BACKGROUND: There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock (HS). The aim of this study was to explore the potential of the histone deacetylase 6 (HDAC6)-specific inhibitor tubastatin A (TubA) to suppress nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation in macrophages under hypoxia/reoxygenation (H/R) conditions.
METHODS: The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8 (CCK8) assay. Briefly, 2.5 μmol/L TubA was used with RAW264.7 cells under H/R condition. RAW264.7 cells were divided into three groups, namely the control, H/R, and TubA groups. The levels of reactive oxygen species (ROS) in the cells were detected using fluorescence microscopy. The protein expression of HDAC6, heat shock protein 90 (Hsp90), inducible nitric oxide synthase (iNOS), NLRP3, gasdermin-D (GSDMD), Caspase-1, GSDMD-N, and Caspase-1 p20 was detected by western blotting. The levels of interleukin-1β (IL-1β) and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay (ELISA).
RESULTS: HDAC6, Hsp90, and iNOS expression levels were significantly higher (P<0.01) in the H/R group than in the control group, but lower in the TubA group than in the H/R group (P<0.05). When comparing the H/R group to the control group, ROS levels were significantly higher (P<0.01), but significantly reduced in the TubA group (P<0.05). The H/R group had higher NLRP3, GSDMD, Caspase-1, GSDMD-N, and Caspase-1 p20 expression levels than the control group (P<0.05), however, the TubA group had significantly lower expression levels than the H/R group (P<0.05). IL-1β and IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group (P<0.01), but significantly lower in the TubA group compared to the H/R group (P<0.01).
CONCLUSIONS: TubA inhibited the expression of HDAC6, Hsp90, and iNOS in macrophages subjected to H/R. This inhibition led to a decrease in the content of ROS in cells, which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1β and IL-18.

HDAC6 is an essential factor in mouse oocyte maturation. However, the roles of HDAC6 in porcine oocyte maturation are still unclear. Therefore, we analyzed the roles of HDAC6 in porcine oocyte maturation by treatment with Tubastatin A (TubA) which is an HDAC6 inhibitor. Our results showed that treatment with 10 μg/ml TubA significantly decreased the rate of porcine oocyte maturation, but it did not influence the rate of germinal vesicle breakdown (GVBD). Then, we found that TubA treatment disrupted spindle organization by increasing the α-tubulin acetylation level during porcine oocyte maturation. Moreover, TubA treatment significantly increased H4K16 acetylation, which may compromise kinetochore and microtubule (K-MT) attachment during meiosis in porcine oocytes. We also analyzed the effects of TubA on meiosis-related (H3T3pho and H3S10pho) and transcription-related histone modifications (H3K4me3, H3K9me3 and H3K4ac) during porcine oocyte maturation. The results showed that TubA treatment increased H3S10pho and H3K4ac levels, but no influence was seen in H3T3pho, H3K4me3 and H3K9me3 levels in porcine oocytes. TubA treated oocytes also showed a compromised ability to develop after parthenogenetic activation. Finally, we found that HDAC6 exhibited higher mRNA levels and lower DNA methylation levels in porcine oocytes than it did in porcine embryonic fibroblasts (PEFs). These results indicate that the low level of DNA methylation in HDAC6 promoter ensures high expression. HDAC6 regulates the deacetylation of α-tubulin and H4K16, which promotes correct spindle organization and meiotic apparatus assembly during porcine oocyte maturation. This study illustrates a new pathway by which HDAC6 modulates mammalian oocyte maturation.

Blood-brain barrier (BBB) disruption after intracerebral hemorrhage (ICH) significantly induces neurological impairment. Previous studies showed that HDAC6 knockdown or TubA can protect the TNF-induced endothelial dysfunction. However, the role of HDAC6 inhibition on ICH-induced BBB disruption remains unknown.
Hemin-induced human brain microvascular endothelial cells (HBMECs) and collagenase-induced rats were employed to investigated the underlying impact of the HDAC6 inhibition in BBB lesion and neuronal dysfunction after ICH.
We found a significant decrease in acetylated α-tubulin during early phase of ICH. Both 25 or 40 mg/kg of TubA could relieve neurological deficits, perihematomal cell apoptosis, and ipsilateral brain edema in ICH animal model. TubA or specific siRNA of HDAC6 inhibited apoptosis and reduced the endothelial permeability of HBMECs. HDAC6 inhibition rescued the degradation of TJ proteins and repaired TJs collapses after ICH induction. Finally, the results suggested that the protective effects on BBB after ICH induction were exerted via upregulating the acetylated α-tubulin and reducing stress fiber formation.
Inhibition of HDAC6 expression showed beneficial effects against BBB disruption after experimental ICH, which suggested that HDAC6 could be a novel and promising target for ICH treatment.

