OBJECTIVE: Acute liver failure (ALF) is characterized by severe liver dysfunction, rapid progression and high mortality and is difficult to treat. Studies have found that sulforaphane (SFN), a nuclear factor E2-related factor 2 (NRF2) agonist, has anti-inflammatory, antioxidant and anticancer effects, and has certain protective effects on neurodegenerative diseases, cancer and liver fibrosis. This paper aimed to explore the protective effect of SFN in ALF and it possible mechanisms of action. METHODS: Lipopolysaccharide and D-galactosamine were used to induce liver injury in vitro and in vivo. NRF2 agonist SFN and histone deacetylase 6 (HDAC6) inhibitor ACY1215 were used to observe the protective effect and possible mechanisms of SFN in ALF, respectively. Cell viability, lactate dehydrogenase (LDH), Fe²⁺, glutathione (GSH) and malondialdehyde (MDA) were detected. The expression of HDAC6, NRF2, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) were detected by Western blotting and immunofluorescence. RESULTS: Our results show that NRF2 was activated by SFN. LDH, Fe²⁺, MDA and ACSL4 were downregulated, while GSH, GPX4 and SLC7A11 were upregulated by SFN in vitro and in vivo, indicating the inhibitory effect of SFN on ferroptosis. Additionally, HDAC6 expression was decreased in the SFN group, indicating that SFN could downregulate the expression of HDAC6 in ALF. After using the HDAC6 inhibitor, ACY1215, SFN further reduced HDAC6 expression and inhibited ferroptosis, indicating that SFN may inhibit ferroptosis by regulating HDAC6 activity. CONCLUSION SFN has a protective effect on ALF, and the mechanism may include reduction of ferroptosis through the regulation of HDAC6. Please cite this article as: Zhang YQ, Shi CX, Zhang DM, Zhang LY, Wang LW, Gong ZJ. Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity. J Integr Med. 2023; 21(5): 464-473.
Humans ; Ferroptosis ; NF-E2-Related Factor 2/genetics* ; Liver Failure, Acute/drug therapy* ; Isothiocyanates/pharmacology* ; Glutathione ; Histone Deacetylase 6

Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin's lymphoma. A total of 10%‒15% of DLBCL cases are associated with myelocytomatosis viral oncogene homolog(MYC) and/or B-cell lymphoma-2 (BCL2) translocation or amplification. BCL2 inhibitors have potent anti-tumor effects in DLBCL; however, resistance can be acquired through up-regulation of alternative anti-apoptotic proteins. The histone deacetylase (HDAC) inhibitor chidamide can induce BIM expression, leading to apoptosis of lymphoma cells with good efficacy in refractory recurrent DLBCL. In this study, the synergistic mechanism of chidamide and venetoclax in DLBCL was determined through in vitro and in vivo models. We found that combination therapy significantly reduced the protein levels of MYC, TP53, and BCL2 in activated apoptotic-related pathways in DLBCL cells by increasing BIM levels and inducing cell apoptosis. Moreover, combination therapy regulated expression of multiple transcriptomes in DLBCL cells, involving apoptosis, cell cycle, phosphorylation, and other biological processes, and significantly inhibited tumor growth in DLBCL-bearing xenograft mice. Taken together, these findings verify the in vivo therapeutic potential of chidamide and venetoclax combination therapy in DLBCL, warranting pre-clinical trials for patients with DLBCL.
Aminopyridines ; Animals ; Benzamides ; Biological Phenomena ; Bridged Bicyclo Compounds, Heterocyclic ; Down-Regulation ; Histone Deacetylase Inhibitors/therapeutic use* ; Humans ; Lymphoma, Large B-Cell, Diffuse/pathology* ; Mice ; Neoplasm Recurrence, Local ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Proto-Oncogene Proteins c-myc/therapeutic use* ; Sulfonamides ; Tumor Suppressor Protein p53/metabolism*

