BackgroundMicroglial 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.MethodsThe 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.ResultsThe 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.ConclusionsThese 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.

BackgroundMicroglial activation and the subsequent inflammatory response in the central nervous system play important roles in secondary damage after traumatic brain injury (TBI). High-mobility group box 1 (HMGB1) protein, an important mediator in late inflammatory responses, interacts with transmembrane receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs) to activate downstream signaling pathways, such as the nuclear factor (NF)-κB signaling pathway, leading to a cascade amplification of inflammatory responses, which are related to neuronal damage after TBI. Omega-3 polyunsaturated fatty acid (ω-3 PUFA) is a commonly used clinical immunonutrient, which has antioxidative and anti-inflammatory effects. However, the effects of ω-3 PUFA on HMGB1 expression and HMGB1-mediated activation of the TLR4/NF-κB signaling pathway are not clear.MethodsThe Feeney DM TBI model was adopted to induce brain injury in rats. Modified neurological severity scores, brain water content, and Nissl staining were employed to determine the neuroprotective effects of ω-3 PUFA supplementation. Assessment of microglial activation in lesioned sites and protein markers for proinflammatory, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, and HMGB1 were used to evaluate neuroinflammatory responses and anti-inflammation effects of ω-3 PUFA supplementation. Immunofluorescent staining and western blot analysis were used to detect HMGB1 nuclear translocation, secretion, and HMGB1-mediated activation of the TLR4/NF-κB signaling pathway to evaluate the effects of ω-3 PUFA supplementation and gain further insight into the mechanisms underlying the development of the neuroinflammatory response after TBI.ResultsIt was found that ω-3 PUFA supplementation inhibited TBI-induced microglial activation and expression of inflammatory factors (TNF-α, IL-1β, IL-6, and IFN-γ), reduced brain edema, decreased neuronal apoptosis, and improved neurological functions after TBI. We further demonstrated that ω-3 PUFA supplementation inhibited HMGB1 nuclear translocation and secretion and decreased expression of HMGB1 in neurons and microglia in the lesioned areas. Moreover, ω-3 PUFA supplementation inhibited microglial activation and the subsequent inflammatory response by regulating HMGB1 and the TLR4/NF-κB signaling pathway.ConclusionsThe results of this study suggest that microglial activation and the subsequent neuroinflammatory response as well as the related HMGB1/TLR4/NF-κB signaling pathway play essential roles in secondary injury after TBI. Furthermore, ω-3 PUFA supplementation inhibited TBI-induced microglial activation and the subsequent inflammatory response by regulating HMGB1 nuclear translocation and secretion and also HMGB1-mediated activation of the TLR4/NF-κB signaling pathway, leading to neuroprotective effects.

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate), or its salt form, phytate, is commonly regarded as the major anti-nutritional component in cereal and legume grains. Breeding of low phytic acid (lpa) crops has recently been considered as a potential way to increase nutritional quality of crop products. In this study, eight independent lpa rice mutant lines from both indica and japonica subspecies were developed through physical and chemical mutagenesis. Among them, five are non-lethal while the other three are homozygous lethal. None of the lethal lines could produce homozygous lpa plants through seed germination and growth under field conditions, but two of them could be rescued through in vitro culture of mature embryos. The non-lethal lpa mutants had lower PA content ranging from 34 to 64% that of their corresponding parent and four of them had an unchanged total P level. All the lpa mutations were inherited in a single recessive gene model and at least four lpa mutations were identified mutually non-allelic, while the other two remain to be verified. One mutation was mapped on chromosome 2 between microsatellite locus RM3542 and RM482, falling in the same region as the previously mapped lpa1-1 locus did; another lpa mutation was mapped on chromosome 3, tightly linked to RM3199 with a genetic distance of 1.198 cM. The latter mutation was very likely to have happened to the LOC_Os03g52760, a homolog of the maize myo-inositol kinase (EC 2.7.1.64) gene. The present work greatly expands the number of loci that could influence the biosynthesis of PA in rice, making rice an excellent model system for research in this area.

