TCM Syndrome Characteristics Of Subclinical Hypothyroidism And MiRNA Studies In Different TCM Syndromes

BackgroundSubclinical hypothyroidism(SCH)is defined as a state of increased serum thyroid-stimulating hormone(TSH)levels,with total thyroxine(TT4)and free thyronine(FT4)concentrations within the population reference range.In recent years,the prevalence of SCH has increased obviously,and has become a focus in the field of endocrinology.TCM has unique advantages in the diagnosis and treatment of SCH.but at present,there is no standard in the TCM syndrome differentiation,which limits the formation of the TCM diagnosis and treatment project of SCH to a certain extent.MicroRNAs(miRNAs)have been shown to play important regulatory roles in a variety of biological processes,and have specificity and time-space,which are intrinsic to the concept of TCM syndromes and may be involved in the occurrence and dynamics of syndromes.At present,miRNA has been applied in the description of TCM syndromes of various diseases,and as far as I know,there is no research related to SCH and its TCM syndromes about miRNA.The previous study of our group showed that the TCM syndromes of SCH patients were mainly composed of three syndrome types:spleen-kidney qi deficiency syndrome,liver-yin deficiency syndrome and liver-qi stagnation syndrome.Therefore,the search for SCH and TCM syndrome-related miRNAs can help to further reveal the microscopic mechanism of SCH syndrome formation,thus providing an objective basis for TCM standardized syndrome differentiation.ObjectiveThis study analyzed the questionnaires of 60 patients with SCH.and initially explored the TCM syndrome characteristics of SCH.MiRNA high-throughput sequencing technology was used to detect the expression of miRNA in three different syndrome types of SCH and normal control group.then screened differential miRNA expression profiles,predicted target gene and performed GO analysis of differential miRNAs,attempting to elucidate the miRNAs involved in the formation of different syndromes of SCH and their biological functions.Methods1 Characteristics of TCM syndromes in 60 patients with subclinical hypothyroidism:According to the inclusion and exclusion criteria,the SCH patients who were treated in the endocrinology clinics of the 3 centers from March 2018 to March 2019 were selected to fill out the "Traditional Chinese Medicine Syndrome and Physical Fitness Questionnaire",including items as follows:general information,medical history,clinical symptoms,laboratory test,TCM constitution and other information needed.A database of SCH was established,and statistical analysis was performed with SPSS24.0 to explore the characteristics of TCM syndromes in SCH.2 Differentially expressed miRNAs between different syndrome types of SCH:The patients with SCH who were treated in our hospital were randomly selected according to the results of TCM syndrome differentiation at the time of enrollment.3 cases were selected respectively from spleen-kidney qi deficiency,liver-qi stagnation and liver-yin deficiency syndrome.At the same time,3 healthy controls were selected.The plasma was taken from the subjects for RNA extraction and high-throughput sequencing to obtain differential miRNA expression profiles between SCH and normal population,predict target genes,and analyze the biological processes in which differential miRNAs may be involved.Results1 Distribution of TCM syndrome in patients with SCH:In the 60 cases of SCH patients,the most common TCM syndrome is spleen and kidney qi deficiency syndrome,followed by liver yin deficiency syndrome,liver qi stagnation syndrome and other syndromes.There was no significant difference in the distribution of SCH syndromes in gender and age.The distribution of syndromes in SCH has a certain relationship with the disease course.The patients whose disease duration is less than 6 months are mainly liver qi stagnation and other syndromes.The incidence of spleen-kidney qi deficiency syndrome increases with the course of the disease2 Correlation between TCM Syndromes and laboratory test:Correlation analysis between TCM syndrome types and laboratory test shows that there is a difference between TSH values in different syndrome types,which can be used as a indicator for TCM syndrome differentiation.There was no significant correlation between FT3.FT4,TT3.7T4.anti-TPO.anti-TG.blood lipid levels with the classification TCM syndromes3 In the constitution survey of 60 cases.the single constitution only accounted for 18.3%,81.7%of the patients had 2 and more constitutions.In terms of the proportion of single constitution in SCH patients,the qi stagnation is the most,followed by qi deficiency,yang deficiency,and yin deficiency4 Differentially expressed miRNAs in SCH patients:Compared with the control group,the SCH group showed significant differential expression of miRNA2136(fold change>2 ie |log2FC|>1 and Q value?0.001),of which 1233 miRNA expression was significantly up-regulated and 903 was significantly down-regulated.Compared with the control group,the spleen and kidney qi deficiency group screened 1585 differentially expressed miRNAs,of which 568 miRNA expression was significantly up-regulated and 1017 was significantly down-regulated.Compared with the control group,the liver qi stagnation group screened 1534 differentially expressed miRNAs,of which 439 miRNA expression was significantly up-regulated and 1095 miRNA expression was significantly down-regulated.Compared with the control group,the liver yin deficiency group screened 1687 significant differentially expressed miRNAs,of which 713 miRNA expression was significantly up-regulated and 974 miRNA expression was significantly down-regulated.5 GO analysis of differentially expressed miRNAs:GO analysis showed that the biological processes involved in differential miRNA target genes are mainly retinoid metabolic process,response to organic substance,keratinization.response to biotic stimulus,diterpenoid metabolic process,response to external biotic stimulus,response to other organism,terpenoid metabolic process,defense response,response to external stimulus,etc.Conclusion1 In the survey of 60 cases of SCH patients,the most common TCM syndrome is spleen and kidney qi deficiency syndrome,followed by liver yin deficiency syndrome,liver qi stagnation syndrome and other syndromes.The distribution of various TCM syndrome types has a certain relationship with the length of the disease course.The short-term disease is mainly characterized by liver qi stagnation and other syndromes,and the spleen-kidney qi deficiency syndrome increases with the course of the disease.2 Comparison of different syndromes and clinical biochemical indicators of SCH patients shows that there is a difference between TSH values in different syndrome types,which can be used as a reference index for TCM syndrome differentiation in SCH.There was no significant correlation between FT3,FT4,TT3,TT4,anti-TPO,anti-TG,blood lipid levels and and the classification of TCM syndromes.3 The constitution distribution of SCH patients is more common in 2 or more complex constitutions.In terms of the proportion of single constitution in SCH patients,the qi stagnation is the most common,followed by qi deficiency,yang deficiency,and yin deficiency.4 There are differentially expressed miRNA in plasma between three different TCM syndromes of SCH patients and control group,which may be used as specific markers for the diagnosis of SCH and one of the objective basis for clinical syndrome differentiation,and it is expected to become a potential therapeutic targets.