Study On The Mechanism Of Heart-Kidney Related Theory In The Yang Deficiency Of Type ? Cardio-Renal Syndrome

BackgroundType ? cardiorenal syndrome(Type ? CRS)is a type of chronic renal damage caused by chronic heart failure(CHF)with high morbidity and mortality.which has become a critical proble worldwide.It is now widely accepted that theinflammatory response involved in the whole process of the developmentalof type II CRS.And MicroRNAs(miRNAs)play an important role in the regulation of inflammatory response.MiRNAs regulate the expression of inflammatory reaction proteins and produces heart and kidney damage,which is an important pathogenesis of type II CRS.However,the miRNAs related to type ? CRS are not definitecurrently.Our previous studies showed that there was a significant correlation and similar long-term prognosis betweenYang-deficiency syndrome and CRS.Therefore,we put forward thatheart-kidney yang deficiency is the main syndrome characteristic of type II CRS.The development process form CHF to CRS is the process of heart-yang deficiency,heart gradually damage kidney and simultaneous illness both in the heart and kidney.Heart-kidney related condition is the pathological basis that the heart and the kidneyprogressively produce functional and material damage while the micro-material basis is probably related to the miRNAswhich regulates the generation and development of type ? CRS.Therefore,there will be great significance for seeking the target miRNAswhich relating to the generation and development of type II CRS,and regulating downstream pathways and inflammation factors.Not only is of great important to deepen the pathogenesis of type II CRS and carry out the effective disease control and prevention,but also is conducive to reveal the micro-material basis for heart-kidney related theory and provide the scientific basis for the construction of heart-kidney related theory.ObjectTo preliminarily outline the impact of miRNAs as the core regulatory chain on the pathogenesis of type II CRS,the differencesof the miRNAs,inflammatory response markers and clinical data between the patients with CHF of Yang-deficiency syndrome and the patients with type ? CRS were compared.And the correlation between the different data was compared.To prove that during the development from CHF to CRS,the heart and kidney closely regulate and control the inflammatory response from gene level and protein level,so as to affect the organs and integral and show the development from heart Yang deficiency to heart-kidney Yang deficiency.Which would be taken as one of the objective bases for the heart-kidney related theory,and the pathogenesis of type ? CRS with Yang deficiency syndrome.And then,it can help to find possible targets for preventing or delaying the development of CHF to CRS from the theoretical level of Chinese medicine,and to intervene in a targeted way to prevent the transmission of disease.At the same time,through this study,the regulation and control chain with syndrome differentiation value between heart and kidney can be possibly clarified,which has a certain significance in enriching and developing the theory of traditional Chinese medicine.Methods65 patients with CHF and Yang-deficiency syndrome and 82 patients with type ?CRS and Yang-deficiency syndrome were enrolled in the Department of Cardiology,Guang'anmen Hospital,China Academy of Chinese Medical Sciences from January 2017 to January 2018,and 20 healthy subjects were collected.1.The collection of basic clinical datain the two groupsThe basic clinical data of the patients was collected,including name,gender,age,course of disease,comorbidity,drug combination and laboratory examinations.which were blood routine examination(RBC,WBC,Hb,PLT),biochemical test(blood lipid,UA,electrolyte,hepatic and renal function),animal spirits,thyroid function,and indices of heart function(NYHA,NT-proBNP,UCG).At the same time,blood routine and biochemical indicators of 20 healthy people were collected as controls,and the differences in basic clinical data between the groups were compared.2.The inflammatory factors detection in the two groupsFifty patients with CHF,50 with CRSand 20 healthy subjects were selected to test the