Oogenesis is the basic reproductive process of female mammals and is essential for fertilization and embryo development. Recent studies have shown that epigenetic modifications play an important role in the regulation of mammalian reproductive processes (such as oogenesis, spermatogenesis, preimplantation embryo development and sex differentiation). Taking histone acetylation as an instance, the dynamic changes of histone acetyltransferases (HATs) and deacetylases (HDACs) are involved in the regulation of gene activation and inactivation when numerous key physiological events occur during reproduction. Thereinto, HDAC1 and HDAC2, which are highly homologous in terms of both structure and function, play a pivotal role in murine oogenesis. HDAC1 and 2 jointly regulate the global transcription and the incidence of apoptosis of growing oocytes and affect its subsequent growth and development, which reflects their compensatory function. In addition, HDAC1 and 2 also play a specific part in oogenesis respectively. It has shown that HDAC2 is more critical than HDAC1 for oocyte development, which regulates de novo DNA methylation and chromosome segregation. Reciprocally, HDAC1 is more critical than HDAC2 for preimplantation development. Deficiency of HDAC1 causes the decreased proliferation of embryonic stem cells and the smaller embryoid bodies with irregular shape. In this review, we summarized the role and the current research progress of HDAC1/2 in murine oogenesis, to provide a reference for further understanding the relationship between epigenetic modifications and reproductive regulation.
Acetylation ; Animals ; Embryonic Development ; Female ; Histone Deacetylase 1/metabolism* ; Histone Deacetylase 2/metabolism* ; Histone Deacetylases/metabolism* ; Male ; Mice ; Oocytes ; Oogenesis

OBJECTIVE: To study the role and mechanism of histone deacetylase 1 (HDAC1) and histone deacetylase 2 (HDAC2) in mouse neuronal development. METHODS: The mice with Synapsin1-Cre recombinase were bred with RESULTS: The mice with CONCLUSIONS Deletion of
Animals ; Blotting, Western ; Histone Deacetylase 1/genetics* ; Histone Deacetylase 2 ; Histone Deacetylases/genetics* ; Immunohistochemistry ; Mice ; Neurons/metabolism* ; Signal Transduction

OBJECTIVES: To investigate glucocorticoid receptor (GR) and histone deacetylase 2 (HDAC2) gene expression and protein levels in peripheral blood mononuclear cells (PBMCs) of patients with severe or profound sudden sensorineural hearing loss (SSNHL) and to explore the roles of GRs and HDAC2 in glucocorticoid (GC) insensitivity. METHODS: Fifty-five severe or profound SSNHL patients were enrolled in the study. According to hearing improvement after GC treatment, patients were assigned into two groups: GC-sensitive and GC-resistant. A normal reference group included 20 healthy volunteers without hearing loss. Quantitative real-time polymerase chain reaction and Western blot analyses were used to detect the relative expression of GRα, GRβ, and HDAC2 in PBMCs at the mRNA and protein levels. RESULTS: The protein levels of GRs and HDAC2 in PBMCs of SSNHL patients were lower than the normal reference values before GC treatment. Compared with the GC-resistant group, both the mRNA and protein levels of GRα and HDAC2 were significantly increased in the GC-sensitive group after GC treatment. CONCLUSION: A lack of GRα and HDAC2 induction following steroid treatment in GC-resistant SSNHL patients may play a fundamental mechanistic role in GC insensitivity. Response of GRα and HDAC2 to steroid treatment may, thus, predict the prognosis of hearing improvement in SSNHL patients.
Blotting, Western ; Gene Expression ; Healthy Volunteers ; Hearing ; Hearing Loss ; Hearing Loss, Sensorineural ; Histone Deacetylase 2 ; Histone Deacetylases ; Histones ; Humans ; Prognosis ; Real-Time Polymerase Chain Reaction ; Receptors, Glucocorticoid ; Reference Values ; RNA, Messenger

