Effects of electro-needling at Zusanli on stress gastric ulcer in rats: Changes in nitric oxide and catecholamine

Abstract

AIM: To investigate the effects of electro-needling at Zusanli (ST36) on changes of nitric oxide (NO), dopamine (DA) and norepinephrine (NE) in rats with stress gastric ulcer.

METHODS: By biochemical methods the changes in contents of NO, DA and NE in gastric antral mucosa, corporal ventriculi mucosa and in blood after electro-needling (EN) were analysed. For observation all animals were randomly divided into four groups: stress group, EP after stress group, stress after EP group and control group.

RESULTS: NO content in serum of stress gastric ulcer rats (5.78 ± 1.49 μmol/L) was significantly decreased, as compared with the control group (13.30 ± 2.75 μmol/L; P < 0.01). DA content in gastric antral mucosa was markedly decreased (3.31 ± 0.67 ng/mg vs 6.78 ± 4.65 ng/mg, P < 0.05). DA in corporal ventriculi mucosa showed a tendency to increase. EN at Zusanli made NO level recover in stress gastric ulcer rats (7.91 ± 1.11 μmol/L), as compared with the stress group (P < 0.01). EN regulated DA and NE contents in antrum and corpora ventriculi mucosa bi-directionally, i.e., the contents were decreased when they formerly rose, and increased when formerly fell, respectively, as compared with the stress group (P < 0.01).

CONCLUSION: EN has a protective effect on the gastric mucosa. EN bi-directionally regulates DA and NE, and the vascular relaxation partially brought about by NO regulates the gastric mucosal blood flow, and strengthens the defensive function of the gastric mucosa.

Key Words: Nitric oxide, Dopamine, Norepinephrin, Zusanli, Electro-needling, Gastric ulcer

INTRODUCTION

Clinical practice has proved that acupuncture is effective in the treatment of gastrointestinal diseases, for which Zusanli is always first selected, but its mechanism is not yet clear completely. It is known that nitric oxide (NO) plays an important role in gastrointestinal physiology and pathology, while dopamine (DA) and norepinephrine (NE) serve as important neuro-transmitters in the gastrointestinal tract[1]. How these bioactive molecules exert their action in the course of electro-needling (EN) has not been reported. In this study, we investigated the changes of contents of NO and catecholamines (DA and NE) in stress gastric ulcer rats after EN at Zusanli.

MATERIALS AND METHODS

Model of stress gastric ulcer

Healthy male Wistar rats, weighing 160-230 g, were used to develop a model of stress gastric ulcer using restraint-cold method. The damaged gastric mucosa showed spotty or patchy hemorrhagic necrosis. The gastric antral mucosa and corpora ventriculi mucosa samples were taken, absorbed out of water, weighed, and stored in a freezer (-20 °C) until analysis. The serum samples were taken from eyeball blood.

Experimental groups

All rats were randomly divided into four groups: stress group, EN after stress group, stress after EN group and control group.

EN method

In the experiments, EN at Zusanli was applied to both sides of the rat using Model PCG-1 acupuncture therapy instrument, with a frequency of 10 Hz, stimulus strength of 10-20 V up to trembling of double lower limbs, totally given for 10 min.

Assay of biochemical indexes

Using the method described by Green et al[2], the contents of NO in serum were assayed with a 752 model ultraviolet grating spectrophotometer. The contents of DA and NE were assayed with a HITACHI MPE-4 model fluorescence spectrophotometer using the method described by Shellenberger et al[3]. All values are given as mean ± SD, and the data were statistically analyzed by t-test.

RESULTS

The effects of EN on contents of NO, DA and NE in stress gastric ulcer rats are shown in the Table 1.

Table 1 Effects of electroneedling on contents of nitric oxide, dopamine and norepinephrine in stress gastric ulcer rats (serum: μmol/L; tissue: ng/mg; wet weight).

	Control		Stress		EN after stress		Stress after EN
	n	Content	n	Content	n	Content	n	Content
NO	7	13.30 ± 2.75	6	5.78 ± 1.49b	6	42 ± 1.31	6	7.91 ± 1.11cb
DA
Gastric antrum	9	6.78 ± 4.65	9	3.31 ± 0.67a	6	4.78 ± 0.89d	6	3.14 ± 0.80
Corporal ventriculi	9	3.70 ± 1.62	9	4.66 ± 1.71	6	3.24 ± 0.77	6	2.33 ± 0.92d
Serum	6	10.32 ± 3.25	6	6.94 ± 4.15	6	4.74 ± 2.59b	3	5.06 ± 1.95a
NE
Gastric antrum	9	6.33 ± 3.77	9	6.63 ± 1.72	6	7.11 ± 4.46c	6	3.14 ± 0.80
Corporal ventriculi	9	2.65 ± 1.51	9	3.73 ± 1.81	6	2.03 ± 1.52	6	2.05 ± 1.19
Serum	6	2.84 ± 0.88	6	2.72 ± 1.00	6	2.07 ± 1.18	3	2.60 ± 1.42

^aP < 0.05,

^bP < 0.01, vs control group;

^cP < 0.05,

^dP < 0.01, vs stress group. NO: Nitric oxide; DA: Dopamine; NE: Norepinephrine; EN: Electroneedling.

