Study On The Co-change Mechanism Of Intestinal Flora And Host NF-?B Signaling Pathway In Females With Yin Deficiency

Objective:The aging of human body has been the focus of medical research,and health care and prevention of aging are also the most concerned topics.To clarify the mechanism of aging,and then to clarify the physiological and pathological changes of human tissues,organs,cells and molecules during aging,which helps to explain the principles of various diseases.At the same time,it is also conducive to the prevention of disease and health care in humans.Physical theory of TCM is one of the main theories in the theoretical system of TCM.By analyzing the mechanism of aging as an entry point,we can further improve the understanding of aging in TCM.There is a correlation between human constitution and aging.Biased physique is more likely to lead to aging.Among them,the aging characteristics of Yin-Deficiency constitution are the most obvious.At the same time,the intestinal flora has certain influence on the aging process of human body,and has common changes with the related genes of the host.Previous studies have suggested that there is an imbalance of intestinal microorganisms in Yin-deficiency constitution,as well as an abnormal expression of the mRNA of factors related to the classical pathway of NF-kB leading to cell senescence.The Yellow Emperor's Classic of Internal Medicine records that women are characterized by gradual aging from the age of 35 to 49,which is the "declining period" of women.Whether the intestinal flora in the declining period of women with Yin-deficiency constitution is involved in the regulation of gene co-changes has not been confirmed.Based on Constitution-aging relation theory,this study reveals the unique physiological characteristics and characteristics of intestinal flora of women with Yin-deficiency constitution through the study of multiple blood biochemical indicators and the characteristics of intestinal flora in the decay period of women with Yin-deficiency constitution.At the same time,based on this,the expression of the host's NF-kappa B signaling pathway-related mRNA and the common change characteristics of intestinal microorganisms were studied.Method:Constitution determination was carried out according to the criteria of Classification and Judgment of Constitution of Traditional Chinese Medicine issued by the Chinese Society of Traditional Chinese Medicine in 2009.Thirty female volunteers aged 35 to 49 years with Yin-deficiency constitution and peace constitution were selected,and their blood and feces were collected as samples.A part of the blood samples were tested by biochemical methods for blood biochemical indicators.The other part used Trizol reagent to isolate total RNA.After their reverse transcription,the cDNA and primers were analyzed by qRT-PCR with premix.The relative expression levels of TAK1,NFKBIA,CCL4,BCL2A1 and IL8 mRNAs were then investigated.After stool collection,the genomic DNA of the samples was extracted,and the purity and concentration of DNA were detected by agarose gel electrophoresis,and then processed by PCR.Based on the concentration of the PCR products,an equal proportion of the amount of mixing was carried out,then the PCR products were purified by agarose gel electrophoresis with 1 x TAE concentration of 2%,and then the target bands were cut and recovered.After the construction of the relevant library,16S rDNA sequencing was started.Finally,Pearson correlation coefficients were calculated by quantifying bacterial abundance and transcriptome mRNA expression in each group of samples by intestinal microbial detection.Then their correlation was found and the relationship between common changes in intestinal flora and genes was studied.Result:1.Compared with the balance constitution group,the values of TG,HGB,PDW,MPV,RDW-SD and P-LCR in Yin-deficiency constitution group were lower,but the values of WBC and RDW-CV were higher(P<0.05).The indexes of PDW,MPV,RDW-CV,RDW-SD and P-LCR were significantly different(P<0.01).2.At all levels,Yin-deficiency group has unique characteristics of intestinal flora compared with Pinghe group,and there are differences in relative abundance,diversity,functional annotation and metabolic pathways of the flora.The difference in relative abundance of intestinal flora species is the material basis for the differences in all aspects of intestinal flora between the two groups,among which the relative abundance of Yin-deficiency group is larger(p<0.05),the microbial categories are:Desulfovibrionaceae,Unidentified Cyanobacteriaceae.Cyanobacteria,Synergistaceae,Methanobacteriaceae,Unidentified Victivallales,Bacillaceae and Eubacteriaceae,Epulopiscium and Unidentified Cyanobacteria,Cloacibacillus,Methanobrevibacter,unidentified Victivallales,Sanguibacteroides.The relative abundances of the Yin-deficiency group were significantly higher(p<0.01),and the microbial categories were Cyanobacteria,Synergistetes and Euryarchaeota,Deltaproteobacteria and Unidentified Cyanobacteria,Synergistia and Methanobacteria;Desulfovibrionales,Unidentified Cyanobacteria,Synergistales,Methanobacteriales and Rhizobiales;Unidentified Rhizobiales;and Oxalobacter.The relative abundances of the Yin-deficiency group were smaller(p<0.05),and the microbial categories were Vibrionales,Vibrionaceae,Coprobacillus and Vibrio.The relative abundance of the Yin-deficiency group was significantly smaller(p<0.01),and the microbial categories were:Merdibacter.3.Compared with balance constitution,the expression of NFKBIA and CCL4 mRNA was significantly up-regulated and BCL2A1 mRNA was significantly down-regulated in Yin-deficiency constitution group(P<0.05).At the same time,the expression of related genes was positively correlated with the microorganisms in the intestinal tract of Yin-deficiency constitution group,and negatively correlated with the microorganisms in the intestinal tract of balance constitution group.Conclusion:This topic takes the physiological characteristics of women from 35 to 49 years old in TCM theory as the starting point,combines the aging process theory of women from 35 to 49 years old in TCM theory with the TCM physique theory,and focuses on the molecular biological characteristics of the declining period from 35 to 49 years old in women with Yin deficiency constitution.We comprehensively used genetic genetics,microbial molecular ecology and multivariate statistical analysis methods to associate the expression of genes reflecting heredity with intestinal microbes reflecting external influences,and clarified the part of regulation mechanism of intestinal flora and gene co-change in Yin-deficiency constitution women in the declining period.Compared with the balance constitution,Yin deficiency constitution has certain differences in blood biochemical indicators and intestinal flora.The characteristics of blood biochemical indicators such as yin deficiency constitution may reflect the role of pro-inflammatory factors in the process of inflammatory aging,and their aging degree is more important than the calm nature.On the other hand,compared with the balance constitution,the difference of intestinal flora of Yin-Deficiency constitution is also one of the reasons why the aging characteristics of Yin-Deficiency constitution are more obvious than other constitutions.Combined with the analysis of the expression results of NF-kappa B signaling pathway-related mRNAs,it was confirmed that the related genes did exist in the mechanism of common changes with intestinal flora.And compared with the balance constitution,this common change characteristic of Yin-Deficiency constitution is one of the reasons for the higher tendency of organism aging.