AGK2 pre-treatment protects against thioacetamide-induced acute liver failure via regulating the MFN2-PERK axis and ferroptosis signaling pathway.

BACKGROUND: Acute liver failure (ALF) is an unpredictable and life-threatening critical illness. The pathological characteristic of ALF is massive necrosis of hepatocytes and lots of inflammatory cells infiltration which may lead to multiple organ failure. METHODS: Animals were divided into 3 groups, normal, thioacetamide (TAA, ALF model) and TAA + AGK2. Cultured L02 cells were divided into 5 groups, normal, TAA, TAA + mitofusin 2 (MFN2)-siRNA, TAA + AGK2, and TAA + AGK2 + MFN2-siRNA groups. The liver histology was evaluated with hematoxylin and eosin staining, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6ß (ATF6ß), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylated-PERK (p-PERK). C/EBP homologous protein (CHOP), reactive oxygen species (ROS), MFN2 and glutathione peroxidase 4 (GPX4) were measured with Western blotting, and cell viability and liver chemistry were also measured. Mitochondria-associated endoplasmic reticulum membranes (MAMs) were measured by immunofluorescence. RESULTS: The liver tissue in the ALF group had massive inflammatory cell infiltration and hepatocytes necrosis, which were reduced by AGK2 pre-treatment. In comparison to the normal group, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ in the TAA-induced ALF model group were significantly increased, which were decreased by AGK2 pre-treatment. The levels of MFN2 and GPX4 were decreased in TAA-induced mice compared with the normal group, which were enhanced by AGK2 pre-treatment. Compared with the TAA-induced L02 cell, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ were further increased and levels of MFN2 and GPX4 were decreased in the MFN2-siRNA group. AGK2 pre-treatment decreased the apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ and enhanced the protein expression of MFN2 and GPX4 in MFN2-siRNA treated L02 cell. Immunofluorescence observation showed that level of MAMs was promoted in the AGK2 pre-treatment group when compared with the TAA-induced group in both mice and L02 cells. CONCLUSIONS: The data suggested that AGK2 pre-treatment had hepatoprotective role in TAA-induced ALF via upregulating the expression of MFN2 and then inhibiting PERK and ferroptosis pathway in ALF.

Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity.

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.

Interventions for non-alcoholic liver disease: a gut microbial metabolites perspective.

Over the past two decades, non-alcoholic fatty liver disease (NAFLD) has become a leading burden of hepatocellular carcinoma and liver transplantation. Although the exact pathogenesis of NAFLD has not been fully elucidated, recent hypotheses placed more emphasis on the crucial role of the gut microbiome and its derivatives. Reportedly, microbial metabolites such as short-chain fatty acids, amino acid metabolites (indole and its derivatives), bile acids (BAs), trimethylamine N-oxide (TMAO), and endogenous ethanol exhibit sophisticated bioactive properties. These molecules regulate host lipid, glucose, and BAs metabolic homeostasis via modulating nutrient absorption, energy expenditure, inflammation, and the neuroendocrine axis. Consequently, a broad range of research has studied the therapeutic effects of microbiota-derived metabolites. In this review, we explore the interaction of microbial products and NAFLD. We also discuss the regulatory role of existing NAFLD therapies on metabolite levels and investigate the potential of targeting those metabolites to relieve NAFLD.

Sirtuin1 attenuates acute liver failure by reducing reactive oxygen species via hypoxia inducible factor 1α.

