山莨菪碱对利血平大鼠胃粘膜损伤的影响
作者：万军利
单位：万军利(台师范学院生物系，山东 烟台 264025)
关键词：胃粘膜；利血平；一氧化氮
中国病理生理杂志000313
[摘 要] 目的：研究山莨菪碱对利血平大鼠胃粘膜损伤的影响及其作用机制。方法：利用利血平大鼠胃粘膜损伤模型，观察腹注山莨菪碱对利血平大鼠胃粘膜损伤、胃酸分泌、胃粘液分泌、胃运动、胃粘膜血流量及胃粘膜一氧化氮含量和一氧化氮合成酶活性变化的影响。结果：山莨菪碱能抑制利血平大鼠胃粘膜损伤灶的形成；山莨菪碱能抑制利血平大鼠胃酸分泌，但对胃液分泌的量无影响；山莨菪碱能促进利血平大鼠胃粘液分泌和胃粘膜血流量，抑制胃的运动；山莨菪碱能抑制利血平导致的大鼠胃粘膜一氧化氮含量的降低和一氧化氮合成酶活性的降低。结论：山莨菪碱抑制利血平大鼠胃粘膜损伤的形成与其抑制胃酸分泌、胃的运动，促进胃粘液分泌、增加胃粘膜血流量有关；NO可能在介导山莨菪碱抑制利血平大鼠胃粘膜损伤形成中有重要作用。
, 百拇医药
[中图分类号] R 961 R 961 [文献标识码] A
[文章编号] 1000-4718(2000)03-0237-06
Effect of anisodamine on the reserpine-induced gastric mucosal lesion in rats
WAN Jun-li
(Department of Biology, Yantai Teachers College, Yantai 264025， China)
[Abstract] AIM: To determine the effects of anisodamine (Ani) administered intraperitoneally on the gastric mucosal lesion induced by reserpine.METHODS:In reserpine-treated rats, gastric mucosal lesion, gastric acid secretion, gastric barrier mucus secretion, gastric contraction, gastric mucosal blood flow (GMBF), gastric mucosal nitric oxide synthase (NOS) activity and nitric oxide (NO) content were examined.RESULTS:Ani in doses of 1,5 and 10 mg/kg significantly inhibited the formation of gastric lesions induced by reserpine, with the suppressive rate of 60.0%, 66.7% and 76.6%, respectively. Ani (10 mg/kg) significantly inhibited the secretion of gastric acid, but had no effect on the volume of gastric juice. Ani (10 mg/kg) significantly prompted the secretion of gastric barrier mucus. Our findings also showed that Ani (10 mg/kg) significantly suppressed the frequency and amplitude of gastric contraction. Ani (10 mg/kg) significantly prompted GMBF. In reserpine treated rats, gastric mucosal NOS activity and NO content were decreased and Ani (10 mg/kg) could inhibit the decrease in NOS activity and NO content.CONCLUSIONS:The protective effect of Ani may results in part from inhibiting gastric acid secretion, prompting gastric barrier mucus secretion, suppressing gastric contraction and improving GMBF. NO seems to play an important mediator role in the Ani protective mechanisms.
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[MeSH] Gastric mucosa; Reserpine; Nitric oxide
[CLC number] R961 [Document code] A
INTRODUCTION
Anisodamine (Ani) is an alkaloid initially isolated from Anisodus tanguticus Pasch in China. As an analog of atropin, Ani has numerous pharmacological effects such as antiacetylcholine, antishock, improving micro-circulation, inhibiting platelet aggregation, antiarrhythmia and calcium antagonistic action. It has been extensively used to treat endotoxin shock, spasm of gastrointestinal and other visceral smooth muscle etc.
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Yong et al[1] reported that Ani administered orally was found to antagonize the gastric mucosal damage induced by indomethacin, restraint, pyloric ligation or absolute ethanol ingestion in rats. Our previous study demonstrated that intraperitoneal Ani protected the rats from contracting gastric lesion induced by restraint water-immersion[2]. The effect of Ani on gastric lesion induced by reserpine, as well as its underlying mechanism, however, remains to be studied.
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Recent studies have shown that reduction in endogenous nitric oxide (NO) seems to be responsible for the gastric mucosal injury induced by ethanol[3], ischemia-reperfusion[4] and cold restraint stress[5]. Nevertheless, whether NO reduction is implicated in the reserpine-induced gastric lesion has not yet been investigated.
Therefore, the aims of this study are to investigate:(1) whether or not Ani administered intraperitoneally can protect against gastric lesion induced by reserpine in rats, (2)if Ani does give protection, by what mechanisms? and (3) the possible implication of NO in the Ani protection.
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MATERIALS AND METHODS
1. Animal preparation Sprague-Dawley rats of either sex (150～220 g)， kept in individual cages with a raised mesh bottom each, were deprived of food but allowed free access to water for 24 h prior to the experiments.
2. Experimental procedure
Experiment I. Production of gastric lesion and measurement of gastric lesion indices The animals were divided into four groups. Group I (control) received saline (5 mL/kg) intraperitoneally. Group Ⅱ，Ⅲ, and Ⅳ received Ani in doses of 1,5 and 10 mg/kg intraperitoneally, respectively. Thirty minutes later, the rats were given reserpine in a dose of 5 mg/kg for each animal intraperitoneally. The rodents were killed by cervical dislocation 6 h after the reserpine injection. Their stomachs were immediately removed, inflated by injecting 10 mL of 10% formalin and immersed in 10% formalin for 10 min. The stomachs were then opened along the greater curvature. The sum of the length of each lesion was expressed as gastric lesion index (mm), as described by Guth et al.
