TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival.

Background and aims: TLR9 deletion protects against steatohepatitis due to choline-amino acid depletion and high-fat diet. We measured TLR9 in human non-alcoholic steatohepatitis (NASH) livers, and tested whether TLR9 mediates inflammatory recruitment in three murine models of non-alcoholic fatty liver disease (NAFLD). Methods: We assayed TLR mRNA in liver biopsies from bariatric surgery patients. Wild-type (Wt), appetite-dysregulated Alms1 mutant (foz/foz), Tlr9-/-, and Tlr9-/-foz/foz C57BL6/J mice and bone marrow (BM) chimeras were fed 0.2% cholesterol, high-fat, high sucrose (atherogenic[Ath]) diet or chow, and NAFLD activity score (NAS)/NASH pathology, macrophage/neutrophil infiltration, cytokines/chemokines, and cell death markers measured in livers. Results: Hepatic TLR9 and TLR4 mRNA were increased in human NASH but not simple steatosis, and in Ath-fed foz/foz mice with metabolic syndrome-related NASH. Ath-fed Tlr9-/- mice showed simple steatosis and less Th1 cytokines than Wt. Tlr9-/-foz/foz mice were obese and diabetic, but necroinflammatory changes were less severe than Tlr9+/+.foz/foz mice. TLR9-expressing myeloid cells were critical for Th1 cytokine production in BM chimeras. BM macrophages from Tlr9-/- mice showed M2 polarization, were resistant to M1 activation by necrotic hepatocytes/other pro-inflammatory triggers, and provoked less neutrophil chemotaxis than Wt Livers from Ath-fed Tlr9-/- mice appeared to exhibit more markers of necroptosis [receptor interacting protein kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] than Wt, and ∼25% showed portal foci of mononuclear cells unrelated to NASH pathology. CONCLUSION: Our novel clinical data and studies in overnutrition models, including those with diabetes and metabolic syndrome, clarify TLR9 as a pro-inflammatory trigger in NASH. This response is mediated via M1-macrophages and neutrophil chemotaxis.

Acute atorvastatin is hepatoprotective against ischaemia-reperfusion injury in mice by modulating eNOS and microparticle formation.

BACKGROUND & AIMS: Steatosis accentuates the severity of hepatic ischaemia-reperfusion injury (IRI); 'statins' (HMG-CoA reductase inhibitors) protect the heart and brain against post-ischaemic injury. We tested whether short-term administration of atorvastatin protects fatty livers in obese mice against IRI. METHODS: Mice with dietary or genetic simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) were subjected to 60 min partial hepatic ischaemia/24 h reperfusion. Atorvastatin was injected intravenously (5 mg/kg) 1 h before IRI. Liver injury, Toll-like receptor-4 (TLR4), cytokines/chemokines, iNOS/eNOS expression, eNOS activity and thromboxane B2 (TXB2) production were determined. RESULTS: Ischaemia-reperfusion injury was exaggerated by two- to five-fold in SS and NASH compared with lean liver. Atorvastatin pretreatment conferred 70-90% hepatic protection in all animals. Atorvastatin increased post-ischaemic eNOS mRNA/protein and strikingly enhanced eNOS activity (by phospho-eNOS). It also attenuated microparticle (MP) production, NF-κB activation, significantly dampened post-ischaemic thromboxane B2 production, induction of TNF-α, IL-6, MIP-1a, MCP-1, GM-CSF and vascular cell adhesion molecule-1 (VCAM), with a resultant reduction on macrophage and polymorphonuclear neutrophil recruitment. Up-regulation of HMGB1 and TLR4 after IRI was marked in fatty livers; 1 h pretreatment with atorvastatin reduced HMGB1 and TLR4 expression in all livers. CONCLUSIONS: Acute (1 h) atorvastatin administration is highly hepatoprotective against IRI in NASH, fatty and lean livers. Key mechanisms include suppression of inflammation by prevention of NF-κB activation, microvascular protection via eNOS activation and suppression of TXB2 and MP release. Short-term intravenous statin treatment is a readily available and effective preventive agent against hepatic IRI, irrespective of obesity and fatty liver disease, and merits clinical trials in at-risk patients.

Microparticles mediate hepatic ischemia-reperfusion injury and are the targets of Diannexin (ASP8597).

BACKGROUND & AIMS: Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis. METHODS: C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min-24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function. RESULTS: Microparticles were detected in the circulation 15-30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8+ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-α increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory properties of microparticles involved activation of NF-κB and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin. CONCLUSIONS: Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.

