Regulating And Controlling Actions Of The Spleen-Invigorating Recipe And Herbal Drugs On Gastrin Receptors And Intracellular Signal Transduction In Gastric Parietal Cells Of Spleen-Deficiency Rats In Vitro

Spleen-deficiency is a kind of comprehensive deficient dysfunctions concerning many systems and organs but mainly digestive system and spleen-deficiency syndrome is a current disease in internal department of TCM. Clinical research has shown that patients of spleen-deficiency are subject to ulcerative diseases, the formation of which has been proved to have a close relation to the dysfunction of acid secretion of gastric parietal cells. Since the method of isolation and purification of gastric parietal cells of rats was established by our department in 1999, for many times we did some research on the relationship between spleen-deficiency and gastric parietal cells and the results suggested that the pathogenic micro-mechanisms of spleen-deficiency was related to the dysfunction of acid secretion of gastric parietal cells. To further explore the molecular mechanisms of spleen-deficiency, this subject observed the binding actions of gastrin receptors and intracellular signal transduction functions of spleen-deficiency rats and that of spleen-deficiency rats treated with spleen-invigoratinging recipe and herbal drugs in vitro by applying the method of serum pharmacology.1. Isolation and Purification of Gastric Parietal Cells of RatsTo further promote the purity and viability of the parietal cells isolated from rat sacs, we tried the method of non-linear density gradient to enrich parietal cells by using percoll as the medium based on the Lewin method applied with the method of pronase-EDTA to isolate gastric mucous cells from everted gastric sacs of rats. By using this way, (1.5±0.44) ×10⁶ parietal cells were achieved with viability and purity of over 95% and 70%respectively from one rat.2. Effect of the Spleen-Invigorating Recipe and Herbal Drugs Serum on the Binding of Gastrin with Its Receptors on Parietal Cells of the Spleen-Deficiency Rats in VitroTo observe the effect of the spleen-invigorating recipe and herbal drugs on the binding of gastrin with its receptors on parietal cells of the spleen-deficiency rats and investigate its relevant mechanisms in vitro, the method of radio-ligand binding assay was used and the results showed that the ligand-binding number of gastrin receptors on parietal cells in the spleen-deficiency rats was less than that in normal and the binding number on parietal cells was promoted after action of the spleen-invigorating recipe and herbal drugs serum in vitro, suggesting that spleen-invigorating recipe and herbal drugs can recover the spleen-deficiency conditions in rats through a probable mechanism that competes the binding sites with gastrin on gastric parietal cells.3. Effect of the Spleen-Invigorating Recipe and Herbal Drugs Serum on the Intracellular [Ca²⁺]i of Gastric Parietal Cells of the Spleen-Deficiency Rats in VitroThe last study results showed that the ligand-binding number of gastrin receptors on parietal cells in spleen-deficiency rats was less than that in normal and the binding number on parietal cells was promoted after action of the spleen-invigorating recipe and herbal drugs serum in vitro. To further investigate pathogenic mechanisms of the Spleen-deficiency rats in molecular level and effect of the spleen-invigorating recipe and herbal drugs serum on it, we took the method of serum pharmacology and the [Ca²⁺]i was measured by double-wavelength fluorescence spectrometer with Fura-2 as fluorescence indicator. The following results were made: (1) The resting [Ca²⁺]i of gastric parietal cells in the model group was significantly lower than that in the normal group(P<0.05), while the percentage of gastrin-induced elevated [Ca²⁺]i magnitude was on the contrary (P<0.01); (2) The resting [Ca²⁺]i of gastric parietal cells in the model group were promoted while the gastrin-induced elevated percentage of [Ca²⁺]i magnitude lowered to a certain extent after treatment of the spleen-invigorating recipe and herbal drugs serum. From the results we can conclude that the intracellular calcium of gastric parietal cells of the spleen-deficiency rats may be responsible for the formation of spleen-deficiency syndrome at molecular level and abnormal mediation of the calcium is one of the possible pathogenic mechanisms of it.4. Effect of the Spleen-Invigorating Recipe and Herbal Drugs Serum on Dynamic Changes of the Intracellular [Ca²⁺]i of Gastric Parietal Cells of the Spleen-Deficiency Rats in VitroCacium plays a key role in the intracellular signal transduction and the dynamic changes of intracellular [Ca²⁺]i are closedly associated with the cells' physiologicalfunctions. To investigate the pathogenic mechanisms of the spleen-deficiency rats in molecular level and effect of the spleen-invigorating recipe and herbal drugs serum on it, again we applied the method of serum pharmacology and the dynamic changes of [Ca2+]i in the gastric parietal cells were measured by confocal microscopy with Fluo-3/AM as fluorescence indicator. The results showed that the percentage of gastrin-induced elevated [Ca2+]i magnitude in gastric parietal cells of the spleen-deficiency group was significantly lower than that in gastric parietal cells of the normal group(P<0.05) and the percentage of gastrin-induced elevated [Ca ]i magnitude in gastric parietal cells of the spleen-deficiency group were lowered to some extent after treatment of the spleen-invigorating recipe and herbal drugs serum, suggesting that the intracellular calcium of gastric parietal cells of the spleen-deficiency rats maybe responsible for the formation of spleen-deficiency in molecular level and the abnormal mediation of the calcium is one of the possible pathogenic mechanisms of spleen-deficiency.5.Effect of the spleen-invigorating Recipe and Herbal Drugs Serum on CaM Activity in Parietal Cells of the Spleen-Deficiency Rats in VitroIt is documented that CaM is an very important kind of intracellular receptor proteins and the changes of its activity are closely related to the cells' dysfunctions. To further observe effects of the spleen-invigorating recipe and herbal drugs on CaM activity in parietal cells of the Spleen-deficiency rats and investigate its relevant mechanisms in vitro, we took the PDE method to detect the intracellular CaM activity. And the results showed that the CaM activity in parietal cells of spleen-deficiency rats was lower than that in normal and the CaM activity was promoted after action of the spleen-invigorating recipe and herbal drugs serum on parietal cells in vitro. So we can conclude that the lowered CaM activity is a possibly pathogenic mechanism of spleen-deficiency and the spleen-invigorating recipe and herbal drugs can recover the spleen-deficiency conditions to some extent.6.Effect of the Spleen-Invigorating Recipe and Herbal Drugs Serum on PRO Activity in Parietal Cells of the Spleen-Deficiency Rats in VitroCa2+/CaM-PKII is a kind of multi-functional calcium-regulated kinases and important CaM-activated downstream receptor proteins. The last experimental study results showed there was increased CaM activity in gastric parietal cells of the spleen-deficiency rats. To further explore the effect of the the spleen-invigorating recipe and herbal drugs on PKII activity in parietal cells of spleen-deficiency rats and investigate its relevant mechanisms in vitro, we studied the PKII activity in gastric parietal cells of spleen-deficiency rats and that treated with the spleen-invigorating recipe and herbal drugs serum. The results showed that...