inflammatory factors.,including CRP(lmmunoturbidimetry),CA125(chemiluminescenceimmunoassay);Renin,ATII,ALD(radioimmunoassay);IL-1?,IL-18,TNF-?,NF-?B and TLR4 expression levels(ELISA).RT-PCR was used to detect theNF-?BmRNA and TLR4mRNA expression level in PBMC of 16 cases in CHF group,16 cases in CRS group and 8 cases in healthy subjects.Furthermore,the difference of inflammatory factors between groups were compared.3.Detection of miRNA expression profiles and bioinformatics analysis in peripheral blood of two groupsFive patients in CHF group,5 patients in CRS group and 5 heathy subjects were selected.And the Illumina HiSeq X Ten instrument was used to carry out the high-throughput sequencing;miRNA expression profiles of peripheral blood of patients in CHF group,CRS Group and Health Group within the scope of whole genome were screened.miRNA expression difference between CHF group and CRS was analyzed and the new miRNA was predicted.Then,RNAhybrid and Miranda software were used to predict the target genes of differential expression miRNA,and the GO annotation and KEGG path analysis were carried out on the candidate target genes of different miRNAs.The main biochemical metabolism pathway and signal transduction pathway involved by major biological function of candidate target gene were preliminarily understood.Results1.Clinical characteristics of the patients in two groupsThe results of clinical data analysis showed that patients with CRS were significantly different from the patients in CHF group in age,gender(female)and history of hypertension(P<0.01),but,was not related to the history of atrial fibrillation,diabetes,HLP or the course of CHF(P>0.05).The venerable age,female and accompanied with hypertension patients were more likely to suffer from CRS;The results of biochemical analysis showed that the serum creatinine,urea nitrogen,blood platelet,direct bilirubin,serum phosphorus,total cholesterol,triglyceride,low density lipoprotein and very low density lipoprotein levels of patients in CRS group were obviously higher than that of the CHF group(P<0.01 or 0.05),and blood calcium,PH value,HC03 level of patients in CRS group were obviously lower than that of the CHF group(P<0.01 or 0.05);The results of heart function analysis showed that compared with CHF group,NYHA functional calss,NT-proBNP level and the left ventricle wall thickness of patients in CRS group were obviously increased,and LVEF was significantly decreased(P<0.01 or 0.05).2.Inflammatory factors levels of the patients in two groups(1)Compared with the healthy group,the contents of CRP,Renin,ATII,ALD,CA125,IL-1 ?,IL-18,TNF-a,NF-?Bp65 and TLR4 in peripheral blood of CHF group and CRS group were significantly increased(P<0.01 or 0.05);(2)Compared with CHF group,the CRP,Renin,AT?,ALD,CA125,IL-1?,IL-18,TNF-?,NF-?Bp65,TLR4 expression in peripheral blood of CRS group were significantly increased(P<0.01 or 0.05).Howerver,there was no statistic difference in Renin,ATII,ALD and CA125 level in two groups(P>0.05);(3)Compared with the healthy group,the NF-KBmRNA and TLR4mRNA expression increased significantly both of CHF group and CRS group(P<0.01or0.05).Compared with CHF group,the NF-?BmRNA and TLR4mRNA expression level of CRS group were increased obviously(P<0.05).3.MicroRNA expression profile and bioinformatics results of the patients in two groupsThrough the high-throughput sequencing,miRNA expression profiles of CHF group?CRS group and heath group were successfully constructed.(1)Compared with health group,1366 differential miRNAs were found in the CHF group.There are 238 miRNAs with significant differences(variation>2 times,P<0.05),of which 116 were up-regulated and 122 were down-regulated.There were 177 miRNAs whose differential expression levels were more than 10 times(P<0.01);(2)Compared with health group,1084 differential miRNAs were found in the CRS group.There were 265 miRNAs with significant differences(variation>1.5 times,P<0.05),of which 161 were up-regulated and 104 were down-regulated.There were 187 miRNAs that differ by more than 10 times in their expression levels.