Study On The High-risk TCM Constitutional Types Of NONFH And The Screening Of Its Specific Expressed MicroRNA

Objective: To explore the situation of the constitutional types of traditional Chinese medicine(TCM)in the sufferers of non-traumatic osteonecrosis of femoral head(NONFH)through literature research and epidemiological investigation,and to analyze the distributional characteristics of the constitutional types of TCM in the patients with NONFH,so as to obtain the frequent constitutional types of TCM in the patients with NONFH in Longyuan area.At the same time,the specific expression of microRNA(miRNA)in plasma of patients with the frequent constitutional types of TCM in the patients with NONFH was screened by biochip technology.The results were verified by real-time PCR.Further analysis of its bioinformatic content was carried out to find potential markers of molecular biology with diagnostic value,which could provide theoretical basis and therapeutic targets for the follow-up intervention of TCM and prevention of NONFH.METHODS: 1.Literature research: By searching PubMed,Embase,Cochrane,CNKI,VIP,Wanfang and CBM databases,a comprehensive collection of the literature of clinical studies(including cross-sectional,case-control and cohort study)on the distribution of the constitutional types of TCM in the patients with NONFH in these databases from inception to August 24,2018 was conducted to explore the distributional characteristics of the constitutional types of TCM in the patients with NONFH.2.Clinical research: A questionnaire survey was conducted among patients diagnosed as NONFH in orthopaedic clinic and inpatient department of Gansu Traditional Chinese Medicine Hospital in August 2016 and May 2018.A database of the distributional characteristics of the constitutional types of TCM in the patients with NONFH in Longyuan area was established by conducting questionnaire survey among patients that met the criteria of inclusion and exclusion and were definitely diagnosed as NONFH from August 2016 to May 2018 from orthopaedic outpatient and inpatient in Gansu Provincial Hospital of TCM in order to find the frequent constitutional types of TCM in the patients with NONFH in Longyuan area and analysis the reasons of suffer from easily.3.Experimental study: miRNA of the specific expression in plasma of the frequent blood stasis constitutional patients with NONFH in Longyuan area was screened by biochip technology.Real-time PCR was used to verify the results.The bioinformatic content was analyzed further.RESULTS: 1.Altogether 340 articles were searched out from the databases initially,and five articles were included finally according to the criteria of inclusion and exclusion.All of which were cross-sectional studies,and the five articles all were Chinese articles.The number of subjects included in our study was 516.The constitution of phlegm-dampness,yang-deficiency and damp-heat are the top three constitutional types of TCM in the patients with NONFH nationally with 23% [95% CI(11%~36%),Q = 134.43,Z = 3.68,P = 0.000],20% [95% CI(6%~33%),Q = 88.25,Z = 2.76,P = 0.006],15% [95% CI(12%~18%),Q = 7.46,Z = 9.51,P = 0.000] respectively.The difference was statistically significant(P<0.05).But the heterogeneity is high.2.A total of 112 subjects were included in the study.The number of males and females was 74 and 38 respectively.The sex ratio between men and women is about 1.95:1.The number of patients that were single constitution was ninety-eight,while multiple constitution was ten.Among the single constitution cases,the constitution of blood stasis(32.65%),phlegm-dampness(13.27%)and qi-deficiency(12.24%)were the most common constitution types of TCM,while qi-deficiency(50.00%)and yang-deficiency(21.43%)were distributed mainly among multiple constitution.The proportion of blood stasis constitution(34.35%)was the highest,followed by Qi deficiency constitution(15.27%),Yang deficiency constitution(12.98%)and phlegm-dampness constitution(11.45%)after merging.The proportion of four constitution accounted for 74.05% of the total constitution.3.There were 5 specific expressed miRNAs in the patients of blood stasis constitution with NONFH.The expression of has-miR-4270 was up-regulation,and the expression of hsa-miR-129-1-3p,hsa-miR-365a-3p,hsa-miR-483-3p and hsa-miR-6731-3p were down-regulation.The results of real-time PCR showed that the expression of hsa-mir-365a-3p in NONFH group was down-regulated compared with the normal group,and the expression of hsa-mir-365a-3p in group of blood stasis constitution was down-regulated compared with group non-blood stasis constitution.The results of real-time PCR were consistent with the results of miRNA chip.The databases of Target Scan,PITA and microRNAorg were used to predict the target genes of specific expressed miRNAs in the patients of blood stasis constitution with NONFH.The results showed that 11 intersectional target genes were detected in hsa-mir-365a-3p,and no intersectional target genes were detected in the rest.A total of 111 annotational descriptions were obtained by GO analysis,including 23 molecule functions,65 biological processes and 23 cellular components.Further analysis of molecular function showed that the specific expressed miRNAs in the patients of blood stasis constitution with NONFH was mainly related to receptor binding and the activity of protein and enzyme.Pathway analysis showed that target genes were significantly enriched in axon guidance,Rap1 signaling pathway,Lysine degradation,Fc epsilon RI signaling pathway,Glycerophospholipid metabolism,T cell receptor signaling pathway,Platelet activation,Osteoclast differentiation and so on.CONCLUSION: 1.The constitutional types of TCM in the patients with NONFH have definitely distributional characteristics,but the frequent constitutional types of TCM and their distributional characteristics in the patients with NONFH in different regions will be affected by many factors such as diet and environment.2.The constitutional types of the patients suffer from NONFH easily are blood stasis constitution,qi-deficiency,yang-deficiency constitution and phlegm-dampness constitution in Longyuan area.And blood stasis constitution is the high-risk constitutional type.3.There are five specific expressed miRNAs in the patients of blood stasis constitution with NONFH.These miRNAs may participate in the occurrence and development of NONFH by regulating Rap1 signaling pathway,lysine degradation,glycerophospholipid metabolism,platelet activation,osteoblast differentiation,Ras signaling pathway,PI3K-Akt signaling pathway and other signaling pathways.