| ObjectiveThis study evaluated whether CDH1 SNPs,its mRNA and protein expression had a role in gastric mucosa lesions and syndromes of traditional Chinese medicine(TCM)in subjects with H.pylori-related gastric diseases(HPGD),aiming to explore the mechanisms of gastric mucosa lesions and biological similarities of TCM syndromes in HPGD.In addition,the relationship between CDH1 SNPs and mRNA levels was analyzed to seek the relevant mechanism of gene polymorphism affecting disease susceptibility.MethodsThere were 668 subjects enrolled in this study.All of them were in collected from the first affiliated hospital of Guangzhou university of TCM during September 2012 to December 2017.Clinical data involving subjects'symptoms and signs were obtained by a professional medical staff,who filled in the clinical observation form according to the subjects'information,and identified subjects' syndromes by the diagnostic criteria of TCM syndromes.The results were reviewed by senior doctors.All subjects underwent electronic gastroscopy,and four pieces of biopsy tissue were taken at the lesion locus.For subjects with gastric ulcer,biopsy specimens should be collected at the peri-ulcer mucosa.The morphological changes of gastric mucosa and degrees of pathological changes were observed in all samples by H&E staining.The evaluation criterion referred to related literatures.Gastric mucosal pathology included gastric mucosal inflammation,activity,atrophy,intestinal metaplasia,dysplasia and carcinogenesis.Rapid urease method and methylene blue staining method were used to detect H.pylori.Both positive results can be diagnosed as H.pylori positive.In addition,degrees of H.pylori infection were assessed by methylene blue staining method.CDH1-160 and-347 gene polymorphisms in gastric mucosa were performed by direct sequencing method.Expression levels of CDH1 mRNA were examined by fluorescence quantitative PCR technology.Immunohistochemistry method was performed to observe expression of E-cadherin protein in gastric mucosa.Qualitative analysis and location analysis of E-cadherin protein were conducted in four different areas of gastric mucosa,namely epithelial surface area,gastric fovea area,gland area in the lamina propria and deep gland area.The qualitative criteria were the percentage of positive cells combined with the staining intensity,and the localization included three categories:only cell membrane expression,only cytoplasmic expression,cell membrane and plasma coexpression.All subjects were evaluated for gastric mucosa lesions and TCM syndrome identification.Based on this,we set up two categories of grouping methods:the first one was pathological grouping method:subjects were divided into normal group(NOR),gastric inflammation group(GI),gastric atrophy group(GA),gastric premalignant lesion group(GPL),gastric severe dysplasia group(GSD),and gastric cancer group(GC);the second one was TCM syndrome grouping method:groups included syndrome of dampness-heat syndrome in the spleen and stomach group(DHSS),liver-stomach disharmony syndrome group(LSD),spleen qi deficiency syndrome group(SQD),syndrome of blood stasis in the stomach collateral group(BSSC)and non-syndrome(NON).This study protocol was approved by ethics committee of the First affiliated hospital of Guangzhou university of TCM.The informed consent documents were signed by all subjects.Results1.General information(1)General information in different pathological groups:the proportion of male in subjects with gastric cancer was significantly higher than that in subjects with normal gastric mucosa.The ages of subjects in NOR group,GI group,GA group and GPL group were significantly lower than that in GC group.Both NOR group and GI group had significantly lower H.pylori infection rates than GA group,GPL group,GSD group and GC groups.There were significantly positive correlations between degree of H.pylori infection and degree of gastric inf lammation or inflammatory activity.In contrast with normal gastric group,multinomial logistic regression analysis showed that male with GI,GSD and GC was separately about 2.062,2.621,5.857 times more likely than female;Those with H.pylori infection in GI,GA,GPL,GSD and GC were separately about 1.784,7.913,10.173,14.462,16.835 times more likely than those without H.pylori infection;incidences of GC for 55-64-year and more than 65-year old people were about 3.750 and 5.542 times than those for under 45-year old people.(2)General information in different TCM syndrome groups:the distribution ratio of male in subjects with SQD was significantly lower than that in those with BSSC group.The age of subjects in SQD and BSSC groups was significantly higher than that in NON group.Among H.pyloripositive subjects,H.pylori infection rate in NON group was significantly lower than that in the other syndrome groups.In contrast with NON-syndrome group,we further performed multinomial logistic regression analysis,and the results showed that those with H.pylori infection were about 16.256,16.973,10.497,17.412 times more likely to be DHSS,LSD,SQD and BSSC than those without H.pylori infection;those with 55-64-year old were about 10.039,12.804,and 12.094 times more likely to be DHSS,SQD and BSSC than those with under 45-year old.2.Relationship between TCM syndromes and gastric mucosal histopathologyAmong H.pylori negative subjects,those with normal gastric mucosa were most likely to show non-syndrome(38.89%),while the possibility of normal gastric mucosa in those with non-syndrome was the highest(84%).There