by Huang Yuxin,Sun Dayong,Chu Zihong Gao Wei,and Wang Qingli 
　　(Dep. of Gastroenterology,*Dep. of TCM, Tangdu Hospital, the Fourth Military Medical University, Xi'an 710038, China)
　　Abstract Aim of the study: To observe the effect of electroacupuncture (EA) on canine pyloric pressure and its relation with contents of motilin (MTL), somatostatin (SS ) and nitric oxide synthetase (NOS) in the gastric mucosal tissues. Methods: The total and basic pressure of the pyloric sphincter, and the frequency of the high-pressure waves were measured by using a gastrotonometer; and the contents of MTL, SS and NOS in tissues of the gastric body and gastric antrum mucosa were detected by using radioimmunoassay(RIA) and biochemical methods in 20 dogs. Results: After EA of Zusanli (ST 36), the total and basic pressure of the pyloric sphincter, and the frequency of the high-pressure waves, the content of SS in the gastric body mucosa, MTL and SS in the gastric antrum mucosa all decreased significantly (P<0.05) and the level of NOS increased clearly ( P<0.05). While after EA of Xiajuxu (ST 39), all the indexes had not any striking changes except significant decrease of SS content in the gastric body mucosa (P< 0.05). Conclusions: EA has a significant modulating action on gastrointestinal functional activities by lowering canine pyloric pressure and contracted frequency, which is also related with its influence on contents of some brain-gut peptides (BGP) and is of specificity in meridians and acupoints.

Key Words EA modulation Pyloric pressure Brain-gut peptide

Modern clinical observations and experimental studies demonstrate that EA is of favorable modulating action on gastrointestinal functional activities but its mechanisms remain unknown. It was reported that EA could regulate the pressure of the pyloric sphincter[1]and affect synthesis and release of some BGP[2], but its mechanisms and the possible connection between the functional regulation and changes of BGP level have not been explained clearly. Up to now, any synchronous observations on EA's effect on pyloric pressure and its relation with changes of some BGP have never been reported in journals both at home and abroad. In the present study, we conducted synchronous observations on the effect of EA on the canine pyloric pressure and the resultant changes of MTL, SS and NOS contents in the gastric mucosal tissues.
　　Material and Methods
　　1. Materials Healthy mongrel dogs weighing 15 to 20 kg were used in the experiments. Twenty dogs were randomly and evenly divided into 4 groups: Zusanli (ST 36), Xiajuxu(ST 39), Non-acupoint and control groups. Before experiments, these animals were fasted for 24 hrs but could drink water freely. On the day of experiment,each dog was anesthetized with intravenous injection of pentobarbital sodium 1mg/kg. An abdominal median incision was performed to expose the gastric body, followed by making a small incision about 0.5 cm in diameter at the front wall of the stomach body and inserting a Teflon catheter connected with a pressure transducer and then with a gastrotonometer (WYY1 Type, made by the Institute of Aerospace Medical Engineering) to the pyloric canal part and fixing it. The pyloric pressure was recorded.
　　2. Methods Bilateral Zusanli (ST 36) and Xiajuxu (ST 39)[3]of the Stomach Meridian of the Foot Yangming were selected. The non meridian non-acupoints were located at the bilateral midpoint between the small head of the fibula and the lateral malleolus, about 1 cm to the lateral edge of the fibula, i.e. between Zusanli (ST 36 ) and Xiajuxu (ST 39). Animals in the blank control group were not needled and not stimulated electrically but the above-mentioned indexes were recorded or detected. A 6805-A Electroacupuncture Therapeutic Apparatus (made by Shanghai Medical Instrument Factory) was used for stimulating the acupoint by setting a stimulating frequency of 50 Hz, voltage of 20 V, intermittent waves and a duration of 30 min. The stimulating strength was adjusted to the degree which could induce slight tremor in the lower limb. The pyloric pressure was detected and recorded continuously for 2 min before EA, once every 10 min in 90 minutes' observation after EA. At the same time, about 3g of the mucosal tissues from the stomach body and the gastric antrum was taken and boiled in normal saline for 5 min at 100°C. The mucosal tissues were dissected to be weighed, then ground and homogenized after adding 1N acetic acid (HAc) 1ml. Two or three hours later, the homogenate was added with the same volume of 1N NaOH to be centrifuged at 3500 r/min for 15 min at 4°C. The supernatant was stored at -70°C until used. Contents of MTL and SS were measured by using radioimmunoassay with duplicate tubes and FJ-2003-50G γ Counter (made by State-run 262 Factory). The radioimmunoassay reagent kits were provided by Beijing East Asian Immune Technique Institute and the Department of Neurobiology of the Second Military Medical University. NOS contents were measured by using biochemical method and the reagent kit was supplied by the Radio-medical Institute of the Military Medical Academy. OD values were read and recorded by using 752 Type Spectrophotometer.
　　3. Statistics data were processed by employing NOSA software in a computer and expressed as mean±SD. A value of P<0.05 was accepted as statistically  significant. Results
　　Changes of pyloric pressure and concentrations of MTL, SS and NOS in the mucosal tissues of the gastric body and antrum before and after EA in every groups were listed in the following Table. 
