Histone Deacetylase 3 Inhibition Ameliorates Microglia-Mediated Neuro-Inflammation Via the SIRT1/Nrf2 Pathway After Traumatic Spinal Cord Injury.

BACKGROUND: Microglial-induced inflammation plays a crucial role in the pathophysiological process of nervous system injury, however, still lacks effective therapeutic agents. Previously, we discovered that the inhibition of histone deacetylase 3 (HDAC3) exerts anti-inflammatory effects after traumatic spinal cord injury (SCI), whereas little is known about its underlying mechanism. Therefore, the present study aimed to explore the effects and potential mechanisms of HDAC3 on neuroinflammation and microglial function. METHODS: Rats were randomized into 4 groups: sham group, SCI group, SCI + vehicle group, and SCI + RGF966 group. To examine the effect of HDAC3 on neurological deficit after SCI, we gathered data using the Basso Beattie Bresnahan locomotion scale, the inclined plane test, the blood-spinal cord barrier, junction protein expression, and Nissl staining. We also evaluated microglial activation and inflammatory factor levels. Immunofluorescence analysis, immunohistochemical analysis, western blotting, and quantitative real-time polymerase chain reaction were performed to examine the regulation of the Sirtuin 1 (SIRT1)/nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. RESULTS: The results showed that HDAC3 inhibition significantly ameliorated Basso-Beattie-Bresnahan (BBB) permeability, brain edema, and improved neurological functions and junction protein levels. Additionally, HDAC3 inhibition significantly inhibited microglial activation, thereby reducing the levels of SCI-induced pro-inflammatory factors. Moreover, HDAC3 inhibition dramatically enhanced the expression of SIRT1 and increased both Nrf2 nuclear accumulation and transcriptional activity, thereby increasing downstream heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1 expression. CONCLUSIONS: The results of this study suggest that HDAC3 inhibition protects the spinal cord from injury following SCI by inhibiting SCI-induced microglial activation and the subsequent inflammatory response via SIRT1/Nrf2 signaling pathway, highlighting HDAC3 as a potential therapeutic target for the treatment of SCI.

[Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells].

Objective: To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). Methods: The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. Results: The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05). Conclusion: Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-κB pathway dependent of HDAC3.

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)-1ß, 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. 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.