Suppression Of Hepatocyte Apoptosis Research Articles

Uncovering the anti-inflammatory mechanisms of phenolic-enriched maple syrup extract in lipopolysaccharide-induced peritonitis in mice: insights from data-independent acquisition proteomics analysis.

Our group has previously reported on the phytochemical composition and biological activities of a phenolic-enriched maple syrup extract (MSX), which showed promising anti-inflammatory effects in several disease models including diabetes and Alzheimer's disease. However, the efficacious doses of MSX and its molecular targets involved in the anti-inflammatory effects are not fully elucidated. Herein, the efficacy of MSX in a peritonitis mouse model was evaluated in a dose-finding study and the underlying mechanisms were explored using data-independent acquisition (DIA) proteomics assay. MSX (at 15, 30 and 60 mg kg-1) alleviated lipopolysaccharide-induced peritonitis by reducing the levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) in the serum and major organs of the mice. Furthermore, DIA proteomics analyses identified a panel of proteins that were significantly altered (both up- and down-regulated) in the peritonitis group, which were counteracted by the MSX treatments. MSX treatment also modulated several inflammatory upstream regulators including interferon gamma and TNF. Ingenuity pathway analysis suggested that MSX may modulate several signaling pathways in the processes of initiation of cytokine storm, activation of liver regeneration, and suppression of hepatocyte apoptosis. Together, these proteomic and in vivo findings indicate that MSX could regulate inflammation signaling pathways and modulate inflammatory markers and proteins, providing critical insight to its therapeutic potential.

Recql5 protects against lipopolysaccharide/D-galactosamine-induced liver injury in mice.

To investigate the effects of Recql5 deficiency on liver injury induced by lipopolysaccharide/D-galactosamine (LPS/D-Gal). Liver injury was induced in wild type (WT) or Recql5-deficient mice using LPS/D-Gal, and assessed by histological, serum transaminases, and mortality analyses. Hepatocellular apoptosis was quantified by transferase dUTP nick end labeling assay and Western blot analysis of cleaved caspase-3. Liver inflammatory chemokine and cytochrome P450 expression was analyzed by quantitative reverse transcription-PCR. Neutrophil infiltration was evaluated by myeloperoxidase activity. Expression and phosphorylation of ERK, JNK, p65, and H2A.X was determined by Western blot. Oxidative stress was evaluated by measuring malondialdehyde production and nitric oxide synthase, superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase activity. Following LPS/D-Gal exposure, Recql5-deficient mice exhibited enhanced liver injury, as evidenced by more severe hepatic hemorrhage, higher serum aspartate transaminase and alanine transaminase levels, and lower survival rate. As compared to WT mice, Recql5-deficient mice showed an increased number of apoptotic hepatocytes and higher cleaved caspase-3 levels. Recql5-deficient mice exhibited increased DNA damage, as evidenced by increased γ-H2A.X levels. Inflammatory cytokine levels, neutrophil infiltration, and ERK phosphorylation were also significantly increased in the knockout mice. Additionally, Recql5-deficient mice exhibited increased malondialdehyde production and elevated inducible nitric oxide synthase, superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase activity, indicative of enhanced oxidative stress. Moreover, CYP450 expression was significantly downregulated in Recql5-deficient mice after LPS/D-Gal treatment. Recql5 protects the liver against LPS/D-Gal-induced injury through suppression of hepatocyte apoptosis and oxidative stress and modulation of CYP450 expression.

Protective effect of MDL28170 against thioacetamide-induced acute liver failure in mice.

Liver injury is known to often progress even after the hepatotoxicant is dissipated. The hydrolytic enzyme calpain, which is released from dying hepatocytes, destroys the surrounding cells and results in progression of injury. Therefore, control of calpain activation may be a suitable therapeutic intervention in cases of fulminant hepatic failure. This study evaluated the effects of a potent cell-permeable calpain inhibitor, MDL28170, and its mechanisms of action on thioacetamide (TAA)-induced hepatotoxicity in mice. We found that MDL28170 significantly decreased mortality and change in serum transaminase after TAA administration. The necroinflammatory response in the liver was also suppressed. Furthermore, a significant suppression of hepatocyte apoptosis could be found by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. The upregulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha), both of which are known to mediate the propagation of inflammation, was abolished. MDL2810 also effectively blocked hepatic stellate cell activation, which is assumed to be the early step in liver fibrosis. These results demonstrated that MDL28170 attenuated TAA-induced acute liver failure by inhibiting hepatocyte apoptosis, abrogating iNOS and TNF-alpha mRNA upregulation and blocking hepatic stellate cell activation.

Role of hepatic non-parenchymal cells in the response of rat hepatocytes to the peroxisome proliferator nafenopin in vitro.

