Study on the Protective Effects of Astragalosides in Gastric Mucosa Injury

Research BackgroundCarrying on the research of traditional Chinese medicine effective parts has become one of the most important directions in traditional Chinese medicine, crude drug and new drug development in recent years. It is our research group’s persistent research direction to develop pharmacology study of nourishing spleen and reinforcing qi TCM for gastric mucosal protective effect basing on TCM theory and combining modern scientific experiment technology.In earlier research work, members of our research group have studyed the effects of AstragaIosides (AST) on function and morphology of gastric parietal cells of spleen deficiency rats. We have found that AST as the main effective parts has an important role on regulating the functions of gastric parietal cells, protecting the gastric mucosal barrier, maintaining the integrity of structure and modulating gastric acid secretion. As the mixture of Astragalus saponins compounds, AST is one of the effective parts of Astragalus. As the monomer of Astragalus saponins compounds, AstragalosideⅣ(AST-Ⅳ) is formulated as the quality evaluation index of Astragalus by TCM Pharmacopeia. As we know, the physical base of TCM multiple targets effects often root in the synergistic effect of multiple similar compounds in effective parts. The activity of single compound is not strong. Therefore, Carry on contrast study of TCM effective parts and monomer using efficacy as standard, which can reveal the difference of TCM effective parts and monomer and clarify the science significance of effective parts synergistic effects. In view of this research ideas, this experiment carry on further study based on the gastric mucosal protective effects of AST and AST-Ⅳ. At present, the related research reports has not yet been seen. ObjectiveThis paper is aimed at studying the gastric mucosal protective effects of Astragalus saponins effective part (AST) and the monomer (AST-Ⅳ) basing on TCM theory and combining with modern scientific experiment technology. It mainly includes the following aspects:First, solid phase extraction-high performance liquid chromatography-evaporative light scattering detection (SPE-HPLC-ELSD) was established for determining the serum concentration of AST-Ⅳin rats after separately intraperitoneal injection of AST and AST-Ⅳat the same time and finding the peak time of AST and AST-Ⅳserum concentration. Thus to lay a foundation for establishing molding time in the experimental study of AST and AST-IV to acute gastric mucosal injury in rats in late, and also provide the basis of pharmacokinetic parameters for the pharmacodynamic difference of the two medicine to resisting acute gastric mucosa injury.Second, study the protective effect of AST and AST-Ⅳdifferent doses on absolute alcohol induced acute gastric mucosal injury in rats from the degree of gastric mucosal pathological morphology. Meanwhile, we observe pharmacodynamics separately from medication one time and medication four times in order to observe the cumulative effect, and expect to study the difference of AST and AST-Ⅳfrom multilevel.Third, probe the protective effects of AST and AST-IV on chronic gastric mucosal injury after prevention medicine and treatment medicine from the gastric mucosal epithelial cells ultrastructural changes.Fourth, observe the effects of AST and AST-Ⅳon acute injury gastric mucosal cells’ apoptosis and proliferation in rats from the degree of cytology and expect to formulate the gastric mucosal protective effects mechanisms and difference of these two drugs.MethodsFirst, investigate AST-Ⅳserum concentration and it’s peak time of AST and AST-IV in rats. Extract the AST-IV in serum by SPE. Investigate the linear relationship and minimum detection concentration, specificity, precision, stability and recovery by HPLC-ELSD to establish the AST-IV content test methodology. Serum AST-Ⅳcontent was detected and compared after separately intraperitoneal injection of AST and AST-Ⅳ(equivalent of AST-Ⅳ20 mg/kg) for 30,60,75,90,105 and 120min. Then the peak time of plasma concentration of each medicine was found.Second, with the AST-Ⅳas the control index, we set AST and AST-Ⅳdifferent doses and ensure the AST-Ⅳcontent of their corresponding large, medium and small doses consistent in the experimental research of AST and AST-Ⅳdifferent doses protecting on acute gastric mucosal injury in rats. SD rats were randomly divided into model group, AST large, medium and small dose group, AST-Ⅳlarge, medium and small dose group and western medicine positive control group, these groups were respectively given intraperitoneal injection of solvent, AST large (200mg/kg), medium (100mg/kg), small (50mg/kg) doses injection and AST-Ⅳlarge (4.08mg/kg), medium (2.04mg/kg), small (1.02mg/kg) doses injection and cimetidine (200mg/kg) injection. Then we used absolute alcohol to induce acute gastric mucosal injury in rats after medication one time and last time of medicine four times 90 min and 75 min (which are the peak time of serum concentrion of AST and AST-Ⅳrespectively). The gland stomach tissues were taken to measure the gastric mucosal injury scores after one hour and paraffin sections were stained with HE and pathomorphological change of gastric mucosal was observed under an optical microscope.Third, with the AST-IV as the control index, we still set AST and AST-Ⅳhigh and low doses and ensure the AST-Ⅳcontent of their corresponding high and low doses consistent in the experiment research of AST and AST-Ⅳinfluence on gastric mucosal epithelial cells ultrastructure of chronic gastric mucosal injury rats. SD rats were randomly divided into normal group, model group, AST high and low dose prevention group, AST-Ⅳhigh and low dose prevention group and model natural recovery group, AST high and low dose treatment group, AST-Ⅳhigh and low dose treatment group. The model of chronic gastric mucosa injury in rats was established by giving Rheum into stomach. These groups were respectively given intraperitoneal injection of solvent, AST high (200mg/kg) and low (100mg/kg) doses injection and AST-Ⅳhigh (4.08mg/kg) and low (2.04mg/kg) doses injection. Prevention groups were given 14 days dosing continuously and treatment groups were given 6 days cure after molding. Then we observed the ultrastructural changes of gastric mucosal surface epithelial cells and the effects of AST and AST-Ⅳprevention and treatment medication on these model rats by scanning electron microscope.Fourth, in the experimental research of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa in rats, the SP method of immunohistochemistry was used to detect the proteins expressions of early apoptosis related gene (Bcl-2/Bax) and proliferating cell nuclear antigen (PCNA) in acute injury gastric mucosa of rats. CMIASWIN image analysis system was used to quantitatively analysis immunohistochemical results and then we do statistical processing.Fifth, single factor analysis variance (One-Way ANOVA) was been adopted to statistic analysis and LSD test was been used to compare among groups.ResultsFirst, the SPE-HPLC-ELSD was used to detect serum AST-IV concentration in rats with AST and AST-Ⅳ. The results showed that:The method of SPE to extract and separate serum AST-Ⅳin rats was easy and simple to handle, the effects of which to remove protein and to enrich AST-IV were very well. HPLC-ELSD to detect serum AST-Ⅳconcentration in rats could solve the quantitative analysis problem of micro-componential and complicated serum sample. The linear relationship was good. The linear equation was Y=1.272X+2.98, r=0.9995. The minimum detection concentration was 1.536μg/mL. With the specificity, precision, stability, recovery and extraction recovery were all desirable, we established the AST-Ⅳcontent test methodology successfully.We measured the peak time of AST serum concentration was about 90 min and AST-Ⅳwas about 75 min after equivalent of AST-Ⅳintraperitoneal Injection. At any time point, the serum concentration of AST-Ⅳin rats after intraperitoneal injection for AST was three times as much as that in rats after intraperitoneal injection for AST-Ⅳ, and even arrived 3.5 times in peak time. The serum concentration of AST-Ⅳwas 33.05±5.36μg/mL when AST reached it’s peak time, while AST-Ⅳwas 9.29±3.63μg/mL when AST-IV reached it’s peak time.Second, the results of AST and AST-Ⅳprotective effects on acute gastric mucosa injury in rats indicated that:Each group except AST-Ⅳsmall dose group could take different degree effect on reducing gastric injury score and gastric injury index under the light microscope in medication one time and four times (P<0.05). AST each dose group was superior to AST-Ⅳ(P<0.05), AST large dose group which had the same effect with western medicine positive control group (P>0.05) was obviously superior to the rest groups (P<0.05). The groups of medication one time compare with medication four times had no significant difference (P>0.05). The effect of medication one time of AST each dose group was superior to medication four times of AST-Ⅳeach dose group (P<0.05)Third, in the experiment research of AST and AST-IV influence on gastric mucosal epithelial cells ultrastructural of chronic gastric mucosal injury rats, We found that:The gastric mucosal epithelial cells of chronic gastric mucosal injury rats became generally necrosis, fragmentation, fuzzy and incomplete form, disorganized arrangement, swelling and applanation, no delimitation among cells, a number of cells squeeze and tangle into