Study on the mechanism of glucocorticoid receptor mitochondrial translocation and glucocorticoid-induced apoptosis in macrophages.

OBJECTIVE: Our research aimed to investigate the therapeutic effects of Tubastatin-A, a glucocorticoid receptor (GR) mitochondrial translocation inhibitor, and mitoquinone (MitoQ), an antioxidant, on attenuating dexamethasone (DEX)-induced macrophage apoptosis. METHODS: We treated RAW264.7 macrophages with different combinations of DEX and either Tubastatin-A or MitoQ. Parameters such as mitochondrial GR translocation, mitochondrial reactive oxygen species levels, mitochondrial membrane potential, mitochondrial permeability transition pore opening, cytochrome C efflux to the cytosol, and apoptosis were subsequently evaluated in the different treatment groups via qRT-PCR, western blotting, and immunofluorescence assays. RESULTS: DEX intervention increased the translocation of GRs into the mitochondria, while reducing the expression of the mitochondrial gene MT-CO1 and the activity of mitochondrial respiratory chain complex IV in macrophages. In addition, DEX administration increased mtROS levels, mitochondrial permeability transition pore opening, and mitochondrial cytochrome C release in macrophages, which promoted their apoptosis. We found that Tubastatin-A inhibited mitochondrial GR translocation and reversed the DEX-induced increase in GR levels within the mitochondria. Furthermore, Tubastatin-A mitigated various mitochondrial changes induced by DEX, including reducing the efflux of mitochondrial cytochrome C and inhibiting macrophage apoptosis. Similarly, MitoQ exerted its effects on macrophage apoptosis by reducing mtROS levels through the mitochondrial pathway. CONCLUSIONS: The DEX-mediated translocation of GR into mitochondria disrupts the mitochondrial function of macrophages, which induces their apoptosis. By inhibiting mitochondrial translocation of GR and reducing mtROS levels, Tubastatin-A and MitoQ can effectively attenuate macrophage apoptosis, which has clinical implications for reducing the notable side effects associated with glucocorticoid use.

HDAC6 inhibitor promotes reactive oxygen species-meditated clearance of Staphylococcus aureus in macrophage.

Staphylococcus aureus (S. aureus) pneumonia has become an increasingly important public health problem. Recent evidence suggests that epigenetic modifications are critical in the host immune defence against pathogen infection. In this study, we found that S. aureus infection induces the expression of histone deacetylase 6 (HDAC6) in a dose-dependent manner. Furthermore, by using a S. aureus pneumonia mouse model, we showed that the HDAC6 inhibitor, tubastatin A, demonstrates a protective effect in S. aureus pneumonia, decreasing the mortality and destruction of lung architecture, reducing the bacterial burden in the lungs and inhibiting inflammatory responses. Mechanistic studies in primary bone marrow-derived macrophages demonstrated that the HDAC6 inhibitors, tubastatin A and tubacin, reduced the intracellular bacterial load by promoting bacterial clearance rather than regulating phagocytosis. Finally, N-acetyl-L- cysteine, a widely used reactive oxygen species (ROS) scavenger, antagonized ROS production and significantly inhibited tubastatin A-induced S. aureus clearance. These findings demonstrate that HDAC6 inhibitors promote the bactericidal activity of macrophages by inducing ROS, an important host factor for S. aureus clearance and production. Our study identified HDAC6 as a suitable epigenetic modification target for preventing S. aureus infection, and tubastatin A as a useful compound in treating S. aureus pneumonia.

Pharmacological blockade of HDAC6 attenuates cancer progression by inhibiting IL-1ß and modulating immunosuppressive response in OSCC.

