Identification and Functional Analysis of Serum Specific miRNAs in Recurrent Aphthous Stomatitis Patients with Excess-heat or Yin-deficiency

Background:The treatment of Traditional Chinese Medicine(TCM)in recurrent aphthous stomatitis(RAS)based on syndrome differentiation has won the international acceptance,but the molecular mechanism of excess-heat syndrome and yin-deficiency syndrome of RAS remains unclear.Objective:To clarify specific microRNAs(miRNAs)and their functions in RAS patients with excess-heat or yin-deficiency.Methods:Serum samples were collected from patients meeting the RAS diagnostic criteria of excess-heat or yin-deficiency syndrome and healthy individuals.Core miRNAs were then identified under miRNA microarray anal-yses.Target prediction and bioinformatic analyses were carried out and gene-pathway-networks were visualized to better understand the relationship between different genes and pathways.Results:(1)90 individuals meeting the inclusion criteria were collected in this study.Among them,9 miRNAs were screened out in excess-heat syndrome group(EH group),with 1 upregulated and 8 downregulated.And four miRNAs(hsa-miR-20b-5p,hsa-miR-122-5p,hsa-miR-483-5p and hsa-miR-3197)were validated by real-time PCR method.14 miRNAs were screened out in yin-deficiency syndrome group(YD group)(7 upregulated and 7 downregulated).And hsa-miR-17-5p,hsa-miR-106-5p and hsa-miR-20b-5p were validated.(2)A total of 4,776 target genes were identified in EH group which enriched in GO categories including nervous system development and calcium ion binding and pathway such as PI3K-Akt signaling pathway and Calcium signaling pathway.10,172 target genes were identified in YD group which enriched GO categories included protein binding and positive regulation of transcription from RNA polymerase II promoter and pathway included MAPK signaling pathway and Ras signaling pathway.Conclusion:Hsa-miR-20b-5p in patients with RAS could act as the novel target for the classification of the syndrome.It is upregulated in RAS patients with excess-heat syndrome while downregulated in patients with yin-deficiency syndrome.The PI3K-Akt and MAPK signaling pathways and their related target genes may provide new insights into the molecular mechanisms of RAS with excess-heat syndrome or yin-deficiency syndrome,respectively.

Clinical Observation on the Endocrinal and Immune Functions in Subjects with Yin-deficiency Constitution

Objective： To explore the relationship between yin-deficiency constitution （YDC） and biochemical indexes by way of observing the endocrinal and immune functions in subjects with YDC. Methods： On the basis of epidemiological investigation, 60 subjects with YDC and 50 with gentle constitution （GC） were selected according to the pertinent criteria. From each subject, 8 mL of fasting venous blood was drawn at 8：00-9：00 in the morning, with the serum separated by centrifugation 3 000 r/min for 5 min and preserved at -70℃ in a freezer. Serum levels of corticosterone, cortisol, adrenocorticotrophic hormone （ACTH）, cyclic adenosine monophosphate （cAMP）, cyclic guanosine monophosphate （cGMP）, free triiodothyronine （FT3）, free thyroxine （1=-1-4）, throtropic stimulation hormone, interleukin 1β （IL-1β ） and interleukin 2 （IL-2） were detected by doubleantibody sandwich ELISA; cAMP/cGMP ratio was calculated, and the difference between the two constitutions in terms of these indexes was analyzed. Results： Serum FT3 was 4.16 ± 1.38 pmol/L in subjects with YDC, which was higher than that in subjects with GC （3.71 ±0.55 pmol/L）, but levels of cortisol （124.58± 45.36 ng/mL）, ACTH （58.92± 14.55 pg/mL）, cGMP （66.00 ± 18.02 pmol/mL） and FT4 （12.33 ± 3.12 pmol/L） in YDC were lower than those in GC （13.43±2.31 pmol/L）, showing significant difference （P〈0.05）. Conclusion： YDC is related to some extent with the disturbances in the hypothalamus-pituitary-adrenal axis, hypothalamus-pituitary-thyroid axis, cyclic nucleoside system and immune function.

Consistency between traditional Chinese medicine constitution-based classification and genetic classification

Background:We studied the consistency between two classification systems for categorizing patients:traditional Chinese medicine(TCM)constitution-based methods,versus genetic clustering.Genetic classification in constitutional identification was also evaluated.Methods:A TCM physician evaluated the constitution of each patient,according to four examinations(inspection,auscultation-olfaction,interrogation,and palpation).Those who met the criteria for Yang-deficient,Yin-deficient,and balanced constitutions were enrolled in the study.Peripheral blood samples were obtained from the participants,and peripheral blood mononuclear cells were separated from the samples within 2 hours.Total RNA extraction from the white blood cells was performed;and an Affymetrix HG-U133 Plus2.0 array was used to determine the peripheral blood gene expression profiles.The samples were classified using a support vector machine genetic classifier,and the“leave-one-out”method was used for validation.Results:The global gene expression profiles of 32 samples were grouped into three categories,and the samples in each of the gene categories corresponded with the three constitution categories.The three constitution types were distinguished using the genetic classifier with 165 genes.The accuracy of the prediction classification was greater than 95%using mathematical method.Conclusions:Participants with Yin-deficient,Yang-deficient,and balanced constitutions have varying physical characteristics and gene expression patterns.Additionally,the results from TCM constitution classification matched those obtained by genetic classification.Finally,our preliminary gene classifier distinguishes among Yin-deficient,Yang-deficient,and balanced constitutions,and provides a methodological basis for identifying the different constitutions.