Study On The Expression Profiles Of NcRNAs In Coronary Heart Disease With Blood Stasis And Toxin Syndrome And Intervention Mechanism Of Active Ingredients Of Red Peony Root And Fructus Aurantii

There are a large number of RNAs which do not code for protein in mammalian genomes called non-coding RNAs （ncRNAs）. They were regarded as’dark matter’in genomics initially, but recent studies suggest that ncRNAs can regulate genes expression from multiple biological process and ncRNAs are closely related to many diseases. microRNAs and long non-coding RNAs（lncRNAs） are the main components of ncRNAs which become research hotspots recently. They can modulate the pathological process such as inflammation, autophagy, cell proliferation and so on; they can expressed in the peripheral blood and specific tissue stably. Thus, ncRNAs become reliable biomarker in diagnosis, treatment and prognosis of disease, while they can also provide new targets for drug treatment. In addition, ncRNAs may reveal the material basis of TCM syndrome. It can provide evidence to support the objective of TCM syndrome. Our group previously propsed ’acute cardiovascular events were induced by blood stasis and toxin’, thus recognize and treat patients with blood stasis and toxin syndrome in early phase can further reduce cardiovascular mortality. Therefore, the different expression of ncRNAs may play a key role in coronary artery disease with blood stasis and toxin syndrome.We use gene chips to establish different expression profiles of ncRNAs in coronary artery disease with blood stasis and toxin syndrome in this study. Then qRT-PCR was used to verify the expression of ncRNAs. Inflammatory macrophages induced by LPS derived from RAW264.7 cell lines as vitro model to explore the function of effective components of Red paeony root and fructus aurantii（paeoniflorin, naringin） in the expression of miR-155. This suty will provide experimental basis for the pharmacology of traditional Chinese medicine with the effect of promoting blood circulation and detoxifying meanwhile provide direction for the biomarker research of blood stais and toxin syndrome.This study is divided into three parts:literature review, clinical and experimental research.1. Literature review:In this part we reviewed the non-coding RNA and coronary atherosclerotic heart disease, research progress in coronary heart disease with blood stasis and toxin syndrome and pharmacology of traditional Chinese medicine with the effect of blood activating and detoxifying.2. Clinical Research:The First Research:Build the expression profiles of microRNAs in blood stasis and toxin syndromes of coronary heart diseaseObjective:Explore the differential expression of microRNAs in blood stasis and toxin syndrome using gene chip. Then Go analysis and pathway analysis used to predict the function of microRNAs. The aim is to find that whether microRNAs can be related with blood stasis and toxin syndrome in coronary heart disease.Methods:The diagnosis of coronary heart disease refer to<The diagnosis and treatment guidelines of chronic stable angina>,< The diagnosis guidelines of unstable angina and non-ST segment elevation acute myocardial infarction> formulated by the 2007 Chinese Medical Association cardiovascular disease branch, Chinese Journal of Cardiology Editorial Committee.2013 ESC published<Guidelines for the management of stable coronary artery disease>.Chinese Medical Association cardiovascular disease branch, editorial board of Chinese Journal of cardiovascular disease in 2010 published<Guidelines for the diagnosis of acute ST segment elevation myocardial infarction> and 2013ACCF/AHA guidelines for ST segment elevation myocardial infarction. Meanwhile, consult the diagnosis of blood stasis syndrome and toxin syndrome. And the total score less than 11 defined as the mild case of blood stasis and toxin syndrome, more than 11 defined as severe case of blood stasis and toxin syndrome. Screening of blood stasis syndrome, blood stasis and toxin syndrome enrolled in blood stasis syndrome group, mild case of blood stasis and severe case of toxin syndrome group with 5 cases in each group,4 subjects served as control group as the research object. The patients with coronary heart disease were drawing blood before coronary angiography. The control group was drawing blood in the circumstance of limosis. Add the red blood cell lysis buffer into the blood to obtain the white blood cells. Gene chip was applied to explore the differential expression of microRNAs in different syndrome of coronary artery disease.Results:The three syndromes of coronary heart disease and control group had no significant difference in age, gender. There is no significant difference on complicating disease and drug combination, coronary lesions and other aspects. Go analysis showed that the difference expression of microRNAs in blood stasis syndrome and toxin syndrome of coronary heart disease were mainly involved in the transcriptional regulation of DNA binding protein and play a key role in the process of biological function. Pathway analysis showed that different microRNAs in blood stasis syndrome of coronary