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Platelet production and destruction in liver cirrhosis

Paola Pradella
Gabriele Pozzato
DOI: http://dx.doi.org/10.1016/j.jhep.2010.08.018

Background & Aims


Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The
purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet
production and destruction.

Methods
Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25
subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood
donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP)
were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using
monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow
cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting.

Results
Serum TPO was significantly lower in LC (29.918.1pg/ml) compared to controls (82.3 47.6 pg/ml).
The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of
RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum antiplatelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p
<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV +LC, while it was
not found in HBV+ or alcoholic LC.

Conclusions
Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet
production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased
platelet clearance and impaired thrombopoiesis.

Endotoxaemia in the pathogenesis of cytopenias in


liver cirrhosis. Could oral antibiotics raise blood
counts?
Georgios Kalambokis
DOI: http://dx.doi.org/10.1016/j.mehy.2010.08.043
Cytopenias are frequently observed in patients with cirrhosis and are associated with increased morbidity.
In particular, thrombocytopenia can impact routine care of patients with cirrhosis by potentially postponing
or interfering with diagnostic and therapeutic procedures including liver biopsy and medically indicated or
elective surgery. The pathogenesis of cytopenias in cirrhosis remains largely unknown. Historically, the
concept of hypersplenism has long been associated with the cirrhosis-related hematological disorders but
was never proven. On the other hand, intestinal bacterial overgrowth and altered gut permeability in
cirrhotic patients lead to increased translocation of bacteria and endotoxin into the portal circulation. The
impaired phagocytic function of the reticuloendothelial system together with the portosystemic shunting
allow endotoxin to reach the systemic circulation and high concentrations of circulating endotoxin are
found in cirrhotic patients even with no clinical evidence of infection and correlate with the severity of liver
disease. Endotoxin activates monocytes and promotes the release of proinflammatory cytokines. Indeed,
serum levels of interleukin-1, interleukin-6, tumor necrosis factor-, and interferon- are elevated in
patients with cirrhosis in proportion to the severity of liver disease. Endotoxaemia stimulates the vascular
production of nitric oxide (NO) directly or indirectly via the cytokine cascade, and correlates with serum
NO metabolite levels in cirrhosis. Several lines of evidence strongly suggest that endotoxaemia may
reduce peripheral blood counts either directly or through the release of cytokines and NO. Previous
studies in experimental models of cirrhosis and cirrhotic patients have demonstrated that long-term
administration of oral antibiotics such as trimethoprim-sulfamethoxazole, norfloxacin, and rifaximin can
reduce bacterial translocation and circulating levels of endotoxin, TNF-, IL-6, and NO. We hypothesize
that endotoxaemia plays a pivotal role in the pathogenesis of cytopenias in cirrhosis and that intestinal
decontamination could raise peripheral blood counts by the suppression of endotoxaemia and the
inhibition of cytokine and NO production.

Suppression of oxidative stress and improvement of liver


functions in mice by ursolic acid via LKB1-AMP-activated protein
kinase signaling.
Yang Y1, Zhao Z, Liu Y, Kang X, Zhang H, Meng M.

Author information

Abstract
BACKGROUND AND AIM:
Hepatic cirrhosis is the final stage of liver dysfunction, characterized by diffuse fibrosis, which is
the main response to the liver injury. This study is to investigate the effects of ursolic acid (UA)
on liver functions and fibrosis in bile duct ligation (BDL) mice and to determine the underlying
mechanisms.
METHODS:
Cultured hepatocytes were treated with lipopolysaccharide (LPS) in the presence or absence of
UA. The reactive oxygen species (ROS) level, protein levels of IB, iNOS and Cox-2, and NFB activation were detected, respectively. C57/BL6 and AMP-activated protein kinase
(AMPK)2(-/-) mice were subjected to BDL for 14 days. UA was administered by gavage. The
markers of liver function and oxidative stress, and liver histopathology were analyzed after
treatment.
RESULTS:
Treatment of hepatocytes with UA dose-dependently activates AMPK, which is abolished by
silence of liver kinase B1 (LKB1). LPS significantly increased ROS productions, apoptosis, NFB activation, and expressions of iNOS and Cox-2 in cultured hepatocytes. All these effects
were blocked by co-incubation with UA. Importantly, silence of LKB1, AMPK, or iNOS/Cox-2 by
small interference RNA transfection reversed UA-induced effects in cultured cells. In an animal
study, 14-day BDL induced liver fibrosis and liver injury, accompanied with increased oxidative
stress and protein expressions of iNOS and Cox-2 in liver. Treatment of UA significantly
attenuated the BDL-induced detrimental effects in wild-type mice but not in AMPK2(-/-) mice.
CONCLUSION:
UA via LKB1-AMPK signaling offers protective effects on BDL-induced liver injury in mice, which
may be related to inhibition of oxidative stress.

Ursolic acid ameliorates hepatic fibrosis in the rat by specific


induction of apoptosis in hepatic stellate cells.
Wang X1, Ikejima K, Kon K, Arai K, Aoyama T, Okumura K, Abe W, Sato N, Watanabe S.

