Abstract: BACKGROUND: Much of the current research on M2 macrophage-derived exosomes focuses on their effects on wound healing and osteoblast proliferation and differentiation, while few studies have focused on their role in regulating microglia phenotype. 
OBJECTIVE: To discuss the role and molecular mechanisms of M2 macrophage-derived exosomes in the phenotypic regulation of microglia.
MERHODS: (1) Bone marrow primary macrophages were extracted and then stimulated with 50 ng/mL interleukin 4 for 24 hours to promote macrophage M2-type polarization. Flow cytometry and cellular immunofluorescence were used to identify the M2-type macrophage marker CD206. (2) M2 macrophage-derived exosomes were extracted and identified. (3) Microglia BV2 were randomly divided into three groups: control group, lipopolysaccharide group, and treatment group. No treatment was done in the control group. 500 ng/mL lipopolysaccharide was added to the intervention for 24 hours in the lipopolysaccharide group. 500 ng/mL lipopolysaccharide and 25 μg/mL M2 macrophage-derived exosomes were added simultaneously to the treatment group for 24 hours. ELISA was performed to detect the secretion of tumor necrosis factor α and interleukin 10 in the culture supernatant. qRT-PCR was performed to detect the mRNA expression of inducible nitric oxide synthase, arginase 1, interleukin 1β, and interleukin 10 in the cells. Western blot assay was performed to detect the protein expression of inducible nitric oxide synthase, arginase 1, and nuclear factor-κB signaling pathway related protein expression.
RESULTS AND CONCLUSION: (1) ELISA results showed that the secretion of tumor necrosis factor α was significantly increased in the lipopolysaccharide group compared with the control group. The secretion of tumor necrosis factor α was reduced and the secretion of interleukin 10 was increased in the treatment group compared with the lipopolysaccharide group. (2) The qRT-PCR results showed that compared with the control group, the mRNA expression of interleukin 1β and inducible nitric oxide synthase increased in the lipopolysaccharide group. Compared with the lipopolysaccharide group, the mRNA expression of interleukin 1β and inducible nitric oxide synthase decreased, and the mRNA expression of interleukin 10 and arginase 1 increased in the treatment group. (3) Western blot assay results showed that the expression of inducible nitric oxide synthase protein was increased in the lipopolysaccharide group compared with the control group. The expression of inducible nitric oxide synthase protein was decreased and the expression of arginase 1 protein was elevated in the treatment group compared with the lipopolysaccharide group. (4) Compared with the control group, the expression of p65 and p-IκB-α proteins in the nuclear factor-κB signaling pathway was reduced in the lipopolysaccharide group, whereas the expression of p65 and p-IκB-α proteins was elevated in the treatment group compared with the lipopolysaccharide group. The results showed that M2-type macrophage-derived exosomes could significantly inhibit lipopolysaccharide-induced inflammatory responses in microglia, enhance the expression of the anti-inflammatory factor interleukin 10, suppress the expression of the pro-inflammatory factors tumor necrosis factor α and interleukin 1β, and promote microglial cell phenotypes polarized from the M1-type to the M2-type. The mechanism may be related to the inhibition of nuclear factor-κB signaling pathway activation by M2-type macrophage-derived exosomes.

Key words: M2 macrophages, exosomes, microglia, inflammation, nuclear factor-κB signaling pathway, neuropathic pain, polarization