A large body of evidence has demonstrated that neuronal apoptosis is involved in the pathological process of secondary brain injury following intracerebral hemorrhage (ICH). Additionally, our previous studies determined that the inhibition of HDAC6 activity by tubacin or specific shRNA can attenuate neuronal apoptosis in an oxygen-glucose deprivation reperfusion model. However, whether the pharmacological inhibition of HDAC6-attenuated neuronal apoptosis in ICH remains unclear. In this study, we used hemin-induced SH-SY5Y cells to simulate a hemorrhage state in vitro and adopted a collagenase-induced ICH rat model in vivo to assess the effect of the HDAC6 inhibition. We found a significant increase in HDAC6 during the early stages of ICH. As expected, the acetylated α-tubulin significantly decreased in correlation with the expression of HDAC6. Medium and high doses (25, 40 mg/kg) of TubA, a selective inhibitor of HDAC6, both reduced neurological impairments, histological impairments, and ipsilateral brain edema in vivo. TubA or HDAC6 siRNA both alleviated neuronal apoptosis in vivo and in vitro. Finally, HDAC6 inhibition increased the level of acetylated α-tubulin and Bcl-2 and lowered the expression of Bax and cleaved caspase-3 post-ICH. In general, these results suggested that the pharmacological inhibition of HDAC6 may act as a novel and promising therapeutic target for ICH therapy by up-regulating acetylated α-tubulin and reducing neuronal apoptosis.

In this study, a novel series of histone deacetylases 6 (HDAC6) inhibitors containing polycyclic aromatic rings were discovered and evaluated for their pharmacological activities. The most potent compound 10c exhibited high HDAC6 inhibitory activity (IC50 = 261 nM) and excellent HDAC6 selectivity (SI = 109 for HDAC6 over HDAC3). 10c also showed decent antiproliferative activity in vitro with IC50 of 7.37-21.84 μM against four cancer cell lines, comparable to that of tubastatin A (average IC50 = 6.10 μM). Further mechanism studies revealed that 10c efficiently induced apoptosis and S-phase arrest in B16-F10 cells. In addition, 10c markedly increased the expression of acetylated-α-tubulin both in vitro and in vivo, without affecting the levels of acetylated-H3 (marker of HDAC1 inhibition). Furthermore, 10c (80 mg/kg) exhibited moderate antitumor efficacy in a melanoma tumour model with a tumour growth inhibition (TGI) of 32.9%, comparable to that (TGI = 31.3%) of tubastatin A. Importantly, the combination of 10c with NP19 (a small molecule PD-L1 inhibitor discovered by us before) decreased tumour burden substantially (TGI% = 60.1%) as compared to monotherapy groups. Moreover, the combination of 10c with NP19 enhanced the anti-tumour immune response, mediated by a decrease of PD-L1 expression levels and increased infiltration of anti-tumour CD8+ T cells in tumour tissues. Collectively, 10c represents a novel HDAC6 inhibitor deserving further investigation as a potential anti-cancer agent.

