| Aim:Heart failure(HF),as the end stage of various cardiovascular diseases,we aimed to control the phenotype and mobilization mechanism of heart-spleen monocytes/macrophages as the key entry point.The HF models were prepared by left anterior descending coronary artery(LAD)ligation,and we explored the relationship between the spleen and its released inflammatory cells and cardiac function from the aspects of function and structure.We aimed to identify the mechanisms of Qishen granules(QSG)regulate the "heart-spleen axis"monocytes/macrophages phenotype inhibits HF through splenic AT1-cardiac monocyte chemotactic protein-1(MCP-1)/CC chemokine receptor 2(CCR2)pathway.Methods:1.To explore the role of the spleen mobilization mechanism in HF,the HF models were prepared by LAD ligation and then rats were randomly divided into the model group,splenectomy group,and the positive drug fosinopril group.The sham operation group was also established as a control.The splenectomy group underwent total splenectomy before LAD ligation.The cardiac function of rats in each group was evaluated by echocardiography.Haematoxylin-eosin(HE)staining was used to detect inflammatory cell infiltration in myocardial tissue and spleen basic structure.The changes of cell table type were detected by immunofluorescence(IF)staining.2.To explore the mechanism of QSG "treating from the spleen" inhibited heart failure,the HF models were prepared by LAD ligation and then studied by continuous intragastric administration for 21 d.The cardiac function of rats in each group was evaluated by echocardiography.HE staining was used to detect the changes of monocytes in the spleen tissue.Radioimmunoassay was used to detect the content of Ang Ⅱ,Western blot(WB)was used to detect the expression of AT1,MCP-1 proteins in the spleen tissue.3.To further verify QSG inhibited HF through regulating the balance of M1 and M2 macrophages in the heart tissue,M1 and M2 macrophages were detected by IF staining.The deposition of collagen were detected by Masson staining and Sirius red staining.Immunohistochemical(IHC)staining of CD31 showed that the number of microvessels.The expression of MCP-1,CCR2,AT1,transforming growth factor-β1(TGF-β1)/Smad3 fibrosis-related proteins and angiogenesis-related protein were detected by WB.4.QSG on Phenotype Changes of monocytes/macrophages in "heart-spleen axis" of heart failure mice,because mouse flow cytometry antibodies are more comprehensive,therefore,the HF mouse models were prepared by LAD ligation,after 24 h,7 d and 14 d in each group,cardiac function measured by echocardiography.Serum levels of creatine kinase-MB(CK-MB)and lactate dehydrogenase 1(LDH1)content were detected by blood biochemical tests within each group of mice.Based on the above results,the efficacy of QSG in different periods of HF was evaluated.Since QSG have the most significant regulation of cardiac function during the recovery period of heart failure(7 days after administration),this time point was used to further explore the specific mechanism of spleen-heart monocytes/macrophages migration and differentiation after HF.The monocytes of spleen mice were detected by HE staining.Changes in phenotype and number of monocytes in the spleen and circulation,and different macrophages in myocardial tissue were detected by Flow cytometry(FCM).ELISA was used to detect the expression of Ang Ⅱ in mice.The above results explain the effect of QSG on phenotype changes of monocytes/macrophages in"heart-spleen axis" after heart failure.Results:1.Echocardiography showed the ejection fraction(EF)and fractional shortening(FS)were decreased significantly in model group.The splenectomy group and the positive drug fosinopril group can significantly improve the heart function of rats.HE staining showed that the inflammatory cells in the spleen was decreased after treatment by positive drug fosinopril compared with the model group.HE staining showed that splenectomy and the positive drug fosinopril could significantly down-regulate inflammatory cell infiltration in myocardial tissue.IF staining results showed that the number of M1 macrophages was increased significantly in the model group relative to the sham group.After splenectomy,the number of M1 macrophages was reduced significantly and M2 macrophages remained unchanged relative to the model group,the positive drug fosinopril group could inhibited Ml macrophages and increased M2 macrophages.2.Echocardiography showed that QSG could effectively