Interleukin-1-beta (IL-1ß) one of the biomarkers for oral squamous cell carcinoma (OSCC), is upregulated in tumor-microenvironment (TME) and associated with poor patient survival. Thus, a novel modulator of IL-1ß would be of great therapeutic value for OSCC treatment. Here we report regulation of IL-1ß and TME by histone deacetylase-6 (HDAC6)-inhibitor in OSCC. We observed significant upregulation of HDAC6 in 4-nitroquniline (4-NQO)-induced OSCC in mice and 4-NQO & Lipopolysaccharide (LPS) stimulated OSCC and fibroblast cells. Tubastatin A (TSA)-attenuated the OSCC progression in mice as observed improvement in the histology over tongue and esophagus, with reduced tumor burden. TSA treatment to 4-NQO mice attenuated protein expression of HDAC6, pro-and-mature-IL-1ß and pro-and-cleaved-caspase-1 and ameliorated acetylated-tubulin. In support of our experimental work, human TCGA analysis revealed HDAC6 and IL-1ß were upregulated in the primary tumor, with different tumor stages and grades. We found TSA modulate TME, indicated by downregulation of CD11b+Gr1+-Myeloid-derived suppressor cells, CD11b+F4/80+CD206+ M2-macrophages and increase in CD11b+F4/80+MHCII+ M1-macrophages. TSA significantly reduced the gene expression of HDAC6, IL-1ß, Arginase-1 and iNOS in isolated splenic-MDSCs. FaDu-HTB-43 and NIH3T3 cells stimulated with LPS and 4-NQO exhibit higher IL-1ß levels in the supernatant. Interestingly, immunoblot analysis of the cell lysate, we observed that TSA does not alter the expression as well as activation of IL-1ß and caspase-1 but the acetylated-tubulin was found to be increased. Nocodazole pre-treatment proved that TSA inhibited the lysosomal exocytosis of IL-1ß through tubulin acetylation. In conclusion, HDAC6 inhibitors attenuated TME and cancer progression through the regulation of IL-1ß in OSCC.

Selective HDAC6 inhibition protects against blood-brain barrier dysfunction after intracerebral hemorrhage.

BACKGROUNDS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

HDAC6 decreases H4K16 and α-tubulin acetylation during porcine oocyte maturation.

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.

Selective HDAC6 inhibitor TubA offers neuroprotection after intracerebral hemorrhage via inhibiting neuronal apoptosis.

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.

Discovery of novel benzohydroxamate-based histone deacetylase 6 (HDAC6) inhibitors with the ability to potentiate anti-PD-L1 immunotherapy in melanoma.

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.

HDAC6 inhibition alleviates acute pulmonary embolism: a possible future therapeutic option.

INTRODUCTION: Acute pulmonary embolism (APE) is a clinical syndrome of pulmonary circulation disorder caused by obstruction of the pulmonary artery or its branches. Histone deacetylase 6 (HDAC6) has been reported to play an important role in lung-related diseases. However, the functional role of HDAC6 in APE remains unclear. MATERIAL AND METHODS: Male Sprague Dawley rats were used. The APE model was constructed by inserting an intravenous cannula into the right femoral vein and injecting Sephadex G-50 microspheres (12 mg/kg; 300 µm in diameter). After 1 h, the control and APE rats were intraperitoneally injected with tubastatin A (TubA) (40 mg/kg, an inhibitor of HDAC6) and sampled at 24 h after modeling. H&E staining, arterial blood gas analysis, and wet/dry (W/D) weight ratio were used to evaluate the histopathological changes and pulmonary function in APE rats. ELISA, Western blot, and immunohistochemistry were used to explore the potential mechanism of HDAC6-mediated inflammation in APE. RESULTS: The results indicated that HDAC6 expression was significantly increased in lungs of APE rats. TubA treatment in vivo decreased HDAC6 expression in lung tissues. HDAC6 inhibition alleviated histopathological damage and pulmonary dysfunction, as evidenced by decreased PaO2/FiO2 ratio and W/D weight ratio in APE rats. Furthermore, HDAC6 inhibition alleviated APE-induced inflammatory response. Specifically, APE rats exhibited increased production of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, IL-6, and IL-18, however, this increase was reversed by HDAC6 inhibition. Meanwhile, the activation of the NLRP3 inflammasome was also observed in lungs of APE rats, while HDAC6 inhibition blocked this activation. Mechanically, we demonstrated that HDAC6 inhibition blocked the activation of the protein kinase B (AKT)/extracellular signal-regulated protein kinase (ERK) signaling pathway, a classic pathway promoting inflammation. CONCLUSIONS: These findings demonstrate that the inhibition of HDAC6 may alleviate lung dysfunction and pathological injury resulting from APE by blocking the AKT/ERK signaling pathway, providing new theoretical fundamentals for APE therapy.

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms. / ç»„è›‹ç™½è„±ä¹™é °é ¶6特异性抑制剂tubastatin Aå¯¹å¤§é¼ è››ç½‘è†œä¸‹è ”å‡ºè¡€çš„ä¿æŠ¤ä½œç”¨åŠå ¶æœºåˆ¶.