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Experiment Ⅱ. Measurement of gastric acid secretion Gastric acid secretion was measured in pylorus-ligated rats. The animals were divided into three groups. Group I: saline group; group Ⅱ： reserpine group; group Ⅲ： Ani group. Saline (5 mL/kg) was given intraperitoneally in group Ⅰ and Ⅱ； Ani (10 mg/kg) was given intraperitoneally in group Ⅲ， respectively. Thirty minutes later, the pyloric ends of the stomachs were tied off under ether anesthesia. Immediately thereafter, reserpine (5 mg/kg for each rat) was administered intraperitoneally in group Ⅱ and Ⅲ, and saline was given in group I. Six hours after pylorus ligation, the animals were killed by cervical dislocation, then the stomachs were removed and the gastric contents were separately drained into graduated centrifuge tubes. After centrifugation at 3 000 r/min for 10 min, the supernatants were carefully decanted and assayed for the volume of the gastric juice and its H+ concentration. The H+ concentration was determined by titrating the gastric juice to pH 7.0 with 0.01 mol/L NaOH. Volumes of gastric secretion, total acid output and titratable acidity were expressed as mL of gastric juice/100 g body weight, μmol of H+/100 g body weight and mmol of H+/L, respectively.
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Experiment Ⅲ. Measurement of gastric barrier mucus Gastric barrier mucus was determined according to the modified procedure of Bolton et al. The animals were divided into three groups and treated as in experiment Ⅱ. Six hours after reserpine injection, the animals were killed and the stomachs removed. The stomachs were opened along the lesser curvature and free mucus was gently removed from the surface of the gastric mucosa. Each of the stomachs was then placed separately in 20 mL Alcine Blue solution (20 mg Alcian Blue was dissolved in 100 mL McIlvaine buffer, pH 5.8) and incubated for 2 h at 20℃, followed by centrifugation at 4 000 r/min for 10 min and the supernatants were used for measuring absorbance at 615 nm on a spectrophotometer. Gastric barrier mucus was calculated using the following formula:
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gastric barrier mucus (mg)=4-4×(test absorbance)/(standard absorbance)
The content of gastric barrier mucus was expressed as mg/per stomach.
Experiment Ⅳ. Measurement of gastric contraction Gastric contraction was recorded according to the method described by Takeuchi et al. A miniature gastric balloon (5 mm in diameter) fashioned from condom rubber was connected to one end of a polyethylene tube with two side holes on this end. The air in the balloon was driven out by filling it with water. The water was allowed to escape to zero pressure, at which time the flaccid balloon contained approximately 0.3 mL of water. Under ether anesthesia, an incision on the abdominal wall of the rat was made and the balloon was inserted through a cautery hole into the greater curvature of the forestomach about 5 mm from the limiting ridge. The balloon was then tied in place so that it lay in the glandular part of the stomach, care being taken not to damage the balloon with the tube. The balloon and tube system was connected to a pressure transducer which was joined to a physiological recorder. Then the incision on the abdominal wall was sutured with silk. Rats were divided into two groups. Group Ⅰ: reserpine group; group Ⅱ: Ani (10 mg/kg) group. Saline or Ani was given intraperitoneally, respectively. Thirty min later, reserpine was given intraperitoneally in all rats and gastric contractions were recorded for a total of 6 h. All waves with amplitudes greater 4 cm H2O and lasting longer then 2 s were noted as contractions. Frequency of contraction and amplitude of contraction were analyzed in a 1-h recording block.
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Experiment V. Measurement of gastric mucosal blood flow Rats were divided and treated as in experiment Ⅱ. Gastric mucosal blood flow (GMBF) was measured using neutral red clearance technique according to the procedure modified by Zhan et al. Briefly, animals were anesthetized with urethane given intraperitoneally (1.25 g/kg). Saline (5 mL/kg) was given in group I and Ⅱ，Ani (10 mg/kg) was given in group Ⅲ. Thirty minutes later, the pyloric ends of stomachs were tied off. After a bolus dose of 1 mL neutral red was given intravenously, constant plasma neutral red level was maintained by continous intravenous infusion of neutral red (3 mg*kg-1*h-1) at a rate of 4 mL/h, and thereafter saline was administered in group I and reserpine (5 mg/kg) was given intraperitoneally in group Ⅱ and Ⅲ immediately, respectively. Six hours later, samples of blood (1 mL) and gastric content were collected. The content of neutral red in the plasma and gastric juice were measured and GMBF were calculated as previously described by Zhan. The results were expressed as mL/min.
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Experiment Ⅵ. Measurement of gastric mucosal NOS activity and NO content The animals were divided and treated as in experiment Ⅱ. Six hours after reserpine injection, the animals were killed by cervical dislocation and the stomachs were removed. The stomachs were opened along the greater curvature and washed with cold saline. The mucosal layer was separated from the muscular layer with a sharp blade, the mucosal material was weighed and homogenized in 10 volumes of cold saline. Following centrifugation at 6 000 r/min for 15 min, the supernatant was used for measuring gastric mucosal NOS activity and NO content. The gastric mucosal NOS activity was determined by using the NOS activity measuring kit. One unit (U) of activity was defined as the amount of enzyme producing 1 nmol of NO per min and NOS activity was expressed as U/mg protein. The content of gastric mucosal NO was measured according to the method described by Rachmilewitz et al. NO was quantified by measurement of the NO metabolite nitrite with the use of the Griess reaction. Briefly, 50 μL of supernatant was mixed with an equal volume of 1% sulfanilamide in 0.5 mol/L HCl. After 5 minutes, 50 μL of 0.02% N-1-naphthylethylene diamine dihydrochloride was added; 10 minutes later, absorbence was measured at 540 nm. NO content was expressed as nmol/mg protein.
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Reagents Anisodamine (Shanghai, China), Alcian Blue (Fluka), reserpine (Shanghai, China), NOS activity measuring kit (Nanjing Jiancheng Bioengineering Institute, China).