was a significant difference for distribution of TCM syndromes between NOR group and GI group.Compared with the subjects with normal gastric mucosa,subjects with gastric inflammation had a higher possibility to show SQD than other syndromes.Among the H.pylori positive subjects,only 5%(3/60)of the subjects with normal gastric mucosa showed non-syndrome,while the proportion of the subjects with DHSS was the most(46.67%).The possibility of LSD and DHSS were much higher than other syndromes in subjects with gastric mucosa inflammation,atrophy and precancerous lesions.The proportion of DHSS and SQD were much higher than other syndromes in subjects with gastric mucosa severe dysplasia and gastric cancer.There were significant differences about distribution of TCM syndromes between GC group and NOR group,and between GC group and GPL group.The proportion of SQD and BSSC in subjects with gastric cancer was significantly higher than that in subjects with normal gastric mucosa and premalignant lesions.3.Relationship between CDH1 SNPs and gastric mucosa histopathology,and between CDH1 SNPs and TCM syndromes(1)CDH1 SNPs and gastric mucosa histopathologyThere was no significant difference in H.pylori infection rates among different genotypes and alleles of CDH1-160 and-347.No matter H.pylori infection or not,there were no statistically significant correlation between CDH1-160 and-347 loci and gastric mucosal pathological changes.Although the p-value didn' t reach the adjusted test level,there were still some trends.Among H.pylori positive subjects,the possibilities of gastric mucosal premalignant lesions,severe dysplasia and carcinogenesis were increased than that of normal gastric mucosa in subjects with CDH1347 G/GA genotype.The possibilities of gastric mucosal precancerous lesions,severe dysplasia,and carcinogenesis were down-regulated in subjects with CDH1-347 G/G genotype.Subjects with CDH1-347 GA allele had more possibility to suffer from gastric mucosal atrophy and precancerous lesions.Among H.pylori negative subjects,the possibility of gastric mucosa severe dysplasia for subjects with CDH1-160 C/A combined with-347 G/GA genotype was higher than that of normal gastric mucosa,inflammation and premalignant lesions.For H.pylori positive subjects,ones with CDH1160 C/C genotype combined with CDH1-347 G/G genotype had more possibility to have normal gastric mucosa than gastric mucosal inflammation,premalignant lesions and carcinogenesis.To exclude the effect of gender,age,H.pylori and TCM syndrome on gastric mucosal pathology,we performed multinomial logistic regression analysis.The results showed that subjects with CDH1-160 C/A genotype more likely suffered from gastric inflammation than those with CDH1-160C/C genotype;while the possibility of gastric severe dysplasia for subjects with CDH1-347 G/GA genotype was about 4.821 times than that for those with CDH1-347 G/G genotype.These further identified that there were associations between gastric inflammatory and CDH1-160 locus,and between gastric severe dysplasia and CDH1-347 locus.(2)CDH1 SNPs and TCM syndromesNo matter H.pylori infection or not,there was no statistically significant correlation between CDH1-160 and-347 loci and TCM syndromes.However,there were still some trends.Among subjects with H.pylori infection,subjects with CDH1-160 A/A genotype were more likely to form SQD than DHSS.Subjects with CDH1-160 A/A genotype combined with-347 G/G genotype were more likely to show SQD than DHSS.Multinomial logistic regression analysis showed that there still were no associations between TCM syndrome and CDH1-160\-347 loci when we excluded effect of gender,age,H.pylori and gastric pathology,which may be caused by small samples.4.Level of CDH1 mRNA in gastric mucosal pathology and TCM syndromesAmong subjects with normal gastric mucosa,there was significantly higher for the expression of CDH1 mRNA in gastric mucosa in H.pylori positive group than H.pylori negative group.Among H.pylori positive subjects,the expression level of CDH1 mRNA in GPL group was significantly higher than that in GC group.By Kruskal-Wallis H test,either with or without H.pylori infection,no statistical difference was found about CDH1 mRNA expression levels of gastric mucosal tissues in subjects with different TCM syndromes.5.Qualitative and location analysis of E-cadherin protein in different gastric mucosa pathological changes and TCM syndromes(1)Qualitative and location analysis of E-cadherin protein in different gastric pathological groups ?Epithelial surface area:In H.pylori negative subjects,no statistical correlation was found between gastric pathological changes and the expression of E-cadherin protein in this area.There were significant differences about the expression of Ecadherin protein among different gastric pathological groups in subjects with H.pylori infection.Possibilities of E-cadherin cytoplasmic expression and membrane-cytoplasmic co-expression in GSD and GC groups were significantly higher than those in other pathological groups.?Gastric pits:No significant correlations were found between expression of Ecadherin protein in gastric pits and pathological changes of gastric mucosa in H.pylori negative subjects.Among subjects with H.pylori infection,there were significant differences about expression of E-cadherin protein in this area among different pathological changes.Compared with NOR,GI and GA groups,the expression of E-cadherin protein in GPL,GSD and GC groups was most located in cytoplasm.Meanwhile,the