MSC Exosomes Inhibit Preeclampsia By Blocking Neutrophil Ccl3-Ccr1 Signaling Axis And Its Mechanism

Objective: To clarify the therapeutic effects of uc MSC-exosome on preeclampsia,and to explore the target cell and response molecular mechanism of uc MSC-exosome regulating the placental microenvironment of preeclampsia.Methods:(1)The animal model of preeclampsia was established by intraperitoneal injection of nitroso-L-arginine methyl ester into sexually mature SD rats,and low-dose uc MSC-exosome,high-dose uc MSC-exosome and normal saline were injected into each group through the tail vein on the 14-19 days of pregnancy.The blood pressure,urine protein,fetal rats and placenta in each group of rats were observed.(2)The placental cell types of the three groups of SD rats were sorted by single cell sequencing and target cells were identified.(3)222 patients with preeclampsia and 396 normal pregnant women without complications of the Affiliated Hospital of Jining Medical College from September 2018 to June 2021 were selected as the study subjects.The difference of neutrophil count in the last blood routine before delivery was analyzed.(4)The neutrophils in the placental microenvironment were isolated and cultured,and uc MSC-exosome was added for co-culture in vitro to analyze their chemotaxis.(5)The single cell sequencing results of SD rat placenta were analyzed by Cell Phone DB cell communication,and the signal communication axis and intensity of receptors and ligands between neutrophils and cells in placenta(including neutrophils themselves)were analyzed.Results:(1)By comparing the general conditions of rats in each group,it was found that uc MSC-exosome could significantly reduce the hypertension and proteinuria of preeclampsia rats(P<0.05);there was also a significant improvement in the development of fetal litters.(2)The results of single cell sequencing that were analyzed by Marker gene showed that the most obvious cells in the three groups were neutrophils.The number of neutrophils in the preeclampsia model group was significantly increased,while the number of neutrophils in the uc MSC-exosome treatment group was significantly lower than that in the preeclampsia model group.(3)Analysis of the routine blood results of preeclamptic patients and normal pregnant women showed that the neutrophil count in peripheral blood was significantly higher in preeclamptic patients than in normal pregnant women(P<0.05).(4)In vitro experiments with neutrophils in the placental microenvironment revealed that uc MSC-exosome significantly inhibited neutrophil chemotaxis.(5)The Ccl3-Ccr1 signaling axis was found to be the most important core signaling pathway of neutrophils by Cell Phone DB cell communication analysis.The m RNA expression analysis of neutrophils in the three groups showed that Ccr1 and Ccl3 were significantly increased in the preeclampsia model group.After uc MSC-exosome transplantation,the expression of Ccr1 was significantly decreased,but the expression of Ccl3 was not decreased.Conclusion: In this study,we found that(1)uc MSC-exosome significantly reduced blood pressure and urinary protein in rats with pre-eclampsia and effectively improved the development of fetuses.(2)Neutrophils may be the key target cells of uc MSC-exosome in the treatment of preeclampsia.(3)uc MSC-exosome may block the activation of the Ccl3-Ccr1 axis by targeting the expression of Ccr1 to exert its blocking effect on neutrophil chemotaxis and migration.In summary,we believe that uc MSC-exosome inhibits preeclampsia by blocking the neutrophil Ccl3-Ccr1 signaling axis.