BACKGROUND: The occurrence and development of acute liver failure (ALF) is closely related to a series of inflammatory reactions, such as the production of reactive oxygen species (ROS). Hypoxia inducible factor 1α (HIF-1α) is a key factor that regulates oxygen homeostasis and redox, and the stability of HIF-1α is related to the ROS level regulated by Sirtuin (Sirt) family. The activation of Sirt1 will lead to a powerful antioxidant defense system and therapeutic effects in liver disease. However, little is known about the relationship between HIF-1α and Sirt1 in the process of ALF and the molecular mechanism. AIM: To investigate whether HIF-1α may be a target of Sirt1 deacetylation and what the effects on ALF are. METHODS: Mice were administrated lipopolysaccharide (LPS)/D-gal and exposed to hypoxic conditions as animal model, and resveratrol was used as an activator of Sirt1. The cellular model was established with L02 cells stimulated by LPS. N-acetyl-L-cysteine was used to remove ROS, and the expression of Sirt1 was inhibited by nicotinamide. Western blotting was used to detect Sirt1 and HIF-1α activity and related protein expression. The possible signaling pathways involved were analyzed by immunofluorescent staining, co-immunoprecipitation, dihydroethidium staining, and Western blotting. RESULTS: Compared with mice stimulated with LPS alone, the expression of Sirt1 decreased, the level of HIF-1α acetylation increased in hypoxic mice, and the levels of carbonic anhydrase 9 and Bcl-2-adenovirus E1B interacting protein 3 increased significantly, which was regulated by HIF-1α, indicating an increase of HIF-1α activity. Under hypoxia, the down-regulation of Sirt1 activated and acetylated HIF-1α in L02 cells. The inhibition of Sirt1 significantly aggravated this effect and the massive production of ROS. The regulation of ROS was partly through peroxisome proliferator-activated receptor alpha or AMP-activated protein kinase. Resveratrol, a Sirt1 activator, effectively relieved ALF aggravated by hypoxia, the production of ROS, and cell apoptosis. It also induced the deacetylation of HIF-1α and inhibited the activity of HIF-1α. CONCLUSION: Sirt1 may have a protective effect on ALF by inducing HIF-1α deacetylation to reduce ROS.

Epigenetic Regulation of Hepatic Stellate Cell Activation and Macrophage in Chronic Liver Inflammation.

Chronic liver inflammation is a complex pathological process under different stress conditions, and the roles of stellate cells and macrophages in chronic liver inflammation have been widely reported. Moderate liver inflammation can protect the liver from damage and facilitate the recovery of liver injury. However, an inflammatory response that is too intense can result in massive death of hepatocytes, which leads to irreversible damage to the liver parenchyma. Epigenetic regulation plays a key part in liver inflammation. This study reviews the regulation of epigenetics on stellate cells and macrophages to explore the new mechanisms of epigenetics on liver inflammation and provide new ideas for the treatment of liver disease.

Efficacy of a Chinese herbal formula on hepatitis B e antigen-positive chronic hepatitis B patients.

BACKGROUND: No guideline recommends antiviral therapy for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients with persistently normal alanine aminotransferase levels and a high hepatitis B virus (HBV) DNA viral load. AIM: To evaluate the feasibility and safety of a Chinese herbal formula as a therapeutic option for chronic HBV infection. METHODS: In total, 395 patients (30-65 years old) with confirmed HBeAg-positive chronic hepatitis B infection and persistently normal alanine aminotransferase were randomized to receive either Chinese herbal formula or placebo for 96 wk. Endpoints to evaluate therapeutic efficacy included: (1) HBV DNA levels decreased to less than 4 log10 IU/mL at weeks 48 and 96; and (2) HBeAg clearance and seroconversion rates at weeks 48 and 96. RESULTS: HBV DNA levels ≤ 4 log10 IU/mL were 10.05% at week 48 and 18.59% at week 96 in the treatment group. The HBeAg clearance and conversion rates were 8.54% and 8.04% at week 48 and 16.08% and 14.57% at week 96, respectively. However, HBV DNA levels ≤ 4 log10 IU/mL were 2.55% and 2.55% at weeks 48 and 96, respectively, and the HBeAg clearance rates were 3.06% and 5.61% at weeks 48 and 96, respectively, in the control group. The quantitative hepatitis B surface antigen and HBeAg levels at baseline and changes during the treatment period as well as the alanine aminotransferase elevation at weeks 12 and 24 were strong predictors of HBeAg clearance. CONCLUSION: High rates of HBV DNA reduction, HBeAg clearance and seroconversion could be achieved with Chinese herbal formula treatments, and the treatments were relatively safe for HBeAg-positive chronic hepatitis B-infected patients with persistently normal alanine aminotransferase. The ability of the compound to modulate host immune function probably contributed to this effect.

