Gastrin, somatostatin, G and D cells of gastric ulcer in rats

Abstract

AIM: To investigate the relationship among gastrin, somatostatin, G and D cells in gastric ulcer and in its healing process in rats.

METHODS: Fourty-nine Wistar rats were divided into 7 groups. The gastric ulcer model was induced by acetic acid successfully. The gastrin and the somatostatin in rat plasma, gastric fluid and antral tissue were measured by radioimmunoassay (RIA). G and D cells in antral mucosa were analyzed with polyclonal antibody of gastrin and somatostatin by immunohistochemical method and Quantimet 500 image analysis system.

RESULTS: In gastric ulcer, the level of gastrin in plasma, gastric fluid, and antral tissue increased, that of somatostatin declined, and the disorder gradually recovered to the normal level in the healing process. Immunohistochemical technique of G and D cells in antral mucosa demonstrated that the number of G cells increased and that of D cells decreased, both areas of G and D cells declined, the ratio of number and area of G/D increased in gastric ulcer, and the disorder gradually recovered in the healing process.

CONCLUSION: In gastric ulcer, the increased gastrin secreted by G cells, the declined somatostatin secreted by D cells, and the disordered G/D cell ratio can lead to gastrointestinal dysfunction.

Key Words: $[Keywords]

INTRODUCTION

Gastrin is secreted in G cells, while somatostatin is secreted in D cells. Gastrin, somatostatin and other gastrointestinal hormones regulate the function of gastrointestinal tract such as secretion, movement, absorption, circulation and nutrition of cells[1-12]. The nerve system and the endocrine system participate in the healing process of gastric ulcer and regulate the absorption of inflammantory filtration, hyperplasia of granulation tissues, and regeneration of epithelial tissues. Obvious regular change takes place in many endocrine tissues[13-24]. In order to investigate the relationship between gastrin, somatostatin, G and D cells in the period of gastric ulcer and its healing process in rats, the gastrin and somatostatin in plasma, gastric juice and antral tissue were tested in rats. At the same time, the number of G and D cells was measured in the antral mucosa.

MATERIALS AND METHODS

Animal

Forty-nine healthy male Wistar rats weighing from 200 g to 260 g were obtained from the Experimental Animal Center of Sun Yat-Sen University of Medical Sciences. The rats were divided into seven experiment groups: 4, 7, 10, 14, 21, 28 d groups, and a control group. The rats of six experimental groups were anesthetized with 30 g•L⁻¹ sodium pentobarbital intraperitoneally at a dose of 30 mg•kg^-1. The abdomen was opened and its stomach was found, 0.05 mL acetic acid was injected into rat's antral tissues. Omentum majus and antral tissue of the injection site were stitched. The peritoneum, parietal abdomen and ventral muscle, and skin were stitched continually. After operation, the rats were raised separately, and fasted overnight with free access to water one day before sacrifice. No treatment was given to normal control group.

Plasma sampling

The rats' abdomen and chest chambers were opened and 4 mL blood was directly withdrawn from the heart ventricle. Fourty μL (500 u) aprotinin (Livzon Libao Biochemical&Pharmacertical Co. Ltd) and 60 μL of 100 g•L⁻¹ EDTA was added to each blood sample. The samples were centrifuged at 3500 r•min^-1 for 15 min to obtain plasma. The plasma samples were then stored at -70 °C until assay.

Gastric juice

After the rats' abdomen was opened, a plastic catheter was inserted into the stomach through pylorus and another catheter was inserted through oral cavitas and esophagus into stomach. Two mL saline (pH7.0, 35 °C) was infused into the stomach at a flow velocity of 12 mL/h. The gastric fluid was collected and 40 μL (500 u) aprotinin was added. The samples were centrifuged at 3500 r•min^-1 for 15 min to obtain gastric fluid. The gastric fluid samples were then stored at -70 °C until assay.

Antral mucosa

The rats' stomach was separated and was split from the greater curvature of stomach. The antral tissues in the ulcer area and non-ulcer area were separately taken with ophthalmic scissors. The tissue was quickly weighed by an electric analytical balance and was boiled in a microwave stove. The boiled tissue was homogenized into homogenate in a homogenizer with 1 mL of 1 mol•L^-1 acetic acid. Then 1 mL of 1 mol•L^-1 NaOH was added to neutralize it. The homogenate was centrifuged at 3500 r•min^-1 for 15 min and the supernatant was collected. The samples were then stored at -70 °C until assay. The stomach was separated and was split from the greater curvature of stomach. About 1.0 × 0.5 cm antral tissues in the ulcer area and non-ulcer area was separately taken with ophthalmic scissors. The specimen was fixed in 100 mL•L⁻¹ neutrally buffered formalin. It was embedded with paraffin 24 h later and was serially sectioned at 4 μm. The sections were mounted onto histostick-coated slides. Adjacent ribbons were collected for immunohistochemical staining.