Atorvastatin protects obese mice against hepatic ischemia-reperfusion injury by Toll-like receptor-4 suppression and endothelial nitric oxide synthase activation.

BACKGROUND AND AIM: Steatosis accentuates the severity of hepatic ischemia-reperfusion injury (IRI). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors ("statins") protect the heart and brain against post-ischemic injury, without necessarily lowering serum cholesterol. We tested whether 10-day or 1-day atorvastatin administration protects livers with fatty change or non-alcoholic steatohepatitis (NASH) against IRI. METHODS: Mice with dietary or genetic simple steatosis (SS) or NASH were subjected to 60 min of partial hepatic ischemia/24-h reperfusion, with/without atorvastatin administered with food (5 mg/kg body weight) for 10 days, or injected intravenously (5 mg/kg) 24 h before ischemia. Liver injury, Toll-like receptor-4 (TLR4), cytokines/chemokines, endothelial nitric oxide synthase (eNOS), activation and thromboxane B2 production were determined. RESULTS: Atorvastatin conferred 70-90% hepatic protection against IRI in obese animals with SS or NASH, in which IRI was accentuated twofold to fivefold. IRI markedly upregulated TLR4 and activated nuclear factor-κB (NF-κB); atorvastatin abrogated these effects, as well as activating eNOS. Atorvastatin dampened the post-ischemic induction of thromboxane B2, macrophage inflammatory protein-1a, monocyte chemotactic protein-1, tumor necrosis factor-α, interleukin (IL)-12 p40, γ-interferon, IL-6, and adhesion molecules (vascular cell adhesion molecule-1, E-selectin, vascular endothelial-cadherin), and reduced macrophage and neutrophil recruitment. There was no reduction in serum cholesterol that could explain these effects, and hepatic cholesterol was normal in these mice. A single 24-h injection of atorvastatin conferred equivalent hepatoprotection. CONCLUSION: Statins exert major hepatoprotection against IRI in lean, fatty, and NASH livers that is not due to cholesterol removal. Rather, statins downregulate TLR4 to prevent NF-κB activation, with resultant suppression of adhesion molecules, chemokines/cytokines, and thromboxane B2 production. Short-term statin treatment is an effective, readily-available preventive agent against hepatic IRI, irrespective of obesity and fatty liver disease.

Antioxidant effects of xanthohumol and functional impact on hepatic ischemia-reperfusion injury.

Therapeutic effects of dietary flavonoids have been attributed mainly to their antioxidant capacity. Xanthohumol (1), a prominent flavonoid of the hop plant, Humulus lupulus, was investigated for its antioxidant potential and for its effect on NF-kappaB activation. To examine the biological relevance of 1, a hepatic ischemia/reperfusion model was chosen as a widely accepted model of oxidative stress generation. The impact of 1 on endogenous antioxidant systems, on the NF-kappaB signal transduction pathway as well as on apoptotic parameters, and on hepatic tissue damage was evaluated. Compound 1 markedly decreased the level of reactive oxygen species in vitro. Furthermore, levels of enzymatic and nonenzymatic antioxidants were restored after pretreatment in postischemic hepatic tissue, and lipid peroxidation was attenuated. NF-kappaB activity was reduced in vitro as well as in hepatic tissue after ischemia/reperfusion upon pretreatment with 1. In addition, the phosphorylation of Akt was markedly inhibited. Surprisingly, 1 decreased the expression of the antiapoptotic protein Bcl-X and increased caspase-3 like-activity, a proapoptotic parameter. Moreover, hepatic tissue damage as well as TNF-alpha levels increased in xanthohumol-pretreated liver tissue after ischemia/reperfusion. In summary, xanthohumol did not protect against ischemia/reperfusion injury in rat liver, despite its antioxidant and NF-kappaB inhibitory properties.

Ischaemic and pharmacological preconditionings protect liver via adenosine and redox status following hepatic ischaemia/reperfusion in rats.