The NO content in serum of stress gastric ulcer rats showed a significant decrease, as compared with the control group (t = 5.97, P < 0.01). DA and NE contents in the gastric antrum mucosa showed a tendency to decrease, and the decrease of DA had statistical significance (t = 2.22, P < 0.05). DA and NE in the corpora ventriculi mucosa showed a tendency to increase.

EN at Zusanli made NO level recover in the stress gastric ulcer rats. The NO level in the stress after EN group was significantly higher compared with the stress group (t = 2.84, P < 0.05). Although it was still markedly lower than that of the control group, it approached the level of the control group. EN regulated DA and NE contents in the gastric mucosa bi-directionally, i.e., DA in the antral mucosa was decreased when it had a rise formerly, and DA content of the EN after stress group was especially increased, as compared with that in the stress group (t = 0.68, P < 0.01). DA in the corpora ventriculi mucosa was increased when it decreased formerly, in the stress after EN group it was especially decreased compared with the stress group (t = 3. 04, P < 0.01), all of these were tending to a level of the control group. DA level in serum was continuously decreased in the EN after stress group compared with the control group (t = 3.20, P < 0.01). The changes of NE in the mucosa were similar to the changes of DA. The level of NE in serum had not changed basically.

DISCUSSION

In long time practice, clinical acupuncturists have treated gastrointestinal diseases by using Zusanli as the main acupoint. The data have shown[4-6] that needling at Zusanli has yielded obvious bi-directional function in regulating gastric motivity. It is no doubt related to the nervous and humoral factors. In recent years the study on NO has become a hot point in medical investigation. Because NO functions as a vasodilator, it acts on regulating the gastric mucosal blood flow and maintaining the mucosal integrity and defense. It is closely related to the protection of the gastric mucosa.

In this study we observed the changes of NO, DA and NE in stress gastric ulcer rats resulting from the damage to the gastric mucosa. DA has drawn more attention as a gastrointestinal neurotransmitter in recent years[7]. A decrease of DA contents in antral mucosa caused a decrease of blood flow and brought about poor perfusion to tissues, so that gastric ulcer occurred. DA produced NE through the action of dopamine-β-carboxylase, while the decrease of DA content led to a corresponding change of NE. NE is one of the agents that induce endotheliocytes to release NO[8]. When the gastric mucosa is damaged, the release of endogenous NO was decreased and normal biochemical metabolism was obstructed. It was disadvantageous for maintaining vessels in a relaxation state. The mechanism of DA to regulate the blood flow of the mucosa suggests that acting on DA₁ receptor in peripheral vascular wall should directly cause the expansion of blood vessels, and that, stimulating DA₂ receptor at sympathetic nerve ending should decrease the release of NE and increase the blood flow. The increase of DA content in the corpora ventriculi mucosa in the ulcer rats may possibly be a factor strengthening the gastric capacity for relaxation, on the other hand, may possibly be a factor yielding actions on different receptors. Therefore, contents of catecholamines (DA and NE) in the gastric antrum and corpora ventriculi showed different change tendencies.

The DA and NE contents in the ulcer rats after EN at Zusanli nearly recovered to normal, which showed a bi-directional regulatory effect. This indicates that acupuncture can protect the gastric mucosa from damage. The effect of acupuncture at Zusanli is closely associated with the nervous system, especially the autonomic nervous system. It may lower down the rising excitation of sympathetic nerve or parasympathetic nerve. Our study showed that the changes of the catecholamine content were consistent with the excitation of the nervous system. It also suggests that the effects of EN at Zusanli are due to the above-mentioned bio-active molecules, and other transmitters such as VIP (intrestinal peptide), ATP (adenosine triphosphate) and GABA (γ-aminobutyric acid) may take part in, but NO may be the final transmitter. From the change of NO, it was revealed that the EN applied before stress was more favourable than that applied after stress in ulcer rats. It further confirmed the protection of EN for the gastric mucosa.

In this study, DA content in serum of ulcer rats was found still continuously decreased after EN, and the NE in serum did not. It suggests that the damage to the gastric mucosa possibly speeds up the metabolic inactivation of peripheral catecholamines. DA decreased but NE remained in kinetic equilibrium, implying that the formation of NE from DA and the further analytic metabolism of NE were speeded up so as to fit the changes of the body in stress state. EN at Zusanli may give rise to the response of the body to the stress state.

To sum up, EN at Zusanli is able to cause the changes of NO and catecholamines in stress gastric ulcer rats, suggesting that EN is effective in protecting the gastric mucosa. It is regarded that the effect is brought about by the bi-directional regulatory effect of acupuncture on DA and NE, the regulation from DA, the changes of NE levels and the vascular relaxation due to NO, which all in turn regulate the gastric mucosal blood flow, and strengthen the defensiveness of the gastric mucosa.