Valproic Acid Attenuates Traumatic

Chen et al. Journal of Neuroinflammation (2018) 15:150 https://doi.org/10.1186/s12974-018-1193-6 RESEARCH Open Access Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-κB pathway dependent of HDAC3 Shoubo Chen1†, Jingfang Ye2†, Xiangrong Chen3* , Jinnan Shi1, Wenhua Wu1, Wenping Lin1, Weibin Lin3, Yasong Li3, Huangde Fu4 and Shun Li5* Abstract Background: Microglial polarization with M1/M2 phenotype shifts and the subsequent neuroinflammatory responses are vital contributing factors for spinal cord injury (SCI)-induced secondary injury. Nuclear factor-κB (NF- κB) is considered the central transcription factor of inflammatory mediators, which plays a crucial role in microglial activation. Lysine acetylation of STAT1 seems necessary for NF-kB pathway activity, as it is regulated by histone deacetylases (HDACs). There have been no studies that have explained if HDAC inhibition by valproic acid (VPA) affects the NF-κB pathway via acetylation of STAT1 dependent of HDAC activity in the microglia-mediated central inflammation following SCI. We investigated the potential molecular mechanisms that focus on the phenotypic transition of microglia and the STAT1-mediated NF-κB acetylation after a VPA treatment. Methods: The Basso-Beattie-Bresnahan locomotion scale, the inclined plane test, the blood-spinal cord barrier, and Nissl staining were employed to determine the neuroprotective effects of VPA treatment after SCI. Assessment of microglia polarization and pro-inflammatory markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and interferon (INF)-γ was used to evaluate the neuroinflammatory responses and the anti-inflammatory effects of VPA treatment. Immunofluorescent staining and Western blot analysis were used to detect HDAC3 nuclear translocation, activity, and NF-κB signaling pathway activation to evaluate the effects of VPA treatment. The impact of STAT1 acetylation on NF-kB pathway and the interaction between STAT1 and NF-kB were assessed to evaluate anti-inflammation effects of VPA treatment and also whether these effects were dependent on a STAT1/NF-κB pathway to gain further insight into the mechanisms underlying the development of the neuroinflammatory response after SCI. (Continued on next page) * Correspondence: [email protected]; [email protected] †Equal contributors 3Department of Neurosurgery, The Second Affiliated Hospital, Fujian Medical University, Quanzhou 362000, Fujian Province, China 5Department of Neurosurgery, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. Journal of Neuroinflammation (2018) 15:150 Page 2 of 14 (Continued from previous page) Results: The results showed that the VPA treatment promoted the phenotypic shift of microglia from M1 to M2 phenotype and inhibited microglial activation, thus reducing the SCI-induced inflammatory factors. The VPA treatment upregulation of the acetylation of STAT1/NF-κB pathway was likely caused by the HDAC3 translocation to the nucleus and activity. These results indicated that the treatment with the VPA suppressed the expression and the activity of HDAC3 and enhanced STAT1, as well as NF-κB p65 acetylation following a SCI. The acetylation status of NF-kB p65 and the complex with NF-κB p65 and STAT1 inhibited the NF-kB p65 transcriptional activity and attenuated the microglia-mediated central inflammatory response following SCI. Conclusions: These results suggested that the VPA treatment attenuated the inflammatory response by modulating microglia polarization through STAT1-mediated acetylation of the NF-κB pathway, dependent of HDAC3 activity. These effects led to neuroprotective effects following SCI. Keywords: Spinal cord injury, Valproic acid, HDAC3, Microglia, Inflammatory, STAT1, NF-κB pathway Background [15–17]. Releases of the NF-κB proteins for intranuclear Traumatic spinal cord injury (SCI) initiates a series of transfer allow them to bind to their target gene pro- cellular and molecular events, including microglial acti- moters that encode chemokines and cytokines, including vation, inflammatory response, and abnormal mitochon- persistently activated STATs [13, 16, 17]. drial activities, which induce neuronal death and lead to Valproic acid (VPA) is commonly used to treat epi- permanent neurological deficits [1–3]. SCI-induced lepsy. VPA exerts its therapeutic benefits through mul- microglial activation and subsequent release of inflam- tiple