Although IPC (ischaemic preconditioning) is considered as a protective strategy in HI/R (hepatic ischaemia/reperfusion), the mechanisms for this effect have not been fully elucidated. In the present study we investigate whether PPC (pharmacological preconditioning) by transient activation of A(1)R (adenosine A(1) receptor) protects against long-term HI/R and whether the protective effects of IPC depend on A(1)R activation and whether both preconditionings affect remote organs. Wistar rats underwent IPC and long-term HI/R. Another set of animals were pharmacologically preconditioned with the A(1)R-agonist CCPA [2-chloro-N(6)-cyclopentyladenosine; 0.1 mg/kg of body weight, i.p. (intraperitoneally)] 24 h before HI/R. In other groups, rats received an A(1)R-antagonist, DPCPX (1,3-dipropyl-8-cyclopentylxanthine; 0.1 mg/kg of body weight, i.p.) 24 h before HI/R. Hepatic damage was evaluated by transaminase [AST (aspartate transaminase), ALT (alanine transaminase)] release; inflammation was assessed by hepatic MPO (myeloperoxidase) and serum TNFalpha (tumour necrosis factor alpha) and NO; oxidative stress was estimated by MDA (malondialdehyde) and 4-HDA (4-hydroxyalkenals), SOD (superoxide dismutase) activity, GSH and ADA (adenosine deaminase) as adenosine metabolism. Both preconditionings protected liver and lung against HI/R as indicated by the reduction in transaminases, MPO, MDA+4-HDA, NO, TNFalpha and ADA activity as compared with HI/R (P<0.05). However, pre-treatment with DPCPX abolished the protective effects of IPC and PPC. Preconditionings induced a significant increase in hepatic MnSOD (manganese SOD) activity and NO generation compared with the sham group, and this activity was abolished by DPCPX pre-treatment. A(1)R activation induced hepatic delayed preconditioning and blockade of A(1)R abolished hepatic IPC. IPC, as well as PPC, were able to prevent lung damage. These protective effects are associated with a reduction in oxidative stress, inflammation and endogenous antioxidant preservation.

Ozone oxidative preconditioning is mediated by A1 adenosine receptors in a rat model of liver ischemia/ reperfusion.

The liver is damaged by sustained ischemia in liver transplantation, and the reperfusion after ischemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the role of A(1) adenosine receptor on the protective actions conferred by OzoneOP in hepatic I/R. By using a specific agonist and antagonist of the A(1) subtype receptor (2-chloro N6 cyclopentyladenosine, CCPA and 8-cyclopentyl-1,3-dipropylxanthine, DPCPX respectively), we studied the role of A(1) receptor in the protective effects of OzoneOP on the liver damage, nitiric oxide (NO) generation, adenosine deaminase activity and preservation of the cellular redox balance. Immunohistochemical analysis of nuclear factor-kappa B (NF-kappaB), tumor necrosis factor alpha (TNF-alpha) and heat shock protein-70 (HSP-70) was performed. OzoneOP prevented and/or ameliorated ischemic damage. CCPA showed a similar effect to OzoneOP + I/R group. A(1)AR antagonist DPCPX blocked the protective effect of OzoneOP. OzoneOP largely reduced the intensity of the p65 expression, diminished TNF-alpha production, and promoted a reduction in HSP-70 immunoreactivity. In summary, OzoneOP exerted protective effects against liver I/R injury through activation of A(1) adenosine receptors (A(1)AR). Adenosine and (.)NO produced by OzoneOP may play a role in the pathways of cellular signalling which promote preservation of the cellular redox balance, mitochondrial function, glutathione pools as well as the regulation of NF-kappaB and HSP-70.

Use of growth-hormone-releasing peptide-6 (GHRP-6) for the prevention of multiple organ failure.

Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer (HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given alone and in combination with the potent protective factor EGF (epidermal growth factor) in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion). In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P<0.01), but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that, compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage (histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 microg/kg of body weight, intraperitoneally) alone truncated these effects by 50-85% (all P<0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments. In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.

Role of protein synthesis in the protection conferred by ozone-oxidative-preconditioning in hepatic ischaemia/reperfusion.

The liver is damaged by sustained ischaemia during liver transplantation, and the reperfusion after ischaemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischaemia/reperfusion (I/R) injury through different mechanisms. The aim of this study was to investigate the influence of the inhibition of protein synthesis on the protective actions conferred by OzoneOP in hepatic I/R. Rats were treated with cycloheximide (CHX) in order to promote protein synthesis inhibition after OzoneOP treatment. Plasma transaminases, malondialdehyde and 4-hydroxyalkenals and morphological characteristics were measured as an index of hepatocellular damage; Cu/Zn-superoxide dismutase (SOD), Mn-SOD, catalase, total hydroperoxides and glutathione levels as markers of endogenous antioxidant system. OzoneOP increased Mn-SOD isoform and ameliorated mitochondrial damage. CHX abrogated the protection conferred by OzonoOP and decreased Mn-SOD activity. Cellular redox balance disappeared when CHX was introduced. Protein synthesis is involved in the protective mechanisms mediated by OzoneOP. Ozone treatment preserved mitochondrial functions and cellular redox balance.

Methylglyoxal administration induces diabetes-like microvascular changes and perturbs the healing process of cutaneous wounds.