OBJECTIVES: 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.

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#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.

Role and mechanism of tubastatin A in alleviating brain injury after cardiopulmonary resuscitation by inhibiting endoplasmic reticulum stress-mediated cell apoptosis in swine / 中华急诊医学杂志

Objective:To explore the role and mechanism of tubastatin A (TubA) in alleviating brain injury after cardiac arrest and cardiopulmonary resuscitation (CA-CPR) by inhibiting endoplasmic reticulum stress-mediated cell apoptosis in swine.Methods:Twenty-three conventional male white swine, weighing 33-40 kg, aged 4 to 6 months, were divided into 3 groups by random number table method: sham group ( n=6), CA-CPR group ( n=9), and TubA group ( n=8). The CA-CPR swine model was established by 9 min of electrically induced CA through pacing catheter in the right ventricle and then 6 min of CPR in the CA-CPR group. The CA-CPR swine model was established by the same method, and then a dose of 4.5 mg/kg of TubA at 5 min after resuscitation was intravenously infused in the TubA group. The serum concentrations of neuron specific enolase (NSE) and S100β protein (S100β) were measured using ELISA before modeling and at 1, 2, 4 and 24 h after resuscitation. Neurological deficit score (NDS) was evaluated at 24 h after resuscitation. Thereafter, the animals were euthanized, and brain cortex tissues were harvested, and the expression levels of caspase-12 and caspase-3 were measured using immunohistochemistry. Cell apoptosis index was detected by TUNEL assay. The variables among the three groups were compared with one-way analysis of variance and the Bonferroni hoc test using SPSS software. Results:Twenty-four h after resuscitation, the serum concentrations of NSE and S100β were significantly increased, and NDS was markedly elevated in the CA-CPR and TubA groups compared with the sham group (all P<0.05). Compared with the CA-CPR group, serum concentration of NSE starting 2 h after resuscitation and serum concentration of S100β starting 1 h after resuscitation were significantly decreased in the TubA group [NSE (ng/mL): (23.1±2.0) vs. (20.2±2.0) at 2 h, (28.4±2.3) vs. (23.7±1.9) at 4 h, (32.1±2.7) vs. (26.6±2.0) at 24 h; S100β (pg/mL): (2239±193) vs. (1923±101) at 1 h, (2817±157) vs. (2360±141) at 2 h, (3384±250) vs. (2691±210) at 4 h, (3965±303) vs. (3119±260) at 24 h, all P<0.05], and NDS was markedly reduced (240±30 vs. 63±44, P<0.05). At 24 h after resuscitation, brain cortex tissue detection showed that the expression levels of caspase-12 and caspase-3 were significantly increased, and cell apoptosis index was markedly elevated in the CA-CPR and TubA groups compared with the sham group (all P<0.05). However, the expression levels of caspase-12 and caspase-3 were significantly decreased [caspase-12:(7.1±0.7) vs. (4.2±0.4); caspase-3: (13.3±1.6) vs. (7.7±0.8), all P<0.05], and cell apoptosis index was markedly reduced in the TubA group compared to the CA-CPR group [(31.1±8.6) vs. (17.3±2.2), P<0.05]. Conclusions:TubA alleviates brain injury and neurological dysfunction after CA-CPR in swine, which may be related to the inhibition of cell apoptosis mediated by endoplasmic reticulum stress.

Pharmacologic Inhibition of Histone Deacetylase 6 Prevents the Progression of Chlorhexidine Gluconate-Induced Peritoneal Fibrosis by Blockade of M2 Macrophage Polarization.

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-ß/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-ß/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.

Pharmacological inhibition of HDAC6 overcomes cisplatin chemoresistance by targeting cancer stem cells in oral squamous cell carcinoma.

