| Modern studies suggest that gastric mucosal defense system is a multi-level complex system that can be divided into three levels. In this three-tier defense system, every level system is related to various cytokines, which play an important role in the gastric mucosal defense and influence on each other.Trefoil factor 1 (TFF1) is a member of tefoil factor family with highly conserved structure, which is quite stable and can endure acid and decomposition caused by heat. Studies have found that the rate of incidence of ulcer is lower in the jejunum in transgenic mice with human TFFl overexpressing, which indicates that TFF1 plays an important role in protection and repairment in the gastrointestinal tract. Mucin is a general name of a group of different highly glycosylated glycoprotein which is of great value in the gastric mucosa defense and repair process.In normal conditions, TFF1 has important physiological functions with he combination of MUC5AC mucin or the corresponding receptors or transporters. Promoting cell migration is an important step in the gastric mucosa repairation and reconstitution. The launch, and inducing the migration of TFF1 mainly depend on the activation of Ras and ERK1/2, which is closely related to the signaling pathway of EGFR tyrosine kinase. EGFR is a kind of cell surface receptors with tyrosine kinase activity. The ligand can be spontaneously activiated after bond with EGFR, which can activate the downstream of proteins siganl pathways, including mitogen-activated protein kinase (MAPK). Ras/Raf/MEK/ERK pathway is one of the MAPK signaling cascade, which is an important pathway that mediates the extracellular signal transmission from the cell surface to the inside. It can cause the expression of specific proteins or activity changes through a cascade reaction, resulting in specific biological response, including the induction of cell migration, proliferation or differentiation. It can be seen that TFF1-MUC5AC-EGFR and MEK/ERK pathway is related to gastric mucosalbarrier.LiDongYuan, a famous doctor in JingYuan Dynasty, created buzhongyiqi decoction, which is a representative prescription, according to his academic thought "all diseases are caused by internal damage of the spleen and stomach". For invigorating spleen-stomach, replenishing qi, elevating yang and raising the drooping, this decoction contains primarily these herbs: Huangqi, Renshen, Baizhu, Danggui, Chenpi, Chaihu, Shengma, Gancao. It is a classical prescription for the Syndrome of Spleen Deficiency. The Syndrome of Spleen Deficiency is one of commonly clinical diseases, such as peptic ulcer, chronic gastritis, etc. It is more common in sub-healthy groups. Spleen Deficiency is a disease which is characterized by abnormal morphology and dysfunction. Gastrointestinal mucosal lesions and its corresponding dysfunction may be one of the pathological basis of Spleen Deficiency. Many studies have found that gastric mucosal tissue does have damage, which provides a morphological proof for the thought of "all diseases are caused by internal damage of the spleen and stomach".Spleen is the source of qi and blood, "No spleen deficiency, no disease", which indicats that the spleen and stomach are closely related to human defense. Spleen can't translate, or can not ship cereal essence, which will inevitably lead to a decline in host defense against function and the emergence of various diseases. That is to say, Spleen Deficiency decreases the defense capabilities of gastrointestinal mucosa. So it is easier for the emergence of various diseases.Now most of the researches of Buzhongyiqitang are about clinical observation. The basic study is rather weak, which is more focused on pharmacodynamics. The protection of Buzhongyiqitang on injuried gastric mucosal is certain and precise, but the relevant mechanisms are lack of depth. Therefore, we observed the TFF1-MUC5AC-EGRFR and ERK/EK pathway in gastric mucosa in rats with Spleen Deficiency, and discussed the effect of Buzhongyiqitang. Our work was to explain the nature of Spleen Deficiency Syndrome, and was expected to reveal the molecular pharmacological mechanism of this prescription. 