　　Table. Changes of Pyloric Pressure and Concentrations of MTL, SS and NOS  in the Gastric Mucosal Tissues before and after EA in Every Groups (M±SD)   Indexes  Zusanli group(n=5) Xiajuxu group(n=5) Non-aupoint group (n=5)Control group
　　(n=5)
　　Pre-EAEA-30min
　　 Pre-EAEA-30min   Pre-EA  EA-30minPre-EA
　　  EA-30minTotal pressure (kpa)  28.7± 2.3 23.3±2.7** 26.1± 3.3 26.3± 4.8 26.9± 0.8 25.3±3.2 28.2±3.2 28.5± 2.7Basic pressure(kpa)
　　 17.7±1.8  13.2±2.9* 15.8± 0.6  16.0±6.9 16.2±2.4 15.6±1.716.7±1.917.3±3.8Frequency (C/min)4.8±0.6 4.1±0.3* 4.7±0.6   4.6±0.95.1±0.7 4.9±1.3 4.6±0.3  4.8±0.5MTL of GB (ng/L)147.2±22.8156.3±18.1 164.7±2 1.5 159.8±15.3127.6± 28.7   129.1±38.4  186.9±21.2  173.5±29.7 SS of GB (ng/L) 169.5±25.3128.2±29.3*   143.4±19.5102.8±21.3* 197.1±18.5  177.3±26.7  149.7±21.5 156.9±36.3 NOS of GB (ng/L) 4.5±0.7 5.5±1.7 4.5±1.2  4.0±2 .7 4.7±0.3 4.9±0.8 5.2±2.0   4.5±2.4MTL of GA (ng/L) 209.4±23.3 156.0±29.2* 235.1±31. 6 210.8±40.3  224.7± 19.8  216.2±28.0   241.2±27.8  235.7±31.3 SS of GA (ng/L) 87.9± 7.7  71.8±10.7*81.2± 9.9   86.9±13.1  75.7± 18.9 68.6±12.7  73.1±21.2  81.8± 26.9NOS of GA (ng/L) 2.7±0.3 2.1±0.4* 2.5±0.7 2.8 ±1.0 3.2±0.4  3.0±0.5 2.5±0.2  2.6±0.4GB: gastric body; GA: gastric antrum; *P<0.05 **P< 0.01 It can be seen clearly that 30 min after EA of Zusanli (ST 36), the total pressure and basic pressure of the pyloric sphincter muscle lower significantly ( P< 0.05), the frequency of the high-pressure waves decreases considerably (P<0.05); SS content of the mucosal tissues of the gastric body, MTL and SS contents of the gastric antrum decrease strikingly (P<0.05) but NOS content of the gastric antrum lowers apparently; while MTL and NOS contents of the gastric body have no significant changes. All the indexes except SS content in the mucosal tissues of the gastric body in Xiajuxu group and those of non-acupoint and control groups have not any considerable changes.
　　Discussion
　　EA stimulation is capable of modulating functional activities of the stomach. Its theoretical basis lies in that the meridians are subordinates to the internal organs in the interior and also connect closely with the body surface including muscles, skin, etc. in the exterior. EA of Renzhong (GV 26) could significantly suppress the contracted amplitude and frequency of the gastric antrum in dogs[4] . EA of Zusanli (ST 36) could cause the gastric motion to increase in patients with weak gastric motility and could also inhibit gastric hyperactivity in patients with enhancement of gastric motility[5]. Results of the present study displayed that EA of Zusanli (ST 36) could significantly lower the total pressure, basic pressure and frequencies of the high-pressure waves of the pyloric sphincter muscle, suggesting that the effect of EA on gastric movement is achieved partially by the way of regulating the function of the pyloric sphincter muscle. It is held in traditional Chinese medicine that converging points often serve to treat diseases of the corresponding viscera, thus EA of Zusanli (ST 36), the lower converging point of the Stomach Meridian could effectively modulate the pyloric movement. While EA of Xiajuxu (ST 39), the lower converging-point of the Small Intestine Meridian or non-acupoint near Zusanli had no significant action on the pyloric pressure, showing a relative specificity of acupoints in regulating gastric functional activity. Mocells secreting MTL mainly distribute in the mucosal tissues of the duodenum and jejunum. MTL functions chiefly in stimulating the mechanical and electrical activities of the upper digestive tract and initiating the migrating motion  complex (MMC) waves in the inter digestive period and speeding gastric emptying of solid food and fluid in the stomach[6] . Most of SS in the stomach distributes in the D type cells of the gastric body and antrum mucosal tissues. It is of a suppressive action on multiple physiological functions of the stomach, including inhibition on the secretion of almost all the BGP and gastric motion[7] . NOS is the only synthetase in the process of production of NO. Its content in the inter muscular nerve plexus is significantly higher than that of its surrounding tissues. NO can result in relax of the gastrointestinal smooth muscle and plays a very important role in regulating pyloric functional activity[8] . In the present study, results showed that 30 min after EA, SS content of the mucosal tissues in the gastric body lowered in both Zusanli and Xiajuxu groups; MTL and SS contents of the gastric antrum in Zusanli group decreased but NOS content increased, while those indexes in Xiajuxu group had no striking changes. It suggests that EA stimulation may lead to changes of levels of MTL,SS, NOS, etc. and some related synthetases, especially in the gastric antrum, which may be due to different distribution of BGP secreting cells in difficult part of the gastrointestinal tract. In addition, different acupoints had a different action on BGP. MTL can cause the gastric smooth muscle to produce stronger contraction, on the contrary, NO may lead to its relaxation. Therefore, after EA of Zusanli (ST 36), decrease of MTL content and increase of NOS level worked in coordination to result in decline of pyloric pressure and decrease of the contracted frequency of the pyloric sphincter muscle. In addition, SS can function in suppressing gastrointestinal movement. When its content declines, the gastric movement should increase. However, this situation did not occur in this study, the reasons probably are: 1) after EA, MTL and NOS change-induced suppression of gastric movement is strong enough to overcome SS decline-caused indirect enhancing action; and 2) EA may have other pathways affecting gastric movement, but its exact mechanisms need to be studied. In conclusion, results of the present study show that the action of EA on regulating gastric functional activity is achieved by the way of affecting pyloric pressure, this change is closely associated with changes of some BGP contents; and the action of EA is of relative specificity in regard to meridians and acupoints.