Measurement of gastrin and somatostatin

Gastrin and somatostatin were detected by using RIA method. The gastrin kits were purchased from Tianjin Qianye Biotech Co. Ltd, and the somatostatin kits were bought from the Department of Neurobiology of the Second Military Medical University. Measurement procedures were performed according to the instruction attached to the kits. The unit of result of plasma and gastric fluid was transformed to ng•L⁻¹, while the unit of result of antral mucosa tissue to ng·g^-1.

Immunohistochemical staining for G and D cells

The anti-gastrin antibody (Sigma Co. USA) and anti-somatostatin antibody (GYMED Co. USA) were used and immunohistochemical staining for G and D cells was performed with the strept-avidin-biotin-peroxidase complex (SABC) (Wuhan Boster Biological Technology Co. Ltd), negative control sections were normal serum blocking and PBS instead of the primary antibody. Images of 5 views randomly selected under microscope (10 × 40) from each anti-gastrin immunohistochemical staining section were input into the Quantimet 500 image analysis system (Leica Co, Germany). The number and area of G cells were calculated by the computer. The mean number and area of G cells in 5 views of the section served as the number and area of G cells of a section. That of D cells was calculated in the same way as the G cells. The ratio of G/D number and G/D area was acquired by separately dividing the number of G cells and the area of G cells by the number of D cells and the area of D cells in adjacent sections.

Statistical analysis

The result of quantitative data was expressed as mean ± SE. Data was analyzed using the variance analysis (ANOVA). Post hoc analysis between factors was performed using least significant difference test. Unpaired data was compared using the Wilcoxon ranksum test and correlated using Pearson's correlation.

RESULTS

The concentration of gastrin and somatostatin in the plasma, gastric juice, antral mucosa is shown in Table 1 and Table 2. The number and area of G and D cells in the rat antral mucosa, and the ratio of G/D number and G/D area is shown in Table 3. The relationship between rat G and D cells, and the relationship between G or D cells with gastrin or somatostatin are shown in Table 4.

Table 1 Gastrin concentration in rat plasma, gastric juice, and antral mucosa (¯x ± s, n = 7).

Group	Plasma (ng·L-1)	Gastric juice (ng·L-1)	Ulcer mucosa (ng·g-1)	Non-ulcer mucosa (ng·g-1)
Control	147 ± 41	44 ± 15	-	3.7 ± 1.1
4 d	397 ± 130a	56 ± 16	2.0 ± 0.7	6.7 ± 2.3a
7 d	364 ± 91a	114 ± 34a	1.3 ± 0.4b	6.1 ± 1.8a
10 d	255 ± 87a	60 ± 20	2.3 ± 0.8	1.9 ± 0.7a
14 d	238 ± 88	45 ± 21	1.9 ± 0.7	1.1 ± 0.4a
21 d	211 ± 65	44 ± 17	1.3 ± 0.4b	1.1 ± 0.7a
28 d	216 ± 95	60 ± 18	1.5 ± 0.5	1.5 ± 0.3a

^aP < 0.05, vs control group.

^bP < 0.05, vs 4 d group.

Table 2 Somatostatin concentration in rat plasma, gastric juice, and antral mucosa (¯x ± s, n = 7).

Group	Plasma (ng·L-1)	Gastric juice (ng·L-1)	Ulcer mucosa (ng·g-1)	Non-ulcer mucosa (ng·g-1)
Control	45 ± 12	64 ± 16	-	1.4 ± 0.4
4 d	13 ± 5a	46 ± 15a	0.3 ± 0.1	0.4 ± 0.1a
7 d	22 ± 8a	42 ± 15a	0.3 ± 0.1	0.4 ± 0.2a
10 d	41 ± 9	54 ± 17	0.2 ± 0.1	0.8 ± 0.2a
14 d	33 ± 8a	54 ± 12	0.9 ± 0.4b	1.3 ± 0.4
21 d	32 ± 9a	49 ± 12	1.1 ± 0.2b	1.1 ± 0.4
28 d	45 ± 12	64 ± 16	1.1 ± 0.3b	1.1 ± 0.3

^aP < 0.05, vs control group.

^bP < 0.05, vs 4 d group.

Table 3 Number and area of G and D cells, and the ratio of G/D number and G/D area (¯x ± s, n = 7).

Group	No. (G cells)	Area of G cells(× 10-6 m2)	No. (D cells)	Area of G cells (× 10-6 m2)	Number ratioof G/D	Area ratio of G/D
Control	33 ± 6	99 ± 7	15 ± 2	70 ± 11	2.3 ± 0.1	1.4 ± 0.1
4 d	50 ± 7a	87 ± 7a	10 ± 2a	56 ± 8a	4.9 ± 0.3a	1.6 ± 0.1a
7 d	69 ± 8a	91 ± 7a	9 ± 2a	60 ± 9a	7.6 ± 0.5a	1.5 ± 0.2
10 d	73 ± 13a	94 ± 7	10 ± 1a	63 ± 7	7.4 ± 0.4a	1.5 ± 0.1
14 d	62 ± 11a	95 ± 9	11 ± 2a	66 ± 11	5.8 ± 0.5a	1.5 ± 0.1
21 d	46 ± 8a	95 ± 8	14 ± 2	68 ± 11	3.4 ± 0.2a	1.4 ± 0.1
28 d	43 ± 6a	95 ± 7	14 ± 2	66 ± 8	3.1 ± 0.1a	1.4 ± 0.1

^aP < 0.05, vs control group.