BACKGROUND: Glucocorticoids have been widely used to treat patients with chronic obstructive pulmonary disease (COPD). Nevertheless, corticosteroid insensitivity is a major barrier to the effective treatment of COPD and its mechanism remains unclear. This study aimed to evaluate the effect of cathelicidin LL-37 on corticosteroid insensitivity in COPD rat model, and to explore the involved mechanisms. METHODS: COPD model was established by exposing male Wistar rats to cigarette smoke combined with intratracheal instillation of lipopolysaccharide (LPS). Inhaled budesonide and LL-37 were consequently applied to COPD models separately or collectively to confirm the effects on inflammatory cytokines (tumor necrosis factor [TNF]-α and transforming growth factor [TGF]-β) by enzyme-linked immunosorbent assay (ELISA) and lung tissue histopathological morphology. Expression of histone deacetylase-2 (HDAC2) and phosphorylation of Akt (p-AKT) in lung were also measured. RESULTS: Briefly, COPD model rats showed an increased basal release of inflammatory cytokines (lung TNF-α: 45.7 ± 6.1 vs. 20.1 ± 3.8 pg/mL, P < 0.01; serum TNF-α: 8.9 ± 1.2 vs. 6.7 ± 0.5 pg/mL, P = 0.01; lung TGF-β: 122.4 ± 20.8 vs. 81.9 ± 10.8 pg/mL, P < 0.01; serum TGF-β: 38.9 ± 8.5 vs. 20.6 ± 2.3 pg/mL, P < 0.01) and COPD related lung tissue histopathological changes, as well as corticosteroid resistance molecular profile characterized by an increase in phosphoinositide 3-kinase (PI3K)/Akt (0.5 ± 0.1 fold of control vs. 0.2 ± 0.1 fold of control, P = 0.04) and a decrease in HDAC2 expression and activity (expression: 13.1 ± 0.4 μmol/μg vs. 17.4 ± 1.1 μmol/μg, P < 0.01; activity: 1.1 ± 0.1 unit vs. 1.4 ± 0.1 unit, P < 0.01), compared with control group. In addition, LL-37 enhanced the anti-inflammatory effect of budesonide in an additive manner. Treatment with combination of inhaled corticosteroids (ICS) and LL-37 led to a significant increase of HDAC2 expression and activity (expression: 15.7 ± 0.4 μmol/μg vs. 14.1 ± 0.9 μmol/μg, P < 0.01; activity: 1.3 ± 0.1 unit vs. 1.0 ± 0.1 unit, P < 0.01), along with decrease of p-AKT compared to budesonide monotherapy (0.1 ± 0.0 fold of control vs. 0.3 ± 0.1 fold of control, P < 0.01). CONCLUSIONS This study suggested that LL-37 could improve the anti-inflammatory activity of budesonide in cigarette smoke and LPS-induced COPD rat model by enhancing the expression and activity of HDAC2. The mechanism of this function of LL-37 might involve the inhibition of PI3K/Akt pathway.
Animals ; Antimicrobial Cationic Peptides ; pharmacology ; therapeutic use ; Glucocorticoids ; metabolism ; Histone Deacetylase 2 ; metabolism ; Humans ; Inflammation ; chemically induced ; drug therapy ; Lipopolysaccharides ; pharmacology ; Male ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; metabolism ; Rats ; Rats, Wistar ; Smoking ; adverse effects ; Tumor Necrosis Factor-alpha ; metabolism

Interferon-γ (IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells (BMECs) in vitro. In this study, we investigated the mechanisms underlying the malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ (10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4 (CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene (c-Abl) and histone deacetylase 2 (HDAC2). The HDAC2 inhibitor, valproate (VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.
Animals ; Carcinogenesis/pathology* ; Cattle ; Cell Cycle Proteins/metabolism* ; Cell Proliferation/drug effects* ; Cell Transformation, Neoplastic/pathology* ; Cells, Cultured ; Epithelial Cells/pathology* ; Female ; Histone Deacetylase 2/metabolism* ; Imatinib Mesylate/pharmacology* ; Interferon-gamma/pharmacology* ; Mammary Glands, Animal/pathology* ; Mammary Neoplasms, Experimental/pathology* ; Proto-Oncogene Proteins c-abl/metabolism* ; Signal Transduction ; Valproic Acid/pharmacology*

