Histone deacetylase inhibitor givinostat attenuates nonalcoholic steatohepatitis and liver fibrosis.

Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease that is increasingly prevalent worldwide. Liver inflammation is an important contributor to disease progression from nonalcoholic fatty liver (NAFL) to NASH, but there is a lack of efficient therapies. In the current study we evaluated the therapeutic potential of givinostat, a histone deacetylase (HDAC) inhibitor, in the treatment of NASH in vivo and in vitro. Liver inflammation was induced in mice by feeding a methionine- and choline-deficient diet (MCD) or a fructose, palmitate, cholesterol diet (FPC). The mice were treated with givoinostat (10 mg·kg-1·d-1, ip) for 8 or 10 weeks. At the end of the experiment, the livers were harvested for analysis. We showed that givoinostat administration significantly alleviated inflammation and attenuated hepatic fibrosis in MCD-induced NASH mice. RNA-seq analysis of liver tissues form MCD-fed mice revealed that givinostat potently blocked expression of inflammation-related genes and regulated a broad set of lipid metabolism-related genes. In human hepatocellular carcinoma cell line HepG2 and human derived fetal hepatocyte cell line L02, givinostat significantly decreased palmitic acid-induced intracellular lipid accumulation. The benefit of givinostat was further confirmed in FPC-induced NASH mice. Givinostat administration significantly attenuated hepatic steatosis, inflammation as well as liver injury in this mouse model. In conclusion, givinostat is efficacious in reversing diet-induced NASH, and may serve as a therapeutic agent for the treatment of human NASH.

Histone deacetylase inhibitor givinostat alleviates liver fibrosis by regulating hepatic stellate cell activation.

Hepatic fibrosis, a common pathological manifestation of chronic liver injury, is generally considered to be the end result of an increase in extracellular matrix produced by activated hepatic stellate cells (HSCs). The aim of the present study was to target the mechanisms underlying HSC activation in order to provide a powerful therapeutic strategy for the prevention and treatment of liver fibrosis. In the present study, a high­throughput screening assay was established, and the histone deacetylase inhibitor givinostat was identified as a potent inhibitor of HSC activation in vitro. Givinostat significantly inhibited HSC activation in vivo, ameliorated carbon tetrachloride­induced mouse liver fibrosis and lowered plasma aminotransferases. Transcriptomic analysis revealed the most significantly regulated genes in the givinostat treatment group in comparison with those in the solvent group, among which, dermokine (Dmkn), mesothelin (Msln) and uroplakin­3b (Upk3b) were identified as potential regulators of HSC activation. Givinostat significantly reduced the mRNA expression of Dmkn, Msln and Upk3b in both a mouse liver fibrosis model and in HSC­LX2 cells. Knockdown of any of the aforementioned genes inhibited the TGF­ß1­induced expression of α­smooth muscle actin and collagen type I, indicating that they are crucial for HSC activation. In summary, using a novel strategy targeting HSC activation, the present study identified a potential epigenetic drug for the treatment of hepatic fibrosis and revealed novel regulators of HSC activation.

Overexpression of the receptor-like protein kinase genes AtRPK1 and OsRPK1 reduces the salt tolerance of Arabidopsis thaliana.

AtRPK1 (AT1G69270) is a leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene in Arabidopsis thaliana. The rice gene Os07g0602700 (OsRPK1) is the homolog of AtRPK1. AtRPK1 and OsRPK1 were overexpressed and the expression of AtRPK1 was inhibited by RNAi in A. thaliana. The functional results showed that the degrees of salt tolerance of the 35S:RPK1 A. thaliana plants were significantly lower than that of the control plants. The AtRPK1-RNAi A. thaliana plants exhibited higher salt tolerance than the wild-type plants (Col). The subcellular localisation results showed that the RPK1 proteins were mainly distributed on the cell membrane and that the overexpressed AtRPK1 proteins exhibited a significantly clustered distribution. The physiological analyses revealed that the overexpression of the RPK1 genes increased the membrane permeability in the transgenic A. thaliana plants. In response to salt stress, these plants exhibited an increased Na(+) flux into the cell, which caused greater damage to the cell. The real-time quantitative PCR analysis showed that the expression of the P5CS1 gene was inhibited and the SOS signalling pathway was blocked in the 35S:AtRPK1 A. thaliana plants. These effects at least partially contribute to the salt-sensitive phenotype of the 35S:RPK1 plants.

