| Objectives1.Part 1:In this part,the effect of He Qi powder on the effect of type 2 diabetes and glycolipid metabolism was studied.The effect of He Qi powder on the treatment of type 2 diabetes patients with phlegm and blood stasis type diabetes and the correlation with the changes of glycolipid metabolism index were discussed.It provides a theoretical basis for the treatment of type 2 diabetes in traditional Chinese medicine,and provides objective clinical practice for the development of new Chinese medicine.2.Part 2:In this part,the role of DNA methylation in peripheral blood cell genome of type 2 diabetic patients before and after the intervention of He Qi powder was observed,and the biological processes and related signaling pathways involved in the treatment of type 2 diabetes by He Qi powder in the treatment of type 2 diabetes were explored,and a new idea and method for the prevention and treatment of type 2 diabetes in traditional Chinese medicine were provided.Methods1.Part 1:A total of 60 inpatients of endocrinology of Shenzhen Chinese Materia Medica from March 2017 to September 2017 were included in the 60 patients eligible for inclusion/exclusion/shedding and termination of type 2 diabetes mellitus and spastic syndrome.For the control group and the treatment group each 30 cases.The control group took the conventional treatment program.The treatment group took the traditional endocrinology clinical experience of He Qi powder on the basis of the routine treatment of the control group.The treatment duration was 3 months in both groups.The basic conditions,clinical efficacy,and TCM syndromes of the two groups were observed and recorded.Waiting for points and indicators related to glucose and lipid metabolism(fasting plasma glucose,2 hours postprandial blood glucose,fasting insulin,glycosylated hemoglobin,total cholesterol,total triglycerides,low-density lipoprotein cholesterol,high-density lipoprotein cholesterol)and safety indicators Data were analyzed statistically.2.Part 2:From the first part of the clinical study,3 patients were randomly selected from the control group and the treatment group.The diagnostic criteria,inclusion/exclusion/abatement,termination criteria,and treatment protocol were the same as above,and 3 healthy volunteers were included in the blank group.A total of 15 peripheral blood samples were collected before and after the intervention in the control group and the treatment group and in the blank group.The methylation level of the whole genome was detected by DNA methylation chip technology(MeDIP-Chip),and the methylation binding sites in the promoter regions of each gene were obtained.Point probe raw data and enrichment peaks were analyzed using the M' method for differential enrichment peak(DEP)analysis.Differential methylation sites in different promoter promoter regions were screened and judged by GO analysis and Pathway analysis.It may participate in biological processes and diabetes-related signaling pathways,and screen for target genes.Results1.Part 1:(1)Compared with general data,there was no significant difference in gender distribution between the two groups(P>0.05),and the average of two groups of patients,There was no significant difference in age,height,weight,BMI and duration of disease(P>0.05).(2)Compared with curative effect index,the total score of TCM syndrome in the two groups was no significant difference before the intervention(P>0.05),and the total score of TCM syndrome in the two groups had significant difference(P<0.05)after the intervention;the difference of total score of TCM syndrome in the two groups was significantly different(P<0.01).In the control group,4 cases were cured,8 cases were markedly effective,8 cases were effective,10 cases were invalid and the total effective rate was 67%.After treatment,8 cases were cured,9 cases were markedly effective,10 cases were effective,and 3 cases were invalid,the total effective rate was 90%,significant difference(P<0.01).(3)Compared with the results of glucose and lipid metabolism,Before intervention,HbAlc,FPG,2hPG,FINS,TG,total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)were not significantly different(P>0.05);after the intervention,the above indicators in the two groups of patients were lower than before,and there were significant differences.The difference was statistically significant(P<0.01).There was a significant difference between the two groups before and after treatment(P<0.05).2.Part 2:(1)Methylation chip preliminary screening results:The original report showed that a total of 46123 genes were detected in the whole genome of 15 samples.The CPG islands in the promoter region showed possible DNA methylation status changes detected,of which 29914 genes were methylated at high CpG levels.On the density promoter region,7798 gene methylation occurred in the middle CpG density promoter region.