Objective: To illustrate the role of epidermal growth factor (EGF) secreted by astrocytes in the process of tacrolimus (FK506) in promoting neurite outgrowth.
Methods: The spinal cord astrocytes and neuronal cells were isolated respectively from 2-day-old Sprague Dawley (SD) rats and 15-day SD pregnant rats, and cultured in vitro and identified by immunofluorescence staining. The spinal cord astrocytes were cultured with 20 µmol/L FK506 medium in the experimental group, and with FK506 free medium in the control group. The supernatant was collected after 24 hours for preparing conditioned medium, and astrocytes were collected. EGF proteins in the conditioned medium were detected with ELISA, and EGF gene expressions of astrocytes were detected with real-time quantitative PCR (RT-qPCR). The spinal cord neurons were cultured respectively with conditioned medium from the experimental group (FK506-CM) and the control group (C-CM) in group A and group B, also with neutralized C-CM and neutralized FK506-CM with anti-EGF neutralizing antibodies in group C and group D. Both the total neurite length and the longest neurite length were measured and compared among groups.
Results: Both astrocytes and neurons were confirmed by immunofluorescence staining. The EGF content of experimental group (0.241 ± 0.044) was significantly higher than that of the control group (0.166 ± 0.014) (t = 3.93, P = 0.01); EGF gene expression of the experimental group (1.12 ± 0.25) was significantly higher than that of the control group (0.46 ± 0.11) (t = 5.78, P = 0.00). The neurite length measurement displayed that the total neurite length and the longest neurite length of groups C and D were significantly shorter than those of groups A and B (P < 0.05). Both the total and longest neurite length of group A were significantly longer than those of group B (P < 0.05), but no significant difference was shown between groups C and D (P > 0.05).
Conclusion: The EGF secreted by spinal cord astrocytes can promote the neurite outgrowth. So spinal cord astrocytes can be used as an important intermediary target of FK506 to promote the recovery of neurological function.