Study Of Protective Action And Mechanism In Gastric Mucosa Induced By Puwei Pill

Puwei Pill(PWP) is a secret recipe for the treatment of gastric ulcer (Gastric Ulcer, GU) with the advantages of low frequency in using, lasting efficacy, few side reactions and low relapse rate.This experiment evaluated the pharmacodynamic action of anti-gastric ulcer and explored the mechanisms for protecting gastric mucosa of PWP,which provided the basis for the further researching PWP.Objective:To evaluate the PWP's pharmacodynamic action of anti-gastric ulcer through several experimental GU models, investigate PWP's effect on analgesia and acute toxicity, and explore the mechanisms for protecting the gastric mucosa of PWP.Methods:1 To evaluate PWP's pharmacodynamic action of resisting GU. The gastric ulcer models were induced by water- immersion stress, acetic acid, and pyloric ligation in rats. The acute gastric mucosa injure in mice were induced by absolute ethyl alcohol and reserpine, respectively.The PWP's pharmacodynamic action of resisting GU was evaluated by calculating ulcer index(UI) and ulcer inhibition ratio (UIR).The analgesic effect of PWP was detected by the hot-plate and torsive malposition test in mice. The acute toxicity was eveluated by measuring the maximal tolerating dose in mice.2 To study PWP's mechanisms for protecting the gastric mucosa.A series of studies, such as the content of gastric juice and gastric acidity being measured by acid-base titration, the activity of pepsin being detected by Matt's method, the integrity of gastric mucosa being abserved using HE staining, the content of endothelin-1 (ET-1) being detected by radioimmunoassay method and the concentrations of nitric oxide (NO), malondialdehyde (MDA), the activities of the superoxide dismutase(SOD) and glutathione peroxidase (GSH-Px) being all determined by spectrophotometry respectively, were performed to study PWP's mechanisms for protecting the gastric mucosa.Results:1 The evaluation of PWP's pharmacodynamic action of resisting GU.1.1 The effect of PWP on resisting acute GU.1.1.1 Study in stress-induced GU experimentThe UI in normal and model group were 0.76±1.61 and 31.56±8.60,respectively.When the rats were treated with PWP p.o. at the doses of 0.12 g/kg, 0.24g/kg and 0.48g/kg, the UI were 15.44±2.88, 12.20±1.79 and 9.75±2.49,and UIR were 51.98%,62.05% and 69.67%,respectively. Compared with the model group, PWP markedly reduced the UI (P<0.01) in a dose-dependent manner(r=0.461, P<0.05). 1.1.2 Study in pyloric ligation GU experimentThe UI in model group is 21.89±5.93. After the rats being treated with PWP p.o. at the doses of 0.12g/kg, 0.24g/kg and 0.48g/kg, the UI were 4.50±3.95, 4.11±2.31 and 3.67±1.88, and the UIR were 79.44%, 81.22% and 83.23%, respectively. Compared with the model group, PWP significantly depressed the UI (P<0.01). These results indicated that PWP has protective effect on the acute GU.1.2 The effect of PWP on the chronic GU.The UI in model group was 96.25±17.15 in the GU induced by acetic acid,and the UI of normal group was 0.After the rats being treated with PWP p.o. at the doses of 0.12g/kg, 0.24g/kg and 0.48g/kg, the UI changed into 40.64±10.60, 21.60±6.24 and 5.75±2.36 and the UIR changed into 57.76%, 77.56% and 94.03%, respectively. Compared with the model group, PWP dose-dependently depress the UI (P<0.01;r=0.858,P<0.01), which suggesting that PWP could heal the chronic GU in a dose-dependent manner.1.3 The protecting effect of PWP on the acute gastric mucosal injury.1.3.1 Study in mucosal injury induced by reserpine The UI of normal group was 0.38±0.52. After the gastric mucosal injury being induced by reserpine in mice, the UI was 5.22±2.38. When the mice were treated with PWP p.o. at the doses of 0.25g/kg, 0.5g/kg and 1.0g/kg, the UI changed into 2.75±1.67,2.71±1.11 and 1.87±0.83, and the UIR were 47.32%, 48.08% and 64.18%, respectively. Compared with the model group, the UI in PWP groups dose-dependently lowed(P<0.01;r=0.315,P<0.05).1.3.2 Study in mucosal injury induced by absolute ethyl alcoholThe UI of normal group was 0.34±0.54.After the mice fed with absolute ethyl alcohol by lavage, the acute gastric mucosal injury was followed, and the UI in model group was 65.80±12.60. When the mice were treated with PWP p.o. at the doses of 0.25g/kg, 0.5g/kg and 1.0g/kg, the UI were 22.11±7.36, 18.10±7.16 and 5.00±1.86, and the UIR were 60.82%, 75.23% and 94.49%, respectively. Compared with the model group, the UI in PWP groups significantly reduced (P<0.01) in a dose-dependent manner (r=0.787,P<0.01). The results showed that PWP could dose-dependently prevent gastric mucous from acute injury induced by some factors, such as reserpine and absolute ethyl alcohol.1.4 The analgesic effect of PWP.1.4.1 When