1、    山莨菪堿對利血平大鼠胃粘膜損傷的影響        摘要目的：研究山莨菪堿對利血平大鼠胃粘膜損傷的影響及其作用機制。方法：利用利血平大鼠胃粘膜損傷模型，觀察腹注山莨菪堿對利血平大鼠胃粘膜損傷、胃酸分泌、胃粘液分泌、胃運動、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變化的影響。結(jié)果：山莨菪堿能抑制利血平大鼠胃粘膜損傷灶的形成；山莨菪堿能抑制利血平大鼠胃酸分泌，但對胃液分泌的量無影響；山莨菪堿能促進利血平大鼠胃粘液分泌和胃粘膜血流量，抑制胃的運動；山莨菪堿能抑制

2、利血平導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。結(jié)論：山莨菪堿抑制利血平大鼠胃粘膜損傷的形成與其抑制胃酸分泌、胃的運動，促進胃粘液分泌、增加胃粘膜血流量有關(guān)；NO可能在介導山莨菪堿抑制利血平大鼠胃粘膜損傷形成中有重要作用。主題詞胃粘膜；利血平；一氧化氮中分類號 R 961文獻標識碼 A文章編號1000-4718(2000)03-0237-06 Effect of anisodamine on the reserpine-induced gastric mucosal lesion in ratsWAN Jun-li(Department of Biology, Yantai

3、 Teachers College, Yantai 264025， China)AbstractAIM: 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, gas

4、tric 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 6

5、0.0%, 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 suppre

6、ssed 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

7、 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.MeSHGastric mucosa; Reserpine; Nitric oxideCLC numberR961Docume

8、nt codeAINTRODUCTIONAnisodamine (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 ca

9、lcium antagonistic action. It has been extensively used to treat endotoxin shock, spasm of gastrointestinal and other visceral smooth muscle etc.Yong et al1 reported that Ani administered orally was found to antagonize the gastric mucosal damage induced by indomethacin, restraint, pyloric ligation o

10、r absolute ethanol ingestion in rats. Our previous study demonstrated that intraperitoneal Ani protected the rats from contracting gastric lesion induced by restraint water-immersion2. The effect of Ani on gastric lesion induced by reserpine, as well as its underlying mechanism, however, remains to

11、be studied.Recent studies have shown that reduction in endogenous nitric oxide (NO) seems to be responsible for the gastric mucosal injury induced by ethanol3, ischemia-reperfusion4 and cold restraint stress5. Nevertheless, whether NO reduction is implicated in the reserpine-induced gastric lesion h

12、as 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 A

13、ni protection.MATERIALS AND METHODS1. Animal preparation Sprague-Dawley rats of either sex (150220 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 procedureExperiment I. Production o

14、f 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 ，, and received Ani in doses of 1,5 and 10 mg/kg intraperitoneally, respectively. Thirty minutes later, the rats were given reser

15、pine 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

16、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. Experiment . Measurement of gastric acid secretion Gastric acid secretion was measured in pylorus-ligated rats. The animals were divided into three groups. Group

17、I: saline group; group ： reserpine group; group ： Ani group. Saline (5 mL/kg) was given intraperitoneally in group and ； Ani (10 mg/kg) was given intraperitoneally in group ， respectively. Thirty minutes later, the pyloric ends of the stomachs were tied off under ether anesthesia. Immediately therea

18、fter, reserpine (5 mg/kg for each rat) was administered intraperitoneally in group and , 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

19、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 s

20、ecretion, total acid output and titratable acidity were expressed as mL of gastric juice/100 g body weight, mol of H+/100 g body weight and mmol of H+/L, respectively.Experiment . Measurement of gastric barrier mucus Gastric barrier mucus was determined according to the modified procedure of Bolton

21、et al. The animals were divided into three groups and treated as in experiment . 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 o

22、f 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 spectr

23、ophotometer. Gastric barrier mucus was calculated using the following formula:gastric barrier mucus (mg)=4-4×(test absorbance)/(standard absorbance)The content of gastric barrier mucus was expressed as mg/per stomach.Experiment . Measurement of gastric contraction Gastric contraction was record

24、ed 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 t

25、o 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 li

26、miting 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 ab

27、dominal wall was sutured with silk. Rats were divided into two groups. Group : reserpine group; group : 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 tota

28、l 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.Experiment V. Measurement of gastric mucosal blood flow Rats were divided and treated as in experi

29、ment . 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 ，Ani (10 mg/kg) was given in

30、group . 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-1RESULTS1. Effect of Ani on reserpine-induced gastr