Induction of liver cancer by peroxisome proliferators such as nafenopin is frequently associated with increased liver growth, increased DNA synthesis and suppression of apoptosis. The cytokine, tumour necrosis factor alpha (TNF alpha), and non-parenchymal liver cells have been implicated in mediating the hepatic response to peroxisome proliferators. Here, we have investigated the dependency of the hepatocyte response to peroxisome proliferators on non-parenchymal cells, a major source of hepatic cytokines. Addition of non-parenchymal cells, or conditioned medium from non-parenchymal cell cultures, increased DNA synthesis (220% and 270% of control, respectively) and suppressed transforming growth factor beta(1)-induced hepatocyte apoptosis (32% and 54% of control, respectively). Removal of non-parenchymal cells from normal hepatocyte cultures prevented both the nafenopin- and TNF alpha-induced increase in DNA synthesis and suppression of hepatocyte apoptosis; this response was restored by returning non-parenchymal cells to the purified hepatocytes. TNF alpha was detected in the medium of non-parenchymal cell (3-15 pg/ml) and normal hepatocyte cultures (25-100 pg/ml) by bioassay using L929 cells. However, the contribution of TNF alpha released from non-parenchymal cells was small compared with that released spontaneously by hepatocytes. Nafenopin significantly increased the release of TNF alpha from non-parenchymal cells to 56 +/- 18 pg/ml, but had little effect on TNF alpha release by hepatocytes. However, the concentration of exogenous TNF alpha required to elicit a response in hepatocytes was 100 pg/ml and above. These data provide evidence that hepatic non-parenchymal cells are permissive for the growth response of hepatocytes in vitro to peroxisome proliferators and this may be mediated, at least in part by TNF alpha. However, the levels of TNF alpha released spontaneously or in response to peroxisome proliferators are insufficient per se to induce a growth response.

Cytokines in non-genotoxic hepatocarcinogenesis.

Many toxicants can cause liver injury. Some, such as diethylnitrosamine, are genotoxic and act principally by damaging DNA (1). A second more diverse group cause liver injury but are non-genotoxic (2). Despite differences in the primary target, genotoxic and non-genotoxic hepatotoxicants frequently cause tumours in the liver of experimental rats and mice (1,2). This review is concerned with evaluating the hypothesis that cytokines may play a role in mediating the biological effects of liver non-genotoxic carcinogens. Non-genotoxic liver toxicants constitute a diverse group of chemicals. Some, such as carbon tetrachloride (3), cause acute liver injury followed by regenerative hyperplasia, whereas others cause proliferation without identifiable tissue damage. The latter group includes barbiturate drugs such as phenobarbitone (PB) (4), dioxins such as 2,3,7,8-tetrachlorodibenzo-pdioxin (5,6) and the largest group, the peroxisome proliferators (PPs) (7). The induction of cell proliferation by toxicants that cause liver injury probably underpins their common ability to cause hepatocellular adenomas and carcinomas after prolonged exposure (8). During chemical-induced carcinogenesis, the tightly controlled balance that exists normally in tissues between cell growth and cell death is disrupted (9–11). Rodent hepatocarcinogenesis in response to PPs is preceded by liver enlargement due to hepatocyte hypertrophy and induction of cell proliferation coupled with suppression of hepatocyte apoptosis (7,12,13). The ability of barbiturates such as PB to stimulate hepatocyte DNA synthesis in vivo has also been demonstrated (14). The capacity of non-genotoxic liver carcinogens to perturb both sides of the growth equation between cell gain and cell loss can be modelled in vitro using hepatocytes prepared from rats or mice. In such models, PPs such as nafenopin can suppress spontaneous apoptosis (15–17) and also that induced by diverse stimuli, including transforming growth factor-β1. Interleukin (IL)-1 can act alongside TNFα to play a role in inflammation (18) and is implicated in the regulation of hepatic lipid metabolism (19), ligation of Fas or DNA damage (20). Thus, non-genotoxic carcinogens may

Suppression of hepatocyte apoptosis and induction of DNA synthesis by the rat and mouse hepatocarcinogen diethylhexylphlathate (DEHP) and the mouse hepatocarcinogen 1,4-dichlorobenzene (DCB).

Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the difference in rat carcinogenicity of these two hepatic mitogens may be due to differences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P = 0.001) and suppressed 10- and 4-fold, respectively both spontaneous (P = 0.0008) and transforming growth factor beta1 (TGFbeta1)-induced (P = 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P = 0.03) and a small (1.7- to 2-fold) suppression of both spontaneous (P = 0.022) and TGFbeta1-induced (P = 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P = 0.023) was less marked than that with DEHP (13.4-fold; P = 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of five animals with DCB and three out of five with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from differences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to effect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis.