山莨菪堿對利血平大鼠胃粘膜損傷的影響

1、山莨菪堿對利血平大鼠胃粘膜損傷的影響關(guān)鍵詞：胃粘膜；利血平；一氧化氮中國病理生理雜志000313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(jī)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(yùn)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(jìn)利血平大鼠胃粘液分泌和胃粘膜血流量，抑制胃的運(yùn)動；山莨菪堿能抑制利血平導(dǎo)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

2、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(yùn)動，促進(jìn)胃粘液分泌、增加胃粘膜血流量有關(guān)；NO可能在介導(dǎo)山莨菪堿抑制利血平大鼠胃粘膜損傷形成中有重要作用。中分類號 R 961 R 961 文獻(xiàn)標(biāo)識碼 A文章編號 1000-4718(2000)03-0237-06Effect of anisodamine on the reserpine-induced gastric mucosal lesion in ratsWAN Jun-li(Department of Biology, Yantai Teachers College, Yantai 264025， China)Abstract

3、 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 (G

4、MBF), 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

5、/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 con

6、traction. 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 gastri

7、c 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.MeSH Gastric mucosa; Reserpine; Nitric oxideCLC number R961 Document code AINTRODUCTIONAnisodamine (Ani) is an

8、 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 exte

9、nsively 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 or absolute ethanol ingestion in rats. Our p

10、revious 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 be studied.Recent studies have shown that r

11、eduction 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 has not yet been investigated.Therefore, the

12、 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.MATERIALS AND METHODS1. Anima

13、l 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 of gastric lesion and measurement of gastric

14、 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 reserpine in a dose of 5 mg/kg for each animal i

15、ntraperitoneally. 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

16、 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 I: saline group; group ： reserpine group; gr

17、oup ： 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 thereafter, reserpine (5 mg/kg for each rat) was a

18、dministered 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 centrifuge tubes. After centrifugation at 3

19、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 a

20、cidity 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 et al. The animals were divided into three g

21、roups 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 of the stomachs was then placed separately in

22、 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 calcu

23、lated 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 recorded according to the method described by Takeuchi e

24、t 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 flacc

25、id 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 s

26、o 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 divi

27、ded 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 total of 6 h. All waves with amplitudes greater 4 cm H

28、2O 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 experiment . Gastric mucosal blood flow (GMBF) was measu

29、red 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 group . Thirty minutes later, the pyloric ends of

30、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?*P0.01, vs saline group2.Effect of Ani on gastric acid secretion Table 2 shows the effect of Ani on

31、 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 grou

32、p, 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.Tab 2 Effect of anisodamine on gastric secretion(s) GroupnVolume ofgastric secretion(mL/100gbody wt.)Total acidoutput(H+

33、mol/100g body wt.)Titratableacidity(H+?#*P0.01, vs group ;#*Amplitude(cm H2*11.902.8821.407.70*P0.05,*P0.01，vs group ; : reserpine group (n=7); : Anisodamine group (n=5)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

34、 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

35、 of anisodamine on gastric mucosal NOS activityand NO-2/NO-3 content in reserpine-treated rats (s) GroupnNOS activity(U/mg protein)NO-2/NO-3*#*P0.01, vs group ;#P0.01, vs group ; : saline group; : reserpine group; : Anisodamine groupDISCUSSIONYong et al1 reported that Ani (12.550 mg/kg) given orally

36、 protected the gastric mucosal damage induced by indomethacin administration, restraint, pyloric ligation or absolute 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 ra

37、ts2. The results of the present study revealed that Ani (110 mg/kg) administered intraperitoneally protected against 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.

38、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 aci

39、d. These data are in agreement with those reported by Yong et al1, who found that Ani (12.525 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 re

40、sult 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 action7. Therefore, the action of Ani to inhibit gastric acid secretion may be medi

41、ated by blocking of the calcium channel.In agreement with our previous study6, 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 gast

42、ric 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 lik

43、ely that the protective effect of Ani results in part from prompting the secretion of gastric barrier mucus.In 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 s

44、tudy, 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

45、 a key role in smooth muscle contraction and Ani is effective in inhibiting calcium channel7. Therefore, Ani may 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

46、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 stud

47、y, 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.Nitric oxide synthesized from L-arginine by nitric oxide synthase is a potent vasodilator and an inhibitory neurotransmitter. It has been

48、 reported that NO is an important mediator in regulation GMBF8， acid secretion9 and gastric motility10. Recent 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

49、 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

50、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 “r

51、escuing” 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.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, pr