| Objective:This research was aimed to find new targets for prevention and adjustment of ventricular remodeling after myocardial ischemia-reperfusion injury. We hope to provide a new way of thinking and powerful experimental evidences for the application of stem cells in the prevention and treatment of heart failure after myocardial infarction.Methods:①M/R models in rats were set up. Neutrophil numbers in blood were counted using automated hematology analyzer. Infiltrations of neutrophils and macrophages in hearts were analyzed using HE staining, immunofluorescence staining and flow cytometry analysis. Thus, time points of ADSCs transplantation were determined.②ADSCs in rats were isolated, cultured and identificated, and then were intravenous infused into MI/R model at different time points. Indications of cardiac function, such as FS, EF,+dp/dtmax, dp/dtmin were measured by echocardiography and hemodynamic detection at 28 d. The areas of myocardial infarction were observed using TTC/Evans blue staining at 48 h. Infiltrations of neutrophils and macrophages and the expressions of CDllb in neutrophils and CD 163 in macrophages at different time points within 72 h were analyzed using HE staining, immunofluorescence staining and flow cytometry analysis. ③The expression of CD 163 in macrophages and macrophage efforcytosis of neutrophils were determined by flow cytometry analysis at 48 h. Macrophages and neutrophils in rats were isolated, cultured and identificated. Neutrophils were labeled with CFSE and their apoptosis were analyzed by flow cytometry analysis. Macrophages and neutrophils were co-cultured with or without ADSCs, and the expression of CD 163 in macrophages and macrophage efforcytosis of neutrophils were determined by flow cytometry analysis and immunofluorescence staining.Results: ①Compared with control group, neutrophil number in blood changed over time that the number of neutrophils in the MI/R group continued to rise after 12 h, and a large number of neutrophils infiltrated into hearts in the MI/R group and peaked at 24h.②Compared with MI/R group, FS、EF、+dp/dtmax and-dp/dtmin in ADSCs groups were significantly improved at 28 d, infarct size and infarct size/AAR were significantly decreased at 48h, and immediate transplantation had a better outcome than that of 24 h later. When ADSCs were transplanted immediately, ratios of neutrophils in hearts were significantly decreased, while CD11b expression of neutrophils were significantly increased 24 h after. When ADSCs were transplanted at 24 h, ratios of WBC and neutrophils in hearts were significantly decreased, while ratio of macrophages and CD 163 expression of macrophages were significantly increased at 72 h.③When ADSCs were transplanted at 24 h, neutrophils’ CD163 positive rate increased significantly, compared with MI/R group. Macrophages’CD 163 positive rate increased significantly when co-cultured with ADSCs in vitro. After adding early apoptotic neutrophils, macrophages’ CFSE positive rate increased obviously and CFSE+CD 163+ cells increased significantly at 24 h, when co-cultured with ADSCs.Conclusions: ① Neutrophil infiltration is a hallmark in early inflammatory response after MI/R. The number of neutrophils changes over time, which peaked at 24 h, meaning that resolution of inflammation begans at 24 h. ②ADSCs transplantation could alleviate ischemia-reperfusion injury, decrease the infarct size, and improve heart function, and immediate transplantation at the beginning of inflammation had a significant outcome. ADSCs mediate neutrophil infiltration, which promote neutrophils’ removal without affecting their adhesion. Thus, inflammatory reaction could be adjusted more properly after MI/R.③ADSCs could stimulate macrophage polarized into M2 type through paracrine mechanism, which enhance their phagocytosis of apoptotic neutrophils, thus promote the resolution of inflammation, accelerate the tissue repair, and eventurally improve heart function after MI/R. |
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