heart disease may be involved in metabolic pathways, ubiquitin mediated protein degradation, endoplasmic reticulum protein processing. Different microRNAs in blood stasis and toxin syndrome may be involved in MAPK signal pathway, the proliferation and apoptosis of cells, actin skeleton protein, Wnt signal pathway and other biological processes, and both of those can affect the immune reaction. The function of those different microRNAs fit with the previous theory of’toxin’. Compared with control group, has-miR-1228-3p, has-miR-3157-3p have significant difference in groups with coronary heart disease （FC>1.5, P<0.05）; has-miR-3162-3p, has-miR-182-5p, has-miR-363-3p, has-miR-455-3p, has-miR-1228-5p, has-miR-4443, has-miR-3064-3p have no significant differences in blood stasis syndrome of coronary heart disease compared with control group （P> 0.05 or FC<1.5）, but they have significant differences in blood stasis and toxin syndrome of coronary heart disease compared with controls （P<0.05, FC≥1.5）. has-miR-155-5p has significant difference in slight case of blood stasis and toxin syndrome group compared with control group （P<0.05, FC>1.5）. has-miR-941, has-miR-1281, has-let-7d-3p have significant differences between severe blood stasis and toxin group and the control group （P<0.05, FC≥1.5）. Further analysis showed that parts of those different microRNAs can participate in pathological processes of atherosclerosis, such as inflammation, immune, platelet activation, cell proliferation, apoptosis, and so on.Conclusion:The expression of microRNAs is different in different syndromes of coronary heart disease. microRNAs may be involved in the development of coronary heart disease with blood stasis and toxin syndrome. Has-miR-3162-3p, has-miR-182-5p, has-miR-363-3p, has-miR-455-3p, has-miR-1228-5p, has-miR-4443, has-miR-3064-3p, has-miR-155-5p and other microRNAs were involved in the immune reaction, inflammation, platelet activation and other pathophysiological processes. Both of those functions of different microRNAs are accordance with the theory of ’blood stasis and toxin syndrome’ in our groups. The different expression of microRNAs verified the relationship between the theory of poison and the events of acute cardiovascular disease via biological function. The different expression of microRNAs can lay the foundation of the study for the biomarker of coronary artery disease with blood stasis and toxin syndrome.The Second Research:Establish the differential expression profiles of lncRNAs in blood stasis and toxin syndromes of coronary heart diseaseObjective:Explore the differential expression profiles of lncRNAs in blood stasis and toxin syndrome using gene chip. Then co-expression analysis with mRNA was used to predict the function of lncRNAs. The aim is to find whether lncRNAs can be associated with blood stasis and toxin syndrome of coronary heart disease.Methods:Screening of blood stasis syndrome, blood stasis and toxin syndrome enrolled in blood stasis syndrome group, mild case and severe case of blood stasis and toxin syndrome group with 5 cases,4 healthy subjects served as control group as the research object. The patients with coronary heart disease were drawing blood before coronary angiography. The healthy control group was drawing blood in the circumstance of limosis. Add the red blood cell lysis buffer into the blood to obtain the white blood cells. Gene chip was applied to explore the differential expression profiles of lncRNAs in different syndrome of coronary heart disease.Results:The three syndromes of coronary heart disease groups and control group had no significant differences in age, gender. There are no significant differences on complicating disease, medication and other aspects （P>0.05）. Pathway analysis showed that the different lncRNAs in blood stasis syndrome of coronary heart disease may be involved in natural killer cell mediated cytotoxicity and antigen presentation; different IncRNAs in blood stasis and toxin syndrome may be involved in cell adhesion molecules, extracellular matrix and calcium signaling pathways, both of those can affect the stability of plaques. Both of those functions were fit with the previous theory of ’toxin’.Compared with control group, NONHSAT118577,FR066195,TCONS₀017783,NONHSAT040728,ENST00000583306 and other total 211 items have significant differences in groups with coronary heart disease （FC>2 and P<0.05）;NONHSAT040728, NONHSAT005533, NONHSAG029753,NONHSAG011835,NONHSAT125298 and other 826 items have no significant differences in blood stasis syndrome of coronary heart disease compared with control group （P>0.05 or FC<2）, but they have significant differences in blood stasis and toxin syndrome of coronary heart disease compared with controls （P<0.05, FC>2）. NONHSAT054767,NONHSAT081325,TCONS₁2₀0007025,NONHSAT096941,FR 156571 and other 1796 items have significant differences between severe blood stasis and toxin group and the control group （P<0.05, FC>2）. Further analysis showed that parts of the different IncRNAs can participate in pathological processes of atherosclerosis, such as inflammation, immune, oxidative stress, autophagy and