Explore Inflammatory Macrophage Subsets Associated With CHF Based On Single-cell Sequencing And Screen Regulatory Components Of Nuanxinkang

Objective1.Established mouse model of chronic heart failure（CHF）induced by pressure overload,to explore the transcriptional landscape and heterogeneity of cardiac macrophages in mice with CHF by using single cell RNA sequence（sc RNA-seq）and bioinformatics analysis technology,and to screen potential inflammatory macrophage subsets associated with CHF,and to explore their source,phenotype and function.At the cellular level,to investigate the effects of new macrophage subsets on cardiomyocytes by establishing a co-culture system.In addition,to study the effect of new macrophage subsets on heart of mouse model of CHF,and to elaborate the mechanism of new macrophage subsets in CHF,so as to provide a new therapeutic target for CHF.2.To explore the effect of Nuanxinkang（NXK）on cardiac function and ventricular remodeling of CHF mice induced by pressure overload.By means of computer and molecular biological experiments,to examine the effect and effective regulatory components of NXK on inflammatory macrophage subsets related to CHF,so as to provide theoretical basis for NXK in the treatment of CHF.Methods:1.Exploration of potential inflammatory macrophage subsets associated with CHF based on sc RNA-seq（1）The CHF mice model was established by the surgery of transverse aortic constriction（TAC）.The cardiac function was detected by echocardiography 4 weeks after operation,which ensured TAC model were established.After the mice were anesthetized excessively,the heart was free and single cell suspension was prepared.The CD45 antibody was added,and CD45+cells were separated by flow cytometry.The single cell transcripts of CD45+cells were obtained by 10X Genomic Chromium.（2）By means of bioinformatics,t-distributed stochastic neighbour embedding（t-SNE）and k-means clustering were performed on transcriptome data.Cell specific gene database established by ourselves was used to identify cell population based on Gene Set Enrichment Analusis（GSEA）.Macrophage subsets associated with inflammation and heart injury were screened,and their markers were identified.The results were verified by animal experiments.（3）The origin of CD72hi-CMφs were explored by Chip-seq,fluorescence tracing,bioinformatics analysis and flow cytometry.The effects of CD72 and Rel over-expression bone marrow derived macrophages（BMDM）on cardiomyocytes were studied by establishing co-culture system.The changes of CD72hi-CMφs levels in WT mice and Rel^-/-mice under Sham and TAC were studied in vivo.The effect of CD72hi-CMφs on heart was explored by over-expression of Rel in monocytes transplantation.（4）Based on the Single-cell transcriptome data of myocardial infarction（MI）and atherosclerosis in mice,the level of CD72hi-CMφs were analyzed,and the transcriptome data of patients with CHF were analyzed.2.Screening of effective components of NXK regulating macrophage subsets related inflammatory injury in CHF（1）The CHF model induced by transverse aortic constriction（TAC）was established.The Sham group and TAC group were treated with normal saline.The NXK group and Peri group were treated with NXK and perindopril respectively.After 6 weeks of administration,the cardiac function was measured by echocardiography,the myocardial hypertrophy was evaluated by heart weight ratio,the myocardial fibrosis was measured by Masson staining,and the myocardial fibrosis was measured by TUNEL staining.The levels of cardiac hypertrophy indexes（ANP,BNP,β-MHC）and inflammatory factors（IL-6,IL-1β,TNF）were measured by Real-time q PCR to evaluate the effects of NXK on cardiac function and ventricular remodeling in mice with CHF.（2）Based on the transcriptome data of NXK established by our research group,the regulatory effect of NXK on CD72hi-CMφs was evaluated by deconvolution method,and the key targets of NXK on CD72hi-CMφs were predicted and verified based on enrichment analysis and luciferase report analysis.