| 1 Research Background and ObjectiveStress ulcer(SU)refers to the acute gastric mucosal lesion characterized by congestion,edema,erosion and bleeding of gastric mucosa. Till now the pathogenesis of SU still remains unclear.The onset of SU is the results of combination of the influences of gastric acid,oxygen free radicals,neuroendocrine factors based on the damage of gastric mucosa barrier.Melatonin(MT)is the best endogenous antioxidant,and there exists the MT receptor in gastrointestinal tract.Large amount of research showed that MT has protective effect on gastric lesion in experiments.So the effect of MT in the gastricintestinal has been given a high interest.The study of MT would conduce to the diagnose and treatment in stress ulcer. SU is classified into blood syndrome and gastric abscess in traditional Chinese medicine(TCM),and its pathogenesis is associated with heat, deficiency and stasis.The traditional chinese medicine has some predominant curative effect in clinic for stress ulcer.How to choose the suitable medicine and reduce the toxic or side effect are the emphasis in the reseach of stress ulcer.Resently investigation showed that MT had a close relation with acute gastric injury.Though the exogenous MT was used in some experiment,the influence of endogenous MT in gastric mucosa has not been reported.Strengthening Qi and activating blood is one of the methods for the treatment of SU,and Radix Astragali and Radix Notoginseng are the representative herbs.The previous research of Professor Wang Rujun indicated that therapy of strengthening Qi and activating blood can counteract gastric mucosal lesion and promote the recovery of gastric mucosa, and improve the function of parietal cells of gastric mucosa. Total saponins of Panax notognseng(PNS)is the main active components of Panax notognseng.including various kinds of monomer ginsenoside.PNS has the pharmacological actions of dilating vessels,decreasing myocardial oxygen consumption,inhibiting the platelet coagulation,prolonging clotting time,improving micro-circulation,decreasing blood lipid, clearing free radicals,counteracting inflammation and oxidation.It is reported that PNS hasa good effect on oxidation-induced damage in heart, liver,lung,kidney and brain,and the preventive mechanism of PNS is related to the clearance of free radicals.Based on the above research achievements, we get the idea of carrying out experimental and clinical research of PNS and Radix Notoginseng for SU.On the base of superiority of the chinese medicine and the experimental study before,the effect of PNS on gastric mucosal MT receptor expression and oxidation stress was observed,and the anti-stress-damage and preventive mechanism of PNS was explored by observing the effect of PNS on endogenous MT receptor.2 MethodsThis study consists of three parts:(1)effect of PNS on gastric mucosal damage and ultrastructure of gastric wall cells in SU rats;(2)effect of PNS on oxygen free radical of in gastric mucosa of SU rats;(3)effect of PNS on MT receptor expression in gastric mucosa of SU rats.2.1 Establishment and evaluation of SU rat modelsWater-immersion restraint stress method:after fasting 24 hours,the rats were immersed into the water at the temperature of(19±1)℃for 6 hours.The models were executed to observe the damage degree of gastric mucosa(by Guth methos).2.2 Observation of ultrastructure of gastric mucosal epithelial ceils under Transmission electron microscopeGastric tissues from gastric antrum were prepared for the slices.After double staining with uranyl acetate and lead citrate,the slices were observed under the electron microscope.2.3 Detection of gastric mucosal MT1 receptor mRNAThe exaction of total RNA from gastric mucosa was performed according to the instructions of RNAiso Reagent.The reverse transcription of the RNA was carried out with RevertAid First Strand cDNA Synthesis Kit.After reverse transcription,the polymerase chain reaction was performed.The primer is as follows:the upstream sequence of MT1 receptor: 5' -GTGCTACGTGTTCCTGATATGG-3',the downstream sequence of MT1 receptor: 5' -GGATCTGAGGCCACAATAAGAC-3',360bp:the upstream sequence of GAPDH: 5' -CTACCCACGGCAAGTTCAAT-3',the downstream sequence of GAPDH: 5' -ACTGTGGTCATGAGC CCTTC-3',382bp.PCR were carried out by 40 cycles of denaturation at 94℃for one minute,annealing at 55℃for one minute, extending at 72℃for 2 min.The product of PCR were detected by agarose gel electrophoresis.The expression level of MT1 receptor mRNA was calculated with the optimal density of ratio of MT1 receptor and GAPDH.2.4 Detection of oxygen stress parameters(MDA,SOD and NO)The contents of malondialdehyde(MDA),superoxide dismutase(SOD)and nitric oxide(NO)in rat serum oand homogenate of gastric mucosa were detected.3 Results and Conclusion3.1 Effect of PNS on gastric mucosal damage and ultrastructure of gastric wall cells in SU ratsExtensive erosion,bleeding and ulceration were found in the gastric mucosa of SU model rats,and PNS can reduce the above pathological changes, indicating the preventive effect of PNS for gastric mucosa of SU rats.Obvious stress-induced changes of ultrastructure were found in the model group:concentrated endocrine tubular villi and disappearance of cytoplasm interior vesicles in gastric parietal cells,which accorded with the changes of macro-structure of gastric mucosal lesion.The results indicated that water-immersion restraint stress can activate the gastric parietal cells and increase the secretion of gastric acid,which was correlated with the degree of gastric mucosal lesion.3.2 Effect of PNS on oxygen free radicals of serum and gastric mucosa of SU ratsNO content in gastric mucosa of the model group was lower(P＜0.05),MDA was higher and SOD activities was lower than those in the normal group after stress for 6 hour,indicating that water-immersion restraint stress can induce the production of oxygen free radicals,increase the SOD consumption and induce the damage of gastric mucosa.After the treatment of PNS,NO content increased,MDA decreased and SOD activity increased,indicating that PNS can clear oxygen free radicals,inhibit the production of peroxides and prevent the gastric mucosa from damage.The decrease of NO content and SOD activity and the increase of MDA in the serum of model group showed the damage induced by oxygen free radicals. After treatment of PNS,NO content increased,but MDA and SOD did not from those in the model group,indicating that stomach is sensitive to the oxidation,and damage induced by oxygen free radicals mainly involved the gastric mucosa.Anti-oxidation action of PNS may be one of its mechanism for counteracting gastric mucosal damage induced by oxygen free radicals.3.3 Effect of PNS on melatonin receptor expression in gastric mucosa of SU ratsA positive band of 360bp was showed in the RT-PCR product of MT1 receptor,which indicated the existence of MT1 receptor mRNA expressed in gastric mucosa.The relative content of MT receptor in the model group showed a decreasing trend as compared with the normal group.However,the relative content of MT receptor in PNS group and ranitidine group was increased as compared with the model group(P＜0.05 or P＜0.01),indicating that PNS and ranitidine can upregulate the level of MT receptor.Its mechanism may be related to the synergism of PNS with MT during the counteraction of damage induced by oxygen free radicals.This will supply experimental data for PNS regulating endogenous MT indirectly and preventing gastric mucosa.The results of this study will supply a new thought for the pathogenesis, prevention and treatment of SU,supply evidence for the clinical safety of MT and PS and expanding their pharmacological actions,will benefit to the exploration of TCM therapeutic theory and development of new anti-SU Chinese preparations. |
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