Sodium phenylbutyrate inhibits Schwann cell inflammation via HDAC and NFκB to promote axonal regeneration and remyelination

Abstract

Background: Epigenetic regulation by histone deacetylases (HDACs) in Schwann cells (SCs) after injury facilitates them to undergo de- and redifferentiation processes necessary to support various stages of nerve repair. Although de-differentiation activates the synthesis and secretion of inflammatory cytokines by SCs to initiate an immune response during nerve repair, changes in either the timing or duration of prolonged inflammation mediated by SCs can affect later processes associated with repair and regeneration. Limited studies have investigated the regulatory processes through which HDACs in SCs control inflammatory cytokines to provide a favorable environment for peripheral nerve regeneration.

Methods: We employed the HDAC inhibitor (HDACi) sodium phenylbutyrate (PBA) to address this question in an in vitro RT4 SC inflammation model and an in vivo sciatic nerve transection injury model to examine the effects of HDAC inhibition on the expression of pro-inflammatory cytokines. Furthermore, we assessed the outcomes of suppression of extended inflammation on the regenerative potential of nerves by assessing axonal regeneration, remyelination, and reinnervation.

Results: Significant reductions in lipopolysaccharide (LPS)-induced pro-inflammatory cytokine (tumor necrosis factor-α [TNFα]) expression and secretion were observed in vitro following PBA treatment. PBA treatment also affected the transient changes in nuclear factor κB (NFκB)-p65 phosphorylation and translocation in response to LPS induction in RT4 SCs. Similarly, PBA mediated long-term suppressive effects on HDAC3 expression and activity. PBA administration resulted in marked inhibition of pro-inflammatory cytokine secretion at the site of transection injury when compared with that in the hydrogel control group at 6-week post-injury. A conducive microenvironment for axonal regrowth and remyelination was generated by increasing expression levels of protein gene product 9.5 (PGP9.5) and myelin basic protein (MBP) in regenerating nerve tissues. PBA administration increased the relative gastrocnemius muscle weight percentage and maintained the intactness of muscle bundles when compared with those in the hydrogel control group.

Conclusions: Suppressing the lengthened state of inflammation using PBA treatment favors axonal regrowth and remyelination following nerve transection injury. PBA treatment also regulates pro-inflammatory cytokine expression by inhibiting the transcriptional activation of NFκB-p65 and HDAC3 in SCs in vitro.

Keywords: HDAC inhibitor; Inflammation; Peripheral nerve injury; Regeneration and myelination; Schwann cells.

Fig. 1

Inflammatory response and the modulation of HDAC expression by LPS in RT4 SCs. A Secretion of TNFα after LPS induction for 24 h were measured by ELISA. B Representative western blot data of changes in the expression of TNFα, c-PARP, and TLR4 expression in a dose-dependent manner, following LPS induction for 24 h. C Expression profile of HDAC1, 2, 3, and 4 after LPS induction for 24 h. D LPS-induced increases of HDAC3 activity were confirmed by HDAC3 activities assay. n = 4. Significance was assessed by one-way ANOVA. The data are presented as the mean ± SEM. *p < 0.05 versus 0 μg/ml LPS. LPS lipopolysaccharide, SCs Schwann cells, TNFα tumor necrosis factor α, c-PARP cleaved poly (ADP) ribose polymerase, HDAC histone deacetylase, TLR4 toll-like receptor 4, ANOVA analysis of variance, SEM standard error of the mean

Fig. 2

LPS induces the phosphorylation and translocation of NFkB-p65 at 1 h after LPS treatment. A Western blot data and quantification showed the phosphorylation and expression pattern of NFκB-p65 at various timepoints after LPS induction. B Time course of p-NFkB-p65 expressions in cytoplasm and nuclear fraction confirmed the translocation of NFkB-p65 after LPS stimuli. C Immunofluorescence staining for NFκB-p65 showing the start of NFκB-p65 nuclear translocation from 1 to 3 h, followed by a decline at 6 h. n = 4. LPS lipopolysaccharide, p-NFκB-p65 phospho-nuclear factor κB p65 subunit

Fig. 3

PBA prevents the LPS-induced NFκB-p65 phosphorylation and translocation to the nucleus. A Western blot analysis and quantification showing a significant decrease in phosphorylation of NFκB-p65 following combined PBA and LPS treatment. n = 4. B Reduction of activated NFκB-p65 into the nucleus was revealed after PBA treatment. C Immunofluorescence staining shows the reduced nuclear translocation of NFκB-p65 after induction with LPS and PBA together and its representative graph. n = 4. Significance was assessed by one-way ANOVA. Data are presented as the mean ± SEM. *p < 0.05 versus no LPS and no PBA. ^#p < 0.05 versus only LPS. PBA sodium phenylbutyrate, LPS lipopolysaccharide, NFκB-p65 nuclear factor κB p65 subunit, ANOVA analysis of variance, SEM standard error of the mean

Fig. 4

PBA reverses LPS-induced inflammatory effects in RT4 SCs. A Western blot analysis shows the decreased expression levels of TNFα, c-PARP, and TLR4 following LPS and PBA co-treatment for 24 h. B LPS-induced secretion of TNFα was inhibited by PBA treatments. C Western blot depicting the changes in HDAC3 expression levels but not in the levels of the other HDACs after 24 h of treatment with LPS and PBA. D HDAC3 activity induced by 24 h of treatment with LPS was also suppressed after PBA treatment. n = 4. Significance was assessed by one-way ANOVA. Data are presented as the mean ± SEM. *p < 0.05 versus no LPS and no PBA. ^#p < 0.05 versus only LPS. LPS lipopolysaccharide, PBA sodium phenylbutyrate, SCs Schwann cells, TNFα tumor necrosis factor α, c-PARP cleaved poly (ADP) ribose polymerase, TLR4 toll-like receptor 4, HDAC histone deacetylase, ANOVA analysis of variance, SEM standard error of the mean

Fig. 5

PBA administration reduces chronic inflammation after peripheral nerve injury. A Representative pictures of the harvested sciatic nerves after 6 weeks from three groups of rats. B Representative images of immunohistochemistry staining against TNFα, IL-1β, and IL-6 in paraffin-embedded sections obtained from the middle region of the nerves in each treatment group and their and their respective quantification for the expression. n = 4 different rats for each group. Scale bar = 20 μm. Significance was assessed by Student’s t test. *p < 0.05 versus hydrogel control group. PBA sodium phenylbutyrate, TNFα tumor necrosis factor α, IL interleukin

Fig. 6

PBA administration enhances axonal regeneration and myelination and prevents muscle atrophy after 6 weeks of sciatic nerve transection injury. A Representative images of immunohistochemistry staining for S100β, PGP9.5, and MBP in the middle region of the regenerating nerve from three groups with quantification of positive-stained cells. B Myelin sheath staining using Luxol fast blue in the middle part of the nerve and quantification of positive-stained myelin sheath. C Graph representing the comparison of RGMW between the hydrogel control and PBA groups. D H&E staining of sections of the left gastrocnemius muscle in three groups. n = 4 different rats for each group. Scale bar = 20 μm. Significance in (C) was assessed by Student’s t test. *p < 0.05 versus hydrogel control group. PBA sodium phenylbutyrate, PGP9.5 protein gene product 9.5, MBP myelin basic protein, LFB luxol fast blue, RGMW relative gastrocnemius muscle weight