Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity / 中西医结合学报

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.

Effects of HDAC6 regulation of HSC70 acetylation level on bone loss in osteoporosis after oophorectomy / 中华内分泌外科杂志

Objective:To investigate the effects of histone deacetylase 6 (histone deacetylase 6, HDAC6) on oopherectomy (OOX) induced osteoporosis (OP) bone loss by binding to the promoter region of heat-shock protein 70 (HSC70) and regulating it’s acetylation.Methods:OP mouse model was established by using OOX methods. Then the mice were divided into sham operation group, OOX group, OOX+shHDAC6 group, OOX+shNC group and OOX+shHDAC6+shHSC70 group. The micro-CT system and Western blot experiment were used to detect the bone microscopic parameters of the mouse right femur and the protein expression levels of osteoblast-specific transcription factors. In vitro experiments, Westwen blot, alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining were used to determine the effects of HDAC6 and HSC70 on the osteogenic differentiation of MC3T3-E1 cells. QRT-PCR was used to detect the expression levels of HDAC6 and HSC70 in tissue or cells. The relationship between HDAC6 and HSC70 was analyzed by ChIP experiment.Results:Compared with sham group, the expression of bone mineral density (BMD) , trabecular bone number (Tb. N) , trabecular thickness (Tb.th) and bone volume fraction (BV/TV) in the right femur of OOX group mice were decreased, the expression of TB. Sp was increased, protein expression of OSX and RUNX2 was increased. At the same time, compared with sham group (1±0.11) , the expression of HDAC6 was increased in OOX group (2.33±0.19) ( t=10.56, P<0.001) . Compared with pcDNA3.1-NC group, the protein level of Osterix (OSX) and runt-related transcription factor 2 (RUNX2) , ALP activity and mineralized area in pcDNA3.1-HDAC6 group were decreased (all P<0.05) . ChIP analysis showed that compared with the pcDNA3.1-NC group (5.26±0.47) , the acetylation level of the HSC70 promoter region in the pcDNA3.1-HDAC6 group (2.37±0.21) was significantly reduced ( t=9.72, P<0.001) . Compared with pcDNA3.1-HDAC6 group, the expression of OSX and RUNX2, ALP activity and mineralization were increased in pcDNA3.1-HDAC6+ pcDNA3.1-HSC70 group (all P<0.05) . Compared with OOX+shHDAC6 group, the expression of OSX and RUNX2 protein, BMD, Tb.N, Tb.th and BV/TV were decreased but the expression of Tb. Sp was increased in OOX+ shHDAC6+ shHSC70 group. Conclusions:HDAC6 regulates the acetylation level of HSC70 and then affects OOX-induced OP bone loss. Inhibition of HDAC6 can significantly improve OP bone loss.

Analgesic effects of miR-362 on bone cancer pain process via targeting HDAC6 in rats / 西安交通大学学报(医学版)

