Effects of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived exosomes on HIBD immunoinflammation in neonatal rats

Objective To investigate the effects of UC-MSCs-EXOS on immune inflammation of microglia caused by HIBD in neonatal rats. To explore the neuroinflammatory repair possibility and mechanism of UC-MSCs-EXOS transplantation in neonates with HIE. Methods In vitro experiments: The Oxygen and glucose deprivation (OGD) model was established. The growth and proliferation status of HAPI microglia was measured by CCK-8 method, the most suitable concentration of UC-MSCs–EXOS for the growth of HAPI cell line after OGD was identified. The expression of inflammatory genes in HAPI cell line after OGD were detected by q-PCR. In vivo experiments:1. The HIBD model was established in vivo, the rats were divided into four groups: Sham operation control group (SHAM group), HIBD group, Exosomes intervention group (EXOS group), miRNA-146a-5p knockdown in EXOS intervention group (miRNA-146a-5p K/d group). the changes of M1 (CD16) and M2 (CD206) of rat microglia and the upstream signaling pathway of inflammatory factors were detected by Western blot after 72 hours of sampling. The gene expressions of M1 (CD16) and M2 (CD206) co-stained with microglia marker Ibal were detected by immunofluorescence. The expression of inflammation factors in rat brain tissue were detected by q-PCR. 2. Finally, the rats underwent a water maze test at 28 days to evaluate their spatial memory. Results Through in vitro experiments, we confirmed that the most suitable concentration of UC-MSCs-EXOS for the growth of HAPI cell line was 25 ng/ml. After OGD 6 hours, the expression of inflammatory factors IL-1β, IL-6 and TNF-α in HAPI cells were upregulated (P<0.05) , whereas downregulated after 25 ng/ml EXOS co-culture for 24 h (P<0.05) . Through in vitro experiments, we confirmed that UC-MSCs-EXOS could reduce microglial inflammation, in vivo experiments were continued to be explored. In vivo experiments: after injection of 50 μg UC-MSCs-EXOSinto the lateral ventricle of rats, Western blot analysis showed that the expression of M1 proinflammatory marker (CD16) was decreased, while the expression of M2 anti-inflammatory marker(CD206) was increased (P<0.05) ; Immunofluorescence results showed the same trend. the expression of inflammatory cytokines IL-1β, IL-6, TNF-α were downregulated ( P<0.05) , the expressions of inflammatory factor upstream proteins IRAK and TRAF6 were downregulated after EXOS intervention ( P<0.05) ; After knockdown of miRNA-146a-5p with siRNA, various proteins and genes showed the opposite trend against unmodified UC-MSCs-EXOS intervention, Western blot analysis showed that the expression of M1 proinflammatory marker(CD16) was increased, while the expression of M2 anti-inflammatory marker (CD206) was decreased (P<0.05) ; Immunofluorescence results showed the same trend. The expression of inflammatory cytokines IL-1β, IL-6, TNF-α were upregulated (P<0.05) , the expression of inflammatory factor upstream proteins IRAK and TRAF6 were upregulated after the intervention of knocked down exosomes (P<0.05). Water maze experiment: Compared with SHAM group, in the space exploration experiment, the number of platform crossings in HIBD group and miRNA-146a-5p K/d group were reduced (P<0.05) . Compared with HIBD group and miRNA-146a-5p K/d group, the number of platform crossings in EXOS group was increased, and the difference was statistically significant (P<0.05). EXOS intervention showed a promoting effect on long-term memory recovery in rats. Conclusion UC-MSCs-EXOS showed stem cell immunoregulation properties, down-regulating inflammatory factors IL-1β, IL-6 and TNF-α in HIBD rats and promoting microglia transformation from M1 proinflammatory phenotype to M2 phenotype. Finally presenting anti-inflammatory properties. which is accomplished by miRNA-146a-5p through IRAK and TRAF6 pathways.