| Objective:Heart failure(HF)is a major public health problem in the 21st century.The continuous inflammatory infiltration of myocardial tissue is an important inducement of heart failure after myocardial infarction.The immune network system of monocyte macrophage in the heart spleen axis is an important part of the inflammatory response in heart failure.Therefore,this study focuses on heart failure.In order to investigate the mechanism of Qishen Granule on regulating the mobilization of spleen monocytes,balancing the inflammatory activation of cardiac macrophages and inhibiting heart failure,animal experiment and cell experiment were used.Method:1.The 21 day rat heart failure model was established by ligation of left anterior descending coronary artery,and the intervention of splenectomy was added.The rats were divided into sham operation group,model group,Qishen granule group and splenectomy group.The cardiac function of rats was evaluated by echocardiography.The migration and phenotype of splenic monocytes/macrophages were analyzed by immunohistochemistry.The regulatory role of splenic monocytes/macrophages mobilization in the occurrence and development of heart failure was further explored.2 Using transcriptomics technology combined with network pharmacology analysis method,partial least squares method was used to screen differential genes and differential callback genes of spleen leukocyte immune regulation in control group,model group and Qishen granule group;Cluster principal component analysis was used to verify the differential genes.The biological characteristics of the differentially expressed genes were comprehensively evaluated by GO biological process analysis combined with KEGG pathway enrichment analysis,and the key differentially expressed proteins were verified by Western blot.Objective to comprehensively evaluate the mobilization mechanism of mononuclear cells in spleen of heart failure rats and the specific biological mechanism of Qishen granule in regulating spleen mobilization.3.RAW264.7 macrophages were used,LPS induced inflammation model was used,combined with Qishen granule administration method,divided into control group,model group,Qishen granule group.M1/M2 macrophage surface specific markers CD86 and cd206 were labeled by immunofluorescence technique to explore the regulation of Qishen Granule on macrophage polarization balance in LPS induced inflammation model;the expression levels of key proteins ALOX15,ALOX12 and CCR2 in each group were detected by Western blot;the expression levels of LPS induced inflammation model,control group and model group were detected by lipid peroxidation assay The level of oxylipin secretion of macrophages in Qishen granule group was higher than that in control group.Objective to comprehensively evaluate the mechanism of Qishen granule in inhibiting heart failure by balancing the inflammatory activation of macrophages.Result:1.The results of echocardiography showed the heart function of rats with heart failure at 21 days.Compared with sham operation group,the left ventricular(Ejection fraction EF)of rats with heart failure in model group was significantly decreased(P<0.001),the left ventricular shortening rate(Fractional shortening FS)value decreased significantly(P<0.001);Compared with the model group,the EF value of splenectomy group was significantly increased(P<0.01),left ventricular EF of Qishen granules group was significantly increased(P<0.05);The results of spleen immunohistochemistry showed that compared with the sham operation,the number of CD68 positive cells in the spleen tissue of the model group was significantly decreased(P<0.01);The expressions of CD 163 and CD68 in Qishen granules group were significantly higher than those in model group(P<0.001,P<0.01).2.Spleen transcriptomics detection and differential gene screening showed that 418 genes were co expressed in the model group and the sham group,among which 235 genes were upregulated and 183 genes were down-regulated(P<0.05);218 genes were co expressed in the Qishen granule group compared with the model group,among these genes,79 genes were upregulated and 139 genes were down regulated(P<0.05);104 genes could be co regulated in model group and Qishen granule group,70 genes could be regulated in Qishen granule group and model group on the same direction,and 34 genes could be reversibly regulated in Qishen granule group;GO biological process analysis showed that cell composition Cellular component CC)was the most important factor There are 12 components involved in blood particles,outer plasma membrane and MHC protein complexes,28 Biological processes(BP)involved in complement activation,immune response,antigen processing and presenting cell transport,There are eight Molecular functions(MF),which mainly involve oxygen transport,binding and antigen binding;The results of KEGG pathway enrichment analysis were ranked as the top 20 by P value,which mainly involved immune-related pathways such as antigen processing and presentation,Staphylococcus aureus infection,herpes simplex infection,graftversus-host reaction,allograft rejection and so on.Western blot assay showed that the expressions of STAT3 and CCR2 in spleen tissue of rats in model group were significantly higher than those in sham group(P<0.001,P<0.0001),the expressions of STAT3 and CCR2 in spleen tissue of Qishen granules group were significantly down-regulated compared with model group(P<0.001,P<0.01).3.Immunofluorescence results of RAW264.7 macrophages showed that the volume of RAW264.7 macrophages in model group was significantly larger than that in control group,and most of the cells were