BackgroundMicroglial polarization and the subsequent neuroinflammatory response are contributing factors for traumatic brain injury (TBI)-induced secondary injury. High mobile group box 1 (HMGB1) mediates the activation of the NF-κB pathway, and it is considered to be pivotal in the late neuroinflammatory response. Activation of the HMGB1/NF-κB pathway is closely related to HMGB1 acetylation, which is regulated by the sirtuin (SIRT) family of proteins. Omega-3 polyunsaturated fatty acids (ω-3 PUFA) are known to have antioxidative and anti-inflammatory effects. We previously demonstrated that ω-3 PUFA inhibited TBI-induced microglial activation and the subsequent neuroinflammatory response by regulating the HMGB1/NF-κB signaling pathway. However, no studies have elucidated if ω-3 PUFA affects the HMGB1/NF-κB pathway in a HMGB1 deacetylation of dependent SIRT1 manner, thus regulating microglial polarization and the subsequent neuroinflammatory response.MethodsThe Feeney DM TBI model was adopted to induce brain injury in rats. Modified neurological severity scores, rotarod test, brain water content, and Nissl staining were employed to determine the neuroprotective effects of ω-3 PUFA supplementation. Assessment of microglia polarization and pro-inflammatory markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and HMGB1, were used to evaluate the neuroinflammatory responses and the anti-inflammatory effects of ω-3 PUFA supplementation. Immunofluorescent staining and western blot analysis were used to detect HMGB1 nuclear translocation, secretion, and HMGB1/NF-κB signaling pathway activation to evaluate the effects of ω-3 PUFA supplementation. The impact of SIRT1 deacetylase activity on HMGB1 acetylation and the interaction between HMGB1 and SIRT1 were assessed to evaluate anti-inflammation effects of ω-3 PUFAs, and also, whether these effects were dependent on a SIRT1-HMGB1/NF-κB axis to gain further insight into the mechanisms underlying the development of the neuroinflammatory response after TBI.ResultsThe results of our study showed that ω-3 PUFA supplementation promoted a shift from the M1 microglial phenotype to the M2 microglial phenotype and inhibited microglial activation, thus reducing TBI-induced inflammatory factors. In addition, ω-3 PUFA-mediated downregulation of HMGB1 acetylation and its extracellular secretion was found to be likely due to increased SIRT1 activity. We also found that treatment with ω-3 PUFA inhibited HMGB1 acetylation and induced direct interactions between SIRT1 and HMGB1 by elevating SIRT1 activity following TBI. These events lead to inhibition of HMGB1 nucleocytoplasmic translocation/extracellular secretion and alleviated HMGB1-mediated activation of the NF-κB pathway following TBI-induced microglial activation, thus inhibiting the subsequent inflammatory response.ConclusionsThe results of this study suggest that ω-3 PUFA supplementation attenuates the inflammatory response by modulating microglial polarization through SIRT1-mediated deacetylation of the HMGB1/...

AKT pathway has a critical role in mediating signaling transductions for cell proliferation, differentiation and survival. Previous studies have shown that AKT activation is achieved through a series of phosphorylation steps: first, AKT is phosphorylated at Thr-450 by JNK kinases to prime its activation; then, phosphoinositide-dependent kinase 1 phosphorylates AKT at Thr-308 to expose the Ser-473 residue; and finally, AKT is phosphorylated at Ser-473 by several kinases (PKD2 and others) to achieve its full activation. For its inactivation, the PH-domain containing phosphatases dephosphorylate AKT at Ser-473, and protein serine/threonine phosphatase-2A (PP-2A) dephosphorylates it at Thr-308. However, it remains unknown regarding which phosphatase dephosphorylates AKT at Thr-450 during its inactivation. In this study, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 (PP-1) is a major phosphatase that directly dephosphorylates AKT to modulate its activation. First, purified PP-1 directly dephosphorylates AKT in vitro. Second, immunoprecipitation and immunocolocalization showed that PP-1 interacts with AKT. Third, stable knock down of PP-1a or PP-1b but not PP-1c, PP-2Aa or PP-2Ab by shRNA leads to enhanced phosphorylation of AKT at Thr-450. Finally, overexpression of PP-1a or PP-1b but not PP-1c, PP-2Aa or PP-2Ab results in attenuated phosphorylation of AKT at Thr-450. Moreover, our results also show that dephosphorylation of AKT by PP-1 significantly modulates its functions in regulating the expression of downstream genes, promoting cell survival and modulating differentiation. These results show that PP-1 acts as a major phosphatase to dephosphorylate AKT at Thr-450 and thus modulate its functions.