BACKGROUND: Chemoresistance is associated with recurrence and metastasis in oral squamous cell carcinoma (OSCC). The cancer stem cell (CSC) subpopulation is highly resistant to therapy, and they are regulated by epigenetic mechanisms. HDACs are histone deacetylase enzymes that epigenetically regulate gene expression. HDAC6 acts on several physiological processes, including oxidative stress, autophagy and DNA damage response, and its accumulation is associated with cancer. Here, we investigate the role of HDAC6 in CSC-mediated chemoresistance in oral carcinoma in addition to its application as a therapeutic target to reverse chemoresistance. METHODS: Wild-type oral carcinoma cell lines (CAL27 WT and SCC9 WT), cisplatin-resistant (CAL27 CisR and SCC9 CisR), and the subpopulations of cancer stem cells (CSC+) and non-stem (CSC-) derived from CisR cells were investigated. HDAC6 accumulation was analyzed by Western blot and immunofluorescence; DNA damage was evaluated by immunofluorescence of phospho-H2A.X; the qPCR for PRDX2, PRDX6, SOD2, and TXN and ROS assay assessed oxidative stress. Apoptosis and CSC accumulation were investigated by flow cytometry. RESULTS: We identified the accumulation of HDAC6 in CisR cell lines and CSC. Cisplatin-resistant cell lines and CSC demonstrated a reduction in DNA damage and ROS and elevated expression of PRDX2. The administration of tubastatin A (a specific HDAC6 inhibitor) increased oxidative stress and DNA damage and decreased PRDX2. Tubastatin A as a monotherapy induced apoptosis in CisR and CSC and reduced the stemness phenotype. CONCLUSION: High levels of HDAC6 sustain CSC subpopulation and chemoresistance in OSCC, suggesting HDAC6 as a pharmacological target to overcome resistance and perhaps prevent recurrence in OSCC.

Tubastatin A suppresses the proliferation of fibroblasts in epidural fibrosis through phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway.

OBJECTIVES: This study was designed to explore the effect of tubastatin A (Tub A) on epidural fibrosis and the underlying mechanism. METHODS: Histone deacetylase 6 (HDAC6)-overexpressed fibroblasts were constructed, and the effect of Tub A on the proliferation of activated fibroblasts was detected by Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine (EdU) and cell cycle assay. Besides, 20 Sprague-Dawley rats were subjected to animal laminectomy model construction and then randomly treated with 4% dimethyl sulfoxide (DMSO) (diluted in 0.9% saline) or Tub A (10 mg/kg/day), separately. The expression of HDAC6 and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway-related proteins was measured in epidural fibrosis tissues. KEY FINDINGS: HDAC6 was overexpressed in activated fibroblasts and epidural scar tissues of rat models. Cell proliferation was remarkably elevated in HDAC6-overexpressed fibroblasts, which was reflected by cell viability, EdU and flow cytometry-based cell cycle assay, and paralleled with the increased expression of phosphorylated PI3K, AKT and mTOR, which was remarkably reversed following Tub A treatment. 740Y-P activator addition significantly reversed the declined fibroblast proliferation induced by Tub A. The expressions of PI3K/AKT/mTOR pathway-related proteins were also reduced in epidural tissues in rat models with Tub A treatment. CONCLUSION: Tub A could prevent epidural fibrosis formation by inhibiting fibroblast proliferation through mediating PI3K/AKT/mTOR pathway.

Tubastatin A Improves Post-Resuscitation Myocardial Dysfunction by Inhibiting NLRP3-Mediated Pyroptosis Through Enhancing Transcription Factor EB Signaling.

Background Myocardial dysfunction is the leading cause of early death following successful cardiopulmonary resuscitation (CPR) in people with cardiac arrest (CA), which is potentially driven by cell pyroptosis mediated by NOD-like receptor pyrin domain 3 (NLRP3) inflammasome. Recently, histone deacetylase 6 (HDAC6) inhibition was shown to exert effective myocardial protection against regional ischemia/reperfusion injury. In this study, we investigated whether tubastatin A, a specific histone deacetylase 6 inhibitor, could improve postresuscitation myocardial dysfunction through the inhibition of NLRP3-mediated cell pyroptosis and its modulation mechanism. Methods and Results Healthy male white domestic swine were used to establish the model of CA/CPR in vivo, and the H9c2 cardiomyocyte hypoxia/reoxygenation model was used to simulate the CA/CPR process in vitro. Consequently, tubastatin A inhibited NLRP3 inflammasome activation, decreased proinflammatory cytokines production and cell pyroptosis, and increased cell survival after hypoxia/reoxygenation in H9c2 cardiomyocytes in vitro. In addition, tubastatin A increased the acetylated levels of transcription factor EB and its translocation to the nucleus, and its protective effect above was partly abrogated by transcription factor EB short interfering RNA after hypoxia/reoxygenation in H9c2 cardiomyocytes. Similarly, tubastatin A promoted cardiac transcription factor EB nuclear translocation, inhibited NLRP3-mediated cell pyroptosis, and mitigated myocardial dysfunction after CA/CPR in swine. Conclusions The inhibition of histone deacetylase 6 activity by tubastatin A limited NLRP3 inflammasome activation and cell pyroptosis probably through the enhancement of transcription factor EB signaling, and therefore improved myocardial dysfunction after CA/CPR.