BACKGROUND: Asthma is a disease of chronic airway inflammation with heterogeneous features. Neutrophilic asthma is corticosteroid-insensitive asthma related to absence or suppression of TH2 process and increased TH1 and/or TH17 process. Macrolides are immunomodulatory drug that reduce airway inflammation, but their role in asthma is not fully known. The purpose of this study was to evaluate the role of macrolides in neutrophilic asthma and compare their effects with those of corticosteroids. METHODS: C57BL/6 female mice were sensitized with ovalbumin (OVA) and lipopolysaccharides (LPS). Clarithromycin (CAM) and/or dexamethasone (DXM) were administered at days 14, 15, 21, 22, and 23. At day 24, the mice were sacrificed. RESULTS: Airway resistance in the OVA+LPS exposed mice was elevated but was more attenuated after treatment with CAM+DXM compared with the monotherapy group (p < 0.05 and p < 0.01). In bronchoalveolar lavage fluid study, total cells and neutrophil counts in OVA+LPS mice were elevated but decreased after CAM+DXM treatment. In hematoxylin and eosin stain, the CAM+DXM-treated group showed less inflammation additively than the monotherapy group. There was less total protein, interleukin 17 (IL-17), interferon γ, and tumor necrosis factor α in the CAM+DXM group than in the monotherapy group (p < 0.001, p < 0.05, and p < 0.001). More histone deacetylase 2 (HDAC2) activity was recovered in the DXM and CAM+DXM challenged groups than in the control group (p < 0.05). CONCLUSION: Decreased IL-17 and recovered relative HDAC2 activity correlated with airway resistance and inflammation in a neutrophilic asthma mouse model. This result suggests macrolides as a potential corticosteroid-sparing agent in neutrophilic asthma.
Adrenal Cortex Hormones ; Airway Resistance ; Animals ; Asthma ; Bronchoalveolar Lavage Fluid ; Clarithromycin ; Dexamethasone ; Eosine Yellowish-(YS) ; Female ; Hematoxylin ; Histone Deacetylase 2 ; Histone Deacetylases ; Humans ; Inflammation ; Interferons ; Interleukin-17 ; Lipopolysaccharides ; Macrolides ; Mice ; Neutrophils ; Ovalbumin ; Th17 Cells ; Tumor Necrosis Factor-alpha

BACKGROUNDPlacental multidrug resistance-associated protein 2 (MRP2), encoded by ABCC2 gene in human, plays a significant role in regulating drugs' transplacental transfer rates. Studies on placental MRP2 regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the roles of epigenetic mechanisms in regulating placental drug transporters are still unclear. This study aimed to investigate the effect of histone deacetylases (HDACs) inhibition on MRP2 expression in the placental trophoblast cell line and to explore whether HDAC1/2/3 are preliminarily involved in this process.

METHODSThe human choriocarcinoma-derived trophoblast cell line (Bewo cells) was treated with the HDAC inhibitors-trichostatin A (TSA) at different concentration gradients of 0.5, 1.0, 3.0, and 5.0 μmol/L. Cells were harvested after 24 and 48 h treatment. Small interfering RNA (siRNA) specific for HDAC1/HDAC2/HDAC3 or control siRNA was transfected into cells. Total HDAC activity was detected by colorimetric assay kits. HDAC1/2/3/ABCC2 messenger RNA (mRNA) and protein expressions were determined by real-time quantitative polymerase chain reaction and Western-blot analysis, respectively. Immunofluorescence for MRP2 protein expression was visualized and assessed using an immunofluorescence microscopy and ImageJ software, respectively.

RESULTSTSA could inhibit total HDAC activity and HDAC1/2/3 expression in company with increase of MRP2 expression in Bewo cells. Reduction of HDAC1 protein level was noted after 24 h of TSA incubation at 1.0, 3.0, and 5.0 μmol/L (vs. vehicle group, all P < 0.001), accompanied with dose-dependent induction of MRP2 expression (P = 0.045 for 1.0 μmol/L, P = 0.001 for 3.0 μmol/L, and P < 0.001 for 5.0 μmol/L), whereas no significant differences in MRP2 expression were noted after HDAC2/3 silencing. Fluorescent micrograph images of MRP2 protein were expressed on the cell membrane. The fluorescent intensities of MRP2 in the control, HDAC2, and HDAC3 siRNA-transfected cells were week, and no significant differences were noticed among these three groups (all P > 0.05). However, MRP2 expression was remarkably elevated in HDAC1 siRNA-transfected cells, which displayed an almost 3.19-fold changes in comparison with the control siRNA-transfected cells (P < 0.001).