(2)Results of differential methylation(DEP)analysis:In the high CpG density promoter region(HCP),there were 841 genes with differentially methylated regions before and after the intervention in the control group,compared with 957 genes in the treated group before and after the intervention.In the regionalized area,there were 658 genes with differentially methylated regions after intervention in the control group and the treatment group,and there were 689 genes with differentially methylated regions in the control group before and after the intervention in the control group.There was a difference between the treatment group and the blank group before the intervention in the treatment group.833 genes had differentially methylated regions;in the CpG density promoter region(ICP),220 genes had differentially methylated regions before and after the intervention in the control group,and there were 251 genes with different methyl groups before and after the intervention in the treatment group.In the area,there were 236 genes with differentially methylated regions after intervention in the control group and the treatment group,and 227 genes in the control group had a difference in methylation areas before and after the intervention in the control group.There was a difference between the treatment group and the blank group before the intervention in the treatment group.267 genes have differentially methylated regions.(3)GO analysis results:Compared with the control group before and after the intervention,there were 678 GO items involved in biological processes with differentially methylated regions.The most significant GO levels in the top 10 were:chromatin silencing,epigenetics,Notch Signalling pathway regulation,muscle relaxation,nervous system development,cAMP signaling pathway regulation,anatomical structure changes,cell development,chromatin condensation,nucleosome assembly and other biological processes;compared with before and after intervention group intervention,genes with differentially methylated regions are involved There are 830 GO items in the biological process.The top 10 GO items with significant enrichment are:anatomical structure development,multicellular organism development,development process,phylogeny,nervous system development,neural production,cell protein modification,protein modification Bioprocesses such as macromolecule modification,cell composition,etc.;compared with control group and treatment group,there were 725 GO items with differentially methylated regions involved in biological processes,and the most significant GO levels were among the top 10 GO items.For:macromolecular location,protein breakdown,proteasome catabolism,heart conduction to heart rate Regulation,sliding filaments,chaperone-mediated transport proteins,molecular targeting,intracellular protein catabolism regulation,cellular localization,microtubule polymerization or depolymerization nd other biological processes.(4)Pathway analysis:Compared with before and after intervention in the control group,diabetes-related signaling pathways involved in the differentially methylated gene expression pathways involved cAMP signaling pathways,Notch signaling pathways,Wnt signal ing pathways,and Rap1 signaling pathways.The genes with significant degree of basification were mainly related to CREB3L4,SSTR5,HDAC2,LFNG,RFNG,FZD10,GPC4,FLT1,and PDGFC.In the treatment group,genes with differentially methylated expression were involved in the diabetes-related signaling pathways.The signaling pathways mainly involve estrogen receptor signaling pathways,Rapl signaling pathways,and MAPK signaling pathways.The genes with more significant methylation levels mainly involve ADCY2,FLT1,FLT3,MAP3K14,PPP3R1,and PRKACA;the control group and therapy After the intervention,the diabetes-related signaling pathways involved in the differentially methylated genes involved in TGF-?signaling pathways,AMPK signaling pathways,insulin signaling pathways,and genes with more significant methylation levels were involved.It mainly involves LTBP1,SMAD3,CCND1,FOX03.(5)Gene screening:Genes and literature analysis results of NCBI(National Center for Biotechnology Information,National Institute of Biotechnology Information)were performed on related genes.Peak scores and Peak M Value values were used to calculate the difference in methylation of CREB3L4 and FLT1,And participate in a number of diabetes-related signal pathways,with practical research value.Conclusions1.Part 1:He Qi powder can significantly relieve the clinical symptoms of patients with type 2 diabetes mellitus and sputum syndrome,and improve their glucose and lipid metabolism indicators.2.Part 2:Through statistical analysis of DNA methylation microarray technology,it was found that there were differentially methylated gene loci in both the control and treated groups,and they were involved in a variety of biological processes and cellular signal transduction pathways that are closely related to the pathogenesis of type 2 diabetes.Finally,we screened out the functional verification of CREB3L4 and FLT1 genes for the next step of methylation and expression,in order to explore the possibility of studying the mechanism of interaction between He Qi powder and patients with type 2 diabetes mellitus and sputum syndrome as a DNA methylation marker. |
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