lump. Microvillus of cell surface became rare and detached with few secretory granules. The construction gastric pits became malformed. AST high (200mg/kg) and low(100mg/kg) doses as well as AST-IV high dose(4.08mg/kg), prevention and therapy medication, were able to reverse the gastric mucosal epithelial lesions in rats with chronic gastric mucosal injury, the effect of AST each dose was superior to all doses of AST-Ⅳ, and the effect of AST high dose of prevention and therapy medication was superior to the rest doses and equal to the normal group. The effect of AST low dose of prevention and therapy medication was superior to AST-Ⅳhigh dose.Fourth, as we can see from effects of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa, Compared with model group, AST each dose group, AST large and medium dose group and western medicine positive control group, Bax protein positive expression were weaken (P<0.05), Bcl-2 protein positive expression were strengthen (P<0.05). While AST each dose group, AST large dose group and western medicine positive control group, gastric mucosal PCNA protein expression enhanced obviously, which had significant difference compared with model group (P<0.05).The effects of weakening Bax expression, enhancing Bcl-2 and PCNA expression of AST each dose group were superior to AST-IV each dose group (P<0.05). AST large dose group which had close to the western medicine positive control group was obviously superior to the rest groups (P<0.05)ConclusionFirst, the method of SPE-HPLC-ELSD which had been used to detect the AST-Ⅳserum concentration in rats with AST and AST-Ⅳwas correct and reasonable. With AST-Ⅳas the serum concentration index, we could consider that AST was easier to be absorbed than AST-Ⅳ. This result provided powerful evidence that the effects of Astragalus saponins effective parts were superior to the monomer and also laid a foundation for late pharmacodynamics study.Second, the study of AST and AST-Ⅳprotective effected on acute gastric mucosa injury in rats confirmed that:AST and AST-IV had a protective effect on rats’ alcohol-induced acute gastric mucosal injury, the effect of AST was obviously superior to AST-Ⅳ, and with the dosage’s increasing, the superiority of AST was more obviously. The protective effect of AST large dose group had already access to positive control medicine cimetidine. There was no potency stacked phenomenon in AST and AST-Ⅳcontinuous medication four times. The protective effect of AST medication once was better than AST-IV multiple medication.Third, the experiment research of AST and AST-Ⅳinfluenced on gastric mucosal epithelial cells ultrastructure of chronic gastric mucosal injury rats demonstrated:gastric mucosal barrier of chronic gastric mucosal injury rats got damaged, AST and AST-Ⅳhad a protective effect on gastric mucosal barrier. The effect of AST was obviously superior to AST-Ⅳ. With the dosage’s increasing, the superiority of AST was more obviously, the protective effect of AST high dose had already access to the normal group. AST in the case of lower dose could achieve even more than AST-Ⅳhigh dose effect.Fourth, the influence of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa illustrated:AST and AST-IV could descend the expression of Bax, and ascend the expression of Bcl-2 and PCNA. Thus restrained gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation from different degree for alleviating the injury of gastric mucosa. That was one of the mechanisms of these two medicines resisting acute gastric mucosa injury. The effect of AST was obviously superior to AST-Ⅳ. With the dosage’s increasing, the superiority of AST was more obviously. The protective effect of AST large dose had already access to the positive control medicine Cimetidine.We have illustrate the effective parts of Astragalus saponins AST and the monomer AST-Ⅳhave clearly protective effects on gastric mucosa via the above-mentioned experimental study. Thus have certified the scientific meaning of TCM Astragalus "nourishing spleen and reinforcing qi" and "promote new growth of tissues". We have also revealed the effects of Astragalus saponins effective parts are superior to the monomer composition, and the effective parts in the case of lower dose can achieve even more than the monomer high dose effect. Thus state that reasonable compatibility among multicomponent of TCM effective parts play the role of synergistic function. This function is similar to TCM compound which is the result of multiple similar compositions combined action by influencing each other. This research results provide a new thought on the prevention and treatment for gastric mucosal injury, and also provide theory basis for carrying on medicine development of Astragalus from medicine components level.