[Expression and correlation of angiotensin-converting enzyme 2 in CCl4-induced rat liver fibrosis].

OBJECTIVE: To investigate the expression and pathogenic relevance of angiotensin-converting enzyme 2 (ACE2) in liver fibrosis by using the rat model of CCl4-induced liver fibrosis. METHODS: The liver fibrosis model was generated by delivering subcutaneous injections of CCl4 (dissolved in olive oil at a 2:3 ratio; injection dose: 3 ml/kg) every three days for six weeks into male Sprague-Dawley rats. Another group of rats that received simultaneous injections of olive oil alone (3 ml/kg) were used as controls. At week 0, 2, 4, or 6 after the first injection, a subset of rats from each group was sacrificed to obtain liver tissues and serum samples. Pathological analyses were carried out to detect the presence and extent of liver cell degeneration, necrosis, inflammatory cell infiltration, and collagen deposition. ACE2 and ACE gene and protein expressions were measured by real-time PCR and Western blotting, respectively. The significance of differential expression between groups and time points was assessed by t-test and one-way ANOVA or Kruskal-Wallis tests, and correlation with fibrosis was assessed by Spearman's rank correlation coefficient. RESULTS: CCl4 administration led to significantly up-regulated ACE2 mRNA levels at week 2 (3.055+/-1.034), 4 (3.545+/-1.947), and 6 (6.448+/-1.836) (vs. controls; H = 23.224, P less than 0.001). Similarly, hepatic ACE mRNA was significantly increased after the CCl4 injections (week 2: 3.055+/-1.034, week 4: 3.545+/-1.947, week 6: 6.448+/-1.836; vs. controls: F = 12.982, P less than 0.001). There was a significant correlation between the ACE and ACE2 gene expression levels (r = 0.750, P less than 0.001). Protein levels of ACE2 also showed an increasing trend following CCl4 administration (week 0: 0.034, week 2: 0.097, week 4: 0.356, week 6: 0.512). The hepatic ACE2 gene expression strongly correlated with levels of alanine aminotransferase (r = 0.669, P less than 0.0001) and aspartate aminotransferase (r = 0.815, P less than 0.0001), and with the Ishak fibrosis score (r = 0.850, P less than 0.001). Finally, there was a significant correlation between circulating ACE2 and the Ishak fibrosis score (r = 0.730, P less than 0.001). CONCLUSION: A significant relationship exists between ACE2 gene expression and extent of liver fibrosis. ACE2 may play a crucial role in liver fibrogenesis.

Expression of angiotensin-converting enzyme 2 in CCL4-induced rat liver fibrosis.

The renin angiotensin system (RAS) plays a major role in liver fibrosis. A novel homologue of angiotensin converting enzyme, ACE2, was identified as a negative regulator of RAS as it degrades Ang II to Ang1-7. We investigated in vivo the expression of ACE2 in liver fibrosis. We evaluated the relationship between biochemical variables and liver tissue expression of ACE2, the correlation between a histological assessment of liver fibrosis and liver tissue expression of ACE2. Male SD rats were randomly divided into a CCL4 group which received injections of CCL4 and the control group which received injections of olive oil. Liver pathology was examined by H&E and Sirius red staining, and real-time PCR was performed to determine the gene expression levels of ACE2 and ACE. Real-time PCR analysis revealed that ACE2 mRNA was higher at the two-, four-, and six-week time points, respectively (p<0.01). Similarly, hepatic ACE mRNA was significantly increased after CCL4 injection. There was a significant correlation between ACE and ACE2 gene expression (r=0.750, P<0.001). ACE2 gene expression strongly correlated with ALT (r=0.669, P<0.0001) and AST levels (r=0.815, P<0.0001). There was a significant correlation between circulating ACE2 and histological scores of liver fibrosis. ACE2 and ACE gene expression correlated with the ISHAK score (r=0.850, P<0.001; r=0.806, P<0.001). There was a significant relationship between ACE2 gene expression and the degree of liver fibrosis. ACE2 plays a crucial role in liver fibrogenesis.