| Regulation of gastrointestinal hormone receptor and signal transduction pathway is a previous topic of life science, which investigates the signal transduction in a single cell or between cells of organism. It has been proved that gastrin (GAS) was closely related with spleen-deficiency syndrome. Our laboratory studies showed that the binding capacity of gastrin receptor (GR) markedly decreased in gastric mucosa and isolated gastric parietal cells of spleen-deficiency rats, the metabolism and function of GR and signal transmission of gastric parietal cells with spleen-deficiency rats markedly decreased too, and the buzhongyiqi recipe and huangqizhusheye for invigorating spleen would improve the decreased functions of spleen-deficiency syndrome. Radix Astragali plays an important role on this action. Astragalosides (AST) is the main effective constituent of Radix Astragali. Because AST has similar pharmacologic effects as huangqizhusheye, in this subject, we will further study the effects of AST on function and morphology of gastric parietal cells of spleen-deficiency rats by some new methods such as Radioligand Binding Assay (RBA). Meanwhile we explored the essence of spleen-deficiency syndrome from the level of GR and molecular signal transduction (the binding capacity of GR, the free intracellular [Ca2+]i, intracellular activity of CaM, )and morphology (cell modality of gastric mucosa including epithelial cells, principal cells and parietal cells) of gastric mucosa with spleen-deficiency rats.1. Effect of AST on the Binding Capacity of GR in the Parietal Cells of Rat Model with Spleen-Deficiency SyndromeGastrin (GAS) exerts actions of regulating gastric acid secretion, and cellular growth, nourishing gastric mucosa, regulating gastrointestinal movement andcentral nervous activity. Binding with its receptor is the premise of normal GAS function and activation of GR is also the prerequisite of intracellular signal transmission, so the influence on the binding capacity of GR is probably an important intermediate link. The model rat with spleen-deficiency syndrome was made by feeding 100% Rhei liquid, lml/lOOg, twice a day for 14 days. The isolation and purification of gastric parietal cells were achieved by-Lewin1 s method and nonlinear density gradient centrifugation using 40% percoll and 60% percoll. The gastric parietal cells were incubated with AST(containing astragaloside I 10.21%) under 37°C for 30 minutes immediately after isolated and stired meanwhile. We used the method of RBA single point binding experiment to test the GR binding capacity of parietal cells. The results showed that the binding capacity of GR decreased markedly in parietal cells of spleen - deficiency model group than normal control group, and increased significantly after incubated with AST in a dose-dependent manner. 2. Effect of AST on the Concentration of Free [Ca2+]i in the Parietal Cells of Rat Model with Spleen-Deficiency SyndromeCalcium is a basic substance for maintaining cellular physiologic function. It has been proved that [Ca2+]i is the second signal messenger during acid secretion in parietal cells. After being digested, isolated and purified, the changes of [Ca2+]i in parietal cells of rat were measured with Fura-2/AM fluorescence indicator. The results showed that in the resting state, the concentration of free [Ca2t]i in parietal cells of spleen-deficiency rats were significantly lower than that of the normal control group rats. And the elevating concentration of [Ca2+]i stimulated by [Leu'5]-gastrin was also markedly lower than that of the normal control group. Incubated with AST in vitro, the concentration of free [Ca2+]i of parietal cells in spleen-deficiency rats would recover to normal level under the resting state and stimulated by [Leu' 5]-gastrin. Comparing high-dose group with spleen-deficiency model group, the difference was significant, but not significant as compared with low-dose group.3. Effect of AST on the Activity of Calmodulin (CaM) in the Parietal Cells of Rat Model with Spleen-Deficiency SyndromeCaM is a chief receptor of intracellular [Ca2+]i and elaborate biological activity through the combination with Ca2+. In parietal cell, CaM is an important substance for GR signal transduction. After being digested, isolatedand purified, the activity of CaM in parietal cells of every group rat was detected by PDE method. And the results showed that the activity of CaM increased obviously in parietal cells of spleen-deficiency rat, higher than normal control group, and AST decreased the enhanced activity in parietal cells of spleen-deficiency rat significantly, and shown in a dose-dependent manner.4. Effects of AST on the Morphogical Changes of Gastrointestinal Mucosa in Rat Model with Spleen-Deficiency SyndromeThe functions of digestive system were decreased in spleen-deficiency state and its histomorphology may change correspondingly. After paraffin embedding and HE staining, the sample was observed with light microscope to discover the changes of chief cell, parietal cells and tissue morphology in gastrointestinal mucosa of spleen-deficiency rats. The result showed that obvious pathological changes appeared in gastrointestinal mucosa of spleen-deficiency rats. Under naked eye, the gastrointestinal mucosa appeared pale and reduced light intensity, with plenty of sticky substance in the intestinal cavity. Under light microscope, the gastric mucosa was thinned, the chief cells and parietal cells became degenerated obviously, the parietal cells were vacuolated, the number of chief cells markedly decreased, and the intestinal mucosa became thinned with sparse and shortened villi. After preventive medication and treatment with AST, the above morphological changes were improved, being more perminant than spleen-deficiency model group and spontaneous recover group.5. Effect of AST on the Ultrastructural Changes of Epithelial Cells in Gastric Mucosa of Rat Model with Spleen-Deficiency SyndromeIn self-protection of gastric mucosa, the barrier