Table 4 Relationship between rat G and D cells, and between G or D cells and gastrin or somatostatin.

Group	No. of G cells vsNo. of D cells	Area of G cells vs area of D cells	No.of G cells vsgastrin in non-ulcer mucosa	No.of D cells vssomatostatin in non-ulcer mucosa	Area of G cellsvsgastrin in non-ulcer mucosa	Area of D cells vssomatostatin in non-ulcer mucosa
Control	0.97b	0.95b	0.95b	0.93b	0.94b	0.93b
4 d	0.92b	0.93b	0.87a	0.90b	0.98b	0.86a
7 d	0.97b	0.93b	0.98b	0.98b	0.93b	0.94b
10 d	0.97b	0.96b	0.93a	0.88b	0.93b	0.95b
14 d	0.91b	0.99b	0.87a	0.89b	0.78a	0.99b
21 d	0.97b	0.88b	0.90b	0.68	0.98b	0.99b
28 d	0.99b	0.94b	0.95b	0.89b	0.92b	0.96b

^aP < 0.05,

^bP < 0.01, vs control group.

DISCUSSION

Both gastrin and somatostatin are gastrointestinal hormones closely related to the function of gastrointestinal system. Gastrin is mainly secreted in G cells in antral and upper small intestines. It has several kinds of molecules and distributes in plasma, tissues, gastric juice, and intestinal juice. Somatostatin is a 14 amino peptide. It also has several kinds of molecules and distributes vastly in the body. The somatostatin in gastrointestinal system is secreted in D cells. It is mainly in intestinal nerve plexus, stomach and pancreas, and it is also in gastric and intestinal fluid. In the antrum there are many G and D cells, most of which belong to the open type endocrine cells that can directly secrete gastrin or somatostatin into gastric fluid. Elevated gastrin level can increase the secretion of basal and peak gastric acid, pepsin and inner factor, increase the amount of circulating blood in the mucosa of upper gastrointestinal tract, improve the nutrition of gastric mucosa cells. Somatostatin can obstruct the secretion of basal gastric acid and pepsin and inhibit the invoking function of gastrin to the secretion of gastric acid[25-38]. Our experiment is to reveal the change of gastrin and somatostatin in gastric ulcer rats. The result shows that when the rat had gastric ulcer, the gastrin in plasma, gastric juice and antral mucosa tissue increased, and the somatostatin declined. With the healing of ulcer, gastrin and somatostatin gradually recovered to the normal level.

G and D cells have interactive relation in the function[39-49]. In order to study their morphology we used the immunohistochemical method to identify the G and D cells and to measure the number and area of G and D cells by the medical image analysis system. We found that with ulcer the number of G cells increased as compared with the normal control group, the D cells decreased, and both areas of G and D cells declined. And with the healing of the ulcer, the number and area of G and D cells gradually recovered to the normal level. This suggests that in the ulcer stage, the function of synthesis and secretion of G cells increased, while that of D cells declined. This inclined us that the increased secretion of gastrin in ulcer rat might be related to the increased number of G cells. The declined area of G cells may be related to the increased secretion of gastrin. The decline of somatostatin secretion in gastric ulcer is likely related to both the decreased number of D cells and the reduced area of D cells. It is known that somatostatin can inhibit G cells to secrete gastrin. Because in the gastric ulcer, the somatostatin secretion of D cells in rats lowered its inhibitory effect to G cells, gastrin secretion of G cells increased.

G and D cells are interrelated in morphology. In normal gastrointestinal mucosa, the ratio of G/D number and G/D area is stable. This is useful for keeping the normal gastrointestinal function. The ratio of G/D cells can be an important index for knowing the change of the two kinds of cells and its effect on focal endocrine function[50-58]. Our result showed that in gastric ulcer, the ratio of G/D number and the ratio of G/D area increased than that in normal control group. In the healing process, the disorder recovered gradually to the normal level. This proves that the number of G cells increased and the number of D cells declined in gastric ulcer. It also shows that in gastric ulcer rat, the decrease of the D cell area is more obvious than that of G cells. The statistical analysis indicates that there is a relationship between the number of G and D cells, and between the area of G and D cells. There is also a relationship between the number and area of G cells and gastrin in antral mucosa, and between the number and area of D cells and somatostatin in antral mucosa. In summary, our result further proves histologically that in gastric ulcer rat, the G cell secreted gastrin increased, and the D cell secreted somatostatin reduced, the ratio and function of G/D cells were imbalanced.