1. Effect of Buzhongyiqitang on the expression of gastric mucosa TFF1mRNA and protein in rats with the Syndrome of Spleen DeficiencyAs a member of the trefoil factor family, TFF1 is secreted by the gastric mucous cells, which is one kind of molecule peptides, and plays an important role in the gastric mucosal barrier protection and repair. The expression of gastric mucosa TFF1mRNA and protein in the Spleen Deficient rats and the effect of Buzhongyiqitang were observed, then the related mechanisms were discussed. Methods:The rats were randomly divided into the spleen deficiency with injury group, the Buzhongyiqitang group, the blank control group, and the non-spleen deficiency with injury group according to their weight. The spleen deficiency with injury group and the Buzhongyiqitang group were administrated by 100% DaHuang solution, twice a day, for 10 days, to copy the model of spleen deficiency. The other two groups were administrated by distilled water. Then the Buzhongyiqitang group was treated by Buzhongyiqitang for 7 days, twice a day, and the other three groups were administrated by distilled water. In addition to the control group, the other three groups were fed with indomethacin solution to cause mucosa injruy after treatment.7 hours later, all the rats were killed, gastric mucosa was scraped on the ice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assay TFF1mRNA, ELISA was used to detect the expression of TFF1 protein. Results:Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa TFF1mRNA and protein in the spleen deficiency with injury group were decreased significantly (P<0.05, P<0.01); Compared with the spleen deficiency with injury group, the expression of gastric mucosa TFF1mRNA and protein in the Buzhongyiqitang group were increased significantly (P<0.05). Conclusion: The expression of gastric mucosa TFF1mRNA and protein in the spleen deficiency with injury group were decreased significantly, suggesting the decreasing mucosal defense capabilities in rats with spleen deficiency. Buzhongyiqitang could increase their expression greatly, which suggested the prescription raise the expression of TFF1 to enhance gastric mucosal barrier and promote the reconstruction of damaged gastric mucosal, which may be one of the mechanisms of reducing gastric mucosal's vulnerability of Buzhongyiqitang.2.Effect of Buzhongyiqitang on the expression of gastric mucosa MUC5ACmRNA and protein in rats with the Syndrome of Spleen DeficiencyGastric mucus belongs to important gastric mucosa protective system, the main component is mucin with high molecular weight. Widely in the digestive tract, mucin is to maintain the mucus layer thickness and colloidal, which plays an important role in the gastric mucosa defense and repair process. MUC5AC mucin is a major component of the gastrointestinal tract colloidal, play a more important role in gastric mucosa protection with the combination of TFFl in physiological conditions. In this experiment, the expression of gastric mucosa MUC5ACmRNA and protein in the Spleen Deficient and the effect of Buzhongyiqitang were observed, then the related mechanisms were discussed. The rats grouping, the model copying,, the drug treatment and indomethacin treatment and reciping essay were the same as the Experiment One. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assay MUC5ACmRNA, ELISA was used to detect the expression of MUC5AC protein. Results: Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa MUC5AC mRNA and protein in the spleen deficiency with injury group were decreased in some degree (P>0.05); Compared with the spleen deficiency with injury group, the expression of gastric mucosa MUC5AC protein in the Buzhongyiqitang group was increased significantly (P<0.05). Conclusion: The expression of gastric mucosa MUC5AC protein in the spleen deficiency with injury group was decreased significantly, suggesting the decreasing mucosal defense capabilities in rats with spleen deficiency. Buzhongyiqitang could increase the expression of MUC5AC protein greatly, to promote the formation of gastric mucosal gellayer. MUC5AC was bound to TFF1, which may be one of gastric mucosa's protective mechanisms of Buzhongyiqitang.3. Effect of Buzhongyiqitang on the expression of gastric mucosa EGFR protein in rats with the Syndrome of Spleen DeficiencyEGFR is one kind of cell surface receptors with tyrosine kinase activity. It starts signal transduction inside the cells to adust transcription factors and direct cell migration, proliferation or differentiation through the cascade of adapter proteins and enzymes after its ligand is activated. The expression of gastric mucosa EGFR protein in the Spleen Deficient rats and the effect of Buzhongyiqitang were observed, then the related mechanisms were discussed.The rats grouping,the model copying,, the drug treatment and indomethacin treatment were the same as the Experiment One. All the rats were killed after the administration of indomethacin for 7 hours, and the same site of gastric mucosa was collected and put into 10% neutral formalin solution to