The Effect Of Astrocytes Stimulated By LPS On Neurites Growth And Mechanism

Background and Objective Traumatic brain injury(TBI) is a common clinical disease with extremely high mortality and disability rate, whose disappointed recovery is closely related with axonal impaired regeneration. Inflammation brings significant changes of micro-environment after injury. The increase of inhibitory factors and the lack of promoting factors restrict axonal regeneration. Ephrin A3(one of axonal guiding molecules) that is mainly expressed on astrocytes is the key activator of neuronal membrane protein Eph A4. It is unclear whether astrocytes affect neurites growth through the change of ephrin A3 under inflammation. Moreover, recent studies showed that anaphase promoting complex(APC) and its subunit Cdh1 are involved in the regulation of axonal growth. Effect induced by the knockout of Cdh1 is similar with the downregulation of Eph A4, both of which promote neurite growth. However, it is unknown that whether Cdh1 interacts with Eph A4 in regulating neurite growth. In this study, in order to investigate the change of ephrin A3 expression and its influence on neurites growth of astrocytes under inflammation, LPS is used to sitimulate astrocytes imitating inflammation. Through the downregulation of Eph A4 by si RNA and Cdh1 by Lv-cdh1-si RNA, we will explore the role of APC-Cdh1 in the process of ephrin A3/Eph A4 regulating neurites growth.Methods and Results Methods: Experiment 1(The change of ephrin A3 expression and the influence on neurites growth of astrocytes in inflammatory condition): Astroytes cultured in vitro were randomly divided into C group and LPS group(1, 5, 10, 20, 40 μg/ml), to determine the optical stimulated concentration by cell counting and Western blot examing of PCNA. The optical concentration of LPS sitimulating astrocytes, expression of ephrin A3 at different time points(12 h, 24 h, 48 h, 72 h) was analyzed by Western blot between C group and LPS group; Neurons(DIV 6 d) were planted on the astrocyte surface of C goup and LPS group or cultured by the medium of both groups respectively for 24 h. Lengths of the longest neurites were calculated by immunoflurescence. After downregulation of Eph A4 by si RNA, neurons were planted on astrocytes of C group and LPS group, dividing into contol-C, control-LPS, si RNA-C, si RNA-LPS group.Immunoflurescence was used to measure lengths of the longest neurites. Experiment 2:(The role of APC-Cdh1 in the process of ephrin A3/Eph A4 restricting neurites growth):Recombinant ephrin A3-Fc-Ig G and Fc-Ig G clustered with goat anti-human Ig G-Fcγ were adopted to culture neurons replacing astrocytes of LPS group and C group respectively and were divided into A3-Fc group and Fc group. Lengths of the longest neurites were calculated by immunoflurescence while the activation of Eph A4 and the interaction among APC, Cdh1 and Eph A4 were detected by co-immunoprecipitation. After downregulation of Cdh1 by Lv-cdh1-si RNA, neurons were planted on the cell culture cluster coated by clustered ephrin A3-Fc-Ig G or Fc-Ig G, dividing into Lv-control-Fc group, Lv-control-A3-Fc group, Lv-cdh1-Fc group and Lv-cdh1-A3-Fc group. Immunoflurescence was used to measure neurite growth. After downregulating Cdh1, neurons were planted on astrocytes of C group and LPS group, divided into Lv-control-C group, Lv-control-LPS group, Lv-cdh1-C group and Lv-cdh1-LPS group. Lengths of longest neurites were measured by immunoflurescence.Resluts: Cell counting and Western blot analysis demonstrated that the concentration of 20 μg/ml was the optical one to stimulate astrocytes proliferating and that compared with C group, the expression of ephrin A3 increased gradually for at least 72 h(P<0.05). Immunoflurescence showed that neurites growth of neurons on LPS group was significantly inhibited, in contrast with Cgroup(P<0.001). However there is no difference between both groups cultured with the medium of C group and LPS group(P>0.05). After downregulation of Eph A4, the neutite lengths of si RNA-C group did not differ significantly from si RNA-LPS group(P>0.05). When recombinant molecules were used instead of astrocytes, lengths of A3-Fc group decreased compared with Fc group(P<0.001). IP analyses showed that ephrin A3-Fc-Ig G induced a significant increase in Eph A4 phosphorylation. APC2 and Cdh1 were co-immunoprecipitated with Eph A4. After downregulating Cdh1 by Lv-cdh1-si RNA, neurites of Lv-control-A3-Fc group shortened compared with Lv-control-Fc group(P<0.001), meanwhile neurites of Lv-cdh1-A3-Fc group lengthened, compared with Lv-control-A3-Fc group(P<0.001). Similarly, after downgulation of Cdh1, lengths of Lv-control-LPS group neurites significantly decreased compared with Lv-control-C group(P<0.001), while lengths of Lv-cdh1-LPS group increased in contrast with Lv-control-LPS group(P<0.001).Conclusion: Astrocytes stimulated by LPS inhibit the growth of neurons through ephrin A3/Eph A4 signaling, which can be ameliorated by the downregulation of neuronal cdh1.