【Objective】 To detect the expressions of microRNA（miR）-362 and histone deacetylase 6 （HDAC6） in bone cancer pain （BCP） rats and investigate the analgesic effect of miR-362 and its potential analgesic mechanism. 【Methods】 The BCP model was developed by injecting Walker 256 mammary gland carcinoma cells into bone marrow cavity. Plasmid transfection was used to regulate the expressions of miR-362 and HDAC6. The Van Frey filaments and radiant heat instrument were used to detect the paw withdrawal threshold （PWT） and paw withdrawal latency （PWL）. qRT-PCR was used to detect mRNA expression levels of miR-362 and HDAC6， and Western blotting was used to detect protein expression of HDAC6 and nuclear factor kappa-B p65 （NF-κB p65）. ELISA assay was used to detect the protein levels of interleukin （IL）-1β， IL-6 and tumor necrosis factor α （TNF-α）. Luciferase activity assay was used to determine the relationship between miR-362 and HDAC6. 【Results】 Compared to sham group， the significant decrease of PWT and PWL， decrease of miR-362 and the increase of HDAC6 mRNA and protein in the spinal were detected in BCP group （P<0.05）. Compared to BCP group， the significantly increase of PWT and PWL and decrease of HDAC6 mRNA and protein in the spinal were detected in BCP+LV-miR-362 group （P<0.05）. Compared to BCP+LV-miR-362 group， PWT and PWL significantly decreased in BCP+LV-miR-362+LV-HDAC6 group （P<0.05）. In addition， compared to BCP group， significant decreases of NF-κB p65， IL-1β， IL-6 and TNF-α in spinal were detected in BCP+LV-HDAC6 siRNA group （P<0.05）. Moreover， compared to mimic miR-362+HDAC6 3’UTRMUT group， the luciferase activity significantly decreased in mimic miR-362+HDAC6 3’UTRWT group （P<0.05）. 【Conclusion】 As a key factor regulating the mechanism of BCP through “HDAC6-NF-κB p65” signal pathway in rats， targeting miR-362 may be a novel therapeutic method for BCP.

Effect of ginsenoside Rh2(S) on apoptosis of human leukemia cell by inhibiting HDAC6 / 中草药

Objective To explore the specific mechanism of ginsenoside Rh2(S) inducing the apoptosis of leukemia cells. Methods The effect of Rh2(S) on proliferation of leukemia cells K562 and KG1a was measured by cell counting kit-8 assay (CCK-8 assay). The growth states of cells were observed under the inverted phase microscope, and cell cycle distribution, and apoptosis were determined by flow cytometry (FCM). The expression levels of HDAC6, HSP90, Akt, GSK-3β, β-catenin, and cell cycle apoptosis related proteins were ascertained by Western blotting. Results The results of CCK-8 showed that Rh2(S) had the most obvious inhibitory effect on the proliferation of leukemia cells. Rh2(S) significantly induced apoptosis and led to cell cycle arrest at G0/G1 phase of leukemia cells by FCM. While the microscope observation showed that the number of cells was decreased and normal cell morphology changed. Rh2(S) decreased the expression of Bcl-2, Cyclin D1, HDAC6, HSP90, p-Akt, and β-catenin and increased the expression of Bax, Ac-α-tubulin, and GSK-3β by Western blotting. Conclusion Rh2(S) can effectively inhibit the proliferation and promote the apoptosis of K562 and KG1a cells, its specific mechanism may relate to inhibitting the expression of HDAC6, resulting in a decline in the expression of HSP90, so as to further inhibit the activity of Akt activation of GSK-3, and finally inhibit Wnt/β-catenin pathway.

Effect of HDAC-6 on PD cell induced by lactacystin

Objective: To explore the effects of histone deacetylase 6(HDAC-6) on the PD cell model induced by proteasome inhibitor lactacystin. Methods: Human neuroblastoma SK-N-SH cells were cultured. The wild type pcDNA3.1-alpha-synuclein eukaryotic expression plasmid was transferred into the cells which then were divided into control group, group L, group T and group T+L. The cells of group L were added with 5 μmol/L lactacystin dissolved indimethylsulfoxide (DMSO) to induce PD cell model with abnormal protein aggregation, the cells of control group were treated with 5 μmol/L DMSO, the cells of group T were treated with 5 μmol/L selective HDAC-6 inhibitor tubacin dissolved in DMSO, and the cells of group T+L were treated with 5 μmol/L lactacystin and 10 μmol/L tubacin dissolved in DMSO. The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 were detected by Western blot and the cell survival rate of all the groups was detected by MTT colorimetric assay, and compared 24 h after the cells were treated. Results: The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 of the cells of group L were significantly higher than the control group, and the cell survival rate was significantly lower (P 0.05). Conclusions: The expression level of alpha-synuclein oligomers can be improved and the cell survival rate can be reduced by the PD cell model induced by lactacystin and treated with selective HDAC-6 inhibitor tubacin, which means that alpha-synuclein oligomers of the PD cell model induced by lactacystin can be inhibited and the cell survival rate can be improved by HDAC-6, and the mechanism may be related to the increased of HSP-27 and HSP-70.

