Association between Golgi apparatus morphological alteration and A1 astrocytes

AimTo investigate the relationship between Golgi morphology and astrocyte activation.MethodsCTX-TNA2 cells were divided into the following groups: the control group, the astrocyte conditioned medium (ACM) group, the ACM + 10 μmol·L-1 calycosin (CAL) group, the ACM + 25 μmol·L-1 CAL group, and the ACM + 50 μmol·L-1 CAL group. ACM, a pre-prepared culture medium, was used for inducing A1 astrocyte. In the ACM group, cells were stimulated with ACM for 24 h. In the CAL+ACM groups, cells were pretreated with the corresponding concentration of CAL for 6 hours before being stimulated with ACM for an additional 24 h. First, the cells in the control group and the ACM group were incubated with DMEM high-glucose medium containing Golgi apparatus fluorescent probes. The morphology of the Golgi apparatus was subsequently observed using the MICA full-scene microscopic imaging analysis platform. Next, real-time fluorescence quantitative PCR and cellular immunofluorescence were employed to measure the indicators related to A1 astrocyte activation to verify the regulatory effect of CAL on A1 astrocyte activation. Third, after ACM-stimulated AS were treated with CAL, the gradual changes in Golgi apparatus morphology upon inhibition of astrocyte activation were observed by MICA full-scene microscopic imaging analysis platform. Meanwhile, using Western blot or immunofluorescence, we further examined the expression alterations of proteins associated with Golgi apparatus morphology in ACM-stimulated astrocytes after treatment with CAL, and the changes in the signaling pathways associated with Golgi fragmentation.ResultsThe observation of Golgi apparatus morphology revealed that, compared with the control group, the ACM group exhibited dissociation of the Golgi structure and the formation of a large number of dispersed vesicles. PCR and immunofluorescence analyses demonstrated that CAL intervention dosedependently attenuated the ACM-induced C3, iNOS, and IL-1β mRNA expressions and the activation of signal molecules related to A1 astrocyte activation. Further fluorescence probe staining of the Golgi apparatus suggested that, CAL treatment of A1 astrocytes could dose-dependently diminish the formation of Golgi apparatus vesicles within astrocytes. Additionally, Western blot and immunofluorescence results indicated that CAL could dose-dependently prevent the decrease in the expression of proteins associated with Golgi apparatus morphology induced by ACM, and block the activation of the UNC-5-like kinase 1/Ras-like GTPase 9 signaling pathway.ConclusionsThe fragmentation of the Golgi apparatus exhibits a synchronous correlation with the A1 astrocyte activation and may play a role in the evolutionary development of A1 astrocytes.