formed by tight arrangement of epithelial cell and mucous layer secreted by epithelial cells exerts an important protective effect. Scanning electron microscopy was applied to observe the changes of ultrastructure of gastrointestinal mucosa in spleen-deficiency rats. The result showed that epithelial cell of gastric mucosa necrotized, brokened and cellular structure incomplete, free microvilli sparse, dropped and secretory granule rare, intracellular space widened, cells fused and twisted, gastric pits unclear, deformed and widened. Both preventive medication and treatment with AST can lessen the above pathological changes, the high dose being better than the low dose. The impaired integrity of gastric mucosa epithelium of spleen-deficiency rats,scarce and dropped microvilli and reduced secretory granules reflect indirectly the decreased epithelial secretion of neutral glycoprotein, indicating that the two important anatomic bases of gastric mucosal barrier are damaged, and AST can protect barrier action of gastric mucosa. 6.Influence of AST on Ultrastructures of Gastric Parietal Cells and Chief Cells in Rat Model with Spleen-Deficiency SyndromeThe characteristic morphological structure of parietal cells determines the simultaneous occurrence of cellular function and morphology and dynamic change of ultrastructures during the course of acid secretion. The model rats of spleen-deficiency syndrome were made by feeding 100% Rhei liquid, lml/lOOg, twice a day for 14 days. After the model made, the rat was killed by cervical dislocation, and the stomach was removed. The samples were cut from the stomach tissue, operated according to the method of transmission electron microscope. The ultrastructures of the parietal cells and chief cells were observed by transmission electron microscope. The results showed that secretory tubules of parietal cells of spleen-deficiency rats dilated significantly, intra-cavity microvilli decreased and dropped, microresicles reduced and irregular in shape, and mitochondria normal in number and shape. The nuclei of chief cells became atrophic and irregular, metachromatin increased, euchromatin decreased, the space between cytoplasm and nuclear membrane widened with racuolar formation, rough endoplasmic reticulum broadened and dilated, ribosome granules dropped and decreased, limiting membrane of zymogen gtanule unclear, but the change of mitochondriae not obvious. Preventive medication and treatment of AST after model made can reverse the pathological change of ultrastructures in parietal cells and chief cells of spleen-deficiency rats.7. Effect of AST on the Function of Gastric Acid Secretion in Rat Model with Spleen-Deficiency SyndromeIn the occurrence and development of gastric mucosal diseases, low protective effect of gastric mucosa and enhanced aggressive factor are the two main pathogruic factors, gastric acid being the main aggressive factor. It was known that AST can regulate the binding capacity of GR and the post-receptor signal transmission, and maintain the integrity of gastric mucosa, cell and cellular ultrastructures. In this research, the influence of AST on the function of gastric acid secretion and the sensitivity to acid secretion after GASstimulation in spleen-deficiency rats was further studied. The animals were randomly divided into normal control group, model group and model group treated with AST. The pH of the gastric fluid in basal state and that after stimulated by GAS was observed. The results showed that the basal secretion of gastric acid in model group was higher and the sensitivity to GAS was lower than normal control group, indicating that AST can inhibit the hyperactive basal acid secretion in spleen-deficiency rats, increase the sensitivity to GAS stimulation and adjust gastric acid secretion as a whole. In this study, the effects of AST on gastric parietal cells of model rat with spleen-deficiency syndrome were investigated by methods of molecular biology, pharmaco dynamics and morphology. It is summarised as follows: (1)AST can regulate the binding capacity of GR and post-receptor signal transmission pathway in the gastric parietal cells with spleen-deficiency model rats. In Rhei-induced spleen-deficiency rats, the binding capacity of GR in parietal cells decreased, Ca2+decreased in the resting state and after GAS stimulation, and AST can increase its concentration and also regulate its activity of CaM in a dose-dependent manner. (2) AST protects tissue and cellular pathomorphological changes of spleen-deficiency rats in multiple aspects. The integrity of gastric mucosal barrier was destroyed, the shape and ultrastruvturec of epithelial cell of gastric mucosa, chief cells and parietal cells were changed pathologically. Preventive medication and treatment of AST can alleriate the histomorphological changes, protects the gastric mucosal barriers and reverse the diseased ultrastructure of epithelial cells, parietal cells and chiefs cells in spleen-deficiency rats. (3)AST can regulate gastric acid secretion as a whole, inhibit the hyperactive basal acid secretion in spleen-deficiency model rats, and increase its sensitivity to GAS stimulation. The results suggested that gastric parietal cells of spleen-deficiency model rats appea- rs with pathological condition in signal transmission pathway of GR and obvious pathologic basis in the tissue of gastric mucosal barrier, cells and cellular ultrastructure. Its basal acid secretion is high and the sensitivity to GAS stimulation is low. As a chief effective constituent of astragali, AST plays 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. And the results may be useful for scientifically demonstrating the function of astragali ininvigorating spleen and replenishing qi, and for preventing and treating chronic digestive diseases such as gastric mucosal destructive and inflammatory diseases. It is valuable theoretically and practically for further development of AST as a protective agent of gastric mucosa.
|
PDF Full Text Request