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1、 22 World J Acu-Moxi Vol. 16， No, 2， June， 2006 Experimental Research EXPERIMENTAL STUDY ON MOXIBUSTION AT ZUSANLI (ST 36) AND LIANGMEN (ST 21) INDUCING HEAT SHOCK PROTEIN 70 (HSP70) EXPRESSION TO RESIST OXIDATIVE INJURY OF GASTRIC MUCOSA PENGNa(彭娜） CHANGXiao-rong(常 小 荣） YI Shou-xiang(易受乡） PENGYan(彭
2、艳） YANJie(严洁 ） Acupuncture and Massage College, Hunan University of Traditional Chinese Medicine, Changsha 410007, China ABSTRACT Objective: To observe effect of moxibustion at Zusanli (足三里 ST 36) and Liangmen (梁门 ST 21) on expression of heat shock protein 70 (HSP70) in gastric mucosa of the rat of
3、stress ulcer (SU) to explore the mechanism of moxibustion in resisting oxidative injury of the gastric mucosa. Methods： Sixty SD rats were evenly randomized into 4 groups, a blank group, a model group, an acupoint moxibustion group and a non-acupoint moxibustion group. Water restraint stress (WRS) m
4、ethod was used to make stress gastric ulcer rat model. The ulcerative index (Ul) of gastric mucosa was evaluated by using GUTH method, the gastric mucosa blood flux (GMBF) was detected by a laser Doppler bloodflow monitor, and HSP70 expression and mal- ondialdehyde (MDA) content in the gastric mucos
5、a were determined respectively with immunohistochemical and thiobarbiturate methods. Results： Moxibustion at Zusanli (ST 36) and Liangmen (ST 21) significantly decreased Ul, up-regulated HSP70 expression, increased GMBF, and decreased MDA content in the gastric mucosa in the rat of stress gastric ul
6、cer, with significant differences as compared with the model group and the non-acupoint moxibustion group (P 2 mm, the point was double. 1. 7. 2 Gastric mucosa blood flux (GMBF ) After anesthesia in the rats， the abdominal cavity was open from the ventral median line and the stomach was explored. A
7、laser Doppler s blood flowmeter (LDF Flowmeter? made in BIOP AC Company? USA) was used to detect GMBF with a Mini-type contact probe， and the signs collected by the blood flowmeter were converted as blood perfusion unit (BPU) by a MP150 type analogue-digital converter? and then the values were enter
8、ed a computer and a curve was graphed by Acqknowledgev 3. 5 Software. For detection of GMBF， a small stoma of 2 cm was incised on the greater curvature of stomach, and the laser probe was placed at the four points successively? the gastric antrum, the fundus of stomach, the greater curvature of stom
9、ach and the lesser curvature of stomach. After the display device showed stability of the measure? the values were taken 3 times at each point in each rat, 15 s each time， and the stable curve of 10 s was calculated the average for statistical analysis. 1.7.3 HSP70 S-P method was adopted. (1) The pa
10、raffin-embedded tissue samples were made continuous section in thickness of 4 with interruption, which were baked on slides and routinely dewaxed? and then put in freshly prepared 3 % H2O2 for 10 min at room temperature， followed by rinsing 3 times with distilled water； (2) The sections were put in
11、0. 01 M citrate buffer and heated to boil with a microwave oven in a high power? which was repeated twice with an interval of 10 min； (3) Drip normal goat serum blocking solution and humidify for 20 min at a room temperature； (4) Drip proper diluted rabbit HSP70 antibody? humidify for 2 h at room te
12、mperature； (5) Drip biotinylated sheep anti-rabbit HSP70 IgG， humidify 30 min at room temperature ； (6) Drip horse radish peroxidase-labeled strepto-ovalbumin work solution， humidify for 30 min at room temperature. After (4) (6) World J Acu-Moxi Vol. 16, No. 2, June, 2006 25 procedures? all of secti
13、ons were washed 3X3 min with 0. 01 MPBS； (7) DAB development at room temperature with developing time controlled under microscope； (8) Light re-staining with compeachy. The first antibody was replaced by PBS for negative control. Staining results were analyzed with a MIAS medical imaging systemu- nd
14、er microscope 1 OX 40， with 5 visual fields in each sectionrandomly taken for analysis of area density and the average was taken. 1.7.4 MDA MDA levels were determined with thiobarbital method according to the directions of the assay kit. 1. 8 Statistical method The experimental results were expresse
15、d as mean 士 standard deviation (x 士 5)， and SPSS 11. 5 Software was used for analysis of data? and the one-way ANOVA was adopted for statistical analysis. 2 RESULTS 2. 1 Effects of moxibustion on UI， GMBF， MDA in the gastric mucosa After various treatments? UI in the group C was very significantly l
16、ower than that in the group B and the group D (P 0. 05)? and in the group B was very significantly higher than that in the group A (P 0. 05)? and in the group B was significantly lower than that in the group A (P 0. 01). See Table 1. Table 1. Comparison of UI, GMBF and contents of MDA among the grou