Increased formation of MG (methylglyoxal) and related protein glycation in diabetes has been linked to the development of diabetic vascular complications. Diabetes is also associated with impaired wound healing. In the present study, we investigated if prolonged exposure of rats to MG (50-75 mg/kg of body weight) induced impairment of wound healing and diabetes-like vascular damage. MG treatment arrested growth, increased serum creatinine, induced hypercholesterolaemia (all P < 0.05) and impaired vasodilation (P < 0.01) compared with saline controls. Degenerative changes in cutaneous microvessels with loss of endothelial cells, basement membrane thickening and luminal occlusion were also detected. Acute granulation appeared immature (P < 0.01) and was associated with an impaired infiltration of regenerative cells with reduced proliferative rates (P < 0.01). Immunohistochemical staining indicated the presence of AGEs (advanced glycation end-products) in vascular structures, cutaneous tissue and peripheral nerve fibres. Expression of RAGE (receptor for AGEs) appeared to be increased in the cutaneous vasculature. There were also pro-inflammatory and profibrotic responses, including increased IL-1beta (interleukin-1beta) expression in intact epidermis, TNF-alpha (tumour necrosis factor-alpha) in regions of angiogenesis, CTGF (connective tissue growth factor) in medial layers of arteries, and TGF-beta (transforming growth factor-beta) in glomerular tufts, tubular epithelial cells and interstitial endothelial cells. We conclude that exposure to increased MG in vivo is associated with the onset of microvascular damage and other diabetes-like complications within a normoglycaemic context.

Similar protective effect of ischaemic and ozone oxidative preconditionings in liver ischaemia/reperfusion injury.

Many studies indicate that oxygen free-radical formation after reoxygenation of liver may initiate the cascade of hepatocellular injury. It has been demonstrated that controlled ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage induced by reactive oxygen species (ROS) and protecting against liver ischaemia-reperfusion (I/R) injury. On the basis of those results we postulated that ozone treatment in our experimental conditions has biochemical parameters similar to the ischaemic preconditioning (IscheP) mechanism. Four groups of rats were classified as follows: (1) sham-operated animals subjected to anaesthesia and laparotomy, plus surgical manipulation; (2) I/R animals were subjected to 90 min of right-lobe hepatic ischaemia, followed by 90 min of reperfusion; (3) IscheP, previous to the I/R period (as in group 2): animals were subjected to 10 min of ischaemia and 10 min of reperfusion; (4) ozone oxidative preconditioning (OzoneOP), previous to the I/R period (as in group 2): animals were treated with ozone by rectal insufflation 1 mg kg (-1). The rats received 15 ozone treatments, one per day, of 5-5.5 ml at the ozone concentration of 50 microg ml (-1). The following parameters were measured: serum transaminases (AST, ALT) and 5'-nucleotidase (5 '-NT), with morphological determinations, as indicators or hepatocellular injury; total sulfhydryl groups, calcium levels and calpain activity as mediators which take part in xanthine deshydrogenase (XDH) conversion to xanthine oxidase (XO) (reversible and irreversible forms, respectively); XO activities and malondialdehyde + 4-hydroyalkenals as indicators of increased oxidative stress. AST, ALT levels were attenuated in the IscheP (130 +/- 11.4 and 75 +/- 5.7 U l (-1)) with regard to the I/R group (200 +/- 22 and 117 +/- 21.7 U l (-1)) while the OzoneOP maintained both of the enzyme activities ( 89.5 +/- 12.6 and 43.7 +/- 10 U l (-1)) without statistical differences (P< 0.05) in comparison with the sham-operated ( 63.95 +/- 11 and 19.48 +/- 3.2 U l (-1)). Protective effects of both the preconditioning settings on the preservation of total sylfhydryl groups (IscheP: 6.28 +/- 0.07, OzoneOP: 6.34 +/- 0.07 micromol mg prot (-1)), calcium concentrations (IscheP: 0.18 +/- 0.09, OzoneOP: 0.20 +/- 0.06 micromol mg prot (-1)), and calpain activity (IscheP: 1.04 +/- 0.58, OzoneOP: 1.41 +/- 0.79 U mg prot (-1)) were observed. Both of the preconditionings attenuated the increase of total XO associated to I/R injury. Generation of malondialdehyde + 4 hydroxyalkenals was prevented by IscheP and OzoneOP without statistical differences between the two protective procedures. These results provide evidence that both of the preconditioning settings share similar biochemical mechanisms of protection in the parameters which were measured. Although there were no differences from a biochemical point of view between Ischaemic and OzoneOPs, the histological results showed a more effective protection of OzoneOP than IscheP in our experimental conditions.