Vitamin E Attenuates the Progression of Non-Alcoholic Fatty Liver Disease Caused by Partial Hepatectomy in Mice.

BACKGROUND AND AIM: The progression of non-alcoholic fatty liver disease (NAFLD) likely involves a 'multiple hit' mechanism. We hypothesized that partial hepatectomy, a procedure performed frequently in patients with NAFLD, would accelerate the progression of disease. METHODS: C57BL/6JolaHsd mice were fed a choline-deficient L-amino acid-defined diet (CD-AA) or a choline-sufficient L-amino acid-defined control diet (CS-AA). Part of the mice in the CD-AA group received a diet enriched in vitamin E (~20 mg /day). Two weeks after the start of the diet, mice underwent a partial hepatectomy or a sham operation. RESULTS: In the CD-AA group, NAFLD activity scores were significantly higher at 7 days after partial hepatectomy compared to the sham operated mice (3.7 ± 1.3 vs. 1.8 ± 0.7; P<0.05). In addition, TBARS, a measure for oxidative stress, in liver tissue of the CD-AA group were significantly higher at day 1, 3 and 7 after partial hepatectomy compared to the sham operated mice (P<0.05). Vitamin E therapy significantly reduced TBARS level at day 7 after partial hepatectomy compared to the CD-AA diet group (P< 0.05). Vitamin E suppletion reduced NAFLD activity score at day 7 after partial hepatectomy compared to the CD-AA group (2.3 ± 0.8 vs. 3.8 ± 1.0; P<0.05). CONCLUSION: Partial hepatectomy accelerates the progression of NAFLD. Disease progression induced by partial hepatectomy is substantially attenuated by vitamin E.

Diffuse reflectance spectroscopy accurately quantifies various degrees of liver steatosis in murine models of fatty liver disease.

BACKGROUND: A real-time objective evaluation for the extent of liver steatosis during liver transplantation is currently not available. Diffuse reflectance spectroscopy (DRS) rapidly and accurately assesses the extent of steatosis in human livers with mild steatosis. However, it is yet unknown whether DRS accurately quantifies moderate/severe steatosis and is able to distinguish between micro- and macrovesicular steatosis. METHODS: C57BL/6JolaHsd mice were fed wit a choline-deficient L-amino acid-defined diet (CD-AA) or a choline-sufficient L-amino acid-defined control diet (CS-AA) for 3, 8, and 20 weeks. In addition B6.V-Lepob/OlaHsd (ob/ob) mice and their lean controls were studied. A total of 104 DRS measurements were performed in liver tissue ex vivo. The degree of steatosis was quantified from the DRS data and compared with histopathological analysis. RESULTS: When assessed by histology, livers of mice fed with a CD-AA and CS-AA diet displayed macrovesicular steatosis (range 0-74 %), ob/ob mice revealed only microvesicular steatosis (range 75-80 %), and their lean controls showed no steatosis. The quantification of steatosis by DRS correlated well with pathology (correlation of 0.76 in CD-AA/CS-AA fed mice and a correlation of 0.75 in ob/ob mice). DRS spectra did not distinguish between micro- and macrovesicular steatosis. In samples from CD-AA/CS-AA fed mice, the DRS was able to distinguish between mild and moderate/severe steatosis with a sensitivity and specificity of 86 and 81 %, respectively. CONCLUSION: DRS can quantify steatosis with good agreement to histopathological analysis. DRS may be useful for real-time objective evaluation of liver steatosis during liver transplantation, especially to differentiate between mild and moderate/severe steatosis.

Horizontal RNA transfer mediates platelet-induced hepatocyte proliferation.

Liver regeneration is stimulated by blood platelets, but the molecular mechanisms involved are largely unexplored. Although platelets are anucleate, they do contain coding or regulatory RNAs that can be functional within the platelet or, after transfer, in other cell types. Here, we show that platelets and platelet-like particles (PLPs) derived from the megakaryoblastic cell line MEG-01 stimulate proliferation of HepG2 cells. Platelets or PLPs were internalized within 1 hour by HepG2 cells and accumulated in the perinuclear region of the hepatocyte. Platelet internalization also occurred following a partial hepatectomy in mice. Annexin A5 blocked platelet internalization and HepG2 proliferation. We labeled total RNA of MEG-01 cells by incorporation of 5-ethynyluridine (EU) and added EU-labeled PLPs to HepG2 cells. PLP-derived RNA was detected in the cytoplasm of the HepG2 cell. We next generated PLPs containing green fluorescent protein (GFP)-tagged actin messenger RNA. PLPs did not synthesize GFP, but in coculture with HepG2 cells, significant GFP protein synthesis was demonstrated. RNA-degrading enzymes partly blocked the stimulating effect of platelets on hepatocyte proliferation. Thus, platelets stimulate hepatocyte proliferation via a mechanism that is dependent on platelet internalization by hepatocytes followed by functional transfer of RNA stored in the anucleate platelet. This mechanism may contribute to platelet-mediated liver regeneration.