cytoplasmic expression of E-cadherin protein in GSD group was significantly higher than that in GPL group.?Lamina propria gland region:In subjects without H.pylori infection,no statistical correlations were found between gastric mucosa pathology and E-cadherin protein expression in this region.Among H.pylori positive subjects,possibilities of E-cadherin cytoplasmic expression in lamina propria gland cells of gastric mucosa in GPL,GSD,and GC groups were significantly much higher than that in NOR group.Meanwhile,possibility of E-cadherin cytoplasmic expression in this area in subjects with GSD was significantly higher than that in subjects with GI.Compared with GA group,much more E-cadherin protein in this region was located on the cytoplasm in GSD and GC groups.In addition,the cytoplasmic expression of E-cadherin in lamina propria gland cells was significantly higher in GC group than that in GPL group.?Deep gland region:In subjects without H.pylori infection,there was no statistical difference in the expression of E-cadherin protein in deep gland region of gastric mucosa among different gastric pathological groups.For subjects with H.pylori infection,possibility of E-cadherin cytoplasmic expression in this region in GC group was significantly higher than that in NOR group.(2)Qualitative and location analysis of E-cadherin protein in different TCM syndrome groups?Epithelial surface area and gastric pits:No statistical correlations were found between TCM syndromes and the expression of E-cadherin protein in gastric mucosal epithelial surface cells and gastric pits.?Lamina propria gland region:There was no statistical difference in E-cadherin protein expression of this area in H.pylori negative subjects with different TCM syndromes.Amon'g subjects with H.pylori infection,the cytoplasm expression or membrane and cytoplasm coexpression of E-cadherin in this region was more common in subjects with BSSC than that in those with NON.?Deep gland region:Similar with lamina propria gland region,there was no statistical difference in the expression of E-cadherin protein in the deep gland region of gastric mucosa in H.pylori negative subjects with different TCM syndromes.Among subjects with H.pylori infection,the possibility of cytoplasmic expression of Ecadherin protein in this region was significantly higher in subjects with BSSC than that in those with DHSS and LSD.6.Relationship between CDH1 SNPs and mRNA expressionKruskal-Wallis H test showed that there were no statistical correlations between CDH1-160 locus and relative expression level of CDH1 mRNA between subjects with H.pylori and those without H.pylori infection.Among subjects without H.pylori,compared with those with CDH1-347 G/G genotype,the relative expression level of CDH1 mRNA in gastric mucosal tissues was significantly higher in subjects with CDH1-347 G/GA genotype.However,in subjects with H.pylori infection,there was no statistical difference in CDH1 mRNA expression level among different genotypes of CDH1347 locus.No statistical correlation was found between CDH1-160 combined with-347 gene polymorphism and the relative expression level of CDH1 mRNA.Conclusion1.Male,advanced age and H.pylori infection are risk factors for gastric mucosa malignant lesions.The formation of TCM syndromes in HPGD are associated with age and H.pylori infection.There also is a certain correlation between pathological changes of gastric mucosa and TCM syndromes.There also is a certain correlation between pathological changes of gastric mucosa and TCM syndromes.2.In H.pylori-negative subjects,the expression of CDH1 mRNA was higher in gastric tissue of those with CDH1-347 G/GA genotype.CDH1-347 G/GA genotype with-160 C/A genotype is the risk factor for gastric severe dysplasia.Those with CDH1-347 G/GA genotype and H.pylori infection,more easily suffer from gastric intestinal metaplasia,dysplasia and cancer,while CDH1 mRNA changed from high to low expression in this process.The formation of gastric mucosal premalignant lesions mainly involves in the ectopic expression of E-cadherin protein in the gastric pits and lamina propria glands,suggesting that gastric mucosal intestinal metaplasia or dysplasia may be caused by the normal differentiation of stem cells through abnormal expression of E-cadherin protein.In the gastric cancer tissues,E-cadherin protein was mainly located in the cytoplasm in all of the above four regions,verifying that E-cadherin protein plays a crucial role in the occurrence and development of malignant tumors.These imply that CDH1-347 locus may be associated with the susceptibility of gastric malignant pathology,as well as this gene transcriptional activity.E-cadherin ectopic expression may have a precaution function on gastric malignant pathology.3.Subjects with CDH1-160 locus and H.pylori infection maybe more possibly show the syndrome of spleen qi deficiency.The differences among TCM syndromes in subjects with normal gastric mucosa may be related to different degrees of E-cadherin protein expression in gastric tissue.The formation of syndrome of blood stasis in the stomach collateral may be partially caused by cytoplasmic expression of E-cadherin protein in gastric glandular cells.These indicate that CDH1-160 locus with H.pylori infection may be responsible for the susceptibility to spleen qi deficiency syndrome in subjects with HPGD,and E-cadherin ectopic expression may be one of general characteristics for HPGD subjects with syndrome of blood stasis in the stomach collateral. |
PDF Full Text Request