Statistical analyses The results were expressed as
±s. Comparisons between two groups were made using the Student's t test. One-factor ANOVA and Duncan multiple comparison test were used for comparisons among three groups.
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RESULTS
1. Effect of Ani on reserpine-induced gastric lesion Intraperitoneally administered reserpine (5 mg/kg) resulted in gross mucosal lesion in the stomach within 6 hours. These lesions were hemorrhagic and were linear or dotted in shape. Pretreatment with Ani (1,5 and 10 mg/kg) intraperitoneally dose dependently reduced the formation of gastric lesion induced by reserpine, with a suppression rate of, 66.0%, 66.7% and 77.6%, respectively (Table 1). These data indicated that Ani pretreatment protected against reserpine-induced gastric lesion in rats.
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Tab 1 Effect of anisodamine on reserpine induced
gastric lesions in rats (±s) Treatment
Dose(mg/kg)
n
Lesion index
Saline
7
27.00±3.32
Ani
, 百拇医药 1
5
10.08±1.36
5
7
9.00±3.40
10
6
6.33±0.33
*P<0.01, vs saline group
2.Effect of Ani on gastric acid secretion Table 2 shows the effect of Ani on gastric acid secretion. The differences in the volume of gastric secretion, total output and titratable acidity between the saline group and reserpine group were not significant. In the Ani (10 mg/kg) group, total acid output and titratable acidity were decreased as compared with the reserpine group, but the differences in the volume of gastric secretion were not significant in these two groups. These results indicate that Ani pretreatment can inhibit gastric acid secretion.
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Tab 2 Effect of anisodamine on gastric secretion(±s) Group
n
Volume of
gastric secretion
(mL/100g
body wt.)
Total acid
output
(H+μmol/
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100g body wt.)
Titratable
acidity
(H+
mmol/L)
Ⅰ
7
2.62±0.48
145.23±23.06
77.89±7.49
Ⅱ
7
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1.94±0.26
191.31±27.61
98.95±7.88
Ⅲ
6
1.25±0.21
86.60±23.52
65.79±12.08
*P<0.05, vs group Ⅱ; Ⅰ: saline group; Ⅱ: reserpine group; Ⅲ: Anisodamine (10 mg/kg) group
, 百拇医药 3.Effect of Ani on gastric barrier mucus Table 3 shows the effect of Ani on gastric mucus barrier secretion. Reserpine significantly decreased gastric barrier mucus secretion. Pretreatment with Ani (10 mg/kg) significantly inhibited reserpine-induced decrease in gastric barrier mucus.
Tab 3 Effect of anisodamine on gastric
barrier mucus secretion (±s) Group
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n
Gastric barrier mucus
(mg/per stomach)
Ⅰ
7
2.02±0.24
Ⅱ
8
1.10±0.07
Ⅲ
6
1.87±0.06#
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*P<0.01, vs group Ⅰ;#?P<0.01, vs group Ⅱ; Ⅰ: saline group; Ⅱ: reserpine group; Ⅲ: Anisodamine group
4. Effect of Ani on gastric contraction Table 4 shows the effect of Ani on gastric contraction. Ani (10 mg/kg) inhibited the frequency of gastric contractions except for the first three hours. Ani also inhibited the mean amplitude of gastric contractions except for the last two hours.
5. Effect of Ani on GMBF In the reserpine group (n=7)， GMBF (0.10±0.02 mL/min) was significantly lower than that in the saline group (n=6) (0.26±0.04 mL/min) (P<0.05). Ani (10 mg/kg) pretreatment significantly inhibited the decrease in GMBF in reserpine-treated rats (0.24±0.04 mL/min) (n=7) (P<0.05).
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Tab 4 Effect of anisodamine on gastric contraction(±s) Gastric
motility
Group
Time after reserpine treatment (h)
1 st
2 nd
3 rd
4 th
5 th
, 百拇医药 6 th
Frequency
Ⅰ
0.60±0.14
2.30±0.15
2.00±0.47
1.60±0.45
2.10±0.35
2.60±0.26
(contractions/min)
Ⅱ
1.00±0.28
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1.40±0.56
1.10±0.45
0.30±0.13**
0.60±0.22**
0.80±0.36**
Amplitude(cm H2O)
Ⅰ
9.00±1.51
19.60±4.03
25.00±5.79
, 百拇医药 20.10±6.26
23.40±6.39
27.00±6.45
Ⅱ
5.30±0.30*
6.20±0.75*
6.70±1.05*
6.70±0.80**
11.90±2.88
21.40±7.70
*P<0.05,**P<0.01，vs group Ⅰ; Ⅰ: reserpine group (n=7); Ⅱ: Anisodamine group (n=5)
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6. Effect of Ani on gastric mucosal NOS activity and NO content Table 5 shows the effect of Ani on gastric mucosal NOS activity and NO content. In the reserpine group, gastric mucosal NOS activity and NO content were significantly lower as compared with those in the saline group. Ani (10 mg/kg) pretreatment significantly inhibited the decrease in gastric mucosal NOS activity and NO content in reserpine-treated rats.
Tab 5 Effect of anisodamine on gastric mucosal NOS activity
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and NO-2/NO-3 content in reserpine-treated rats (±s) Group
n
NOS activity
(U/mg protein)
NO-2/NO-3 content
(nmol/mg protein)
Ⅰ
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5
0.50±0.02
8.57±1.19
Ⅱ
5
0.32±0.02*
2.67±0.80*
Ⅲ
5
0.52±0.05#
9.87±1.32#
, http://www.100md.com *P<0.01, vs group Ⅰ;#P<0.01, vs group Ⅱ; Ⅰ: saline group; Ⅱ: reserpine group; Ⅲ: Anisodamine group
DISCUSSION
Yong et al[1] reported that Ani (12.5～50 mg/kg) given orally protected the gastric mucosal damage induced by indomethacin administration, restraint, pyloric ligation or absolute ethanol ingestion in rats. Our previous study demonstrated that Ani (1～10 mg/kg) injected intraperitoneally protected against gastric lesion induced by restraint water-immersion in rats[2]. The results of the present study revealed that Ani (1～10 mg/kg) administered intraperitoneally protected against gastric mucosal lesion induced by reserpine in rats.
