Screening And Identification Of Biomarkers For Yin-deficiency-heat Syndrome And The Mechanism Of Action Of Zhibai Dihuang Granule

Part I iTRAQ-based Proteomic Analysis to Screen the Biomarkers for Yin-deficiency-heat syndrome rats and the study for Mechanism of Action of Zhibai Dihuang GranuleBackground:Zhibai Dihuang Granule(ZDG)is a traditional Chinese medicine which has been used to treat Yindeficiency-heat(YDH)syndrome for thousands of years in China.However,little work has been conducted to explore the molecular mechanism of ZDG in YDH syndrome,and the processes of YDH syndrome prevention and treatment have been developed slowly.The present study was aimed to explore the therapeutic mechanism of ZDG on YDH syndrome.Methods:The YDH syndrome rats were induced by hot Chinese herbs,then treated by ZDG orally for 1 week.Body weight was measured every 2 days.After sacrifice,blood samples were collected and the thymus,adrenal glands,spleen,and liver were immediately removed and weighed.iTRAQ-based proteomics approach was applied to explore the serum protein alterations with the treatment of ZDG,and to investigate the underlying mechanism of ZDG in treating YDH syndrome.Results:The body weights of YDH syndrome rats were significantly decreased compared with control group,and increased in ZDG treated rats.The relative weights of thymus in YDH syndrome rats were increased compared with the control rats,and significantly decreased in after ZDG treatment.In the proteomic analyses,seventy-one proteins were differentially expressed in the YDH syndrome group and the ZDG treated group,including 10 up-regulated and 61 down-regulated proteins.Gene ontology analysis revealed that the differentially expressed proteins were mostly related to immune response,and pathway enrichment analysis showed that these proteins were enriched in coagulation and complement cascades.Enzyme-linked immunosorbent assay was performed to detect the protein levels in coagulation and complement cascades,and the results showed that C5 and C9 levels were significantly decreased in YDH syndrome group,and C5 was significantly increased after ZDG treatment.While Fga?Fgb?vWF?Fgg levels were significantly increased in YDH syndrome group,and Fgg was decreased after ZDG treatment.Conclusions:We found that ZDG treatment could lead to proteins alteration in immune response,especially in coagulation and complement cascades.ZDG can up-regulate the proteins in the complement cascade to enhance the complement activation to eliminate pathogens,and down-regulate the proteins in the coagulation cascade to suppress inflammation.Our study provides experimental basis to understand the therapeutic mechanism of ZDG and revealed that ZDG can regulate coagulation and complement cascades in treating YDH syndrome.Part II Screening and Identification of miRNAs as Biomarkers for Yin-deficiency-heat syndromeBackground:As a sub-health statuas,Yin-deficiency-heat(YDH)syndrome was caused by congenital endowment,stress,excessive fatigue or unhealty lifestyle.However,the biological nature of YDH syndrome remains unknown.Besides,the current methods for the diagnosis of YDH syndrome are lack of scientific basis.Therefore,novel methods suitable for YDH syndrome diagnosis are urgently needed.Methods:iTRAQ-2D LC-MS/MS and Solexa sequencing were used to screen proteomic and transcriptomic differences between the 85 YDH syndrome individuals and 56 healthy controls.Bioinformatics was used to analyze the biological function of the differentially expressed proteins and miRNAs.Quantitative reverse-transcription polymerase chain reaction(qRT-PCR)were used to validate miRNAs expression.Receiver operating characteristic(ROC)curve and logistic regression analysis was used to evaluate the efficacy of the YDH syndrome diagnostic model.Results:The iTRAQ-2D LC-MS/MS results showed 120 proteins differentially expressed in YDH syndrome group,and GO and KEGG analysis revealed that the differentially expressed proteins mainly involved in complement and coagulation pathway.Solexa sequencing data showed 41 miRNAs differentially expressed in YDH syndrome group,and 12 differentially expressed miRNAs were potentially regulated the proteins in complement and coagulation pathway.The qRT-PCR validation indicated that hsa-miR-27b-3p,hsa-miR-23b-3p,hsa-miR-221-3p,hsa-miR-30b-5p,hsa-miR-142-3p were significantly decreased in YDH syndrome compared to the healthy controls.We established the diagnostic model with a sensitivity of 95.6%and a specificity of 100.00%with the combination of hsa-miR-142-3p,hsa-miR-23b-3p,hsa-miR-27b-3p,and the combination of the three miRNAs may serve as potential biomarkers for YDH syndrome diagnosis.Conclusions:(1)We Screened and identified the differentially expressed porteins and miRNAs by using iTRAQ-2D LC-MS/MS combined Solexa sequencing,and found that the differentially expressed porteins were mostly involved complement and coagulation pathway,indicating the disturbance of complement and coagulation pathway in YDH syndrome.(2)Identification of potential biomarkers such as hsa-miR-27b-3p,hsa-miR-23b-3p,hsa-miR-221-3p,hsa-miR-30b-5p,hsa-miR-142-3p for YDH syndrome diagnosis,and construction of a diagnostic model with 95.60%sensitivity and 100.00 specificity with the combination of hsa-miR-142-3p,hsa-miR-23b-3p,hsa-miR-27b-3p.Our study provides potential biomarkers for YDH syndrome diagnosis and provides a new method for the objectification and quantification of TCM diagnosis.