OBJECTIVE: Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms.
METHODS: Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery.
RESULTS: The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) .
CONCLUSIONS: HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.
目的: 蛛网膜下腔出血(subarachnoid hemorrhage，SAH)是一种严重的脑血管疾病。早期脑损伤(early brain injury，EBI)和脑血管痉挛是导致SAH患者预后不良的主要原因。组蛋白脱乙酰酶(histone deacetylase，HDAC)6特异性抑制剂tubastatin A(TubA)在多种急慢性中枢神经系统疾病的动物模型中被证实有明确的神经保护作用，但其对SAH的神经保护作用尚未明确。本研究旨在探讨HDAC6在SAH早期大脑皮质中的表达和细胞定位，并评估TubA对SAH后EBI和脑血管痉挛的保护作用及其机制。方法: 选取成年健康雄性SD大鼠，采用改良的颈内动脉穿刺法建立大鼠SAH模型。第1部分实验，将大鼠随机分为假手术(sham)组、SAH-3 h组、SAH-6 h组、SAH-12 h组、SAH-24 h组、SAH-48 h组。分别在SAH建模后3、6、12、24 h，取各组大鼠损伤侧大脑皮质样本行蛋白质印迹法检测HDAC6的表达。另取SAH-24 h组大鼠，采用免疫荧光双染法测定HDAC6在损伤侧大脑皮质的分布。第2部分实验，将大鼠随机分为sham组、SAH组、SAH+TubAL组(给予TubA 25 mg/kg)、SAH+TubAH组(给予TubA 40 mg/kg)。建模后24 h，对各组大鼠行神经功能评分，检测各组大鼠脑组织含水量。第3部分实验，将大鼠随机分为SAH组、SAH+TubA组(给予TubA 40 mg/kg)。建模后24 h，取损伤侧大脑皮质组织行蛋白质印迹法检测HDAC6、内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)和诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)的表达水平，末端脱氧核苷酸转移酶介导的dUTP缺口标记(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling，TUNEL)染色检测凋亡，苏木精-伊红(hematoxylin and eosin，HE)染色检测大脑中动脉直径。结果: 损伤侧大脑皮质中HDAC6的蛋白质表达水平在SAH后6 h开始升高(P<0.05)，24 h时达到峰值(P<0.001)，48 h时出现下降，但仍明显高于sham组(P<0.05)。HDAC6主要在神经元的细胞质中表达。与sham组大鼠比较，SAH组大鼠神经功能评分明显降低，脑组织含水量增加(均P<0.01)。与SAH组大鼠比较，SAH+TubAH组神经功能评分明显升高且脑组织含水量明显下降，差异均有统计学意义(均P<0.05)；而SAH+TubAL组神经功能改善和脑组织含水量下降均不明显，差异均无统计学意义(均P>0.05)。与sham组大鼠比较，SAH组eNOS表达显著降低(P<0.01)，iNOS和HDAC6表达均明显上调(分别为P<0.05和P<0.01)；与SAH组相比，SAH+TubA组eNOS表达明显升高，iNOS和HDAC6均明显下调(均P<0.05)。与SAH组大鼠比较，SAH+TubA组TUNEL阳性细胞数明显减少，且大脑中动脉直径明显增加，差异均有统计学意义(均P<0.05)。结论: HDAC6主要表达于神经元，且在SAH早期大脑皮质中的表达上调。HDAC6特异性抑制剂TubA通过减轻SAH早期脑水肿和减少细胞凋亡，对SAH大鼠EBI和脑血管痉挛发挥保护作用。其减轻脑血管痉挛的作用可能与调节eNOS和iNOS的表达有关。.

Peritoneal fibrosis contributes to ultrafiltration failure in peritoneal dialysis (PD) patients and thus restricts the wide application of PD in clinic. Recently we have demonstrated that histone deacetylase 6 (HDAC6) is critically implicated in high glucose peritoneal dialysis fluid (HG-PDF) induced peritoneal fibrosis, however, the precise mechanisms of HDAC6 in peritoneal fibrosis have not been elucidated. Here, we focused on the role and mechanisms of HDAC6 in chlorhexidine gluconate (CG) induced peritoneal fibrosis and discussed the mechanisms involved. We found Tubastatin A (TA), a selective inhibitor of HDAC6, significantly prevented the progression of peritoneal fibrosis, as characterized by reduction of epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) protein deposition. Inhibition of HDAC6 remarkably suppressed the expression of matrix metalloproteinases-2 (MMP2) and MMP-9. Administration of TA also increased the expression of acetylation Histone H3 and acetylation α-tubulin. Moreover, our results revealed that blockade of HDAC6 inhibited alternatively M2 macrophages polarization by suppressing the activation of TGF-β/Smad3, PI3K/AKT, and STAT3, STAT6 pathways. To give a better understanding of the mechanisms, we further established two cell injured models in Raw264.7 cells by using IL-4 and HG-PDF. Our in vitro experiments illustrated that both IL-4 and HG-PDF could induce M2 macrophage polarization, as demonstrated by upregulation of CD163 and Arginase-1. Inhibition of HDAC6 by TA significantly abrogated M2 macrophage polarization dose-dependently by suppressing TGF-β/Smad, IL4/STAT6, and PI3K/AKT signaling pathways. Collectively, our study revealed that blockade of HDAC6 by TA could suppress the progression of CG-induced peritoneal fibrosis by blockade of M2 macrophage polarization. Thus, HDAC6 may be a promising target in peritoneal fibrosis treatment.

