Evaluation of liver fibrosis by investigation of hepatic parenchymal perfusion using contrast-enhanced ultrasound: an animal study.

PURPOSE: To investigate the value of assessing the hepatic parenchymal perfusion in contrast-enhanced ultrasound (CEUS) for evaluating liver fibrosis, using an animal model. METHODS: Seventy Sprague-Dawley rats were divided into experimental (n = 35) and control (n = 35) groups. In the experimental group, liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride. CEUS of the liver was performed at a 2-week interval for 14 weeks. Signal intensity of liver parenchyma was analyzed with time-intensity curves. Histologic examination of liver specimens of the animals was performed to assess the fibrosis stage. RESULTS: The peak signal intensity of hepatic parenchymal perfusion in stage 2-3 fibrosis was significantly lower than that in stage 0-1. The time to peak intensity of hepatic parenchymal perfusion was significantly longer in the experimental group than the control group, and in the stage 3 fibrosis than in stages 0-2 fibrosis. Using time to peak intensity of hepatic parenchymal perfusion to distinguish stage 3 fibrosis and stages 0-2 fibrosis, the optimum cutoff was 75,000 milliseconds with the sensitivity and specificity of 67% and 78%, respectively. CONCLUSIONS: This animal study showed that CEUS has the potential to be a complementary imaging tool in the evaluation of liver fibrosis.

Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis.

Reduced expression of endothelial nitric oxide synthase (eNOS) in chronic liver disease can reduce hepatic perfusion and accelerate fibrosis. The relationship between eNOS expression and liver fibrogenesis remains unclear. We investigated whether L-arginine attenuated chronic liver fibrosis through eNOS expression. Chronic liver injury was induced by administration of carbon tetrachloride (CCl(4)) to mice for 8 weeks. 5-Methylisothiourea hemisulphate (SMT), an iNOS inhibitor, or L-arginine, a NOS substrate were injected subcutaneously. CCl(4)-induced hepatotoxicity, oxidative stress and accumulation of collagen were detected in the liver. The expression levels of inducible NOS (iNOS) and nuclear factor kappa-B (NF-kappaB) activity in the liver after CCl(4) treatment were increased but eNOS expression and activator protein-1 (AP-1) activity were decreased. Both SMT and L-arginine effectively reduced CCl(4) induced oxidative stress and collagen formation, but L-arginine showed a significantly greater suppression of collagen formation, iNOS expression and NF-kappaB activity. L-arginine also restored the level of eNOS and AP-1 activity. L-arginine was more effective than SMT in suppressing liver fibrosis. L-arginine might improve NO production which facilitates hepatic blood flow and thus retards liver fibrogenesis. Our results showed that the reduced eNOS expression in CCl(4)-treated mice was reversed by L-arginine. Furthermore, L-arginine also reversed the reduced AP-1 activity, an eNOS promoter.

Hepatic response to chronic hypoxia in experimental rat model through HIF-1 alpha, activator protein-1 and NF-kappa B.

Chronic liver diseases are commonly associated with tissue hypoxia that may cause inflammation, oxidative stress, liver cell injury and increased nuclear transcriptional regulation. The hepatic response to chronic hypoxia at the molecular level has not yet been clearly understood until now. The aim of this study is to investigate whether nuclear transcription factors [hypoxia-inducible factor-1 (HIF-1α), activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB)] exhibit activity changes during hepatic response to chronic hypoxia. Blood and liver samples were collected from adult Sprague-Dawley rats living in atmospheric air or 10% oxygen for four weeks. Levels of serum alanine aminotransferase (ALT), 8-isoprostane and nitrotyrosine were measured. The activities of nuclear transcription factors and the expression of downstream genes (iNOS, eNOS, ET-1 and VEGF) were measured using RT-PCR, Western blotting and Gel shift analysis. Results showed that serum ALT level, 8-isoprostane level and formation of nitrotyrosine were within normal range at all time-points. In the hypoxic liver, DNA-binding activities of HIF-1α, NF-κB and AP-1 increased significantly. Expression levels of iNOS, VEGF and ET-1 progressively increased from day 7 to day 28. eNOS was also elevated in the hypoxic liver. In conclusion, our study suggests that increased activity of HIF-1α, AP-1 and NF-κB may partly play a significant role in the hepatic response to oxidative stress and liver injury under chronic hypoxia. The increased expression of VEGF, ET-1, iNOS and eNOS may be partly due to the compensatory mechanism in the vascular beds of the liver in response to chronic hypoxia.

Cyclooxygenase inhibitors protect D-galactosamine/lipopolysaccharide induced acute hepatic injury in experimental mice model.

