The Research On The Relationship Between The Damp Heat Syndrome In Spleen And Stomach And The Trefoil Factor-1, Intercellular Adhesion Molecule-1

The theory of the spleen and stomach is the central part of the viscera-state doctrine in the traditional Chinese medicine(TCM). The deficiency syndrome and excess syndrome are the two sides of the essence of the spleen. Nowadays the great achievements have been got in the researches on the essence of the deficiency syndrome of the spleen(DSS). The researches on the damp-heat syndrome of the spleen and stomach(DHSSS) was the erumpent point for the researches on the excess syndrome of the spleen and stomach. It was very important to use the chronic superficial gastritis(CSG) as the effective method to explore the essence of the DHSSS. It had been tested that DHSSS was correlated with the infected inflammation, but the exactly mechanism was not clear. It was probably associated with the disequilibrium between the defensive factors and the aggressive factors in the gastric mucous membrane. This just exactly coincides with the theory of the TCM which is that while the defensive qi exists inside the body and that the pathogenic factors may not invade the body. It was well reported much more on how the Helicobacter pylori(HP) effected on the mechanism of the inflammation in the CSG. However it was rarely reported on the mechanism of the HP negative CSG in the DHSSS. It has been shown that the trefoil factor 1(TFF1) is the defensive factor for the gastric mucosa and that the intercellular adhesion molecule-1(ICAM-1) is the aggressive factor for the gastric mucus membrane. We assumed that the TFF1 and the ICAM-1 might probably be associated with the pathogenesis mechanisms in the HP negative CSG in DHSSS and its subgroups. The mechanisms were that the combination of dampness and heat attacked the body simultaneously and that the wane and wax of the body defensive qi and the invading pathogenic factors would change during their struggling. Part 1. The Literature ReviewThe TFF1 is a kind of low molecular weight polypeptide by which the slime layers are constructed. The TFF1 has the following functions which are to protect the mucous membranes in the gastrointestinal tracts, to repair the injured tissues, to promote the mucous restitution and to inhibit the proliferation and apoptosis. The TFF1 is one of the defensive factors in the gastric mucosa. It belongs to the defensive qi in TCM. The inducing inflammation factors were enhanced and the anti-inflammatory factors were decreased in the gastrointestinal mucosa. This was the main mechanism for the inflammation in the DHSSS in CSG. It was also exactly coincidence with that while the defensive qi exists inside the body and the pathogenic factors may not invade the body. The TFF1 might probably be associated with the defensive mechanism that the defensive qi resisted the pathogenic qi in DHSSS.The ICAM-1 is the glycoprotein which appears in the surfaces of the cell membranes. It belongs to the superfamily members of the immunoglobulin adhesion molecules. It has the functions as participating the inflammatory reactions, the differentiation and development of the cells and the tissues, the immunology responses, the recruiting and recycling of the lymphocytes and the infiltration and metastasis of the tumors. As the aggressive factor in the mucosa of the CSG, the ICAM-1 characterizes as the prolonged expression and maintained for a long time. It just consists with the pathogenic characteristics of the dampness which is viscosity and stagnant. The combination of pathogenic dampness and heat caused the DHSSS in the CSG. Dampness and heat were inseparable and lingering. The ICAM-1 might take part in the molecular pathogenesis mechanism caused by dampness in the DHSSS.The fluorescent quantitative polymerase chain reaction (FQ-PCR) technique was characterized with the high precision, high specificity, short operation time and its safety. The technique was ingeniously combined with the fluorescent energy transfer technology, the classic polymerase chain reaction technique and the mathematical principles. It would show one of the innovations in this thesis by using the FQ-PCR technique to detect the genes of the TFF1 and ICAM-1.Part 2. The Clinical Experimental ResearchesObjective: 1. To examine the geneand protein expression of the TFF1 and ICAM-1 and its effect and significance on the pathogenesis in the gastric mucosa of the DHSSS, the deficiency syndrome of the spleen(DSS) and the three subgroups of the DHSSS in the HP negative CSG. 2. To illustrate the relationship between the expression of the TFF1 and ICAM-1 and the inflammatory degree in the gastric mucosa, the clinical symptoms and the clinical signs. 3. To explore the relationship between TFF1 and ICAW-1 in the DHSSS and its subgroups.Methods:All subjects were chosen from the out-patient department and the wards in the First Affiliated Hospital of the Guangzhou University of TCM during May 2005 and October 2006. The healthy volunteers were the undergraduates from the Guangzhou University of TCM. All participators had signed the informed coasents.In testⅠandⅡ47 subjects were divided into three groups. They were the DHSSS group(27cases), the DSS group(10cases) and the healthy control group(10cases). There were four groups in testⅢandⅣwhich were three subgroups of the DHSSS and one healthy control group(10cases). 