（3）According to the homologous modeling method,the mouse c-Rel protein crystal model was constructed,and the effective small molecules of NXK regulating CD72hi-CMφs were predicted by molecular docking.（4）CCK8 was used to detect the effect of different concentrations of fructose arginine（FA）on BMDM,and the effect of FA on the differentiation of CD72hi-Mφs was studied at the cellular level.The levels of TNF and IL-1βwere determined by Real-time q PCR to evaluate the anti-inflammatory effect of FA.Results:1.Exploration of potential inflammatory macrophage subsets associated with CHF based on sc RNA-seq1.1 Identify inflammatory macrophage subsets and their markers associated with CHF（1）Compared with Sham group,the percentage of CD45+cells in TAC group was significantly increased（P<0.05）.The transcriptome information of 8223 and 5652 cells from Sham and TAC groups were obtained by 10X Genomic Chromium respectively.（2）Through t-SNE dimensionality reduction and k-means clustering,eight different macrophage subsets（cluster 1～cluster 8）can be identified.Cluster 4 was a group of inflammatory macrophages associated with CHF,and its specific marker CD72 was found by bioinformatics analysis.Pearson correlation analysis showed that CD72 was positively correlated with pro-inflammatory cytokines（TNF,IL-1β）,chemokines（Ccl2,Ccl4）and fibrosis factor（Tgfβ1）.GO enrichment analysis showed that CD72hi-CMφs transcriptome was involved in two biological processes:macrophage chemotaxis and macrophage migration.Flow cytometry results showed that compared with Sham group,the level of CD72in cardiac macrophages of TAC group was increased（P<0.05）,and negatively correlated with left ventricular ejection fraction（LVEF）（R²=0.1425）.1.2 Explore the source of inflammatory macrophage subsets associated with CHF（1）Pearson correlation analysis showed that CCR2 mRNA level was positively correlated with CD72 level.The results of flow cytometry showed that the level of CD72 in CCR2+CMφs was higher than that in CCR2-CMφs in Sham group and TAC group（P<0.05）.The results of pseudo times analysis showed that the level of CD72 mRNA increased with the differentiation of monocytes into macrophages.（2）The results of pseudo times analysis,Ch IP-Seq and luciferase reports showed that Rel was the key transcription factor in CCR2-CMφs differentiation.Flow cytometry results showed that the level of CD72 was increased in BMDM with Rel over-expression（P<0.05）.The level of CD72 was significantly increased in mice with Rel over-expression bone marrow transplantation（P<0.05）.Besides,compared with WT mice,the levels of CCR2+CMφs and CD72 were decreased in Rel^-/-mice in Sham and TAC groups（P<0.05）.The results of transcriptome data were consistent.1.3 Explore the role of inflammatary macrophage subsets associated with CHF（1）Compared with the control group,the mRNA levels of cardiac loading markers（ANP and BNP）and oxidase（Cybb,Sod2,Gsr and Prdx5）were increased in cardiomyocytes co-cultured with Rel overexpression BMDM（P<0.05）,but not in cardiomyocytes co-cultured with CD72 overexpression BMDM（P>0.05）.There was no significant difference in MHC mRNA level between the two groups（P>0.05）.The results of phalloidin staining showed that there was no significant change in the size of cardiomyocytes in the two groups（P>0.05）.The results of flow cytometry showed that compared with the control group,the level of reactive oxygen species（ROS）was significantly increased in the cardiomyocytes co-culture with Rel overexpressed BMDM（P<0.05）.However,the ROS level of cardiomyocytes co-cultured with CD72 over-expressed BMDM did not change（P>0.05）.The mitochondrial content of cardiomyocytes co-cultured with Rel over-expression BMDM increased（P<0.05）,but the production of ATP decreased（P<0.05）.CD72 knockout significantly reduced Rel-induced ATP reduction（P<0.05）and apoptosis（P<0.05）.Immunofluorescence showed that the level of mitochondria decreased and the level of lysosome increased after co-culture with Rel overexpressing BMDM.