Activated microglia promoted neuronal apoptosis, whereas ADSCs inhibited neuronal apoptosis.
TUNEL assays were used in this study to evaluate the effects of activated microglia on neuronal apoptosis (Fig. 1A). As described earlier, the microglia were activated via application of 1 µg/mL LPS before co-culturing with neurons. The results of the TUNEL assay clearly demonstrated that neuronal apoptosis was significantly increased at the indicated time points following incubation with activated microglia (Fig. 1B, LPS group vs control group, P < 0.05 after 12, 24, and 48 h). Treatment with ADSCs-CM significantly inhibited neuronal apoptosis (Fig. 1B, ADSCs-CM group vs, control group, P < 0.05 after 12, 24, and 48 h). Compared with the LPS group, the ADSCs-CM + LPS group showed reduced neuronal apoptosis (Fig. 1B, P < 0.05 after 12, 24 and 48 h). These results indicated that inflammation caused by microglial cells exerted a harmful effect on neurons, and that ADSCs or ADSCs-CM could exert protective effects on neurons by reducing the rate of apoptosis.
ADSCs inhibited microglia secretion.
The study further analyzed cytokine and chemokine production by microglia cultured in ADSCs-CM to better characterize the inhibitory effect of ADSCs on microglia inflammation. The supernatants were collected after subjecting microglia to different treatments described earlier. The levels of six cytokines and chemokines (TNF-α, IL-1β, IL-10, MCP-1, RANTES, and MIP-2) were determined using Bio-Plex Pro assays. The results showed that the microglia produced more TNF-α, IL-1β, RANTES, and MIP-2 than those in the control group after incubation in 1 µg/mL LPS (P < 0.001 for each cytokine), while ADSCs-CM significantly suppressed the secretion of these factors (Fig. 2A–2D). The levels of anti-inflammatory cytokines such as IL-10 and chemokines such as MCP-1 were also significantly higher in LPS-treated microglia compared with the control group (Fig. 2E and 2F, P < 0.001). An analysis of the difference between the ADSCs-CM and control groups revealed that the supernatant obtained from ADSCs-CM-treated groups contained more IL-10 and MCP-1, indicating that ADSCs promoted the production of large quantities of these factors. In some ADSCs-CM, SMT was added to inhibit the secretion of NO. The ability of microglia to secrete inflammatory cytokines cultured in these supernatants significantly increased (Fig. 3).
ADSCs inhibited the expression of activated microglia receptors.
Flow cytometry was used to detect the expression of microglia surface receptors CD68 and CD11b (Fig. 4). When microglia in the four groups were treated for 48 h, the expression of the microglia surface receptor CD68 significantly increased in the LPS group compared with the control group (P < 0.05). However, the expression of CD68 was significantly lower in the ADSCs-CM group than in the control group (P < 0.05). In addition, the cells cultured in the CM + LPS group showed lower expression of CD68 compared with the cells cultured in the LPS group (P < 0.05). The study also found that the expression of CD11b on the cell surface was not significantly different among the four groups. CD68 is a typical protein marker of neurotoxic pro-inflammatory M1 microglia. Hence, the results showed that ADSCs therapy was associated with a decrease in the number of brain M1 microglia.
ADSCs significantly decreased distances of microglia migration in wound healing assay.
Microglia are known to be highly migratory cells as other macrophage populations; they can quickly move to the sites of brain damage after detecting small homeostatic disturbances. Microglia migration during wound healing in vivo was mimicked using a wound healing assay, and whether ADSCs could affect this important property of microglia was investigated. As shown in Fig. 5, the control microglia and LPS-treated microglia spontaneously migrated and filled 36.18 ± 4.75% and 59.87 ± 10.44% of the wounded region, respectively, after 24 h of culture. In contrast, the migration ability of microglia cultured with ADSCs-CM was obviously weakened. The distance of cell migration in the ADSCs-CM groups (ADSCs-CM and CM + LPS groups) significantly decreased 24 h after scratching, and the wound closure was reduced by 65.21% and 64.10% compared with control and LPS-treated microglia, respectively. It was speculated that ADSCs might secrete some extracellular effector factors with the capacity to suppress microglial migration.
ADSCs reduced the average number of migrating microglia in the Transwell assay.
Transwell migration assays were performed to further confirm the inhibitory effect of ADSCs on microglial migration. First, the microglia were seeded into the upper chambers and then placed into bottom chambers containing either ADSCs (co-culture group) or no cells (control group). The number of microglia that migrated from the upper surface of the membrane to the lower surface was tested. The bottom wells were also filled with ADSCs-CM instead of ADSCs (ADSCs-CM group) to assess whether the number of migratory microglia could be influenced by molecules secreted by ADSCs into the extracellular media alone. As shown in Fig. 6, fewer cells in the co-culture group passed through the membrane compared with cells in the control group (co-culture group vs control group, P < 0.01 after 12 and 24 h). A sharp decline in chemotactic migration toward the serum was found in cells in the ADSCs-CM group compared with the cells in the control group (ADSCs-CM group vs control group, P < 0.05 after 12 h and P < 0.05 after 24 h).
ADSCs inhibited microglial phagocytosis.
The phagocytosis of latex beads by microglia was observed under a fluorescence microscope. As shown in Fig. 7, microglia activated with 1 µg/mL LPS (LPS group) phagocytosed more green fluorescent latex beads compared with the microglia in the control group. Meanwhile, the microglia cultured in ADSCs-CM phagocytosed fewer latex beads compared with the microglia in the control group. The microglia cultured in ADSCs-CM with LPS (CM + LPS group) showed depressed phagocytic ability compared with the microglia cultured in LPS alone (LPS group).