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.

Genistein attenuates LPS-induced inflammatory injury of rat dorsal root ganglion neuron via down-regulating HDAC6 / 中南大学学报(医学版)

OBJECTIVES@#Neuropathic pain (NP) is a chronic pain caused by somatosensory neuropathy or disease, and genistein (Gen) might be a potential drug for the treatment of NP. Therefore, this study aims to investigate the effect of Gen on lipopolysaccharide (LPS)-induced inflammatory injury of dorsal root ganglion neuron (DRGn) in rats and the possible molecular mechanism.@*METHODS@#The DRGn of 1-day-old juvenile rats were taken for isolation and culture. The DRGn in logarithmic growth phase were divided into a control group, a LPS group, a tubastatin hydrochloride (TSA)+LPS group, a Gen1+LPS group, a Gen2+LPS group, a Gen2+LPS+TSA group, a Gen2+pcDNA-histone deacetylase 6 (HDAC6)+LPS group, and a Gen2+pcDNA3.1+LPS group. The LPS group was treated with 1 μg/mL LPS for 24 h; the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group were treated with 5 μmol/L TSA, 5 μmol/L Gen, 10 μmol/L Gen respectively for 0.5 h, and then added 1 μg/mL LPS for 24 h; the Gen2+TSA+LPS group was treated with 10 μmol/L Gen and 5 μmol/L TSA for 0.5 h and then added 1 μg/mL LPS for 24 h; the Gen2+pcDNA-HDAC6+LPS group and the Gen2+pcDNA3.1+LPS group received 100 nmol/L pcDNA-HDAC6 and pcDNA3.1 plasmids respectively, and 24 h after transfection, 10 μmol/L Gen was pretreated for 0.5 h, and then added 1 μg/mL LPS for 24 h. Real-time RT-PCR was used to detect the HDAC6 mRNA expression in DRGn; CCK-8 method was used to detect cell viability of DRGn; flow cytometry was used to detect cell apoptosis of DRGn; ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α in DRGn culture supernatant; Western blotting was used to detect the protein expression of HDAC6, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 in DRGn.@*RESULTS@#Compared with the control group, the expression levels of HDAC6 mRNA and protein, the expression levels of TLR4 and MyD88 protein in DRGn of LPS group rats were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, and the activity of DRGn was significantly decreased, the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05). Compared with the LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group and the Gen2+TSA+LPS group were significantly down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly decreased, the activity of DRGn was significantly increased, the apoptosis rate was significantly decreased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly decreased (all P<0.05), and the above changes were most obvious in the Gen2+TSA+LPS group. Compared with the Gen2+LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the Gen2+pcDNA-HDAC6+LPS group were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, the activity of DRGn was significantly decreased, and the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05).@*CONCLUSIONS@#Gen can alleviate LPS-induced DRGn inflammatory injury in rats, which might be related to down-regulating the expression of HDAC6 and further inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway.

Characterization of aggrephagy in exercise-induced muscle damage / 中国组织工程研究

BACKGROUND: A high-intensity eccentric exercise may lead to skeletal muscle damage, endoplasmic reticulum stress, increasing misfolded proteins. Aggrephagy works as an effective way of controlling quality of proteins and plays an important role in damage repair. OBJECTIVE: To discover the activation degree of aggrephagy in the damage repair process after an overload eccentric exercise, and to discuss the role of aggrephagy in the exercise-induced muscle damage, which is supplementary to the protein degradation pathway in skeletal muscle damage. METHODS: Sixty-four male adult Sprague Dawley rats were randomly divided into a static control group (n=8) and seven exercise groups according to post-exercise time (0, 3, 6,12, 24,48 and 72 hours, eight rats in each). Damage models were established by downhill running (-16°, 16 m/min, 90 minutes). Subsequently, the histological changes of the rat soleus muscle was observed under an electron microscope and protein expressions of histone deacetylase 6 and microtubule-associated protein 1 light chain 3II/I related to aggrephagy were detected using western bot assay. RESULTS AND CONCLUSION: (1) Under the transmission electron microscope, the rat soleus muscle was disordered and widened and appeared to have transient sarcomere, the Z line was water wave-like, broken and disappeared, and the mitochondria were swollen and unevenly distributed with an unclear structure after high-intensity eccentric exercise. The model rats suffered severest muscle damage at 12 hours and recovered completely at 72 hours after high-intensity eccentric exercise. (2) After one-time eccentric exercise, the expression of histone deacetylase 6 had a transient increase. The histone deacetylase 6 expression reached the peak at 6 hours, then gradually reduced and recovered at 72 hours after exercise. The microtubule-associated protein 1 light chain 3II/I expression was significantly increased at 3 hours after exercise, reached the peak at 12 hours, then reduced significantly and recovered at 72 hours after exercise. All these findings indicate that aggrephagy transiently happens and exerts important roles in the clearance of misfolded proteins, maintenance of cellular environmental homeostasis and recovery of skeletal muscle damage.

The different expression and significance of methyl CpG binding protein 2 and histone deacetylase 6 in human scar formation / 中华整形外科杂志

Objective@#To investigate the expression and significance of fibroblasts methyl CpG binding protein 2(MECP2)and histone deacetylase 6(HDAC6) at different stages of scar tissues and hypertrophic scars.@*Methods@#From August 2016 to May 2017, 118 cases of scar and 33 cases of upper eyelid lasia received from the Department of Plastic and Burn Surgery of the First Affiliated Hospital of Chongqing Medical University, Chongqing were collected, including 33 cases of normal skin (ages 34-68, 12 cases of male and 21 cases of female), 32 cases of normal scar (ages 8-68, 19 cases of male and 13 cases of female), 35 cases of keloid (ages 11-62, 11 cases of male and 24 cases of female) and 51 cases of hypertrophic scar (ages 12-58, 22 cases of male and 29 cases of female) without any treatment.The hypertrophic scar was divided into 4 groups according to the growth time: 10 cases in the 0-3 month group, 11 cases in the 4-6 month group, 13 cases in the 7-12 month group, and 17 cases in the >12 month group. Immunohistochemistry and western blot were used to detect the expressions of MECP2 and HDAC6 in tissues of each group, and real-time quantitative polymerase chain reaction gene amplification fluorescence detection technology was used to detect the expressions of MECP2mRNA and HDAC6 mRNA, and the differences between each group were compared. SPSS20.0 was used for statistical analysis of the data obtained in the experiment, and one-way ANOVAwas used to test the differences between the groups. P<0.05 was considered statistically significant.@*Results@#MECP2 protein is gradually expressed in normal skin (1 326.4±572.3), normal scar (2 341.4±816.2), hypertrophic scar (3 500.7±1407.6) and keloid (4 787.9±1 514.3), F=33.82, P=0.001. Similarly, the expression of MECP2mRNA in normal skin (0.82±0.43), normal scar (1v14±0.45), hypertrophic scar (1.59±0.39) and keloid (2.14±0.53) was also gradually increased, F=23.4, P=0.001. In normal skin (1 332.5±746.7) and normal scar (2 307.7±1 027.4), hypertrophic scar (4 107.4±1 223.1) and keloid, the level of HDAC6 protein expression (5 155.9±1 265.3) were significant different, F = 50.27, P=0.001.There were still statistically significant differences in HDAC6mRNA between normal skin (0.57±0.23), normal scar(1.03±0.35), hypertrophic scar (1.47±0.31) and keloid (1.87±0.45), F=38.06, P=0.001.In hypertrophic scar, there was no significant difference in MECP2 between the 0-3 month group (4 758.4±660.1) and the 4-6 month group (4 602.4 ±583.9), F=1.28, P=0.97), 7-12 month (3 000.7±982.7) group and >12 month (1 990.7 ±992.3)group of MECP2 expression quantity decrease, F=25.8, P=0.001; There was no significant difference in the level of HDAC6 protein in the 0-3 month (5 069.3±1 236.1) group and 4-6 month (5 316.7±1 237.4) group, F=1.02, P=0.98, and the expression was significantly reduced in the 7-12 month (3 084.7±1 685.5) group and the>12 month (2 304.6±1 337.1)group, F=11.41, P=0.001.@*Conclusions@#MECP2 and HDAC6 play a critical role in the formation of scarring in the human body. Epigenetic regulation mediated by MECP2 and HDAC6 is an important target for inhibiting scar formation.