inhibit the decline of cardiac function.HE staining results showed that QSG could significantly increase the number of monocytes in red pulp under the capsule and marginal zone of the spleen.Radioimmunoassay showed that the circulation level of angiotensin Ⅱ(Ang Ⅱ)increased in model group and was inhibited by QSG.Moreover,WB results showed that treatments of QSG could significantly down-regulate the levels of AT1 and MCP-1.3.IF staining results showed that compared with the sham group,the number of M1 macrophages significantly increased in the model group,and the number of M2 macrophages was less.QSG could reduce the number of M1 macrophages and increase the number of M2 macrophages in the border zone of infarcted heart.The results of Masson staining and Sirius red staining showed that QSG could inhibit the excessive deposition of myocardial interstitial collagen.IHC staining of CD31 showed that,the number of microvessels was significantly up-regulated after treatment with QSG WB results showed that the effects of QSG could significantly down-regulate the expressions of AT1,MCP-1 and CCR2,inhibition of TGF-β1/Smad3 fibrosis expression proteins.In addition,QSG could up-regulate the expressions of levels of angiogenesis-related protein.4.Echocardiography showed that EF and FS were decreased significantly in model group by LAD ligation 24 h,after treatment with QSG,EF and FS value were increased.Cardiac function measure by echocardiography after 7 d and 14 d,and prolong the intragastric administration time to 14 d,the EF and FS of mice in QSG group had no significant change compared with the 7 d in QSG group.The results of myocardial zymogram showed that,after 24 h,CK-MB and LDH1 were increased.The changes of CK-MB and LDH1 were not obvious in the 7 d and 14 d model group.24 h after treatment with QSG,cardiomyocytes could inhibit the release of CK-MB and LDH1.Therefore,7 d after treatment with QSG,to explore the specific mechanism of spleen-heart monocytes/macrophages migration and differentiation after heart failure.After 7 d,the general structure of the spleen showed that HF could increase the length of the spleen,after treatment with QSG,the structure of the spleen tended to be normal.HE staining results showed that QSG could increase the number of monocytes in red pulp under the capsule and marginal zone in the spleen by continuous intragastric administration for 7 d.FCM showed that QSG could significantly increased the number of CD11b+ and a large number of Ly6Chigh monocytes were found in the model,while in QSG can inhibit the expression of Ly6Chigh monocytes.Blood FCM showed that QSG could significantly reduced the number of CD11b+.In response to the initiating factors of spleen mobilization,the circulation level of angiotensin Ⅱ increased in the model,while the circulation level of angiotensin Ⅱ inhibited by QSG Cardiac FCM showed that QSG could significantly reduced the number of F4/80+CD11b+.Conclusions:Our results showed that the spleen released inflammatory cells and recruited by damaged myocardium to improve heart function.The mechanism is related to the activation of the AT1 receptor in the spleen.QSG could effectively inhibit the decline of cardiac function,the mechanism is related to the release inflammatory cells by inhibiting the activation of AT1 receptors in the spleen.In myocardial tissues,on the one hand,QSG can inhibit the recruitment of monocytes in the injured myocardium MCP-1/CCR2 pathway,on the other hand,QSG can regulate the phenotype of macrophages.QSG mainly anti-myocardial fibrosis by inhibited the TGF-β1/Smad3 pathway by M1 macrophages,at the same time,it can also promote the differentiation of M2 macrophages and produce vascular endothelial growth factor to promote angiogenesis.Specifically,QSG can inhibit the monocytes mainly released by the spleen,which were mainly pro-inflammatory Ly6Chigh,and then inhibit the M1 macrophages in the myocardial tissue.This study by elucidates the heart-spleen connection and the mechanism of QSG intervening in different heart failure models.Our results not only shows that the spleen releases of inflammatory cells and participates in the formation of HF heart function,but also provides a scientific basis for the traditional Chinese medicine compound QSG to regulate the immune network with monocytes/macrophages phenotype and migration as the core,at the same time,it also provided new target for heart failure drugs. |
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