ABSTRACT
Objective:To investigate the effects of induced pluripotent stem cell-derived exosome (iPSC-Exo) on releasing inflammatory factors from microglia induced by lipopolysaccharide (LPS).Methods:iPSC derived from the tubular epithelial cells of sepsis encephalopathy patients were resuscitated and cultured. The iPSC-Exo was isolated by low-temperature ultracentrifugation and analyzed by transmission electron microscopy, Western blot and high sensitivity flow cytometry (HSFCM). Based on the concentration of iPSC-Exo, human microglia line HMO6 cells activated by LPS (100 ng/mL) were divided into four groups randomly: LPS+ phosphate buffer solution (PBS) group, LPS+iPSC-Exo 10 5 group, LPS+iPSC-Exo 10 6 group and LPS+iPSC-Exo 10 7 group. The control group was added equal PBS but not LPS. After culture for 24 h, the concentrations of malondialdehyde in cells were detected. Quantitative RT-PCR was used to measure the mRNA expression levels of macrophage inflammatory protein 2 (MIP2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in the cells and enzyme-linked immunosorbent assay (ELISA) was used to assess the concentration of these cytokines in the supernatant. Under the same concentration of iPSC-Exo, one-way ANOVA and SNK- q test were used for comparison between groups. Results:The extracts showed spherical membrane structure by transmission electron microscopy. HSFCM showed the mean diameter of the extracts was (74.66±15.60) nm and the concentration around 2.98×10 10/mL. Western blot analysis showed high expression of exosome markers CD63, Alix and TSG101, but not GM130. Intracellular MDA concentration and mRNA expression levels and protein concentration of MIP2, TNF-α, IL-1β and IL-6 in the LPS+PBS group were significantly higher than those in the control group (all P<0.01). With the increase of iPSC-Exo concentration, the intracellular MDA concentration decreased gradually ( P<0.01), the mRNA expression levels of inflammatory factors showed a gradual downward trend (all P<0.05). Each inflammatory cytokine in the supernatant declined in a manner that was almost consistent with mRNA. Concentrations of MDA remained constant in the control group. Conclusions:iPSC-Exo derived from the tubular epithelial cells of sepsis encephalopathy patients alleviate oxidative stress and inflammation effect of microglia induced by LPS, and the modulatory effect is dose-dependent.