周围神经损伤
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Figure 6｜The up-regulations of TFs in DRGs after SNC.
We examined the expression levels of Crem, Fosl1, and Klf6 by immunofluorescence staining (Figure 6). Crem was up-regulated from 12 hours after SNC and localized around the nucleus of DRG neurons (Figure 6A and E). Klf6 was localized in the nucleus of neurons and appeared only at the regeneration stage after injury (Figure 6B and F). While Fosl1 mRNA detected by RNA-seq was up-regulated in the regeneration stage, immunofluorescence staining revealed Fosl1 protein showed little change after SNC (Figure 6C and G). Notably, the function of Arid5a, one of the top TFs, in DRG neurons has not been explored. We found that Arid5a was up-regulated from 12 hours to 7 days and showed a higher expression level at 1 and 3 days, especially in DRG neurons (Figure 6D and H).
Figure 7｜Restricted neurite outgrowth in cultured DRG neurons with down-regulated TFs.
To explore the function of the four TFs in axonal regeneration, we transfected siRNAs targeting the TFs in cultured DRG neurons. After confirming effective knockdown by qPCR, we selected two fragments for each TF to perform neurite outgrowth assay (Additional Figure 4A–D). Measurement of the total length of newborn neurites revealed that axonal regrowth was restrained in cells with down-regulated Crem, Arid5a, Fosl1 and Klf6 (Figure 7 and Additional Figure 4E–H).
Figure 8｜Regulatory network of Atf3.
Atf3, the core TF in the axonal regeneration module, has been reported to participate in neuronal injury and axonal regeneration in both the CNS and PNS (Seijffers et al., 2007; Gey et al., 2016; Renthal et al., 2020). Our results showed that both RNA and protein levels of Atf3 were up-regulated at 12 hours after injury (Figure 8A and B). Atf3 activity was also elevated 12 hours after SNC and sustained until 7 days (Figure 8C). Notably, lower activity of the Atf3 regulon was detected in some cells in the regeneration stage, which is consistent with the results of the cell clustering, that some cells were in the unaltered state after SNC (Figures 2A, B and 8D).