Tubastatin A maintains adult skeletal muscle stem cells in a quiescent state ex vivo and improves their engraftment ability in vivo.

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.

MEC17-induced α-tubulin acetylation restores mitochondrial transport function and alleviates axonal injury after intracerebral hemorrhage in mice.

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.

Selective Inhibitors of Histone Deacetylase 10 (HDAC-10).

Histone acetylation balance is one epigenetic mechanism controlling gene expression associated with disease progression. It has been observed that histone deacetylase 10 (HDAC-10) isozyme contributes to the chemotherapy resistance; in addition, the poor clinical outcome observed in patients with aggressive solid tumors, such as neuroblastoma, has been associated with its overexpression. Moreover, HDAC-10 selective inhibition suppresses the autophagic response, thus providing an improved risk-benefit profile compared to cytotoxic cancer chemotherapy drugs. On these bases, HDAC-10 is becoming an emerging target for drug design. Due to the rapid progress in the development of next-generation HDAC inhibitors, this review article aims to provide an overview on novel selective or dual HDAC-8/10 inhibitors, as new leads for cancer chemotherapy, able to avoid the severe side-effects of several actual approved "pan" HDAC inhibitors. A literature search was conducted in MedLine, PubMed, Caplus, SciFinder Scholar databases from 2015 to the present. Since the disclosure that the HDAC-6 inhibitor Tubastatin A was able to bind HDAC-10 efficiently, several related analogues were synthesized and tested. Both tricyclic (25-30) and bicyclic (31-42) derivatives were considered. The best pharmacological profile was shown by 36 (HDAC-10 pIC50 = 8.4 and pIC50 towards Class I HDACs from 5.2-6.4). In parallel, based on the evidence that high levels of HDAC-8 are a marker of poor prognosis in neuroblastoma treatment, dual HDAC-8/10 inhibitors were designed. The hydroxamic acid TH34 (HDAC-8 and 10 IC50 = 1.9 µM and 7.7 µM, respectively) and the hybrid derivatives 46d, 46e and 46g were the most promising both in terms of potency and selectivity. Literature surveys indicate several structural requirements for inhibitory potency and selectivity towards HDAC-10, e.g., electrostatic and/or hydrogen bond interactions with E274 and complementarity to the P(E,A) CE motif helix.

HDAC inhibitor ameliorates behavioral deficits in Mecp2308/y mouse model of Rett syndrome.

Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder. More than 95% of classic RETT syndrome cases result from pathogenic variants in the methyl-CpG binding protein 2 (MECP2) gene. Nevertheless, it has been established that a spectrum of neuropsychiatric phenotypes is associated with MECP2 variants in both females and males. We previously reported that microtubule growth velocity and vesicle transport directionality are altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared to that of their wild-type littermates suggesting deficit in microtubule dynamics. In this study, we report that administration of tubastatin A, a selective HDAC6 inhibitor, restored microtubule dynamics in Mecp2-deficient astrocytes. We furthermore report that daily doses of tubastatin A reversed early impaired exploratory behavior in male Mecp2308/y mice. These findings are a first step toward the validation of a novel treatment for RTT.

Role of epigenetic regulation in myocardial ischemia/reperfusion injury.

Nowadays acute myocardial infarction (AMI) is a serious cardiovascular disease threatening the human life and health worldwide. The most effective treatment is to quickly restore coronary blood flow through revascularization. However, timely revascularization may lead to reperfusion injury, thereby reducing the clinical benefits of revascularization. At present, no effective treatment is available for myocardial ischemia/reperfusion injury. Emerging evidence indicates that epigenetic regulation is closely related to the pathogenesis of myocardial ischemia/reperfusion injury, indicating that epigenetics may serve as a novel therapeutic target to ameliorate or prevent ischemia/reperfusion injury. This review aimed to briefly summarize the role of histone modification, DNA methylation, noncoding RNAs, and N6-methyladenosine (m6A) methylation in myocardial ischemia/reperfusion injury, with a view to providing new methods and ideas for the research and treatment of myocardial ischemia/reperfusion injury.