We investigated the protective effects of two non-steroid anti-inflammatory drugs, indomethacin (COX-1 and COX-2 inhibitors) and nimesulide (specific COX-2 inhibitor) on the hepatic injury induced by lipopolysaccharide in d-galactosamine sensitized (Gal/LPS) mice. ICR male mice were injected with a single dose of Gal/LPS with or without pre-treatment of 3mg/kg indomethacin or 30 mg/kg nimesulide (single i.p. injection). Sixteen hours later, blood and liver tissues of mice were collected for histological, molecular, and biochemical analyses. Our results showed marked reduction of hepatic necrosis, serum ALT, and tissue TBARS levels in both indomethacin- and nimesulide-pre-treated mice when compared with Gal/LPS-treated mice. Western blot and RT-PCR analysis showed decreased levels of iNOS mRNA, iNOS protein, and nitrotyrosine formation in both COX inhibitor pre-treated groups when compared with Gal/LPS-treated group. There was an inverse relationship between COX-1 and COX-2 expressions, as well as between COX-2 and C/EBP-α expressions in COX inhibitors groups, Gal/LPS and control groups. COX inhibitors reduced the expression of TNF-α mRNA and the activity of NF-κB which were elevated by Gal/LPS treatment. We conclude that COX inhibitors protected the liver from Gal/LPS-induced hepatotoxicity. COX inhibitors could be considered as potential agents in the prevention of acute liver failure and sepsis.

Role of nitric oxide in the regulation of fibrogenic factors in experimental liver fibrosis in mice.

Previously, we have shown that an increased expression level of iNOS but a reduction in the expression of eNOS is associated with increased oxidative stress markers in CCl4-induced experimental liver fibrosis. The present study aimed to investigate the effect of L-arginine and 5-methylisothiourea hemisulfate (SMT) in the expression of profibrogenic factors in chronic liver injury. ICR mice were treated with CCl4 with or without treatment of L-arginine, an NO donor, or SMT, an iNOS inhibitor. The expression of matrix metalloptroteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. The activity of the MMP-2 and MMP-9 were measured by zymography. Our results showed that CCl4-treated mice showed significant up-regulation of expression of pro-fibrogenic factors, TNF-α and COX-2. Treatment with L-arginine or SMT showed a significant reduction in CCl4-induced expression of these pro-fibrogenic factors, TNF-α and COX-2. In conclusion, both SMT and L-arginine effectively attenuated the progression of CCl4-induced liver fibrosis. SMT suppresses iNOS mediated NO production. However, L-arginine augments NO production. The similar effect of the two drugs on liver fibrosis indicates that there may be two distinct pathways of NOS mediated fibrogenesis in chronic liver injury by iNOS and eNOS. Our results suggest that eNOS-mediated liver fibrogenesis may play a more important role than that of iNOS in chronic liver injury. Taken together, these results support the contention that NO plays an active role in the progression of liver fibrosis and hepatocellular damage.

Construction of Multi-dimensional Arterial Health Status Map based on Molecular and Clinical Measurements, Fuzzy System and Data Cubes.

Atherosclerosis results from inflammatory processes involving biomarkers, such as lipid profile, haemoglobin A1C, oxidative stress, coronary artery calcium score and flow-mediated endothelial response through nitric oxide. This paper proposes a health status coefficient, which comprehends molecular and clinical measurements concerning atherosclerosis to provide a measure of arterial health. An arterial health status map is produced to map the multi-dimensional measurements to the health status coefficient. The mapping is modeled by a fuzzy system embedded with the health domain expert knowledge. The measurements obtained from the pilot study are used to tune the fuzzy system. The inferred arterial health coefficients are stored into the data cubes of a multi-dimensional database. Due to this adaptability and transparency of fuzzy system, the health status map can be easily updated when the refinement of fuzzy rule base is needed or new measurements are obtained.

Expression and functions of vasoactive substances regulated by hypoxia-inducible factor-1 in chronic hypoxemia.

The aims of the present review are to summarize and to discuss the role of hypoxia-inducible factor-1 (HIF-1) and the expression and functions of vasoactive substances in chronic hypoxemia with specific focus in the liver and the carotid body. Vascular remodelling and vasoactive substances play important functional roles in the adaptive response to chronic hypoxemia for the maintenance of oxygen homeostasis in all systems in man. HIF-1 regulates the gene expression of vasoactive substances such as vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and enzymes for producing nitric oxide (NO). Recent studies have shown the effect of chronic hypoxia on the expression of HIF-1alpha and HIF-1-target genes in multiple organ systems including the liver and the carotid body. Results are consistent with increases in the hematocrit levels, pulmonary arterial pressure and right heart mass developed during chronic hypoxia. In addition, the carotid body is also hyperplastic and increases in organ mass with increased levels of HIF-1alpha and the vasoactive substances. These molecules increase the mitotic activity and modulate the excitability of the chemoreceptor. Intriguingly, the liver morphology, serum alanine aminotransferase and 8-isoprostane levels are within normal range in chronic hypoxia, suggesting the absence of significant oxidative stress. Yet, the HIF-1alpha is upregulated and the mRNA and protein levels of VEGF, ET-1, inducible and constitutive NO synthases are elevated in the liver during chronic hypoxia. In conclusion, the adaptive response to long-term hypoxemia involves compensatory mechanisms mediated by expressing significant levels of HIF-1alpha and vasoactive substances regulated by HIF-1.