Extracellular Histone H3 Induces Pyroptosis During Sepsis and May Act Through NOD2 and VSIG4/NLRP3 Pathways.

Background: Histones could be released from the nucleus when stimulated. Increasing evidence has shown that extracellular histones are associated with a variety of inflammation and diseases. Nucleotide binding oligomerzation domain 2 (NOD2) belongs to the NOD like receptor (NLR) family and is reported to promote apoptosis and aggravate inflammatory response. And V-set and immunoglobulin domain containing 4 (VSIG4), a B7 family-related protein, has been confirmed to mediate transcriptional inhibition of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3). However, little is known about the impact of extracellular histones on NOD2 or VSIG4 signal transduction. In this study, we aim to explore the effect and mechanism of extracellular histone H3 on pyroptosis. Aim: The purpose of this work was to investigate the mechanism of extracellular histone H3 on pyroptosis in sepsis. Methods: Lipopolysaccharide (LPS) and histone H3 were used to induce sepsis mice model and damage in ANA-1 macrophages. H3 antibody was applied to antagonize the effect of histone H3. NOD2 inhibitor NOD-IN-1 and VSIG4-siRNA were used to investigate the mechanism of histone H3 on pyroptosis. Enzyme linked immune sorbent assay (ELISA) was applied to detect the level of extracellular histone H3. Real-time PCR and Western blotting were employed to detect the key mRNA and protein levels. The pathology of tissues was detected. Results: The level of extracellular histone H3 was increased after LPS stimulation. The mRNA and protein levels of NLRP3, caspase-1, gasdermin D (GSDMD), interleukin (IL)-1ß, IL-18 were increased in LPS group, but suppressed by H3 antibody. And the expression of NOD2, receptor-interacting protein 2 (RIP2) was elevated compared with control group. The expression of VSIG4 was inhibited by LPS and suppression of H3 promoted the protein level of VSIG4. H3 antibody alleviated pathological damages in tissues. Furthermore, the mRNA and protein levels of NOD2 in H3 group was higher compared with control group. The mRNA and protein levels of VSIG4 in H3 group was decreased compared with control group, but up-regulated by NOD-IN-1. Besides, the mRNA and protein levels of VSIG4 in NOD-IN-1 + VSIG4-siRNA group was elevated compared with VSIG4-siRNA group. Conclusions: Extracellular histone H3 induced by LPS could cause pyroptosis during sepsis via NOD2 and VSIG4/NLRP3 pathway.

Histone deacetylase 6 inhibitor ACY1215 offers a protective effect through the autophagy pathway in acute liver failure.

AIM: The purpose of the present study was to elucidate the protective effect of histone deacetylase 6 inhibitor ACY1215 on autophagy pathway in acute liver failure (ALF). MAIN METHODS: Lipopolysaccharide (LPS) and d-galactosamine (D-Gal) were used to induce ALF model in C57BL/6 mice. D-Gal and tumor necrosis factor alpha (TNF-α) were applied in L02 cell. Autophagy inhibitor 3-MA and ACY1215 were conducted to induce 3-MA group, ACY1215 group and ACY1215+3-MA group. RESULTS: ACY1215 improved liver histological and functional changes in ALF mice model, whereas the autophagy inhibitor 3-MA aggravated liver tissue pathological and functional damage in ALF mice model group. The apoptotic levels (including apoptotic index/rate and apoptotic proteins) in ALF mice and L02 cell were ameliorated with treatment ACY1215. 3-MA accentuated the apoptotic levels in ACY1215 group. D-Gal/TNF-α could reduce L02 cell mitochondrial membrane potential (ΔΨm) in control group. ACY1215 increased the ΔΨm in ALF model. 3-MA also further reduced the ΔΨm in ACY1215 group. ACY1215 could induce autophagy in ALF mice and cell model group accompanied with an increase in expression of LC3-II and beclin-1 proteins and down-regulation of p62 protein. Moreover, the expression of LC3-II and beclin1 proteins were greatly reduced and the expression of p62 protein was ascended after intervention with 3-MA in ACY1215 group. SIGNIFICANCE: Histone deacetylase 6 inhibitor ACY1215 could protect acute liver failure mice and L02 cell by inhibiting apoptosis pathway through enhancing autophagy way.