Mechanism of evodiamine inducing cell cycle arrest and apoptosis in human erythroleukemia K562 cells through inhibiting histone deacetylase 6 / 中草药

Objective: To explore the mechanism of evodiamine (Evo) inducing cell cycle arrest and apoptosis in K562 cells. Methods: The effect of Evo on proliferation of K562 cells was measured by Cell Counting Kit-8 assay (CCK-8 assay), and cell cycle distribution and apoptosis were determined by flow cytometry (FCM). Chemical colorimetry assay was used to examine the activity of histone modification enzymes. The expression levels of histone deacetylase 6 (HDAC6), Cyclin D1, CDK4, Bcl-2, Bax, Cleaved Caspase-3, ERK, p-ERK, p38, and p-p38 proteins were ascertained by Western blotting. Results: The proliferation of K562 cells was inhibited by Evo (1-16 μmol/L) in a dose- and time-dependent manner. FCM analyses revealed that Evo induced cell-cycle arrest in G0/G1 phase in K562 cells. The apoptosis rates of K562 cells were (11.47 ± 1.05)%, (12.77 ± 0.79)%, and (18.58 ± 1.37)% respectively after induced by Evo with different concentration (2, 4, and 8 μmol/L), which showed statistically significant difference compared with the control group (2.79 ± 1.01)% (P < 0.01). The activity of HDACs was reduced after treated with Evo (2, 4, and 8 μmol/L). Western blotting assay showed that the expression of Bax, Cleaved Caspase-3, p38, and p-p38 proteins increased, while CDK4, Cyclin D1, Bcl-2, HDAC6, ERK, and p-ERK proteins down-reguation after induced by Evo. Conclusion: Evo can induce cell cycle arrest and apoptosis in K562 cells through the inhibition of HDAC6.

Potential Prognostic Value of Histone Deacetylase 6 and Acetylated Heat-Shock Protein 90 in Early-Stage Breast Cancer / 한국유방암학회지

PURPOSE: Histone deacetylase 6 (HDAC6) is an enzyme that deacetylates heat-shock protein 90 (HSP90). Many studies have investigated the role of HDAC6 and HSP90 in tumorigenesis and in the prognosis of cancer patients. This study aimed to evaluate the prognostic value of HDAC6 and acetylated HSP90 (acetyl-HSP90) in a cohort of breast cancer patients. METHODS: Immunohistochemical analysis of 314 surgical specimens obtained from patients with invasive breast cancer was carried out to assess standard pathologic factors and the expression of HDAC6 and acetyl-HSP90. Statistical analyses were performed to determine the association between HDAC6, acetyl-HSP90, and conventional clinicopathological factors, and the prognostic values of these factors were evaluated. RESULTS: HDAC6 expression did not show any correlation with other clinicopathological factors, but acetyl-HSP90 was significantly correlated with histologic grade (p=0.001) and the Ki-67 index (p=0.015). HDAC6 and acetyl-HSP90 expression were significantly associated with each other (p=0.047). Although HDAC6 was not prognostic for disease-free survival (DFS), some patients with high expression of HDAC6 experienced recurrence 5 years after diagnosis, while there was no recurrent disease after 5 years in those with low expression. Acetyl-HSP90 was significantly associated with the DFS of all patients (p=0.016) and with high HDAC6 expression (p=0.017), but not with low expression. CONCLUSION: Expression of HDAC6 and acetyl-HSP90 are correlated. HDAC6 is proposed to be a possible predictive marker of late recurrence, and acetyl-HSP90 has prognostic value in predicting the DFS of breast cancer patients.

Expression and significance of histone deacetylase 6 in non-small cell lung cancer / 国际肿瘤学杂志

Objective To detect the expression of histone deacetylase 6 (HDAC6) and its significance in non-small cell lung cancer (NSCLC).Methods The expression of HDAC6 was measured by SP staining in 70 cases of NSCLC and 18 cases of normal lung tissues.Results Positive rate of HDAC6 immunostaining in NSCLC was 58.57％,which was significantly higher than 16.67％ in normal lung tissues(P ＜0.01 ).No correlation was found between HDAC6 expression and the age,sex,differentiated level,histological type ( P ＞0.05).But the expression level of HDAC6 was closely related to clinical stage and lymph node metastasis in NSCLC patients ( P ＜ 0.05 ).Conclusion HDAC6 over expression may be related to the pathogenesis and development of NSCLC.