27 cases of the DHSSS were divided into three subgroups. They were the dampness with predominant heat(DPH)group(9cases), the heat with predominant dampness(HPD) group(9cases) and the damp-heat predominant (DHP) group(9cases).All the subjects were checked by clinical examinations for the clinical symptoms and physical signs. They were examined by the gastroscope. The specimens should be taken out from the gastric antral mucosa for the rapid urase test(RUT) and the methylene blue staining method to check the HP.TestⅠ. The gene expression of the TFF1 and ICAM-1 in different types of the syndromes in CSG.1. By the gastroscope the inflammation of the gastric mucosa were observed and three specimens were taken out from the gastric antral mucosa in all 47 subjects. 2. One specimen was for the RUT. 3. Another one was for the methylene blue staining to detect the HP and the regular pathological staining method to examine the inflammatory degree of the gastric mucosa. 4. The last one was for the detection of the TFF1 and ICAM-1 gene expression by the FQ-PCR technique.TestⅡ. The protein expression of the TFF1 and ICAM-1 in different types of the syndromes in CSG.Procedures 1-3 were the same as that in TEST I. 4. The last specimen was for the detection of the TFF1 and ICAM-1 protein expression by the inmmunohistochemistry method.TestⅢ. The gene expression of the TFF1 and ICAM-1 in different subgroups of the DHSSS in CSG.1. By the gastroscope the inflammation of the gastric mucosa were observed and three specimens were taken out from the gastric antral mucosa in all 37 subjects. 2. One specimen was for the RUT. 3. Another one was for the methylene blue staining to detect the HPand the regular pathological staining method to examine the inflammatory degree of the gastric mucosa. 4. The last one was for the detection of the TFF1 and ICAM-1 gene expression by the FQ-PCR technique.TestⅣ. The protein expression of the TFF1 and ICAM-1 in different subgroups of the DHSSS in CSG.Procedures 1-3 were the same as that in TESTⅢ. 4. The last specimen was for the detection of the TFF1 and ICAM-1 protein expression by the inmmunohistochemistry method.Results:The results of RUT and the methylene blue staining method for HP detection were all negative in all cases.TestⅠ.Under the gastroscope the mucosawas congestive and edematous with the adhesion of the mucus in all the HP negative CSG. The degree of the mucosa congestion and edema was more severe in the DHSSS group than that in the DSS group.By pathology examination the inflammatory cells and lymphocytes were shown in the gastric mucosa in all the CSG cases. The inflammatory degree in DHSSS group was much more severe than that in the healthy control group(P＜0.01). The inflammatory degree in DSS group was more severe than the healthy control group(P＜0.05 ). However there was no significant differences between these two types of syndromes(P＞0.05).The TFF1 gene expression in DHSSS group was higher than that in the healthy control group(P＜0.05 ) and it was much higher than that in DSS group(P＜0.01=. Its expression in DSS group was much lower than that in the healthy control group(P＜0.01). Its expression was negatively correlated with the tongue proper(r=-0.554, P＜0.01).The ICAM-1 gene expression in DHSSS group was much higher than that in DSS group(P＜0.01) and its expression in DSS was slightly lower than that in the healthy control group(P＞0.05). The ICAM-1 gene expression was positively correlated with the degree of the gastric inflammation(r=0.521, P＜0.01). It was negatively correlated with the tongue proper and appetite, but was positively correlated with thirst, dry mouth and heavy limbs.The relationship between TFF1 and ICAM-1 in their gene expression in DHSSS and DSS: 1. The LOG(TFF1) was positively correlated with the LOG(ICAM-1)(r=0.453, P＜0.01). 2. The ratio of LOG(TFF1) and LOG(ICAM-1) in DHSSS was slightly bigger than that in the healthy group((P＞0.05)). The ratio in DHSSS was bigger than that in DSS(P＜0.05).TestⅡ.The protein expression of TFF1 in DHSSS and DSS: The TFF1 protein was mainly expressed in the cytoplasm of the gastric adenocytes. The positive stained granules were brownish yellow. The more nearer to the luminal surface, the positive stained granule color became darker. The positive stained granules were light brown in healthy group, their range were limited within the gastric superficial layer. The positive stained granules were light brown and their ranges were superficial in DSS group and even some cases were negative stained. The positive stained granules were dark brownish yellow in DHSSS group, the darkness of the positive stained color and its ranges were strongest and the deepest in all three groups(P＜0.01=. The intensity of the TFF1 positive stained color and its ranges in DSS group was stronger and deeper than that in the healthy control group(P＜0.05). The TFF1 protein expression was negatively correlated with the tongue proper(r=-0.394, P＜0.05), and it was positively correlated with the stool characteristics.The protein expression of ICAM1 in DHSSS and DSS: The yellow positive stained granules of ICAM-1 proteins were mainly appeared in the cell membranes and they were also expressed in cytoplasm. They commonly distributed between cells. There was no positive stained granules in healthy gastric mucosa membrane. The ICAM-1 slightly positive stained granules were light brown in DSS group, their ranges were limited within the