Effect of HDAC-6 on PD cell induced by lactacystin

OBJECTIVE@#To explore the effects of histone deacetylase 6(HDAC-6) on the PD cell model induced by proteasome inhibitor lactacystin.@*METHODS@#Human neuroblastoma SK-N-SH cells were cultured. The wild type pcDNA3.1-alpha-synuclein eukaryotic expression plasmid was transferred into the cells which then were divided into control group, group L, group T and group T+L. The cells of group L were added with 5 μmol/L lactacystin dissolved indimethylsulfoxide (DMSO) to induce PD cell model with abnormal protein aggregation, the cells of control group were treated with 5 μmol/L DMSO, the cells of group T were treated with 5 μmol/L selective HDAC-6 inhibitor tubacin dissolved in DMSO, and the cells of group T+L were treated with 5 μmol/L lactacystin and 10 μmol/L tubacin dissolved in DMSO. The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 were detected by Western blot and the cell survival rate of all the groups was detected by MTT colorimetric assay, and compared 24 h after the cells were treated.@*RESULTS@#The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 of the cells of group L were significantly higher than the control group, and the cell survival rate was significantly lower (P  0.05).@*CONCLUSIONS@#The expression level of alpha-synuclein oligomers can be improved and the cell survival rate can be reduced by the PD cell model induced by lactacystin and treated with selective HDAC-6 inhibitor tubacin, which means that alpha-synuclein oligomers of the PD cell model induced by lactacystin can be inhibited and the cell survival rate can be improved by HDAC-6, and the mechanism may be related to the increased of HSP-27 and HSP-70.

RASSF1A Suppresses Cell Migration through Inactivation of HDAC6 and Increase of Acetylated alpha-Tubulin / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The RAS association domain family protein 1 (RASSF1) has been implicated in a tumor-suppressive function through the induction of acetylated alpha-tubulin and modulation of cell migration. However, the mechanisms of how RASSF1A is associated with acetylation of alpha-tubulin for controlling cell migration have not yet been elucidated. In this study, we found that RASSF1A regulated cell migration through the regulation of histon deacetylase 6 (HDAC6), which functions as a tubulin deacetylase. MATERIALS AND METHODS: The cell migration was assessed using wound-healing and transwell assays. The role of RASSF1A on cell migration was examined by immunofluorescence staining, HDAC activity assay and western blot analysis. RESULTS: Cell migration was inhibited and cell morphology was changed in RASSF1A-transfected H1299 cells, compared with controls, whereas HDAC6 protein expression was not changed by RASSF1A transfection in these cells. However, RASSF1A inhibited deacetylating activity of HDAC6 protein and induced acetylated alpha-tubulin expression. Furthermore, acetylated alpha-tubulin and HDAC6 protein were co-localized in the cytoplasm in RASSF1A-transfected H1299 cells. Conversely, when the endogenous RASSF1A expression in HeLa cells was blocked with RASSF1A siRNA treatment, acetylated alpha-tubulin was co-localized with HDAC6 protein throughout the whole cells, including the nucleus, compared with scramble siRNA-treated HeLa cells. The restoration of RASSF1A by 5-Aza-dC treatment also induced acetylated alpha-tubulin through inhibition of HDAC6 activity that finally resulted in suppressing cell migration in H1299 cells. To further confirm the role of HDAC6 in RASSF1A-mediated cell migration, the HDAC6 expression in H1299 cells was suppressed by using HDAC6 siRNA, and cell motility was found to be decreased through enhanced acetylated alpha-tubulin. CONCLUSION: The results of this study suggest that the inactivation of HDAC6 by RASSF1A regulates cell migration through increased acetylated alpha-tubulin protein.