Author information

Abstract
BACKGROUND & AIMS:
Specific induction of cell death in activated hepatic stellate cells (HSCs) is a promising
therapeutic strategy for hepatic fibrosis. In this study, we evaluated the cell-killing effect of
ursolic acid (UA), a pentacyclic triterpenoid, in activated HSCs both in vitro and in vivo.
METHODS:
Culture-activated rat HSCs were treated with UA (0-40M), and the mechanisms of cell death
were evaluated. The cell killing effect of UA on activated HSCs in rats chronically treated with
thioacetamide (TAA) was detected by dual staining of TdT-mediated dUTP nick-end labeling
(TUNEL) and smooth muscle -actin (SMA) immunohistochemistry, and resolution of hepatic
fibrosis was evaluated. Further, the protective effects of UA on progression of hepatic fibrosis
caused by TAA and bile duct ligation (BDL) were evaluated.
RESULTS:
UA induced apoptotic cell death in culture-activated HSCs, but not in isolated hepatocytes and
quiescent HSCs. Mitochodrial permeability transition (MPT) preceded the cleavage of caspase3 and -9 following UA treatment. UA also decreased phosphorylation levels of Akt, and
diminished nuclear localization of NFB in these cells. In rats pretreated with TAA for 6weeks, a
single injection of UA induced remarkable increases in TUNEL- and SMA-dual-positive cells in
24h, and significant regression of hepatic fibrosis within 48h. Moreover, UA ameliorated hepatic
fibrogenesis caused by both chronic TAA administration and BDL.
CONCLUSIONS:
UA ameliorated experimental hepatic fibrosis most likely through specific induction of apoptosis
in activated HSCs. It is therefore postulated that UA is a potential therapeutic reagent for
resolution of hepatic fibrosis

Protective effects of ursolic acid in an experimental


model of liver fibrosis through Nrf2/ARE pathway
Aim
Liver fibrosis is a reversible wound-healing response that occurs following
liver injury. In this study, we aimed to investigate the possible protective
effects of ursolic acid in liver fibrosis induced by carbon tetrachloride (CCl4).
Methods
ICR mice were randomly divided into six groups (Group 1: normal; Group 2:
CCl4-treated group; Group 3: CCl4 plus ursolic acid 25 mg/kg group; Group 4:
CCl4 plus ursolic acid 50 mg/kg group; Group 5: CCl4 plus colchicine 1 mg/kg
group; Group 6: ursolic acid 50 mg/kg group). Mice were administered with
CCl4 (2 mL of CCl4 in olive oil (1:1, v/v) per kg body weight twice weekly) by
intraperitoneal injection and oral injection of colchicine (1 mg/kg) or ursolic
acid (25, 50 mg/kg) daily. After six weeks, serum aminotransferase activity,
hepatic reactive oxygen species (ROS) production, thiobarbituric acid reactive
substances (TBARS), antioxidase (SOD, CAT, GPx) activity and
histopathological analysis were performed. The levels of nuclear factor E2related factor 2 (Nrf2), NAD(P)H: quinone oxidoreductase-1 (NQO1),
glutathione S-transferase (GST) and heme oxygenase-1 (HO-1), tumor
necrosis factor-alpha (TNF-), prostaglandin E2 (PGE2) and inducible nitric
oxide synthase (iNOS), Bcl-2 and caspase-3 were measured.
Results
Ursolic acid significantly prevented CCl4-induced hepatotoxicity and fibrosis,
indicated by both diagnostic indicators and histopathological analysis. CCl4induced profound elevations of oxidative stress, inflammation and apoptosis in
liver were suppressed by ursolic acid.

Propranolol in the Treatment of Cirrhotic Ascites

Propranolol hydrochloride is reported to lower portal pressure and inhibit renin secretion in
patients with chronic liver disease, actions that might lessen the tendency to ascites formation.
We compared the effect of diuretics with that of the same dose of diuretics plus propranolol on
natriuresis, urine output, and daily weight loss in 13 hospitalized patients with stable chronic
liver disease, sodium retention, and ascites. The propranolol hydrochloride dose was 20 to 160
mg four times a day, titrated to reduce resting pulse by 25% or systolic BP 10 mm Hg. Diuretics
given were furosemide, 80 to 160 mg, and triamterene, 100 or 200 mg/day. Periods of time
when each regimen was received ranged from one to four days. Creatinine excretion
documented complete urine collections. Compared with diuretics alone, diuretics plus
propranolol substantially reduced resting pulse, systolic BP, and urine sodium excretion,
although not creatinine clearance. This antinatriuretic effect may limit the proposed usefulness
of propranolol for prevention of variceal bleeding in patients with cirrhosis and ascites.
Portal Hipertensi

Patients with cirrhosis and portal hypertension eventually develop a hyperdynamic


circulation, with high cardiac output and reduced systemic vascular resistance
(SVR). This haemodynamic alteration is thought to be involved in the pathogenesis
of sodium retention and ascites,[10] which has been observed in 80% of cirrhotic
patients after many years of disease.[11] Whether the hyperdynamic circulation also
occurs in cirrhotic patients with arterial hypertension remains unknown.
Nonselective beta-blockers (i.e. propranolol, nadolol, timolol) are the recommended
first-line therapy for the prophylaxis of variceal haemorrhage in cirrhotic patients with
varices at high risk for bleeding.[12]They reduce portal pressure by decreasing portal
and collateral blood flow. This effect is achieved via the blockade of both the beta-1adrenoreceptors, causing a reduction in cardiac output and the beta-2adrenoreceptors in the splanchnic vasculature, causing splanchnic vasoconstriction.
[13, 14]
Near complete protection from variceal bleeding is reported when the hepatic
venous pressure gradient (HVPG) is reduced to <12 mmHg or 20% reduction from
the baseline values is achieved.[1517]