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Our previous studies have led to the conclusion that gastric lesions induced by reserpine do not result from acid hypersecretion[6]. The present study confirmed our previous findings in that gastric acid secretion of reserpine-treated animals was not significantly increased. Pretreatment with Ani had no effect on the volume of gastric juice in the reserpine-treated animals, but significantly inhibited the secretion of gastric acid. These data are in agreement with those reported by Yong et al[1], who found that Ani (12.5～25 mg/kg) given orally inhibited the secretion of gastric acid in pylorus-ligated rats. Since gastric acid secretion was markedly inhibited by Ani, it may be assumed that lesions induced by reserpine do not result from acid hypersecretion but require the presence of acid in the lumen. The mechanism by which Ani inhibits gastric acid secretion is not clearly understood. There is evidence that Ani has a calcium antagonistic action[7]. Therefore, the action of Ani to inhibit gastric acid secretion may be mediated by blocking of the calcium channel.
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In agreement with our previous study[6], the present study showed that reserpine significantly decreased gastric barrier mucus. This finding further indicated that the decrease in gastric barrier mucus is likely to play an important role in the formation of gastric lesion induced by reserpine. The effect of Ani on gastric barrier mucus secretion has hitherto not yet been determined as yet. In the present study, we demonstrated that pretreatment with Ani significantly inhibited reserpine-induced decrease in gastric barrier mucus. It seems therefore very likely that the protective effect of Ani results in part from prompting the secretion of gastric barrier mucus.
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In our previous studies, we found that reserpine increased the frequency of gastric contraction[6] and that Ani treatment inhibited gastric contraction in water-immersion rats[2]. In the present study, Ani significantly suppressed the frequency and amplitude of gastric contraction in reserpine-treated rats, suggesting that the protective effect of Ani may results in part from suppression of gastric contraction in reserpine treated rats. It is well known that entry of calcium into cells plays a key role in smooth muscle contraction and Ani is effective in inhibiting calcium channel[7]. Therefore, Ani may prevent gastric contraction through this mechanism.
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It is generally accepted that adequate GMBF plays an important role in maintaining gastric mucosal integrity. Local mucosal ischema has clearly been found to be an important factor in ulcerogenesis. It is interesting to know whether reserpine induces a decrease in GMBF. The data presented in this study demonstrated that GMBF did decrease in reserpinetreated rats. Ani has been shown to be a direct vasodilator, and in the present study, Ani was found to inhibit the decrease in GMBF induced by reserpine, suggesting that the beneficial effect of Ani may in part be mediated through this mechanism.
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Nitric oxide synthesized from L-arginine by nitric oxide synthase is a potent vasodilator and an inhibitory neurotransmitter. It has been reported that NO is an important mediator in regulation GMBF[8]， acid secretion[9] and gastric motility[10]. Recent studies have shown that reduction in endogenous NO may be responsible for gastric mucosal injury induced by ethanol[3], ischemia-reperfusion[4] and cold restraint stress[5]. However, NO reduction implicating in reserpine-induced gastric lesion has not yet been elucidated. We, therefore, investigated the change in gastric mucosal NOS activity and NO content in reserpine-treated rats and the effect of Ani on these changes. The results of the present study demonstrate that gastric mucosal NOS activity and NO content decreased in reserpine-treated rats and pretreatment of Ani inhibited the decrease in NOS activity and NO content induced by reserpine. It is likely that the decrease in NO content may play a key role in the lesion induced by reserpine and Ani protection may be mediated by “rescuing” NO which is so important in modulating the gastric acid secretion, mucus secretion, GMBF and gastric motility. The mechanism by which Ani inhibited the decrease in gastric mucosal NOS activity induced by reserpine, however, is not clear and further research in this field is required.
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Taken together, the results of the present study suggested that Ani significantly inhibited the formation of gastric lesions induced by reserpine, probably by inhibiting gastric acid secretion, prompting the secretion of gastric barrier mucus, suppressing gastric motility, and increasing gastric mucosal blood flow. NO may play an important role in Ani protection.
[REFERENCES]
[1] 雍定国，耿宝琴，顾刚果，等. 山莨菪碱抗大鼠实验性胃溃疡作用[J]. 中国药理学报，1991，12：522～525.
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[2] Wan JL. Effect of anisodamine on gastric ulcer induced by restraint water-immersion in rats [J]. Exp Clin Gastroenterol,1993,3:154～158.
[3] Konturek SJ, Brzozowski T, Majka J, et al. Nitric oxide in gastroprotection by sucralfate, mild irritant, and nocloprost: role of mucosal blood flow [J]. Dig Dis Sci,1994,39:593～600.
[4] Andrews FJ, Malcontenti-Wilson C, O'brien PE. Protection against gastric ischemia-reperfusion injury by nitric oxide generators [J]. Dig Dis Sci,1994,39:366～373.
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[5] Coskun T, Yegen BC, Alican I, et al. Cold restrain stress induces gastric mucosal dysfunction: role of nitric oxide [J]. Dig Dis Sci,1996,41:956～963.
[6]Wan JL, Wang CL， Chang QD. Effect of tetramethylpyrazine on reserpine-induced gastric lesion in rats [J]. Dig Dis Sci,1998,43:1652～1656.