(P<0.01);(3)Compared with CHF group,1271 different miRNAswere screened,among them,34miRNA expression levels between the two groups were observably different(variation>1.5 times,P<0.05),and there were 10 genes whose difference value is more than 10 times(Log2FC>3.3 ?<-3.3,P<0.05).Five miRNAs(miR-30c-5p?miR-223-3p,miR-4692,miR-3196,miR-785-3p)were significantly increased and 5 miRNAs(miR-5587-5p,miR-4283,miR-3140-3p,miR-4447,miR-6876-3p)were significantly reduced.At the same time,the RNAhybrid and Miranda software were used to carry out the target genes prediction on 34 miRNAs with significant differences,the intersection of proposed algorithm was took as the final result,and 2692 candidate target genes were gained,including the target genes related to heart and kidney injury reported in the literature,such as NF-?B,TLR4,SMAD6,TIAF1,NRF1,AQP7 and PAK3.Aiming at these predicted target genes,GO and KEGG analysis were carried out,and by comparing with the reference genome,we found that the candidate target genes of differential expressionmiRNA were enriched in 667pathways including Wnt signaling pathway,NF-kappaB singaling pathway and so on.In terms of molecular function,the candidate target genes of miRNA differential expression were enriched in 200 pathways,such as protein binding,kinase activity,metal ion binding and so on.In terms of cell compositions,the candidate target genes of differential expressionmiRNA were enriched in 141 pathways,such as cell junction,synapse,dendrite and so on.Ninety significantly enriched KEGG signaling pathways including MAPK signaling pathway,cGMP-PKG signaling pathway,Wnt signaling pathway,mTOR signaling pathwy,and other related pathways were identified by KEGG analysis.The correlation between the NF-kB pathway and the candidate target mRNA and differential miRNA focused on this project revealed that there were 41 target mRNAs participated in NF-?B signaling pathways,which mainly related to inflammation,including TNF,IL,TLR4,CCL and so on.There were 18 mRNAs participated in the regulation of NF-?B signaling pathway.Among them,there were 16 differential miRNAs obtained by high-throughput sequencing.Seven miRNA expressions were increased,Theywere miR-4692,miR-6126,miR-143-3p,miR-3591-3p,miR-574-3p,miR-345-5p,miR-3196,respectively.Nine miRNAs expressions were decreased,Theywere miR-4447,miR-6876-3p,miR-4458,miR-4485-5p,miR-92b--5p,miR-4283?miR-6841-3p,miR-6819-5p,miR-6512-3p,respectively.Among them,the relationship between increased gene miR-4692,miR-3196 and reduced gene miR-4283,miR-4447 were related significantyly(variation>10 times,P<0.05).Conclusion1.Advanced age,female and history of hypertension are the highrisk factors for the development of type ? CRS.With the increase of blood uric acid,blood lipids,platelets,direct bilirubin,bloodphosphorus and left ventricular wall thickness,and the decrease of hemoglobin,blood calcium,PH value,blood HC03,and deterioration of heart function,the incidence of type ? CRS is gradually increasing.Early recognition and control of the above risk factors should be emphasized to delay the occurrence and development of type ? CRS clinically;2.Changes in hemodynamics after the occurrence of CHF and excessive activation of neuroendocrine factors induced the up-regulated expressionof miR-4692 and miR-3196,and the expression of miR-4283?miR-4447 down-regulated,which led to the over-activation of downstream TLR4/NF-?B signaling pathway.Bringthe release of pro-inflammatory cytokines such as CRP,IL-1?,IL-18,and TNF-a into the blood,which eventually leads to kidney damage and is one of the important inflammatory signaling pathway that mediates the development of type ? CRS;3.MiR-4692,miR-3196,miR-4283,and miR-4447 target the excessive activation of TLR4/NF-?B signaling pathway which are the key links in the inflammatory response mediation of type ? CRS.And it may be the molecular mechanism that the heart failure develops from heart Yang deficiency to kidney Yang deficiency,which causes simultaneous illness in the heart and kidney disease,and it may be the pathogenesis of heart-kidney related theory in type ? CRS with Yang deficiency syndrome;4.The pathogenesis of type ? CRS may also be related to renal fibrosis and remodeling which mediated by the activation of the p38MAPK signaling pathway and the Wnt signaling pathway.