rounded,CD86 fluorescence signal became stronger,the color was bright,and M1 polarization phenomenon was enhanced;compared with the model group,the volume of macrophages in the Qishen granule group increased,The fluorescence signal of CD86 was weaker than that of model group.The fluorescence of macrophage CD206 in model group was not significantly different from that in control group,the cell aggregation growth disappeared,the cell volume increased,most of them became round,and the nucleus increased;compared with the model group,the macrophage aggregation growth disappeared,the cell volume increased,the nucleus volume increased,the cell morphology was Octopus type or spindle type,and some cells showed excessive pseudopodia In the elongated state,the fluorescence signal of CD206 was significantly enhanced and distributed on the cell surface,especially in pseudopodia elongated or spindle cells.Western blot assay results showed that the protein expression of ALOX15 in macrophages in the model group was lower than that in the control group,but there was no statistical difference.Compared with the model group,the expression of ALOX15 in the Qishen granule group was increased(P<0.05);There was no significant difference in the expression of ALOX12 and CCR2 among all groups.The results of oxylipin detection showed that:Compared with the control group,the 15-HETE secretion level of macrophages in the model group was significantly increased(P<0.0001),the 15-HETE secretion level in the QSG group was significantly decreased compared with the model group(P<0.05);There was no significant difference in the level of 12-HETE secreted by macrophages between each group and the model group;The secretion level of lipoxine A5(Lipoxi-5,LXA5)in model group was not changed compared with control group,and the secretion level of LXA5 in Qishen granule group was significantly increased compared with model group(P<0.05);The macrophage 4-HDHA secretion content in model group was higher than that in control group(P<0.05),compared with the model group,the 4-HDHA secretion content of macrophages in Qishen granules group showed a downward trend compared with the model group,the difference was not statistically significant.The content of macrophage 7-HDHA secretion in model group was significantly higher than that in control group(P<0.01),the content of 4-HDHA secretion of macrophages in Qishen granules group was lower than that in model group(P<0.05);Compared with control group,the content of macrophage 14(S)-HDHA secretion in model group increased(P<0.05),the secretion level of 14(S)-HDHA of macrophages in Qishi granules group was decreased compared with that in model group,but the difference was not statistically significant.The secretion of 17-HDHA in macrophages in model group and Qishen granule group was significantly higher than that in control group(P<0.05,P<0.05),but there was no significant difference in 17-HDHA secretion level between Qishen granule group and model group;Compared with control group,the secretion of PGF2-? in macrophages in model group and Qishu granule group was significantly increased(P<0.01,P<0.001),however,there was no statistically significant difference in the secretion level of PGF2-? in Qishu granules group compared with model group;The secretion level of 8,9-EET in macrophages in model group was significantly lower than that in control group(P<0.05),there was no significant statistical difference between Qishen granule group and control and model groups;The 11,12-EET secretion levels of macrophages in control group,model group and Qishu granule group were not significantly different.Compared with control group,14,15-EET secretion levels of macrophages in model group and Qishu granule group decreased(P<0.05),the secretion levels of 14,15-EET in macrophages in Qishi granules group were not significantly different from those in model group.The level of macrophage 12-HEPE secretion in model group was significantly higher than that in control group(P<0.05),there was no statistical difference in the secretion level of macrophages in Qishen granule group compared with control group and model group;Compared with control group,the level of macrophage 18-HEPE secretion in model group and Qishu granule group was significantly increased,and the difference was statistically significant(P<0.001,P<0.05),compared with the model group,the secretion level of 18-HEPE in Qishen granules group was significantly decreased,and the difference was statistically significant(P<0.05)Conclusion1.Qishen granule and splenectomy can effectively improve the heart function of rats with heart failure 21 days after myocardial infarction by LAD ligation,and can effectively prevent the decrease of spleen monocytes/macrophages after myocardial infarction;2.Qishen granule can inhibit the inflammatory infiltration of splenic leukocytes and the migration of monocytes by mediating aloxl5-stat3 pathway,and improve the cardiac function.3.Qishen granule can down regulate M1 polarization level of macrophages,up regulate M2 polarization level of macrophages,and regulate the the inflammatory activation balance of macrophages;Qishen granule regulate the inflammatory balance effect of macrophages,which is related to the upregulation of ALOX15 protein;Qishen granule can down regulate the metabolism level of pro-inflammatory lipid mediator 15-HETE,up regulate the metabolism level of SPMs pro-inflammatory mediator LXA5,and mediate EETs.As well as a variety of SPMs synthesis precursor HDHAs,HEPEs metabolic balance,play on macrophage inflammatory activation regulation. |
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