Sphingosine kinase-1 inhibition protects primary rat hepatocytes against bile salt-induced apoptosis.

Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (-70%, P<0.05) without inducing necrosis. GCDCA increased the S1P levels in hepatocytes (P<0.05). GCDCA induced [Ca(2+)] oscillations in hepatocytes and co-treatment with the [Ca(2+)] chelator BAPTA repressed GCDCA-induced apoptosis. Ski-II inhibited the GCDCA-induced intracellular [Ca(2+)] oscillations. Transcripts of all five S1P receptors were detected in hepatocytes, of which S1PR1 and S1PR2 appear most dominant. Inhibition of S1PR1, but not S1PR2, reduced GCDCA-induced apoptosis by 20%. Exogenous S1P also significantly reduced GCDCA-induced apoptosis (-50%, P<0.05), however, in contrast to the GCDCA-induced (intracellular) SphK1 pathway, this was dependent on S1PR2 and not S1PR1. Our results indicate that SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca(2+)] signaling and activation of S1PR1.

Pertussis toxin, an inhibitor of G(αi) PCR, inhibits bile acid- and cytokine-induced apoptosis in primary rat hepatocytes.

UNLABELLED: Excessive hepatocyte apoptosis is a common event in acute and chronic liver diseases leading to loss of functional liver tissue. Approaches to prevent apoptosis have therefore high potential for the treatment of liver disease. G-protein coupled receptors (GPCR) play crucial roles in cell fate (proliferation, cell death) and act through heterotrimeric G-proteins. G(αi)PCRs have been shown to regulate lipoapoptosis in hepatocytes, but their role in inflammation- or bile acid-induced apoptosis is unknown. Here, we analyzed the effect of inhibiting G(αi)PCR function, using pertussis toxin (PT), on bile acid- and cytokine-induced apoptosis in hepatocytes. Primary rat hepatocytes, HepG2-rNtcp cells (human hepatocellular carcinoma cells) or H-4-II-E cells (rat hepatoma cells) were exposed to glycochenodeoxycholic acid (GCDCA) or tumor necrosis factor-α (TNFα)/actinomycin D (ActD). PT (50-200 nmol/L) was added 30 minutes prior to the apoptotic stimulus. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (sytox green staining) were assessed. PT significantly reduced GCDCA- and TNFα/ActD-induced apoptosis in rat hepatocytes (-60%, p<0.05) in a dose-dependent manner (with no shift to necrosis), but not in HepG2-rNtcp cells or rat H-4-II-E cells. The protective effect of pertussis toxin was independent of the activation of selected cell survival signal transduction pathways, including ERK, p38 MAPK, PI3K and PKC pathways, as specific protein kinase inhibitors did not reverse the protective effects of pertussis toxin in GCDCA-exposed hepatocytes. CONCLUSION: Pertussis toxin, an inhibitor of G(αi)PCRs, protects hepatocytes, but not hepatocellular carcinoma cells, against bile acid- and cytokine-induced apoptosis and has therapeutic potential as primary hepatoprotective drug, as well as adjuvant in anti-cancer therapy.

Angiotensin II protects primary rat hepatocytes against bile salt-induced apoptosis.

UNLABELLED: Angiotensin II (AT-II) is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R) and thereby activates hepatic stellate cells (HSCs). AT-II receptor blockers (ARBs) are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA) and tauro-lithocholic acid-3 sulfate (TLCS), to induce apoptosis. AT-II (100 nmol/L) was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans) and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (Sytox green staining) were quantified. Expression of CHOP (marker for ER stress) and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (-50%, p<0.05) without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (-50%, p<0.05). However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis). CONCLUSION: Angiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte-toxicity of Sartans (angiotensin receptor blockers, ARBs) in some patients with chronic liver injury.

Effect of captopril on TNF-α and IL-10 in the livers of bile duct ligated rats.

BACKGROUND: The renin-angiotensin system has an important role in hepatic inflammation and fibrosis. Renin-angiotensin system blockade by angiotensin-converting enzyme (ACE) inhibitors provides some protective effects against hepatic fibrogenesis. Captopril as an ACE inhibitor can decrease inflammatory mediators and attenuate hepatic fibrosis in the livers of bile duct ligated (BDL) rats. OBJECTIVE: The present study was conducted to investigate the effects of captopril on cytokine production in hepatic fibrosis induced by a bile duct ligation model in rats. METHODS: Male rats were divided into four groups including; control, sham operated, BDL, and BDL plus captopril (10 mg/kg/day, orally). After 28 days of treatment, the livers were removed for cytokine analysis. Hepatic interleukin (IL)-10 and tumor necrosis factor (TNF)-α levels were measured. RESULTS: Captopril treatment decreased the hepatic content of the proinflammatory cytokine TNF-α and increased the anti-inflammatory cytokine IL-10. CONCLUSION: the present study suggests that the protective effect of captopril on hepatic fibrosis is likely to be mediated by cytokine production.