mechanisms, including enhancement of GABAergic matory factors, such as interleukin (IL), tumor necrosis activity, depolarization induced by N-methyl-D-aspartic factor (TNF), and interferon (INF), cause direct neuronal acid (NMDA) receptors, and/or inhibition of calcium death while inducing vascular endothelial cells to ex- channels and voltage-gated sodium [18–21]. Recent press a variety of cell adhesion and chemotaxis mole- studies found that VPA is a class 1/II histone deacetylase cules [3–5]. These pro-inflammatory factors stimulate inhibitor (HDCAi), where it inhibits histone deacetylase nitric oxide synthesis, which leads to increased capillary (HDCA) functions [18, 19]. Acetylated histone proteins permeability and blood-spinal cord barrier dysfunction exert their neuroprotective effects by reducing inflam- while promoting neuronal apoptosis [2, 3]. The inhib- mation and inhibiting neuronal death [22, 23], which im- ition of SCI-induced, the microglial activation, and the prove neurological functions in many neurological subsequent neuroinflammatory response have been diseases, including cerebral ischemia [24], traumatic shown to improve the recovery in SCI patients [6, 7]. brain injury [25], and spinal cord injury [21, 26]. The Neuroinflammatory responses induced by activated role that VPA plays in SCI, as well as if VPA treatment microglia, via the NF-κB pathway, are the critical con- could inhibit the activation of microglia and the subse- tributing factors of secondary injury [8, 9]. The NF-κB quent inflammatory response after TBI, has not been signaling pathway is passively released after an injury by studied or established. necrotic or damaged cells, which activates microglia to The non-histone-binding protein complexes, such as secrete a large number of inflammatory cytokines, as the high-mobility group (HMG) family, the NF-kB, and well as cascade amplification of inflammatory responses the signal transducers and activators of transcription [8–10]. The NF-κB exists in an inactive state sequestered (STATs), are modified by post-translational modifica- in the cytoplasm by the NF-κB(IκB) inhibitor during tions [14, 27]. Recent studies have confirmed that the non-inflammatory conditions. The activation of the NF- transcriptional activities of NF-kB and STATs are closely κB is initiated by IκB kinase (IKK), which degrades cyto- related to their lysine acetylations, which are regulated plasmic IκB protein, which triggers the rapid release of by the balance between histone acetyltransferases NF-κB from IκB and intranuclear translocation [11, 12]. (HATs) and histone deacetylases (HDACs) [28–30]. The activation process co-exists with signal transducers HDCAi seems to inhibit NF-kB transcriptional activity and activators of transcriptions (STATs) [13, 14]. The by maintaining the NF-kB acetylated (inactive) state and DNA-binding STATs enter from the cytoplasm to the repressing the inflammatory response. Studies from Leus nucleus and activate their target genes at chromosome et al. [31] found that the expression levels of NF-kB are 19q13. These genes include B cell lymphoma (Bcl)-3, related to HDAC enzymatic activity and the levels of which encodes elements of IκB from the NF-κB pathway histone acetylation. These findings suggest that HDAC Chen et al. Journal of Neuroinflammation (2018) 15:150 Page 3 of 14 expression may be associated with NF-kB-mediated in- lateral ventricle 24 h after intraperitoneal VPA to inflammation. The activation of STATs could alleviate a hibit STAT1 signaling, which clarified the role of multitude of NF-kB-driven inflammatory and metabolic STAT1inVPAneuroprotection[35]. The remainder of disorders [14, 27, 32]. There is no literature regarding the groups was injected with the same dose of a the STAT-mediated nuclear translocation of NF-kB p65 dimethylsulfoxide, as a control. subunit after a traumatic SCI. In the present study, we investigated if VPA, a class 1/II histone deacetylase in- Behavioral assessment hibitor, attenuates the microglia-mediated neuroinflam- The locomotor recovery was based on the Basso- matory response involving the interactive roles of Beattie-Bresnahan locomotion scale and the inclined HDAC, STATs, and NF-kB following a traumatic SCI. plane test, in accordance with the previous reports [1, 2]. The locomotor recovery was evaluated at 1, 3, Methods 7, and 14 days following

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