| Objective;To explore the molecular regulatory mechanism of Yougui pill's intervention on aging kidney Yang deficiency constitution, to provide scientific basis for the prevention and therapy of clinical kidney Yang deficiency and the use of Yougui pill.Methods:(1) Using the kidney Yang deficiency syndrome differentiation factor score rules table, to evaluate the curative effect of semi-quantitative on the aging kidney Yang deficiency constitution treated for twenty eight days (Yougui pill,9g/times, three times a day)(2)Adopting the miRNA expression profile chip technology, and depend on the miRNAs before and after the treat on aging kidney Yang deficiency constitution and the healthy group, finally to test it, and comparatively analysis differentially expressed miRNAs in three groups.(3)According to the different expresses miRNAs specific expression quantity change in the three groups and the combination of traditional Chinese medicine in kidney Yang deficiency failure and pubmed correlations reported in literatures, to screen out miRNAs, and using qRT-PCR technology for the further study.(4) For the target miRNAs, using argetScan program to predict the target gene, and using Total context+scores<-0.6core, Aggregate PCT>0.8 as the standard of target gene selection.(5) Using pyrophosphate sequencing technology to test the methylation level of the target miRANs prediction target gene and the early screening of kidney Yang deficiency of immune related genes(ten genes), and comparatively analysis differentially methylation level of the ten genes in three groups.(6) Using qRT-PCR technology for the further test and analysis on methylation level of the target miRANs prediction target gene and the early screening of kidney Yang deficiency of immune related genes(ten genes).Results:(1)The Yougui pill is effective to the intervention of aging kidney Yang deficiency constitution, especially, the symptoms like "Waist knee ache", "chills afraid of the cold, cold limbs, chills" back " and "pee frequency more" significantly improved, but the symptoms like "Lower extremity edema", "pond loose stool", "phlegm bai qing loose" improved little obviously.(2) miRNA microarray results showed Yang group and the healthy group a total of 63 differentially expressed miRNAs, Yang group and treatment group consisted of six differentially expressed miRNAs, the treatment group and the healthy group a total of 50 differentially expressed miRNAs.(3) 6 miRNAs were screened for the purpose of miRNAs. qRT-PCR statistics showed that 5 miRNAs statistically significant. Wherein after YouguiWan intervention, there are differences in the expression of two genes.(4) Obtained by a total of six genes for miRNAs target gene prediction purposes.(5) Pyrosequencing statistics showed that four genes statistically significant, wherein after YouguiWan intervention, methylation or demethylation enhancing effect three genes occurred.(6) qRT-PCR statistics show 10 genes six genes statistically significant.Conclusion:(1)Accordng to the effect of Warming Yang and filling lean pulp, Yougui pill obviously effect on "Cold like" and "jing kui", but not that obvious on regulating the body fluid. Healthy subjects with age from the symptoms displayed on also has a certain degree of kidney Yang deficiency.(2)The aging kidney Yang deficiency constitution is related on cancer, brain dysfunction or disease, cardiovascular function or disease, modern or disease, aging, disease, reproductive function, and reflect insufficient kidney Yang deficiency, promote gasification, hide the elaboration, heart kidney and brain kidney.(3)The Yougui pill can regulate the expression of BMP7?DICER1?NF1?FLT1 through regulating the miRNAs expression and DNA methylation. AXIN1and SSTR5 might be the target point of playing a indirect role.(4)Aging kidney Yang deficiency constitution has relative stability, and the total change may require a long process. According to the warming Yang, filling lean pulp, Yougui pill may have reduced the occurrence rate of tumor, dysfunction related on the aging kidney Yang deficiency constitution. And the Yougui pill has played a certain degree of protection on anti-aging and longevity health care. |
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