the mice were treated with PWP p.o. at the doses of 0.25g/kg, 0.5g/kg and 1.0g/kg in hot-plat test, the pain threshold in each group was detectd after 15 minutes.Compared with the control group, the pain threshold had no significant change in each group. Nevertheless, after 30th, 60th and 120th minutes, the hot pain threshold markedly extended (P<0.05 or P<0.01).1.4.2 In the test of torsive malposition, after the mice being treated with PWP p.o. at the doses of 0.25g/kg, 0.5g/kg and 1.0g/kg, the torsive malposition times of each mouse were measured in 20 minutes. Compared with the control group, the torsive malposition times were 24.61±5.61, 14.00±4.20 and 13.82±4.24 in PWP groups, respectively, and dose-dependently reduced (P<0.05;r=0.359,P< 0. 05).These proved that PWP had obviously analgetic effect in a dose-dependent manner.1.5 The test of PWP on the maximal tolerating dose in mice.The mice were administed with PWP p.o. at the dose of 0.1g/ml, 0.8ml (the maximum volume at one time in one day for mouse)for one time,and then were observed for 7days. The death or any other abnormality in mice had not been found. On the 8th day, all mice were autopsied, and the main organs, such as heart, liver, spleen, lung, and kidney all had no any disfunction.The maximal tolerating dose in mice of PWP was calculated as 4g/kg, and was 200 times comparing to the clinical dose(0.02g/kg),which proved that PWP was safe in acute uasage.2 The study of PWP on the mechanisms for protecting gastric mucosa.2.1 The effect of PWP on gastric juice, gastric acidity and the activity of pepsin.In the pyloric ligation test, after the rats being treated with PWP p.o. at the doses of 0.12,0.24,0.48g/kg, the volume of gastric juice, gastric acidity and the activity of pepsin were detected.Compared with the model group, PWP, at any doses, significantly reduced gastric juice secretion (P<0.05), depressed the activity of the pepsin (P<0.01 or P<0.05), and only 0.48g/kg PWP markedly reduced the gastric acid(P<0.05).These indicated that PWP could protect the gastric mucosa by reducing the the gastric juice,acid and pepsin secretion.2.2 The effect of PWP on ET-1, NO in plasma.After the GU model being induced by acetic acid, the content of plasma ET-1 in model group was 25.57±4.05pg/ml, and markedly increased than that in the normal group(P<0.01). When the rats were treated with PWP p.o. at the doses of 0.12g/kg, 0.24g/kg and 0.48g/kg, the content of plasma ET-1 changed into 13.14±3.14pg/ml, 12.85±1.28pg/ml and 12.27±3.47pg/ml, respectively,and were significantly lower than that in the model group(P<0.01).At the same time, the content of plasma NO in model group was 18.83±5.19umol/l, and was lower than that in the normal group (P<0.01). Compared with the model group, PWP increased the content of plasma NO (P<0.05 or P<0.01),which provided that PWP could improve blood flow and enhance the protective effect in gastric mucosa by decreasing the content of ET-1 and increasing the NO in plasma.2.3 The effect of PWP on MDA, SOD and GSH-PX in plasma.In the experimental GU model induced by acetic acid in rats, the content of plasma MDA and the activity of SOD and GSH-Px in the model group were 6.83±0.72nmol/ml, 135.95±16.06U/ml and 607.80±236.80U, respectively. Compared with normal group, plasma MDA increased (P<0.01), and the activity of SOD and GSH-Px decreased (P<0.05 and P<0.01) in model group.These indicated that the antioxidant capacity of gastric mucosa reduced, and free radicals in vivo increased in the process of GU.After the rats being treated with PWP p.o. at the doses of 0.12g/kg, 0.24g/kg and 0.48g/kg, the content of MDA were 5.84±0.93nmol/ml, 5.86±0.55nmol/ml and 4.79±0.56nmol/ml in the PWP groups, respectively, and significantly reduced than that in model group in a dose-dependent manner(P<0.05 or P<0.01; r=0.561,P<0.01). Forthmore, the activity of SOD in PWP groups were 173.87±27.21U/ml, 179.45±31.47U/ml and 213.50±19.68U/ml.Compared with the model group, PWP dose-dependently increased the activity of SOD in plasma (P<0.05 or P<0.01) (r=0.531,P<0.05). At the same time, the activity of GSH-Px in PWP groups changed into 924.80±269.19U, 1001.60±227.39U and 1110.00±143.19U, respectively, and were dose-dependently higher than that in model group (P<0.01; r=0.375,P<0.05). These results indicated that PWP had protective effect on gastric mucosa by enhancing antioxidant capacity. Conclusions:1 PWP has the effect of resisting acute and chronic gastric ulcer. PWP has analgesic effect, and is safe in acute usage.2 The effect of PWP on the protecting gastric mucosa is related to the inhibition of gastric juice and gastric acid secretion, reducion of the activity of pepsin, improving the mucosal blood flow and enhancing the antioxidant capacity.