gastric superficial layer. The positive stained granules in DHSSS were yellow, and the intensity of the positive stained color and its ranges were the strongest and the deepest in all the three groups. The intensity of the ICAM-1 positive stained color and its ranges in DSS group were slightly stronger and deeper than that in the healthy coatrol group(P＞0.05). The ICAM-1 protein expression was positively correlated with the degree of the gastric inflammation(r=0.528, P＜0.01). It was negatively correlated with the tongue proper(r=-0.556, P＜0.01), and it was positively correlated with thirst, dry mouth and heavy limbs.The relationship between TFF1 and ICAM-1 in their protein expression in DHSSS and DSS: The TFF1 protein expression was positively correlated with that of ICAM-1(r=0.404, P＜0.01).TestⅢ.Under the gastroscope the mucosa was significantly congestive and edematous with the adhesion of more mucus in all the three subgroups of DHSSS, mostly erosive mucosa or bleeding areas could be seen. There was no significant difference in the appearance of the mucosa under the gastroscope among all the three subgroups of DHSSS. The results of the pathology examination demonstrated that the inflammatory degree in all subgroups was from midrange to severe. The inflammatory degree in DHP subgroup were slightly more severe than that in the DPH subgroup(P＞0.05), and that in DHP subgroup were slightly more severe than that in the DPH subgroup(P＞0.05). However there was no significant difference between these three subgroups of DHSSS(P＞0.05).The TFF1 gene expression in DPH subgroup of DHSSS was slightly higher than that in HPD subgroup(P＞0.05). The TFF1 gene in HPD subgroup was much higher than that in DHP subgroup(P＜0.01). The TFF1 gene expression in three subgroups of DHSSS was negatively correlated with acid regurgitation and belching.The ICAM-1 gene expression in DPH subgroup of DHSSS was slightly higher than that in DHP subgroup(P＞0.05). The ICAM-1 gene in DHP subgroup was much higher than that in HPD subgroup(P＜0.01). The ICAM-1 gene expression in three subgroup of DHSSS was negatively correlated with tongue proper(r=-0.551, P＜0.01).The relationship between TFF1 and ICAM-1 in their gene expression in three subgroups of DHSSS: The ratio of LOG(TFF1) and LOG(ICAM-1) in HPD subgroups of DHSSS was bigger than that in DPH subgroup(P＜0.05). The ratio in DPH was bigger than that in DHP(P＜0.05).TestⅣ.The TFF1 protein expression in three subgroups of DHSSS: The TFF1 protein positively grained granules were dark brownish yellow. Their ranges were deep. The intensity of positive grained color was from＋＋to＋＋＋＋. The TFF1 protein expression in DPH subgroup of DHSSS was slightly higher than that in DHP subgroup(P＞0.05). The TFF1 protein in DHP subgroup was slightly higher than that in HPD subgroup(P＞0.05). The TFF1 protein expression was negatively correlated with the degree of the gastric inflammation(r=-0.556, P＜0.01), and it was negatively correlated with tongue proper.The ICAM-1 protein positive expression was yellow in all three subgroups of DHSSS: The ranges of the ICAM-1 positively grained granules deep. The intensity of positive grained color was from＋＋to＋＋＋. The ICAM-1 protein expression in DHP subgroup of DHSSS was slightly higher than that in DPH subgroup(P＞0.05). The ICAM-1 protein in DPH subgroup was slightly higher than that in HPD subgroup(P＞0.05).Conclusion:1. In DHSSS the expression of both TFF1 and ICAM-1 were up-regulated and the ratio of LOG(TFF1)/LOG(ICAM-1) increased. It was shown that both the inflammatory aggressive and the defensive reactions inside the body might be in a strong and accentuation state in DHSSS.2. The TFF1 protein expression in three subgroups of DHSSS were negatively correlated with the degree of the gastric inflammation. It was suggested that TFF1 might play an important role in the molecular biological pathogenesis by the way of 'defensive qi struggling with pathogenic qi' in the gastric mucosa in DHSSS.3. Both the gene and protein expression of ICAM-1 was positively correlated with the degree of the gastric inflammation in DHSSS. These data suggested that ICAM-1 might take part in the molecular mechanism of pathogenesis in DHSSS.4. The expression of TFF1 and ICAM-1 and the ratio of LOG(TFF1)/LOG(ICAM-1) changed with the different subgroups of DHSSS. It was suggested that the numbers of the dominant pathogenic factors might influence the expression of TFF1 and the combination of dampness and heat might significantly down-regulated the ratio of LOG(TFF1)/LOG(ICAM-1) in DHSSS.5. The tongue proper in CSG were separately correlated with the gene and protein expression of TFF1 and ICAM-1. It was suggested that the changeable expression of TFF1 and ICAM-1 might be one of the basis of mechanism for the excess and deficiency syndromes in the spleen and stomach in TCM.6. The expression of TFF1 and ICAM-1 and the ratio of LOG(TFF1)/LOG(ICAM-1) were down-regulated in DSS. It was shown that the deficiency of the healthy qi might decrease the inflammatory reactions in DSS.7. The TFF1 gene expression was positively correlated with that of ICAM-1 in both DHSSS and DSS. It was indicated that TFF1 and ICAM-1 might play important roles in the pathogenesis mechanism for the wane and wax of pathogenic qi and defensive qi in DHSSS and DSS.8. It showed one of the innovations in this thesis that the FQ-PCR technique was used to detect the genes of the TFF1 and ICAM-1.