[7] 陈锦明，张明志，谷淑珍，等. 山莨菪碱对兔离体主动脉平滑肌的钙拮抗作用[J]. 中国药理学报，1992，13：445～448.
[8] T Lippe I, Holzer P. Participation of endothelium-derived nitric oxide but not prostacyclin in the gastric mucosal hyperemia due to acid back-diffusion [J]. Br J Pharmacol, 1992,105:708～714.
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[9] Martinez-Cuesta MA, Barrachina MD, Pique JM, et al. The role of nitric oxide and platelet-activating factor in the inhibition by endotoxin of pentagastrin-stimulated gastric acid secretion [J]. Eur J Pharmacol,1992,218:351～354.
[10] Fiorucci S, Distrutti E, Quintieri A, et al. L-arginine/nitric oxide pathway modulates gastric motility and gallbladder emptying induced by erythromycin and liquid meal in humans [J]. Dig Dis Sci,1995,40:1365～1371.
[Received]1999-02-01 [Accepted]1999-07-08, 百拇医药
单位：万军利(台师范学院生物系，山东 烟台 264025)
关键词：胃粘膜；利血平；一氧化氮
中国病理生理杂志000313
[摘 要] 目的：研究山莨菪碱对利血平大鼠胃粘膜损伤的影响及其作用机制。方法：利用利血平大鼠胃粘膜损伤模型，观察腹注山莨菪碱对利血平大鼠胃粘膜损伤、胃酸分泌、胃粘液分泌、胃运动、胃粘膜血流量及胃粘膜一氧化氮含量和一氧化氮合成酶活性变化的影响。结果：山莨菪碱能抑制利血平大鼠胃粘膜损伤灶的形成；山莨菪碱能抑制利血平大鼠胃酸分泌，但对胃液分泌的量无影响；山莨菪碱能促进利血平大鼠胃粘液分泌和胃粘膜血流量，抑制胃的运动；山莨菪碱能抑制利血平导致的大鼠胃粘膜一氧化氮含量的降低和一氧化氮合成酶活性的降低。结论：山莨菪碱抑制利血平大鼠胃粘膜损伤的形成与其抑制胃酸分泌、胃的运动，促进胃粘液分泌、增加胃粘膜血流量有关；NO可能在介导山莨菪碱抑制利血平大鼠胃粘膜损伤形成中有重要作用。
, 百拇医药
[中图分类号] R 961 R 961 [文献标识码] A
[文章编号] 1000-4718(2000)03-0237-06
Effect of anisodamine on the reserpine-induced gastric mucosal lesion in rats
WAN Jun-li
(Department of Biology, Yantai Teachers College, Yantai 264025， China)
[Abstract] AIM: To determine the effects of anisodamine (Ani) administered intraperitoneally on the gastric mucosal lesion induced by reserpine.METHODS:In reserpine-treated rats, gastric mucosal lesion, gastric acid secretion, gastric barrier mucus secretion, gastric contraction, gastric mucosal blood flow (GMBF), gastric mucosal nitric oxide synthase (NOS) activity and nitric oxide (NO) content were examined.RESULTS:Ani in doses of 1,5 and 10 mg/kg significantly inhibited the formation of gastric lesions induced by reserpine, with the suppressive rate of 60.0%, 66.7% and 76.6%, respectively. Ani (10 mg/kg) significantly inhibited the secretion of gastric acid, but had no effect on the volume of gastric juice. Ani (10 mg/kg) significantly prompted the secretion of gastric barrier mucus. Our findings also showed that Ani (10 mg/kg) significantly suppressed the frequency and amplitude of gastric contraction. Ani (10 mg/kg) significantly prompted GMBF. In reserpine treated rats, gastric mucosal NOS activity and NO content were decreased and Ani (10 mg/kg) could inhibit the decrease in NOS activity and NO content.CONCLUSIONS:The protective effect of Ani may results in part from inhibiting gastric acid secretion, prompting gastric barrier mucus secretion, suppressing gastric contraction and improving GMBF. NO seems to play an important mediator role in the Ani protective mechanisms.
, 百拇医药
[MeSH] Gastric mucosa; Reserpine; Nitric oxide
[CLC number] R961 [Document code] A
INTRODUCTION
Anisodamine (Ani) is an alkaloid initially isolated from Anisodus tanguticus Pasch in China. As an analog of atropin, Ani has numerous pharmacological effects such as antiacetylcholine, antishock, improving micro-circulation, inhibiting platelet aggregation, antiarrhythmia and calcium antagonistic action. It has been extensively used to treat endotoxin shock, spasm of gastrointestinal and other visceral smooth muscle etc.
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Yong et al[1] reported that Ani administered orally was found to antagonize the gastric mucosal damage induced by indomethacin, restraint, pyloric ligation or absolute ethanol ingestion in rats. Our previous study demonstrated that intraperitoneal Ani protected the rats from contracting gastric lesion induced by restraint water-immersion[2]. The effect of Ani on gastric lesion induced by reserpine, as well as its underlying mechanism, however, remains to be studied.
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Recent studies have shown that reduction in endogenous nitric oxide (NO) seems to be responsible for the gastric mucosal injury induced by ethanol[3], ischemia-reperfusion[4] and cold restraint stress[5]. Nevertheless, whether NO reduction is implicated in the reserpine-induced gastric lesion has not yet been investigated.
Therefore, the aims of this study are to investigate:(1) whether or not Ani administered intraperitoneally can protect against gastric lesion induced by reserpine in rats, (2)if Ani does give protection, by what mechanisms? and (3) the possible implication of NO in the Ani protection.
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MATERIALS AND METHODS
1. Animal preparation Sprague-Dawley rats of either sex (150～220 g)， kept in individual cages with a raised mesh bottom each, were deprived of food but allowed free access to water for 24 h prior to the experiments.