be fixed. IHC was adopt to detect the protein expression of EGFR. Results: Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa EGFR protein in the spleen deficiency with injury group was decreased significantly (P<0.01); Compared with the spleen deficiency with injury group, the expression of gastric mucosa EGFR protein in the Buzhongyiqitang group was increased significantly (P<0.05). Conclusion: Buzhongyiqitang could increase the expression of EGFR protein greatly,to promote the respairation of gastric mucosa, which maybe activate the downstream of proteins siganl pathways, which may be one of gastric mucosa's protective mechanisms of Buzhongyiqitang.4.Effect of Buzhongyiqitang on the expression of gastric mucosa MER/ERKmRNA and protein in rats with the Syndrome of Spleen DeficiencyMAPK is an important pathway that mediats cellular signal transmission, which widely exists in eukaryotic cells. MAPK/ERK is a kind of transcription channel which is proved first. Various kinds of growth factors or other stimulation can induce the activation of ERK pathway and the expression of specified protein or the change of its activity to influence cell metabolism functions. The expression of gastric mucosa MEK, ERK mRNA and protein in the Spleen Deficient rats and the effect of Buzhongyiqitang were observed,then the related mechanisms were discussed. The rats grouping,the model copying,, the drug treatment and indomethacin treatment and reciping essay were the same as the Experiment One. FQ-PCR was used to examine MEKmRNA,ERKmRNA and ELISA was adopt to detect the expression of ME,ERK protein. Results: Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa MEKmRNA and protein in the spleen deficiency with injury group were decreased significantly (P<0.01); Compared with the spleen deficiency with injury group, the expression of gastric mucosa MEKmRNA and protein in the Buzhongyiqitang group were increased significantly (P<0.05), the expression of gastric mucosa ERK mRNA and protein in the Buzhongyiqitang group were increased significantly (P<0.05, P<0.01). Conclusion:Buzhongyiqitang could increase the expression of TFF1,EGFR to activate the MEK/ERK pathway and promote cell migration, proliferation or differentiation, which may be one of the deeper protective mechanisms of gastric mucosa of Buzhongyiqitang.5. ConclusionTFF1 plays an important protective role in the gastrointestinal tract and promotes the re-establishment and reparation in gastric mucosa.The involved mechanisms included that TFF1 was combinated with MUC5AC and influenced on each other to make mucosal fluid strengthen the resistance to noxious substance, which enhanced the expression of EGFR to activite the MEK/ERK signal pathway, and adust cell metabolism, induce its migration, proliferation or differentiation. We studied the correlated targets of Buzhongyiqitang deeply from the TFF1-MUC5AC-EGFR and MEK/ERK signal pathway, which was of great value in study and application.Our empirical study showed:â‘ Buzhongyiqitang could increase the expression of TFF1mRNA and protein of gastric mucosa in the Spleen Deficient rats;â‘¡Buzhongyiqitang could raise the expression of MUC5AC protein of gastric mucosa in the Spleen Deficient rats;â‘¢Buzhongyiqitang could increase the expression of EGFR protein of gastric mucosa in the Spleen Deficient rats;â‘£Buzhongyiqitang could increase the expression of MEK/ERKmRNA and protein of gastric mucosa in the Spleen Deficient rats.In a word, compared with the non-spleen with injury group, the expression of TFF1mRNA,MEKmRNA. ERKmRNA and their protein, MUC5AC,EGFR protein of gastric mucosa were decreased in the rats of spleen deficient with injury group, which indicated gastric mucosal defense function subsided when the body was deficient,and the gastric mucosa was in high stringent state and was more sensitive to all kinds of attack factors. Their expression was increased through Buzhongyiqitang's administration, which indicated this prescription could increase the expression of TFF1, MUC5AC and reinforce their mutual effect to strengthen the barrier defense protection of mucosal fluid. And this recipe could raise the expression of EGFR, and activite the MEK/ERK signal pathway, and stimulate the migration, proliferation or differentiation of Ep cells in gastric mucosa to quicken the reconstitution and recovery in injuried Ep cells, which may be the corresponding target of protecting gastric mucosa and decreasing its vulnerability of Buzhongyiqitang. In some degree, our study reflected the feature of TCM-multitarget and multistrata.
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