Adult skeletal muscle stem cells (MuSCs) are important for muscle regeneration and constitute a potential source of cell therapy. However, upon isolation, MuSCs rapidly exit quiescence and lose transplantation potency. Maintenance of the quiescent state in vitro preserves MuSC transplantation efficiency and provides an opportunity to study the biology of quiescence. Here we show that Tubastatin A (TubA), an Hdac6 inhibitor, prevents primary cilium resorption, maintains quiescence, and enhances MuSC survival ex vivo. Phenotypic characterization and transcriptomic analysis of TubA-treated cells revealed that TubA maintains most of the biological features and molecular signatures of quiescence. Furthermore, TubA-treated MuSCs showed improved engraftment ability upon transplantation. TubA also induced a return to quiescence and improved engraftment of cycling MuSCs, revealing a potentially expanded application for MuSC therapeutics. Altogether, these studies demonstrate the ability of TubA to maintain MuSC quiescence ex vivo and to enhance the therapeutic potential of MuSCs and their progeny.

Injury to long axonal projections is a central pathological feature at the early phase of intracerebral hemorrhage (ICH). It has been reported to contribute to persistent functional disability following ICH. However, the molecular mechanisms that drive axonal degeneration remain unclear. Autologous blood was injected into the striatum to mimic the pathology of ICH. Observed significant swollen axons with characteristic retraction bulbs were found around the striatal hematoma at 24 h after ICH. Electronic microscopic examination revealed highly disorganized microtubule and swollen mitochondria in the retraction bulbs. MEC17 is a specific α-tubulin acetyltransferase, ablation of acetylated α-tubulin in MEC17-/- mice aggravated axonal injury, axonal transport mitochondria dysfunction, and motor dysfunction. In contrast, treatment with tubastatin A (TubA), which promotes microtubule acetylation, significantly alleviated axonal injury and protected the integrity of the corticospinal tract and fine motor function after ICH. Moreover, results showed that 41% mitochondria were preferentially bundled to the acetylated α-tubulin in identifiable axons and dendrites in primary neurons. This impaired axonal transport of mitochondria in primary neurons of MEC17-/- mice. Given that opening of mitochondrial permeability transition pore (mPTP) induces mitochondrial dysfunction and impairs ATP supply thereby promoting axonal injury, we enhanced the availability of acetylated α-tubulin using TubA and inhibited mPTP opening with cyclosporin A. The results indicated that this combined treatment synergistically protected corticospinal tract integrity and promoted fine motor control recovery. These findings reveal key intracellular mechanisms that drive axonal degeneration after ICH and highlight the need to target multiple factors and respective regulatory mechanisms as an effective approach to prevent axonal degeneration and motor dysfunction after ICH.

Trypanosoma cruzi is a protozoan of great medical interest since it is the causative agent of Chagas disease, an endemic condition in Latin America. This parasite undergoes epigenetic events, such as phosphorylation, methylation and acetylation, which play a role in several cellular processes including replication, transcription and gene expression. Histone deacetylases (HDAC) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. Tubastatin A (TST) is a specific HDAC6 inhibitor that affects cell growth and promotes structural modifications in cancer cells and parasites. In the present study, we demonstrated that T. cruzi epimastigote cell proliferation and viability are reduced after 72 h of TST treatment. The results obtained through different microscopy methodologies suggest that this inhibitor impairs the polymerization dynamics of cytoskeleton microtubules, generating protozoa displaying atypical morphology and cellular patterns that include polynucleated parasites. Furthermore, the microtubules of treated protozoa were more intensely acetylated, especially at the anterior portion of the cell body. A cell cycle analysis demonstrated an increase in the number of trypanosomatids in the G2/M phase. Together, our results suggest that TST should be explored as a tool to study trypanosomatid cell biology, including microtubule cytoskeleton dynamics, and as an antiparasitic drug.