2. Experimental procedure
Experiment I. Production of gastric lesion and measurement of gastric lesion indices The animals were divided into four groups. Group I (control) received saline (5 mL/kg) intraperitoneally. Group Ⅱ，Ⅲ, and Ⅳ received Ani in doses of 1,5 and 10 mg/kg intraperitoneally, respectively. Thirty minutes later, the rats were given reserpine in a dose of 5 mg/kg for each animal intraperitoneally. The rodents were killed by cervical dislocation 6 h after the reserpine injection. Their stomachs were immediately removed, inflated by injecting 10 mL of 10% formalin and immersed in 10% formalin for 10 min. The stomachs were then opened along the greater curvature. The sum of the length of each lesion was expressed as gastric lesion index (mm), as described by Guth et al.
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Experiment Ⅱ. Measurement of gastric acid secretion Gastric acid secretion was measured in pylorus-ligated rats. The animals were divided into three groups. Group I: saline group; group Ⅱ： reserpine group; group Ⅲ： Ani group. Saline (5 mL/kg) was given intraperitoneally in group Ⅰ and Ⅱ； Ani (10 mg/kg) was given intraperitoneally in group Ⅲ， respectively. Thirty minutes later, the pyloric ends of the stomachs were tied off under ether anesthesia. Immediately thereafter, reserpine (5 mg/kg for each rat) was administered intraperitoneally in group Ⅱ and Ⅲ, and saline was given in group I. Six hours after pylorus ligation, the animals were killed by cervical dislocation, then the stomachs were removed and the gastric contents were separately drained into graduated centrifuge tubes. After centrifugation at 3 000 r/min for 10 min, the supernatants were carefully decanted and assayed for the volume of the gastric juice and its H+ concentration. The H+ concentration was determined by titrating the gastric juice to pH 7.0 with 0.01 mol/L NaOH. Volumes of gastric secretion, total acid output and titratable acidity were expressed as mL of gastric juice/100 g body weight, μmol of H+/100 g body weight and mmol of H+/L, respectively.
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Experiment Ⅲ. Measurement of gastric barrier mucus Gastric barrier mucus was determined according to the modified procedure of Bolton et al. The animals were divided into three groups and treated as in experiment Ⅱ. Six hours after reserpine injection, the animals were killed and the stomachs removed. The stomachs were opened along the lesser curvature and free mucus was gently removed from the surface of the gastric mucosa. Each of the stomachs was then placed separately in 20 mL Alcine Blue solution (20 mg Alcian Blue was dissolved in 100 mL McIlvaine buffer, pH 5.8) and incubated for 2 h at 20℃, followed by centrifugation at 4 000 r/min for 10 min and the supernatants were used for measuring absorbance at 615 nm on a spectrophotometer. Gastric barrier mucus was calculated using the following formula:
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gastric barrier mucus (mg)=4-4×(test absorbance)/(standard absorbance)
The content of gastric barrier mucus was expressed as mg/per stomach.
Experiment Ⅳ. Measurement of gastric contraction Gastric contraction was recorded according to the method described by Takeuchi et al. A miniature gastric balloon (5 mm in diameter) fashioned from condom rubber was connected to one end of a polyethylene tube with two side holes on this end. The air in the balloon was driven out by filling it with water. The water was allowed to escape to zero pressure, at which time the flaccid balloon contained approximately 0.3 mL of water. Under ether anesthesia, an incision on the abdominal wall of the rat was made and the balloon was inserted through a cautery hole into the greater curvature of the forestomach about 5 mm from the limiting ridge. The balloon was then tied in place so that it lay in the glandular part of the stomach, care being taken not to damage the balloon with the tube. The balloon and tube system was connected to a pressure transducer which was joined to a physiological recorder. Then the incision on the abdominal wall was sutured with silk. Rats were divided into two groups. Group Ⅰ: reserpine group; group Ⅱ: Ani (10 mg/kg) group. Saline or Ani was given intraperitoneally, respectively. Thirty min later, reserpine was given intraperitoneally in all rats and gastric contractions were recorded for a total of 6 h. All waves with amplitudes greater 4 cm H2O and lasting longer then 2 s were noted as contractions. Frequency of contraction and amplitude of contraction were analyzed in a 1-h recording block.
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Experiment V. Measurement of gastric mucosal blood flow Rats were divided and treated as in experiment Ⅱ. Gastric mucosal blood flow (GMBF) was measured using neutral red clearance technique according to the procedure modified by Zhan et al. Briefly, animals were anesthetized with urethane given intraperitoneally (1.25 g/kg). Saline (5 mL/kg) was given in group I and Ⅱ，Ani (10 mg/kg) was given in group Ⅲ. Thirty minutes later, the pyloric ends of stomachs were tied off. After a bolus dose of 1 mL neutral red was given intravenously, constant plasma neutral red level was maintained by continous intravenous infusion of neutral red (3 mg*kg-1*h-1) at a rate of 4 mL/h, and thereafter saline was administered in group I and reserpine (5 mg/kg) was given intraperitoneally in group Ⅱ and Ⅲ immediately, respectively. Six hours later, samples of blood (1 mL) and gastric content were collected. The content of neutral red in the plasma and gastric juice were measured and GMBF were calculated as previously described by Zhan. The results were expressed as mL/min.