（2）In Sham group and TAC group,flow cytometry and deconvolution results showed that the CD72 level of cardiac macrophages in Rel^-/-mice was lower than that in WT mice（P<0.05）.Under TAC condition,compared with WT mice,Rel knockout improved the decrease of LVEF（P<0.05）,decreased the number of cardiomyocyte apoptosis（P<0.05）,decreased MDA level（P<0.05）,and decreased inflammatory cytokines（IL-6,IL-1βand TNF）level（P<0.05）.（3）In Sham group and TAC group,the CD72 level of cardiac macrophages of mice transplanted with Rel overexpression monocytes（Rel Mos）was higher than that of vehicle monocytes（Vehicle Mo）（P<0.05）.Under the condition of TAC,compared with the mice transplanted with vehicle Mo,the LVEF of the mice transplanted with Rel Mos mice decreased significantly（P<0.05）,increased the number of cardiomyocyte apoptosis（P<0.05）,increased the level of MDA（P<0.05）,increased the levels of TNF and GSEA-1（P<0.05）.There was no significant difference in the area of cardiomyocytes（P>0.05）.（4）The results of single cell transcriptional group of myocardial infarction and atherosclerosis in mice showed that CD72hi-CMφs level was increased.The transcriptome data of chronic heart failure patients showed that the level of CD72hi-CMφs was also increased（P<0.05）.2.Screening of effective components of NXK regulating macrophage subsets related inflammatory injury in CHF（1）Compared with Sham group,LVEF and LVFS of TAC group were significantly decreased（P<0.05）,LVDD and LVDs were increased（P<0.05）,the size of cardiac cell was significantly increased（P<0.05）,there were more collagen fiber deposition（P<0.05）,apoptosis rate was significantly increased（P<0.05）,and inflammatory factors（IL-6,TNF,IL-1β）were increased（P<0.05）.Compared with the TAC group,the LVEF and LVFS increased（P<0.05）,while LVDD and LVDs decreased（P<0.05）after treatment with NXK and perindopril.Besides,NXK and perindopril could significantly reduce the heart weight ratio（P<0.05）,the myocardial fibrosis（P<0.05）,and the level of IL-6,TNF and IL-1β（P<0.05）.（2）Deconvolution results showed that the CD72hi-CMφs signal of TAC group was higher than that of sham group,while NXK could reduce the CD72hi-CMφs signal.Gene Set Enrichment Analusis（GSEA）results showed that NXK positively regulated Oct1,Sp3,Evi1,Chop,Ahrarnt,Gncf,Thra,and negatively regulated Hsd17b8,Zfp82,Rel.The enrichment fraction of Rel up-regulated gene set was-0.397（adjusted P<0.01）,and that of Rel down regulated gene set was 0.649（adjusted P<0.01）.（3）Based on the homologous modeling of c-Rel crystal structure sequence from foreign body species,the structural protein of mouse c-Rel protein was obtained.Molecular docking simulation was carried out with 21 effective small molecules of NXK.The docking fraction of fructose arginine and c-Rel protein crystal was the highest（total score=8.741,crash=-1.3346,polar=8.5449）.Luciferase Report showed that FA reduced the luciferase activity driven by CD72 promoter（P<0.05）.（4）Immunofluorescence results showed that the cytosolic extract of 1 mg/m L mouse cardiomyocytes could significantly increase the CD72 level of BMDM after 24 hours of intervention（P<0.05）,suggesting that myocardial injury could promote the differentiation of CD72hi-Mφs,while 100μg/m L fructose-arginine intervention could significantly reduce the CD72hi-Mφs level（P<0.05）.（5）Real-time q PCR results showed that compared with the control group,after treatment with fructose arginine group for 24 hours,the levels of TNF and IL-1βin 100μg/m L significantly decreased（P<0.05）.Conclusion:1.Rel mediated CD72hi-CMφs from bone marrow can cause heart injury and may play an important role in many cardiovascular diseases.2.Fructose arginine,a small molecular component of NXK,can inhibit the differentiation process of CD72hi-CMφs and the level of related inflammatory factors.