CONCLUSIONSHDACs inhibition could up-regulate placental MRP2 expression in vitro, and HDAC1 was probably to be involved in this process.

Cell Line ; Histone Deacetylase 1 ; metabolism ; Histone Deacetylase 2 ; metabolism ; Histone Deacetylase Inhibitors ; pharmacology ; Histone Deacetylases ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Microscopy, Fluorescence ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; RNA, Messenger ; Trophoblasts ; cytology ; metabolism

OBJECTIVETo investigate the effect of histone acetylation/deacetylation imbalances on embryonic hearts of mice and its effect on key genes of planar cell polarity (PCP) pathway-Vangl2, Scrib and Rac1 in H9C2 cells.

METHODSForty pregnant C57/B6 mice were randomly assigned into three groups: blank group (n=10), vehicle group (n=10), and valproic acid (VPA)-treated group (n=20). In the VPA-treated group, VPA, a histone deacetylase (HDAC) inhibitor, was administered to each individual dam intraperitoneally at a single dose of 700 mg/kg on embryonic day 10.5 (E10.5). The vehicle and blank groups received equivalent saline or no interventions, respectively. Dams were sacrificed on E15.5, and death rates of embryos were evaluated. Subsequently, embryonic hearts of survival fetus were removed to observe cardiac abnormalities by hematoxylin-eosin (HE) staining. H9C2 cells were cultured and allotted to the blank, vehicle, and VPA-treated groups: the VPA treated group received VPA exposure at concentrations of 2.0, 4.0 and 8.0 mmol/L; the vehicle and blank groups received equivalent saline or no interventions, respectively. HDAC1-3 as well as Vangl2, Scrib and Rac1 mRNA and protein expression levels were determined by quantitative real-time PCR and Western blot, respectively. The total HDAC activity was analyzed by colorimetric assay.

RESULTSThe fetus mortality rate after VPA treatment was 31.7%, with a significantly higher rate of cardiac abnormalities in comparison with the controls (P<0.05). In comparison with the blank and vehicle groups, HDAC1 mRNA was significantly increased at various concentrations of VPA treatment at all time points of exposure (P<0.05), together with a reduction of protein level after 48 and 72 hours of exposure (P<0.05). The inhibition of HDAC2 mRNA after various concentrations of VPA incubation was pronounced at 24 hours of exposure (P<0.05), while the protein levels were reduced at all time points (P<0.05). HDAC3 mRNA was prominently induced by VPA (4.0 and 8.0 mmol/L) at all time points of treatment (P<0.05). In contrast, the protein level was inhibited after VPA treatment (P<0.05). In comparison with the blank and vehicle groups, Vangl2 mRNA as well as Scrib mRNA/protein expression levels were markedly reduced after 48 and 72 hours of VPA treatment (P<0.05), together with a reduction of protein level in Vangl2 at 72 hours (P<0.05). Compared with the blank and vehicle groups, a significant repression in the total HDAC activity was observed in the VPA-treated group at concentrations of 4.0 and 8.0 mmol/L after 24 hours of treatment (P<0.05), and the effect persisted up to 48 and 72 hours, exhibiting pronounced inhibition at all concentrations (P<0.05).

CONCLUSIONSVPA might result in acetylation/deacetylation imbalances by inhibiting HDAC1-3 protein expression and total HDAC activity, leading to the down-regulation of mRNA and protein expression of Vangl2 and Scrib. This could be one of the mechanisms contributing to congenital heart disease.

Acetylation ; Animals ; Cell Polarity ; Cells, Cultured ; Fetal Heart ; drug effects ; metabolism ; Heart Defects, Congenital ; etiology ; Histone Deacetylase 1 ; genetics ; Histone Deacetylase 2 ; genetics ; Histones ; metabolism ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; genetics ; RNA, Messenger ; analysis ; Valproic Acid ; pharmacology