Inhibition of HDAC6 attenuates LPS-induced inflammation in macrophages by regulating oxidative stress and suppressing the TLR4-MAPK/NF-κB pathways.

BACKGROUND: Histone deacetylase 6 (HDAC6) has been considered as an important regulator in the development of inflammatory diseases. However, the mechanism of HDAC6 in regulating inflammatory responses has not been fully determined. In the present study, we aim to investigate the role and mechanisms of HDAC6 in regulating inflammation in lipopolysaccharide (LPS)-activated macrophages. METHODS: Flow cytometry was used to determine a suitable treatment dosage of ACY-1215 on lipopolysaccharide (LPS)-activated macrophages for the present study. The RAW264.7 macrophages were divided into normal, LPS-treated, and ACY-1215 treated groups, respectively. For the ACY-1215 group, ACY-1215 (10 µM) was added to the medium 2 h prior to treatment with LPS (1 µg/ml) for 24 h. In this study, ROS, inflammatory cytokines, the ultrastructure of mitochondria, mitochondrial membrane potential, RNA and protein expression assay were detected respectively. Subsequently, the effect of HDAC6 knockdown on inflammatory response in LPS-activated RAW264.7 macrophages was also detected. RESULTS: Inhibition of HDAC6 inhibited the overproduction of ROS and suppressed the expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 in LPS-activated RAW264.7 cells. Pretreatment with ACY-1215 could normalize the ultrastructure of mitochondria and mitochondrial membrane potential in LPS-activated macrophages. Moreover, the protein expression of TLR4, Nrf2, HO-1 and the activation of MAPK and NF-κB signaling pathways were normalized by the inhibition of HDAC6. CONCLUSIONS: Inhibition of HDAC6 exhibited protective role against LPS-induced inflammation in RAW264.7 cells by regulating oxidative stress and suppressing the activation of TLR4- MAPK/NF-κB signaling pathway.

Quantitative Proteomic Analysis Reveals the Sites Related to Acetylation and Mechanism of ACY-1215 in Acute Liver Failure Mice.

Background: ACY-1215 is a well-known selective histone deacetylase 6 (HDAC6) inhibitor, and it has been considered as a potential therapeutic drug in inflammatory diseases, including acute liver failure (ALF). However, little is known about the impact of ACY-1215 treatment on histone lysine acetylation and proteome in ALF. In this study, we aim to investigate whether ACY-1215 has inhibitory effects and mechanism on the necrosis of hepatocytes; moreover, the impact of ACY-1215 treatment on histone lysine acetylation still needs further elucidation. Methods: Male C57/BL6 mice were divided into normal, model, and ACY-1215 groups. ACY-1215 (25 mg/kg) and same amounts of saline were injected intraperitoneally to the mice before the establishment of ALF model induced by lipopolysaccharide (LPS) (100 µg/kg) combined with D-gal (400 mg/kg). All animals were sacrificed after 24 h. In this study, detection programs, including quantitative proteomic analysis, transmission electron microscopy (TEM) micrographs, pathological staining, protein expression, the detection of reactive oxygen species (ROS) as well as glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) measurement. Results: The function of liver and the necrosis of hepatocytes in ALF mice were significantly normalized by ACY-1215 pretreatment. The quantitative proteomic analysis revealed that ACY-1215-restrained oxidative phosphorylation normalized the function respiratory electron-transport chain in the mitochondria. Moreover, pretreatment of ACY-1215 not only normalized the structure of mitochondria but also inhibited the generation of reactive oxygen species (ROS). Conclusions: ACY-1215 was able to inhibit necrosis of hepatocytes in ALF mice through regulating the mitochondrial-mediated oxidative stress, and we identified the common sites related to acetylation level.