31、ic 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

32、 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.TreatmentDose(mg/kg)nLesion indexSaline727.00±3.32Ani1510.08±1.36?579.00±3.40

33、?1066.33±0.33?     *P0.01, 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 grou

34、p 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

35、 gastric acid secretion.GroupnVolume ofgastric secretion(mL/100gbody wt.)Total acidoutput(H+mol/100g body wt.)Titratableacidity(H+mmol/L)72.62±0.48145.23±23.0677.89±7.4971.94±0.26191.31±27.6198.95±7.8861.25±0.2186.60±23.52?65.79±12.08?   &#

36、160; *P0.05, vs group ; : saline group; : reserpine group; : Anisodamine (10 mg/kg) group 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) s

37、ignificantly inhibited reserpine-induced decrease in gastric barrier mucus.GroupnGastric barrier mucus(mg/per stomach)72.02±0.2481.10±0.07?61.87±0.06#     *P0.01, vs group ;#?P0.01, vs group ; : saline group; : reserpine group; : Anisodamine group 4. Effect of Ani

38、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

39、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) (P0.05). Ani (10 mg/kg) pretreatment significantly inhibited the decrease in GMBF in reserpine-treated rats (0.24±0.04 mL/min) (n=7) (P0.05).Gastricmotility Gr

40、oupTime after reserpine treatment (h)1 st2 nd3 rd4 th5 th6 thFrequency0.60±0.142.30±0.152.00±0.471.60±0.452.10±0.352.60±0.26(contractions/min)1.00±0.281.40±0.561.10±0.450.30±0.13*0.60±0.22*0.80±0.36*Amplitude(cm H2O)9.00±1.5119.60±

41、;4.0325.00±5.7920.10±6.2623.40±6.3927.00±6.455.30±0.30*6.20±0.75*6.70±1.05*6.70±0.80*11.90±2.8821.40±7.70     *P0.05,*P0.01，vs group ; : reserpine group (n=7); : Anisodamine group (n=5) 6. Effect of Ani on gastric mucosal NOS acti

42、vity 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 decreas

43、e in gastric mucosal NOS activity and NO content in reserpine-treated rats.Tab 5 Effect of anisodamine on gastric mucosal NOS activity and NO-2/NO-3GroupnNOS activity(U/mg protein)NO-2/NO-3 content(nmol/mg protein)50.50±0.028.57±1.1950.32±0.02*2.67±0.80*50.52±0.05#9.87±

44、1.32#     *P0.01, vs group ;#P0.01, vs group ; : saline group; : reserpine group; : Anisodamine group DISCUSSIONYong et al1 reported that Ani (12.550 mg/kg) given orally protected the gastric mucosal damage induced by indomethacin administration, restraint, pyloric ligation or ab

45、solute ethanol ingestion in rats. Our previous study demonstrated that Ani (110 mg/kg) injected intraperitoneally protected against gastric lesion induced by restraint water-immersion in rats2. The results of the present study revealed that Ani (110 mg/kg) administered intraperitoneally protected ag

46、ainst gastric mucosal lesion induced by reserpine in rats.Our previous studies have led to the conclusion that gastric lesions induced by reserpine do not result from acid hypersecretion6. The present study confirmed our previous findings in that gastric acid secretion of reserpine-treated animals w

47、as 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 al1, who found that Ani (12.525 mg/kg) given oral

48、ly 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 inhibit

49、s gastric acid secretion is not clearly understood. There is evidence that Ani has a calcium antagonistic action7. Therefore, the action of Ani to inhibit gastric acid secretion may be mediated by blocking of the calcium channel.In agreement with our previous study6, the present study showed that re

50、serpine 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

51、 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.In

52、our previous studies, we found that reserpine increased the frequency of gastric contraction6 and that Ani treatment inhibited gastric contraction in water-immersion rats2. In the present study, Ani significantly suppressed the frequency and amplitude of gastric contraction in reserpine-treated rats

53、, 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 channel7. Therefore, Ani ma

54、y prevent gastric contraction through this mechanism.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 indu

55、ces 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

56、 Ani may in part be mediated through this mechanism.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 GMBF8， acid secretion9 and gastric motility10. Recent

57、studies have shown that reduction in endogenous NO may be responsible for gastric mucosal injury induced by ethanol3, ischemia-reperfusion4 and cold restraint stress5. However, NO reduction implicating in reserpine-induced gastric lesion has not yet been elucidated. We, therefore, investigated the c

58、hange 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 N

59、OS 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