Authors: Huang Libin, Xu Guoxing, Xie Maosong, Lin Wen, Cui Yi, Li Jianbing
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Background
Retinal microglia(RMG) plays an important role in the pathogenesis of retinal degenerative diseases, while chemokine CX3CL1 participates in the regulation of steady-state of microglia.It has been determined that bone marrow-derived mesenchymal stem cells (BMSCs) have a remarkable role to modulate the immune response and protect the central nervous system through the release of soluble factors in a paracrine fashion and further affect the functional behavior of cells.However, whether BMSCs are able to interact with RMG and activate related signaling pathway for the maintaining of homeostasis in the retina is still unclear.
Objective
The aim of this study was to investigate the interaction between BMSCs and lipopolysaccharide (LPS)-activated RMG in vitro, and dissect the effects of CX3CL1/CX3CR1 signaling pathway on the biological behavior of BMSCs and RMG.
Methods
RMG was isolated from SD rats, cultured with mixed culture of retinal glial cells and purified by shaking.The cells were identified by detecting the expression of CD11b, Iba1 and glutamamine synthetase (GS) with indirect immunofluorescence assay.LPS (1 mg/ml, 2 μl) was added in the medium for 24 hours to stimulate RMG, and then the cells were divided into LPS control group, BMSCs group (cocultured with BMSCs for 24 hours) and CB-BMSCs group (cocultured with CX3CL1-blocking-BMSCs for 24 hours). The cells without LPS stimulation served as the blank control group.The functions of RMG, including the release content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), the proliferation, phagocytosis, and migration of RMG were examined.
Results
RMG was successfully isolated and harvested from SD rats by using mixed culture of retinal glial cells and purified by shaking.CD11b and Iba1 showed the positive expression with the green fluorescence in the cells and GS was absent.The contents of TNF-α in the cell supernatant were (2.55±0.97)ng/ml, (24.91±3.07)ng/ml, (20.38±2.97)ng/ml and (24.90±1.88)ng/ml in the blank control group, LPS control group, BMSCs group and CB-BMSCs group, respectively, showing a significant difference among the groups (F=119.90, P<0.05). The contents of IL-1β in the cell supernatant were (1.12±0.36)ng/ml, (10.40±2.76)ng/ml, (7.00±1.75)ng/ml and (9.55±1.11)ng/ml in the blank control group, LPS control group, BMSCs group and CB-BMSCs group, respectively, showing a significant difference among the groups(F=34.96, P<0.05). The secretory volume of TNF-α and IL-1β were evidently lower in the BMSCs group than those in the LPS control group (both at P<0.05), and no significant differences were found in the secretory volume of TNF-α and IL-1β between CB-BMSCs group and LPS control group (both at P>0.05). The proliferative rate of RMG was lower in the BMSCs group than that in the LPS control group (P<0.05), while there was no statistical difference between BMSCs group and CB-BMSCs group (P>0.05). The mean fluorescence intensity (MFI) and the number of migrated RMG were considerably different among the four groups (F=70.55, 15.49, both at P<0.05), and those in the BMSCs group were significantly increased in comparison with the LPS control group (both at P<0.05), while there was no significant difference between CB-BMSCs group and LPS control group (both at P>0.05).
Conclusions
BMSCs could suppress the proliferation of LPS-activated RMG.Moreover, BMSCs might inhibit proinflammatory cytokines releasing, enhance phagocytosis and migration capabilities of RMG via CX3CL1/CX3CR1 signaling pathway.