Modulations of Histone Deacetylase 2 Offer a Protective Effect through the Mitochondrial Apoptosis Pathway in Acute Liver Failure.

The purpose of this study was to investigate the modulation of histone deacetylase 2 (HDAC2) on mitochondrial apoptosis in acute liver failure (ALF). The cellular model was established with LO2 cells stimulated by tumor necrosis factor alpha (TNF-α)/D-galactosamine (D-gal). Rats were administrated by lipopolysaccharide (LPS)/D-gal as animal model. The cell and animal models were then treated by HDAC2 inhibitor CAY10683. HDAC2 was regulated up or down by lentiviral vector transfection in LO2 cells. The mRNA levels of bcl2 and bax were detected by real-time PCR. The protein levels of HDAC2, bcl2, bax, cytochrome c (cyt c) in mitochondrion and cytosol, apoptosis protease activating factor 1 (apaf1), caspase 3, cleaved-caspase 3, caspase 9, cleaved-caspase 9, acetylated histone H3 (AH3), and histone H3 (H3) were assayed by western blot. Apoptosis was detected by flow cytometry. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels were also assayed. The openness degree of the mitochondrial permeability transition pore (MPTP) was detected by ultraviolet spectrophotometry. The apoptosis of hepatocytes in liver tissues was determined by tunnel staining. The liver tissue pathology was detected by hematoxylin eosin (HE) staining. The ultrastructure of liver tissue was observed by electron microscopy. Compared with cell and rat model groups, the bax mRNA level was decreased, and bcl2 mRNA was increased in the CAY10683 treatment group. The protein levels of HDAC2, bax, cyt c in cytosol, apaf1, cleaved-caspase 3, and cleaved-caspase 9 were decreased, and the apoptosis rate was decreased (P < 0.05), whereas the protein level of bcl2 and cyt c in the mitochondrion was elevated (P < 0.05) in the CAY10683 treatment group. In the HDAC2 down- or upregulated LO2 cells, the mitochondrial apoptosis pathway was inhibited or activated, respectively. After being treated with TNF-α/D-gal in HDAC2 down- or upregulated LO2 cells, the mitochondrial apoptosis pathway was further suppressed or activated, respectively. The MPTP value was elevated in CAY10683-treated groups compared with the rat model group (P < 0.05). Liver tissue pathological damage and apoptotic index in the CAY10683-treated group were significantly reduced. In addition, AH3 was elevated in both cell and animal model groups (P < 0.05). Downregulated or overexpressed HDAC2 could accordingly increase or decrease the AH3 level, and TNF-α/D-gal could enhance the acetylation effect. These results suggested that modulations of histone deacetylase 2 offer a protective effect through the mitochondrial apoptosis pathway in acute liver failure.

Protective role of AGK2 on thioacetamide-induced acute liver failure in mice.

AIMS: The aim of the present study was to investigate the protective effects of AGK2 as a selective SIRT2 inhibitor on thioacetamide (TAA)-induced acute liver failure (ALF) in mice and its potential mechanism. MAIN METHODS: All male C57BL/6 mice were separated into control, TAA, AGK2 + TAA, and AGK2 groups. The histological changes were observed by hematoxylin and eosin (HE) staining. The apoptosis cells of liver tissues were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to evaluate the damage of liver function. The inflammatory cytokines of iNOS, TNF-α, IL-1ß was detected by Western blotting and RT-PCR assay. The expression of mitogen-activated protein kinase (MAPK), NF-κB, and apoptosis pathways was determined by Western blotting. KEY FINDINGS: AGK2 improved the damage of TAA-induced liver pathology and function. AGK2 pretreatment also reduced the levels of pro-inflammatory cytokines in ALF liver tissues. AGK2 improved the TAA-induced survival rate. Moreover, AGK2 administration suppressed the increase of phosphorylation NF-κB-p65 and the activation of MAPK pathway. In addition, pretreatment alleviated TAA-induced the liver cells apoptosis. SIGNIFICANCE: AGK2 improve TAA-induced survival rate in mice with ALF, suppress the inflammatory responses by inhibition of MAPK and NF-κB signaling pathways, and decrease the hepatocyte necrosis by inhibition of apoptosis. Pharmacologic inhibition of SIRT2 may be a promising approach for the treatment of ALF.