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Experiment Ⅵ. Measurement of gastric mucosal NOS activity and NO content The animals were divided and treated as in experiment Ⅱ. Six hours after reserpine injection, the animals were killed by cervical dislocation and the stomachs were removed. The stomachs were opened along the greater curvature and washed with cold saline. The mucosal layer was separated from the muscular layer with a sharp blade, the mucosal material was weighed and homogenized in 10 volumes of cold saline. Following centrifugation at 6 000 r/min for 15 min, the supernatant was used for measuring gastric mucosal NOS activity and NO content. The gastric mucosal NOS activity was determined by using the NOS activity measuring kit. One unit (U) of activity was defined as the amount of enzyme producing 1 nmol of NO per min and NOS activity was expressed as U/mg protein. The content of gastric mucosal NO was measured according to the method described by Rachmilewitz et al. NO was quantified by measurement of the NO metabolite nitrite with the use of the Griess reaction. Briefly, 50 μL of supernatant was mixed with an equal volume of 1% sulfanilamide in 0.5 mol/L HCl. After 5 minutes, 50 μL of 0.02% N-1-naphthylethylene diamine dihydrochloride was added; 10 minutes later, absorbence was measured at 540 nm. NO content was expressed as nmol/mg protein.
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Reagents Anisodamine (Shanghai, China), Alcian Blue (Fluka), reserpine (Shanghai, China), NOS activity measuring kit (Nanjing Jiancheng Bioengineering Institute, China).
Statistical analyses The results were expressed as
±s. Comparisons between two groups were made using the Student's t test. One-factor ANOVA and Duncan multiple comparison test were used for comparisons among three groups., 百拇医药
RESULTS
1. Effect of Ani on reserpine-induced gastric lesion Intraperitoneally administered reserpine (5 mg/kg) resulted in gross mucosal lesion in the stomach within 6 hours. These lesions were hemorrhagic and were linear or dotted in shape. Pretreatment with Ani (1,5 and 10 mg/kg) intraperitoneally dose dependently reduced the formation of gastric lesion induced by reserpine, with a suppression rate of, 66.0%, 66.7% and 77.6%, respectively (Table 1). These data indicated that Ani pretreatment protected against reserpine-induced gastric lesion in rats.
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Tab 1 Effect of anisodamine on reserpine induced
gastric lesions in rats (
±s) TreatmentDose(mg/kg)
n
Lesion index
Saline
7
27.00±3.32
Ani
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5
10.08±1.36
5
7
9.00±3.40
10
6
6.33±0.33
*P<0.01, vs saline group
2.Effect of Ani on gastric acid secretion Table 2 shows the effect of Ani on gastric acid secretion. The differences in the volume of gastric secretion, total output and titratable acidity between the saline group and reserpine group were not significant. In the Ani (10 mg/kg) group, total acid output and titratable acidity were decreased as compared with the reserpine group, but the differences in the volume of gastric secretion were not significant in these two groups. These results indicate that Ani pretreatment can inhibit gastric acid secretion.
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Tab 2 Effect of anisodamine on gastric secretion(
±s) Groupn
Volume of
gastric secretion
(mL/100g
body wt.)
Total acid
output
(H+μmol/
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100g body wt.)
Titratable
acidity
(H+
mmol/L)
Ⅰ
7
2.62±0.48
145.23±23.06
77.89±7.49
Ⅱ
7
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1.94±0.26
191.31±27.61
98.95±7.88
Ⅲ
6
1.25±0.21
86.60±23.52
65.79±12.08
*P<0.05, vs group Ⅱ; Ⅰ: saline group; Ⅱ: reserpine group; Ⅲ: Anisodamine (10 mg/kg) group
, 百拇医药 3.Effect of Ani on gastric barrier mucus Table 3 shows the effect of Ani on gastric mucus barrier secretion. Reserpine significantly decreased gastric barrier mucus secretion. Pretreatment with Ani (10 mg/kg) significantly inhibited reserpine-induced decrease in gastric barrier mucus.
Tab 3 Effect of anisodamine on gastric
barrier mucus secretion (
±s) Group, http://www.100md.com
n
Gastric barrier mucus
(mg/per stomach)
Ⅰ
7
2.02±0.24
Ⅱ
8
1.10±0.07
Ⅲ
6
1.87±0.06#
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*P<0.01, vs group Ⅰ;#?P<0.01, vs group Ⅱ; Ⅰ: saline group; Ⅱ: reserpine group; Ⅲ: Anisodamine group
4. Effect of Ani on gastric contraction Table 4 shows the effect of Ani on gastric contraction. Ani (10 mg/kg) inhibited the frequency of gastric contractions except for the first three hours. Ani also inhibited the mean amplitude of gastric contractions except for the last two hours.
5. Effect of Ani on GMBF In the reserpine group (n=7)， GMBF (0.10±0.02 mL/min) was significantly lower than that in the saline group (n=6) (0.26±0.04 mL/min) (P<0.05). Ani (10 mg/kg) pretreatment significantly inhibited the decrease in GMBF in reserpine-treated rats (0.24±0.04 mL/min) (n=7) (P<0.05).
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Tab 4 Effect of anisodamine on gastric contraction(
±s) Gastricmotility
Group
Time after reserpine treatment (h)
1 st
2 nd
3 rd
4 th
5 th
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Frequency
Ⅰ
0.60±0.14
2.30±0.15
2.00±0.47
1.60±0.45
2.10±0.35
2.60±0.26
(contractions/min)
Ⅱ
1.00±0.28
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1.40±0.56
1.10±0.45
0.30±0.13**
0.60±0.22**
0.80±0.36**
Amplitude(cm H2O)
Ⅰ
9.00±1.51
19.60±4.03
25.00±5.79
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23.40±6.39
27.00±6.45
Ⅱ
5.30±0.30*
6.20±0.75*
6.70±1.05*
6.70±0.80**
11.90±2.88
21.40±7.70
*P<0.05,**P<0.01，vs group Ⅰ; Ⅰ: reserpine group (n=7); Ⅱ: Anisodamine group (n=5)
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6. Effect of Ani on gastric mucosal NOS activity and NO content Table 5 shows the effect of Ani on gastric mucosal NOS activity and NO content. In the reserpine group, gastric mucosal NOS activity and NO content were significantly lower as compared with those in the saline group. Ani (10 mg/kg) pretreatment significantly inhibited the decrease in gastric mucosal NOS activity and NO content in reserpine-treated rats.
