmol/rat)significantly suppress gastric acid content and increase the pH of gastric juice in sham group rats(P<0.05).Whereas in vagotomy group,the effect of nesfatin-1-induced in decreased gastric secretion was not occurred.4.The expression and activity of H~+/K~+-ATPase was inhibited in sham group rats after i.c.v injection nesfatin-1(50pmol/rat)(P<0.05).Whereas in vagotomy group,the effect of nesfatin-1-induced in down-regulation of H~+/K~+-ATPase was not occurred.[Conclusion]Nesfatin-1 injected intracerebroventricularly induces a dose-dependent decrease in gastric acid secretion in rat and vagal nerve signal pathways may be involved in the process of the effect of nesfatin-1.[Background]NUCB2/nesfatin-1 was initially found to be located in the hypothalamus as satiety molecule.In the center,it was widely distributed in food intake associated brain nucleus.In addition,it was also abundant in the periphery,including the stomach,duodenum,pancreas,and adipocyte.Previous studies on NUCB2/nesfatin-l were more concentrated in regulating food intake,glucose and lipid metabolism.In recent years,with the gradual deepening of the research about NUCB2/nesfatin-1 it has been shown related to the development of a variety of tumors,but the specific mechanism is still unknown.Gastric cancer is the world’s second largest cause of cancer related death,which can lead to 80 million patients die each year,is high morbidity and mortality of malignant tumors in the world.It is also one of the most common gastrointestinal tumor in our country,a serious threat to human health.Its etiology and specific pathogenesis is not yet clear.Gastric cancer development is a multi-gene complex process and it is particular important to reveal the possible mechanism of tumorigenesis and seek the possible therapeutic targets.So,how about the expression of NUCB2/nesfatin-1 in patients with gastric cancer?Is it related with the clinical pathological parameters?By detecting the expression of NUCB2/nesfatin-1 in tissue and sera of patients with gastric cancer,we discuss its relevance to gastric cancer and its clinical significance.[Aim]To determine the expression level of NUCB2/nesfatin-1 in tissues and sera of gastric cancer patients and healthy controls.And evaluate its relationship with clinicopathological parameter.[Methods]1.Case selection:from August 2014 to June 2015,patients who received surgical resection due to gastric lesions in Gastrointestinal Surgery of the First Affiliated Hospital of Nanjing Medical University and pathological diagnosis of gastric cancer.Exclusion criteria:①fat,that the body mass index(BMI)>28;②severe diabetes,thyroid disease and heart disease;③gastric tissue specimens and serum samples were not complete;④receiving preoperative chemoradiotherapy.And select the corresponding healthy control group.2.The concentration of serum NUCB2/nesfatin-1 was determined using enzyme-linked immunosorbent assay(ELISA).3.The expression of mRNA level of NUCB2/nesfatin-1 was examined by Real-time Polymerase Chain Reaction(RT-PCR)4.Collect the clinical pathology data of the patients with gastric cancer and analyze relevant factors affecting the expression levels of NUCB2/nesfatin-1.[Results]1.The difference between the mean mRNA expression of NUCB2/nesfatin-1 in cancers and normal tissues was statistically significant.2.NUCB2/nesfatin-1 mRNA was downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues.3.No correlation between the mRNA expression level and age,sex,tumour location,size,grade or stage was observed.4.Serum nesfatin-1 levels were lower in gastric cancer patients than in healthy subjects.In gastric cancer patients,nesfatin-1 levels were increased after surgery.5.Serum nesfatin-1 levels were negatively correlated with tumor diameter,stage,lymph node metastasis and nerve vascular invasion.[Conclusion]The expression of NUCB2/nesfatin-1 was decreased in tissue and serum of patients with gastric cancer compared with the healthy control group,suggesting that NUCB2/nesfatin-1 may be involved in the development of gastric cancer,but the exact mechanism needs further study. |
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