Histone deacetylases and acetylated histone H3 are involved in the process of hepatitis B virus DNA replication.

AIMS: The aim of this study was to investigate the relationship between anti-HBV treatment and the regulation of HDACs during HBV DNA replication. METHODS: HDAC activities and HBV DNA levels in CHB patients' sera were measured and correlation analysis was made. The changes of HDAC2, HDAC6, AH3 and histone H3 levels in normal control and 4 CHB patient liver tissue samples before and after antiviral treatment were examined. The HDAC inhibitor, TSA, anti-HBV agents, ETV and IFN-α were used to stimulate HepG2.2.15 cells. The levels of HBV DNA, pgRNA in supernatants, and cccDNA in the cells were determined by PCR. The HDAC activity, HDAC6, HDAC2, AH3 and H3 protein levels in cells were tested at days 3, 6, and 9 after treatments. KEY FINDINGS: HDAC activity was positively correlated with HBV DNA in the HBV patients' sera. The levels of HDAC2, HDAC6 and AH3 were notably decreased after antiviral treatment. When compared with antiviral treatment group, the normal liver tissue showed obviously decreased HDAC2, HDAC6 and AH3 protein levels. In vitro study, the level of HBV DNA, the HDAC activity, and the HDAC2, HDAC6 and AH3 protein levels decreased in the ETV, IFN-α and TSA groups compared with the control group. The pgRNA level in supernatants was declined in the IFN-α group and increased in the ETV and TSA groups. cccDNA expression was suppressed by IFN-α. SIGNIFICANCE: The changes of HBV replicative products during antiviral treatment are associated with histone deacetylation. Acetylated histone H3 is involved in the process of hepatitis B virus DNA replication.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates liver fibrosis by suppressing the transforming growth factor-ß1 signal pathway.

BACKGROUND: Histone deacetylases (HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) by suppressing transforming growth factor-ß1 (TGF-ß1) signaling. METHODS: Male Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride (CCl4) and LX2 cell (human hepatic stellate cell line) was stimulated by TGF-ß1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor (CTGF) mRNA levels were detected by real-time polymerase chain reaction (PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3 (H3), Smad7, Smad2/3, Acetyl-Histone H3 (AH3), HDAC2, α-smooth muscle actin (α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-ß1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin (HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. RESULTS: Compared with control group, the TGF-ß1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats (P < 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats (P < 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-ß1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced (P < 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats (P < 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver (P < 0.01). CONCLUSION: The HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-ß1 signaling.

The Protective Mechanism of CAY10683 on Intestinal Mucosal Barrier in Acute Liver Failure through LPS/TLR4/MyD88 Pathway.

The purpose of this study was to investigate the protective mechanism of HDAC2 inhibitor CAY10683 on intestinal mucosal barrier in acute liver failure (ALF). In order to establish ALF-induced intestinal epithelial barrier disruption models, D-galactosamine/LPS and LPS were, respectively, used with rats and NCM460 cell and then administrated with CAY10683. Transepithelial electrical resistance (TEER) was measured to detect the permeability of cells. Real-time PCR and Western blotting were employed to detect the key mRNA and protein levels. The intestinal epithelial tissue pathology was detected. After interfering with CAY10683, the mRNA and protein levels of TLR4, MyD88, TRIF, and TRAF6 were decreased compared with model group (P < 0.05), whereas the levels of ZO-1 and occluding were elevated (P < 0.05). The permeability was elevated in CAY10683-interfered groups, when compared with model group (P < 0.05). And the degree of intestinal epithelial tissue pathological damage in CAY10683 group was significantly reduced. Moreover, CAY10683 significantly decreased the TLR4 staining in animal tissue. The HDAC2 inhibitor CAY10683 could promote the damage of intestinal mucosal barrier in ALF through inhibiting LPS/TLR4/MyD88 pathway.