Attenuation of hepatic fibrosis through captopril and enalapril in the livers of bile duct ligated rats.

Hepatic fibrosis is a common feature in different types of chronic liver injury. The demonstration of the pro-fibrogenesis role of angiotensin II in chronic liver diseases brought up the idea that anti-angiotensin II agents may be effective on improvement of hepatic fibrosis by either blocking the angiotensin II receptor or inhibition of angiotensin converting enzyme (ACE). This study is aimed at comparing the anti-fibrogenesis effects of two ACE inhibitors, captopril and enalapril, in the livers of rats with bile duct ligation through biochemical and histopathological parameters. Male Albino Wistar rats were divided into four groups (n=4-5 each), including sham operated, bile duct ligated, captopril and enalapril treated. After 28 days of treatment, the liver was removed and the levels of hepatic hydroxyproline, glutathione and lipid peroxidation were determined. The degree of the development of fibrosis was evaluated through histopathological numerical scores. The results demonstrated that angiotensin converting enzyme inhibitors increased GSH, decreased lipid peroxidation and improved hepatic fibrosis as shown by histopathology as well as decreased hepatic content of hydroxyproline. Enalapril was significantly more effective than captopril (p<0.001) in improvement of hepatic fibrosis. Also it was shown that enalapril has a significant antioxidative effect (p<0.05) in comparison with captopril. In conclusion, the results of our study suggest that the antifibrotic effect of enalapril may be mostly related to the inhibition of angiotensin converting enzyme.

The effects of bladder neck incision on urodynamic abnormalities of children with posterior urethral valves.

PURPOSE: We evaluated the effects of simultaneous bladder neck incision and valve ablation on urodynamic abnormalities in patients with posterior urethral valves. MATERIALS AND METHODS: A total of 46 patients with posterior urethral valves entered our prospective study between 1998 and 2003. Group 1 consisted of 22 patients who underwent simultaneous valve ablation and bladder neck incision at the 6 o'clock position. Group 2 consisted of 24 age matched patients with comparable prognostic factors who underwent simple valve ablation. Trends in renal function tests, urodynamics and changes in the upper urinary tracts were evaluated throughout followup. RESULTS: Mean patient age at presentation was 1.6 years in group 1 and 1.8 years in group 2. Preoperatively, all patients in both groups had hypercontractile bladders and comparable high maximum voiding detrusor pressures. At the end of followup (mean 4.5 years) no patient in group 1 had bladder hypercontractility or detrusor overactivity, and the mean maximum voiding detrusor pressure was 53 +/- 15 cm H(2)O. In comparison, 9 patients in group 2 had bladder hypercontractility, 6 had detrusor overactivity and the mean maximum voiding detrusor pressure was 87 +/- 45 cm H(2)O (p <0.01). Myogenic bladder failure developed in 5 patients in group 2. The number of patients requiring anticholinergic medication and the duration of treatment were also significantly higher in group 2 compared to group 1. CONCLUSIONS: Valve ablation with bladder neck incision may result in better bladder urodynamic function in comparison to simple valve ablation. However, long-term studies with followup through puberty are required to evaluate the final effects on renal function.

Urodynamic changes in patients with anterior urethral valves: before and after endoscopic valve ablation.

PURPOSE: To retrospectively review a series of children with anterior urethral valves (AUV), with emphasis on patterns of urodynamic change and long-term outcome of endoscopic treatment. PATIENTS AND METHODS: We reviewed the medical records of eight patients who had undergone thorough radiological and urodynamic exams before and after treatment. The diagnosis of AUV was based on radiological imaging and confirmed by urethrocystoscopy. The valves were ablated through either transurethral fulguration or resection. The upper urinary tracts were studied by renal scan and ultrasonography before and after the procedure. Bladder function was assessed urodynamically 3 months after surgery. Uroflowmetry was performed as soon as the children were toilet trained. RESULTS: Endoscopic ablation of AUV was successful in all cases and no surgical complications occurred. The initial symptoms resolved in all boys. VUR disappeared in two out of three patients, and five children had bladder trabeculation that was resolved after surgery. The final outcome was successful in seven patients (88%). The major urodynamic dysfunction was bladder hypercontractility that resolved following valve ablation. The mean maximum voiding detrusor pressure (P(detmax)) decreased from 213.2+/-17.9 cmH(2)O to 80.7+/-9.9 cmH(2)O, 6 months after treatment (P<0.001). None of the patients had low-compliant bladder, detrusor instability or myogenic failure. The voiding pattern in all toilet-trained patients was staccato and of an interrupted shape prior to surgery, but changed to a normal bell-shaped voiding pattern following valve ablation. CONCLUSION: AUV should be considered in the differential diagnosis of patients presenting with infravesical obstruction. We recommend endoscopic valve ablation as the treatment of choice.