Tab 5 Effect of anisodamine on gastric mucosal NOS activity
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and NO-2/NO-3 content in reserpine-treated rats (
±s) Groupn
NOS activity
(U/mg protein)
NO-2/NO-3 content
(nmol/mg protein)
Ⅰ
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5
0.50±0.02
8.57±1.19
Ⅱ
5
0.32±0.02*
2.67±0.80*
Ⅲ
5
0.52±0.05#
9.87±1.32#
, http://www.100md.com *P<0.01, vs group Ⅰ;#P<0.01, vs group Ⅱ; Ⅰ: saline group; Ⅱ: reserpine group; Ⅲ: Anisodamine group
DISCUSSION
Yong et al[1] reported that Ani (12.5～50 mg/kg) given orally protected the gastric mucosal damage induced by indomethacin administration, restraint, pyloric ligation or absolute ethanol ingestion in rats. Our previous study demonstrated that Ani (1～10 mg/kg) injected intraperitoneally protected against gastric lesion induced by restraint water-immersion in rats[2]. The results of the present study revealed that Ani (1～10 mg/kg) administered intraperitoneally protected against gastric mucosal lesion induced by reserpine in rats.
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Our previous studies have led to the conclusion that gastric lesions induced by reserpine do not result from acid hypersecretion[6]. The present study confirmed our previous findings in that gastric acid secretion of reserpine-treated animals was not significantly increased. Pretreatment with Ani had no effect on the volume of gastric juice in the reserpine-treated animals, but significantly inhibited the secretion of gastric acid. These data are in agreement with those reported by Yong et al[1], who found that Ani (12.5～25 mg/kg) given orally inhibited the secretion of gastric acid in pylorus-ligated rats. Since gastric acid secretion was markedly inhibited by Ani, it may be assumed that lesions induced by reserpine do not result from acid hypersecretion but require the presence of acid in the lumen. The mechanism by which Ani inhibits gastric acid secretion is not clearly understood. There is evidence that Ani has a calcium antagonistic action[7]. Therefore, the action of Ani to inhibit gastric acid secretion may be mediated by blocking of the calcium channel.
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In agreement with our previous study[6], the present study showed that reserpine significantly decreased gastric barrier mucus. This finding further indicated that the decrease in gastric barrier mucus is likely to play an important role in the formation of gastric lesion induced by reserpine. The effect of Ani on gastric barrier mucus secretion has hitherto not yet been determined as yet. In the present study, we demonstrated that pretreatment with Ani significantly inhibited reserpine-induced decrease in gastric barrier mucus. It seems therefore very likely that the protective effect of Ani results in part from prompting the secretion of gastric barrier mucus.
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In our previous studies, we found that reserpine increased the frequency of gastric contraction[6] and that Ani treatment inhibited gastric contraction in water-immersion rats[2]. In the present study, Ani significantly suppressed the frequency and amplitude of gastric contraction in reserpine-treated rats, suggesting that the protective effect of Ani may results in part from suppression of gastric contraction in reserpine treated rats. It is well known that entry of calcium into cells plays a key role in smooth muscle contraction and Ani is effective in inhibiting calcium channel[7]. Therefore, Ani may prevent gastric contraction through this mechanism.
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It is generally accepted that adequate GMBF plays an important role in maintaining gastric mucosal integrity. Local mucosal ischema has clearly been found to be an important factor in ulcerogenesis. It is interesting to know whether reserpine induces a decrease in GMBF. The data presented in this study demonstrated that GMBF did decrease in reserpinetreated rats. Ani has been shown to be a direct vasodilator, and in the present study, Ani was found to inhibit the decrease in GMBF induced by reserpine, suggesting that the beneficial effect of Ani may in part be mediated through this mechanism.
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Nitric oxide synthesized from L-arginine by nitric oxide synthase is a potent vasodilator and an inhibitory neurotransmitter. It has been reported that NO is an important mediator in regulation GMBF[8]， acid secretion[9] and gastric motility[10]. Recent studies have shown that reduction in endogenous NO may be responsible for gastric mucosal injury induced by ethanol[3], ischemia-reperfusion[4] and cold restraint stress[5]. However, NO reduction implicating in reserpine-induced gastric lesion has not yet been elucidated. We, therefore, investigated the change in gastric mucosal NOS activity and NO content in reserpine-treated rats and the effect of Ani on these changes. The results of the present study demonstrate that gastric mucosal NOS activity and NO content decreased in reserpine-treated rats and pretreatment of Ani inhibited the decrease in NOS activity and NO content induced by reserpine. It is likely that the decrease in NO content may play a key role in the lesion induced by reserpine and Ani protection may be mediated by “rescuing” NO which is so important in modulating the gastric acid secretion, mucus secretion, GMBF and gastric motility. The mechanism by which Ani inhibited the decrease in gastric mucosal NOS activity induced by reserpine, however, is not clear and further research in this field is required.
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Taken together, the results of the present study suggested that Ani significantly inhibited the formation of gastric lesions induced by reserpine, probably by inhibiting gastric acid secretion, prompting the secretion of gastric barrier mucus, suppressing gastric motility, and increasing gastric mucosal blood flow. NO may play an important role in Ani protection.
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[9] Martinez-Cuesta MA, Barrachina MD, Pique JM, et al. The role of nitric oxide and platelet-activating factor in the inhibition by endotoxin of pentagastrin-stimulated gastric acid secretion [J]. Eur J Pharmacol,1992,218:351～354.
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[Received]1999-02-01 [Accepted]1999-07-08, 百拇医药