Histone Deacetylase 2 Inhibitor CAY10683 Alleviates Lipopolysaccharide Induced Neuroinflammation Through Attenuating TLR4/NF-κB Signaling Pathway.

Neuroinflammation involves in the progression of many central nervous system diseases. Several studies have shown that histone deacetylase (HDAC) inhibitors modulated inflammatory responses in lipopolysaccharide (LPS) stimulated microglia. While, the mechanism is still unclear. The aim of present study was to investigate the effect of HDAC2 inhibitor CAY10683 on inflammatory responses and TLR4/NF-κB signaling pathways in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. The effect of CAY10683 on cell viability of BV2 microglial cells was detected by CCK-8 assay. The expressions of inflammatory cytokines were analyzed by western blotting and RT-PCR respectively. The TLR4 protein expression was measured by western blotting, immunofluorescence, immunohistochemistry respectively. The protein expressions of MYD88, phospho-NF-κB p65, NF-κB-p65, acetyl-H3 (AH3), H3, and HDAC2 were analyzed by western blotting. We found that CAY10683 could inhibit expression levels of inflammatory cytokine TNF-α and IL-1ß in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. It could induce TLR4, MYD88, phospho-NF-κB p65, and HDAC2 expressions. Moreover, CAY10683 increased the acetylation of histones H3 in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. Taken together, our findings suggested that HDAC2 inhibitor CAY10683 could suppress neuroinflammatory responses and TLR4/NF-κB signaling pathways by acetylation after LPS stimulation.

Long noncoding RNA LNC473 inhibits the ubiquitination of survivin via association with USP9X and enhances cell proliferation and invasion in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Recent studies reported that lncRNA LINC00473 (LNC473) was involved in cancer progression. However, the clinical significance and functional role of LNC473 in HCC progression is still unknown. In the present study, we found that the LNC473 expression was markedly elevated in HCC tissues and correlated with bigger tumor size, higher BCLC stage, vascular invasion and poor prognosis. Gain- and loss-of-function assay showed that LNC473 enhanced HCC cell proliferation and invasion and induced epithelial-mesenchymal transition (EMT) process. Mechanistically, LNC473 associated with oncoprotein survivin and regulates its stability. Moreover, LNC473 could recruit deubiquitinase USP9X to inhibit the ubiquitination level of survivin and then increase survivin expression. Therefore, our results suggest that LNC473 exerts its functions as an oncogene in HCC progression and may be a therapeutic target for HCC treatment.

Histone deacetylase 6 inhibitor ACY-1215 protects against experimental acute liver failure by regulating the TLR4-MAPK/NF-κB pathway.

Histone deacetylase 6 (HDAC6) is considered a new target for anticancer, anti-inflammatory, and neurodegenerative treatment. ACY-1215 is a selective histone deacetylase 6 inhibitor, and it has been recognized as a potential anticancer and anti-inflammation drug. The aim of our study was to investigate whether ACY-1215 has protective effects on acute liver failure (ALF) in mice and explore its potential mechanism. Male C57/BL6 mice were divided into normal, model, and ACY-1215 groups. ACY-1215 (25mg/kg) and same amounts of saline were given to mice. After 2h, the ALF models were induced by lipopolysaccharide (LPS, 100µg/kg) combined with D-galactosamine (D-gal, 400mg/kg). All animals were killed after 24h. The expressions of HDAC6 were determined by western blotting and RT-PCR assay. The expression levels of inflammatory cytokines were detected by ELISA and RT-PCR. The protein expression of Toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and nuclear factor κB (NF-κB) species were determined by western blot. The mortality of mice with ALF induced by LPS and D-gal was significantly decreased by ACY-1215 pretreatment. Procedures to manage ALF caused adversely affected liver histology and function; this damage was repaired by pretreatment of ACY-1215. ACY-1215 treatment also attenuated the serum and messenger RNA levels of the proinflammatory cytokines. Pretreatment of ACY-1215 significantly decreased the protein expression of TLR4 and the activation of MAPK and NF-κB signalling pathways. ACY-1215 has potential therapeutic value in mice with ALF by directly inhibiting inflammatory response via regulation of the TLR4-MAPK/NF-kB pathway.

