Animals: After an adjustable feeding period of one week, 155 rats were randomly assigned into two groups: the control group (five normal rats) and the high-fat diet group (300 obese rats). In the control group, rats were fed with basic forage. In the high-fat diet group, rats were fed with high-fat forage (15 g of lard, 50 g of egg yolk, 15 g of whole milk, and 10 drops of concentrated cod liver oil were added into per 100 g basic forage). In order to assess the feasibility of the obese rat model, body length and body weight changes of the rats were recorded. Lee’s index and serum triglyceride levels were calculated after feeding for six weeks.
Implantation of the cannula into the lateral ventricle: Each rat was anesthetized using 7% chloral hydrate (0.5 mL/100 g) by intraperitoneal injection. The fur on the head was then shaved, with each rat being subsequently placed on the stereotaxic apparatus. According to the Paxinos-Watson rat brain stereotaxic coordinates, an incision was made into the skin above the animal’s skull, with a hole then being drilled into the skull. A thin catheter was slowly inserted into the right-cerebral ventricle and fixed with bone cement[20,21]. After surgery, each rat was fed separately and provided with 15000 units of penicillin to avoid infection. Follow-up experiments were carried out after they were fed normally for one week, when the stress reaction was reduced.
Experimental grouping: The 300 obese rats that had a cannula placed in the right lateral-cerebral ventricle were further randomly assigned into two groups: the intracerebroventricular injection group and the intravenous injection group. The treatment dose was as follows: intracerebroventricular injection group, 0 μg/5 μL of nesfatin-1, 0.5 μg/5 μL of nesfatin-1, and 1.0 μg/5 μL of nesfatin-1; intravenous injection group, 0 μg/5 μL of nesfatin-1, 0.5 μg/5 μL of nesfatin-1, and 1.0 μg/5 μL of nesfatin-1. Each group was comprised of 150 rats. Gastrointestinal mobility and gastric acid secretion in the rats were measured at 1, 2, 3, and 4 h after drug administration.
Determination of gastrointestinal mobility: Rats received intragastric administration of 2 mL of phenol red. After 20 min, the rats were anesthetized using diethyl ether and sacrificed. The stomach was then cut along the greater curvature, washed with distilled water to collect the stomach contents, and diluted with water to 20 mL. Next, 20 mL of 0.5 mol/L of NaOH was added into the suspension before standing for 0.5 h at room temperature. Then, 0.5 mL of trichloroacetic acid (200 g/L) was added into 2.5 mL of the supernatant to remove the proteins. The mixture was centrifuged at 3500 rpm for 10 min. The absorbance of the supernatant was measured at 560 mL. Gastric emptying rate was determined using the following formula: gastric emptying rate = (1- measured phenol red absorbance/ standard phenol red absorbance) × 100%.
Determination of gastric acid secretion: The principle of acid-base titrations was applied to determine gastric acid secretion. As previously described, gastric juice was collected from the stomach of rats. Then, 0.5 mL of gastric acid was pipetted into a clean beaker containing 2 mL of distilled water. Two drops of phenolphthalein was added, swirled gently to mix well, titrated by slowly adding the NaOH solution (0.5 mol/L), and then swirled in the beaker. Titration ended when the solution turned a faint and persistent color. The volume of NaOH dispensed was recorded in the following manner: volume of gastric acid secretion= (C [NaOH] ×V [NaOH] ×V [gastric juice] /0.5 mL).
Real-time polymerase chain reaction: Gastric mucosal tissues measuring 20 mg were randomly obtained from different parts. Total RNA was extracted using TRIZOL reagent and converted into cDNAs by reverse transcription. With β-actin as the internal control gene, target gene fragments were amplified by real-time polymerase chain reaction (PCR). RT-PCR products measuring 5 μL were subjected to 2% agarose gel electrophoresis. The absorbance of the target gene strands was detected. The relative mRNA expression level of H+/K+-ATP was determined as the ratio of the absorbance of the target gene to that of β-actin.
The real-time PCR primers are as follows: H+/K+-ATPase, 5’-CTCTGCTTTGCGGGACTT-3’ (forward) and 5’-CCTTGGCTGTGATGGGAT-3’(reverse); β-actin, 5’-AGCTGAGAGGGAAATCGTGCG-3’ (forward) and 5’-GTGCCACCAGACAGCACTGTG-3’ (reverse).
Western blot: Gastric mucosal tissues measuring 20 mg were randomly obtained from different parts and total proteins were extracted from the samples. Protein concentrations were detected using the BCA method. Protein extracts containing the same quality were subjected to SDS-PAGE using 12% polyacrylamide gel, followed by western blotting. Each membrane was blocked in 5% skim milk for 2 h at room temperature. The membrane was then incubated for one hour at room temperature with anti-H+/K+-ATPase antibody. After washing with phosphate-buffered saline/tween three times for five minutes each time, each membrane was incubated with a corresponding secondary fluorescein-labeled antibody for one hour at room temperature. The bands were then visualized and imaged using the Odyssey infrared imaging system (LI-COR, Germany).