17、ps (x zts) Groups n UI GMBF (mL/min) MDA (nmol/ mL) Group A 10 12. 000 5. 944 # 363. 806168. 551 # 2. 925 0.625 # Group B 10 26. 800 9. 807 * * 139. 489 33. 133 * 3. 906 0. 768 i Group C 10 14. 100 5. 425 279. 827 172. 862 2. 586 0. 252 Group D 10 26. 200 7. 729 * * 141. 512 58. 450 x 3. 464 1. 502
18、i Notes： 0. 05)? and the HSP70 expression in the group B was significantly higher than that in the group A (P0. 05). See Table 2. 。 26 。 World J Acu-Moxi Vol. 16， No, 2， June， 2006 Table 2. Comparison of HSP70 expression in gastric mucosa among the groups ( X 士s) Groups n HSP70 Group A 5 0. 021 0. 0
19、10 # Group B 5 0. 077 0. 057 Group C 5 0. 133 0. 035 Group D 5 0. 0590. 038 i 3 DISCUSSION HSPs are a group of stress proteins with high conservation produced by organisms or cultured cells in vitro under action of bad environmental factors. They occur universally in the whole biological universe? a
20、nd may be synthesized almost in all cells. Most HSPs may be induced by changes of environmental conditions? such as heat injury? ischemia， oxidative stress， ray， tissue impairment? the virus? the bacteria and their products? and other injury factors? as well as stress stimulation and some chemical s
21、ubstances 8 . HSP70 has over expression in gastric mucosa in the rats of induced acute and chronic gastric ulcer? chronic atrophic gastritis? human gastric cancer? with the lesion parts being the most obvious 9 11 . The over expression mayprotect cells and promote union of ulcer through increasing v
22、olume of blood flow in the gastric mucosa? promoting proliferation of cells? inhibiting apoptosis of the gastric mucosa? protecting cells from injury of oxygen free radical? strengthening protein synthesis of cells and repair of cells in the gastric mucosa? and other mechanisms， 12 15 . Moxibustion
23、as a physiological warm-heat stimulation can induce production of HSP70? and as an immunogen can activate immunologic system to cure some diseases 16,17 . It can be seen from the results in this experiment that after stress (in the model group)? expression of HSP70 in gastric mucosa of the rat was s
24、trengthened as compared with that in the blank group (P 0. 05)? and after pre-treatment of moxibustion, the expression was more significant than that in the model group (P0. 01)? and the degree of injury of gastric mucosa significantly alleviated (P 0. 01). It is indicated that pre-treatment of moxi
25、bustion can protect gastric mucosa through inducing high expression of HSP70 in gastric mucosa. Oxidative stress is an important factor for stress injury of gastric mucosa? and it is possibly involved in cellular apoptosis. Three hours after stress with Allen method, MD showed significant change? re
26、aching the peak after 6 12 h 18， and with prolongation of stress time， a large number of free radicals in the organism increased, and activities of the clearing system strengthened are not enough to clear away them in time? leading to injury of the gastric mucosa 19 . Electroacupuncture at Zusanli (
27、ST 36) can decrease plasma MDA content， influence metabolic level of oxygen free radical? and resist stress injury20 . In the experiment? MDA content in the gastric mucosa in the group B was significantly higher than that in the groups A and C (P0. 05)? indicating that moxibustion can decrease the i
28、ncreased MDA level of the gastric mucosa after stress? hence anti-oxidative injury. Moxibustion has actions of warmly dredging the channels and collaterals? promoting flow of qi to activate blood circulation， dispelling dampness and cold, subduing swelling and resolving masses, recuperating depleted
29、 yang and rescuing the patient from collapse? preventing disease and health-care. In modern time， a great number of clinical and experimental studies have proved that moxibustion has functions of killing pain, improving blood circulation, regulating metabolic disorder， adjusting immune functions and
30、 functions of internal organs? etc 21 World J Acu-Moxi Vol. 16, No. 2, June, 2006 27 Zusanli (ST 36) is a lower confluent point of the stomach, and a confluent point of The Stomach Channel. It is a first selected point for treatment of gastric diseases. Liangmen (ST 21) also is another important poi
31、nt of The Stomach Meridian of Foot-Yangming. The present results showed that moxibustion at Zusanli (ST 36) and Liangmen (ST 21) significantly strengthened expression of HSP70 in the gastric mucosa? decreased MDA content (P0. 05) and alleviated significantly the degree of injury of the gastric mucos
32、a (Pi0. 01) as compared with moxibustion at non-acupoint control points. It can be seen that Zusanli (ST 36) and Liangmen (ST 21) have a certain specificity of acupoints in anti-oxydative injury of gastric mucosa? which provides scientific basis for clinical application of moxibustion to treatment d
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