Glutamine ameliorates lipopolysaccharide-induced cardiac dysfunction by regulating the toll-like receptor 4/mitogen-activated protein kinase/nuclear factor-kB signaling pathway.

The inflammatory response of sepsis induced by lipopolysaccharide (LPS) may result in irreversible cardiac dysfunction. Glutamine (GLN) has a multitude of pharmacological effects, including anti-inflammatory abilities. Previous studies have reported that GLN attenuated LPS-induced acute lung injury and intestinal mucosal injury. The present study investigated whether GLN exerts potential protective effects on LPS-induced cardiac dysfunction. Male Sprague-Dawley rats were divided into three groups (15 rats per group), including the control (saline-treated), LPS and LPS+GLN groups. Pretreatment with 1 g/kg GLN was provided via gavage for 5 days in the LPS+GLN group, while the control and LPS groups received the same volume of normal saline. On day 6, a cardiac dysfunction model was induced by administration of LPS (10 mg/kg). After 24 h, the cardiac functions of the rats that survived were detected by echocardiography and catheter-based measurements. The serum levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6 were detected by enzyme-linked immunosorbent assay, while the mRNA levels of toll-like receptor (TLR)4, TNF-α, IL-1ß and IL-6 were examined by reverse transcription-quantitative polymerase chain reaction. The protein expression of TLR4, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) were also determined by western blot analysis. The results of echocardiography and catheter-based measurements revealed that GLN treatment attenuated cardiac dysfunction. GLN treatment also attenuated the serum and mRNA levels of the pro-inflammatory cytokines. In addition, the protein levels of TLR4, phosphorylated (p-)extracellular signal-regulated kinase, p-c-Jun N-terminal kinase and p-P38 were reduced upon GLN pretreatment. Furthermore, GLN pretreatment resulted in decreased activation of the NF-κB signaling pathway. In conclusion, GLN has a potential therapeutic effect in the protection against cardiac dysfunction mediated by sepsis through regulating the TLR4/MAPK/NF-κB signaling pathway.

Trichostatin A protects against intestinal injury in rats with acute liver failure.

BACKGROUND: Histone deacetylase (HDAC) inhibitors have been widely applied in the clinic as anticancer drugs against multiple neoplasms and proved their anti-inflammation under different pathology recently. Trichostatin A (TSA) is an HDAC inhibitor specific in class I and II HDAC enzymes. The aim of the present study was to elucidate the protective effects of TSA on acute liver failure (ALF) in rats and its potential mechanism. METHODS: A total of 18 female Sprague-Dawley rats were separated into control, model, and TSA groups. We used Western blotting to determine the expression of HDACs, inflammatory cytokines, and acetylation of histone in liver and small intestine. The gene expression of inflammatory factors and Cox-2 was detected by a polymerase chain reaction. Colonic motility was assessed by spatiotemporal mapping. Histologic analysis and immunohistochemistry were performed. Intestinal permeability examination and levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were also observed. RESULTS: ALF procedure caused harm to histology of liver and small intestine, increased the intestinal permeability and serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin. It also interrupted the normal organization of colonic motor patterns by hurting enteric nervous system and pacemaker cells. Along with the decrease of inflammatory factors in ALF rats by TSA administration, all the damage to the liver, the small intestine, and the colon was repaired. CONCLUSIONS: TSA alleviates the lesion in liver, as well as in small intestine and colon in ALF rats by directly inhibiting inflammatory response.