so on. Conclusion:The IncRNAs expressed differently in different syndrome of coronary heart disease and they were involved in the progress of inflammation, immune reaction. IncRNAs may be involved in the development of blood stasis and toxin syndrome with coronary heart disease. NONHSAT118577, TCONS₀0017783,NONHSAT054767,ENST00000583306,NONHSAT130416 and other IncRNAs involved in the immune reaction, inflammation, autophagy, oxidative stress and other pathophysiological processes. Both of those functions of different IncRNAs are accordance with the theory of’blood stasis and toxin syndrome’.The different expression of IncRNAs verified the relationship between the theory of poison and the events of acute cardiovascular disease via biological function. The different expression of IncRNAs can lay the foundation for the study of the biomarkers for coronary heart disease with blood stasis and toxin syndrome.The Third Research:Clinical validation of the expression of different ncRNAs in blood stasis and toxin syndrome with coronary heart diseaseObjective:The aim is to validate the expression of ncRNAs in the blood stasis and toxin syndrome of coronary artery disease via qRT-PCR. So we can focus on ncRNAs as the potential targets of biomarkers for follow-up study and new targets for drugs.Methods:Screening of blood stasis syndrome, blood stasis and toxin syndrome enrolled in blood stasis syndrome group, mild case and severe case of toxin syndrome group with 10 cases in each group,10 subjects without the evidence of ischemia served as control group. The patients with coronary heart disease were drawing blood before coronary angiography. The healthy control group was drawing blood in the circumstance of limosis. Add the red blood cell lysis buffer into the blood to obtain the white blood cells in order to extract lncRNAs and microRNAs, then validate the expression of ncRNAs via qRT-PCR.Results:The three syndromes of coronary heart disease groups and control group had no significant difference in age, gender. There are no significant differences on complicating disease, medication and other aspects. miR-155-5p was significant difference in slight blood stasis and toxin syndrome of coronary heart disease compared with control group（P<0.05）; miR-182 was differential expressed in blood stasis and toxin light syndrome of coronary heart disease compared with control group （P<0.05）; miR-363, miR-455 was differential expressed in each disease group compared with control group （P<0.05）; miR-941 and miR-1228 only had a significant difference in severe blood stasis and toxin syndrome of coronary heart disease compared with control group（P<0.05）; NONHSAT118577 had no change between coronary artery disease and control group （P>0.05）; NONHSAT130416 had significant difference in blood stasis and toxic syndrome of coronary heart disease（P< 0.05）.Conculsion:miR-155-5p、miR-182、miR-363、miR-455、NONHSAT130416、 miR-941 had differential expression between blood stasis and toxin syndrome of coronary artery disease and control group. Those non-coding RNAs can provide direction for the research of biomarkers of coronary heart disease with blood stasis and toxin syndrome.3. Experimental Research:The function of effective components of Red Paeony Roots and Fructus aurantii in the expression of miR-155 in inflammatory macrophage induced by LPSObjective:The aim is to study the function of effective components of red paeony roots and fructus aurantii on inflammatory reaction and the expression of miR-155 in vitro.Methods:Inflammatory macrophages were induced by LPS used as vitro model. IL-6、TNF-α IL-1β were detected after drug intervention using ELISA kits. qRT-PCR was used to detect the expression of miR-155. The dual luciferase gene report and transfection experiment used to identify whether SHIP1 and SOCS1 are the target gene of miR-155. Then Western Blot was used to test the protein content after drug intervention.Results:Paeoniflorin and naringin can decrease the level of IL-6、TNF-α IL-1β and miR-155（P< 0.05）, and combination of paeoniflorin and naringin can exert synergistic effect of lowering IL-6、TNF-α and miR-155（P<0.05）. The dual luciferase gene report showed that miR-155 can act on 3’UTR of SHIP1 and SOCS1. It confirmed that SOCS1 and SHIP1 are the target genes of miR-155. Paeoniflorin, the combination of paeoniflorin and naringin can increase the expression of SHIP1（P< 0.05）; naringin, the combination of paeoniflorin and naringin can increase the expression of SOCS1（P<0.05）. Down-regulated of miR-155 adopt transfect of miR-155 inhibitor showed that the combination of paeoniflorin and naringin can synergetic increased the expression of SHIP1 （P<0.05）, the rest of groups have not synergetic increased the expression of SHIP1 and SOCS1.Conclusion:Paeoniflorin and naringin can significantly decrease the expression of IL-6、TNF-α IL-1β and miR-155,compatibility of the two medicines can exert synergetic anti-inflammatory effect, meanwhile increased the related target gene SHIP1 and SOCS1 in protein level. Thus, paeoniflorin and naringin may act on other inflammation related target genes to exert pharmacological effects.