FTY720 Attenuates Hepatic Ischemia-Reperfusion Injury in Normal and Cirrhotic Livers

Animals

Male Sprague-Dawley rats (body weight: 220–280 g) were used to isolate hepatocytes for primary culture and in the animal experiments. Liver cirrhosis was induced in the rats (5–6 weeks, body weight: 160–180 g) by subcutaneous injection of 50% carbon tetrachloride diluted with olive oil at a dose of 0.2 mL/100 g of body weight twice a week for 9 weeks. Rats were housed in a standard animal laboratory with free activity and access to water and chow. They were kept under constant environment conditions with a 12-h light–dark cycle. All operations were performed under clean conditions.

Experimental procedure

Sample collection

Liver tissues and blood were sampled at 20 min, 60 min, 90 min, 6 h and 24 h after reperfusion for hepatic gene detection, morphologic examination, systemic cytokine expression and liver function tests. Six rats were included at each time point for the treatment group and control group, respectively. The normal liver tissues were sampled immediately after abdominal wall incision for normal control. To compare the expression levels with or without treatment accurately in the cirrhotic liver, liver biopsies were taken from the right inferior lobe before FTY720 treatment and liver ischemia for basal level in the cirrhotic liver group. As the liver biopsy was a small piece of liver tissue, it would not affect the results.

Liver function by biochemical test

Blood samples (0.5 mL) collected at different time points were used to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin levels (Hitachi 747 Automatic Analyzer, Boehringer Mannheim Gmbh, Mannheim, Germany).

Hepatic gene expression profiles by real-time quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR)

Liver biopsies were stored at −80°C until total RNA extraction. The total RNA was extracted using Rneasy Midi Kit (Qiagen, GmbH, Hilden, Germany). About 0.5 μg total RNA from each sample was used to perform reverse transcription (RT) reaction using TaqMan Reverse Transcription Reagents (17) (Applied Biosystems, Foster City, CA). RT product (1 μL) was used to perform real-time quantitative RT-PCR using TaqMan Core Reagent Kit (Applied Biosystems) by the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) (17). The probes and primers of Egr-1, ET-1, inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-2 (MIP-2), heme oxygenase-1 (HO-1) and A20 were designed under the Primer Express software according to the criteria for real-time PCR (Applied Biosystems) (Table 1). The probes and primers of Hsp70 and interleukin-10 (IL-10) were commercially available from Applied Biosystems Limited. The TaqMan Ribosomal RNA Control Reagent (18S RNA probe and primer pair, Applied Biosystems) was used for internal control in the same PCR plate well to normalize the target gene amplification copies. All samples were detected in triplicate, and the readings from each sample and its internal control were used to calculate the gene expression level. After normalization with the internal control, the gene expression levels after I/R injury were calculated as the percentage of the basal levels in sham operation in the normal liver groups. In the cirrhotic liver group, the gene expression levels were expressed as the ratios to basal levels before hepatic ischemia in the same rat biopsy.

Cell signaling pathways related to cell survival, apoptosis and inflammatory response by Western blot

Western-blot assay was modified from the method described previously (11). The immunoreactive signals were visualized by ECL Plus Western blotting detection reagents (Amersham, Little Chalfont, UK), and quantified by scanning densitometry (Syngene, Cambridge, UK). Anti-cleaved caspase-3 Ab, anti-cleaved caspase-9 Ab, anti-Akt Ab, anti-phospho-Akt (ser473) Ab, anti-phospho-Akt (Thr308) Ab, anti-phospho-glycogen synthase kinase-3β (GSK)-3β Ab, anti-phospho-Bad Ab, anti-phospho-Forkhead transcription factor (FKHR) Ab, anti-phospho-Raf Ab, anti-phospho-MEK1/2 Ab, anti-p44/42 MAP kinase Ab and anti-phospho-p44/42 MAPK Ab were purchased from Cell Signaling Technology, Inc (Beverly, MA). Anti-actin Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The protein expression levels after reperfusion in the cirrhotic liver group were demonstrated together with their paired basal levels.

Morphological study by light and electron microscopy

Liver biopsies were taken at different time points after reperfusion for light microscopy for hematoxylin-eosin staining. The specimens for electron microscopy were immediately cut into 1 mm cubes and fixed in 2.5% glutaraldehyde in sodium carcodylate-hydrochloride buffer overnight at 4°C for electron microscopy section. The sections would be examined under a transmission electron microscope (Philips EM208, Eindhoven, Holland).

Apoptotic cell detection by Terminal Deoxynucleotide Transferase-Mediated dUTP Nick-End Labeling (TUNEL)

The paraffin sections of the liver biopsies at different time points were detected for apoptotic cells by the TUNEL method (11) (In Situ Cell Death Detection Kit, Roche Biochemicals, Mannheim, Germany).

Statistical analysis

Continuous variables were expressed as median and range. Mann-Whitney U test was used for statistical comparison. Significance was defined as p < 0.05. Calculations were made with the help of SPSS computer software (SPSS, Chicago, IL).

FTY720 significantly improved liver function in both normal and cirrhotic livers

FTY720 treatment significantly decreased the serum levels of ALT and AST in both normal and cirrhotic livers at 6 h and/or 24 h after reperfusion (Figure 1). Although the serum total bilirubin level was relatively higher in the cirrhotic liver group receiving FTY720 at 60 min after reperfusion, the median value of total bilirubin was 5 μmol/L (within normal range) and it declined to the same levels as the control group subsequently (Figure 1).

FTY720 down-regulated Egr-1 expression through MAPK pathway in both normal and cirrhotic livers

In the normal liver group, the protein expressions of phospho-c-Raf, MEK and Erk were down-regulated by FTY720 starting at 60 min after reperfusion (Figure 2A). The hepatic mRNA level of Egr-1 was decreased in the FTY720 treatment group during 60 min to 6 h after reperfusion compared to the control group (60 min: 156% (115–390%) vs. 1147 (634–1727%)) relative to basal level, p = 0.048; 90 min: 865% (788–1088%) vs. 1700% (1483–1890%) relative to basal level, p = 0.048; 6 h: 228% (152–288%) vs. 629% (300–1428%) relative to basal level, p = 0.021). Several down-stream genes, such as ET-1 (vasoconstrictor), iNOS and MIP-2 (inflammatory cytokine and chemokine), were also down-regulated by FTY720 treatment, especially at 6 h and 24 h after reperfusion (Figure 3A(b), A(c), A(d)) (ET-1: 6 h—769%[337–2118%] vs. 4679%[3334–6907%] relative to basal level, p = 0.021; iNOS: 24 h—134.5%[56–307%] vs. 297.3%[203.5–429.6%] relative to basal level, p = 0.037; MIP-2: 6 h—96%[21–372%] vs. 1948%[1638–2258%] relative to basal level, p = 0.019, 24 h—89%[60–239%] vs. 814%[357–2577%] relative to basal level, p = 0.014).

In the cirrhotic liver group, up-regulation of Phospho-c-Raf, MEK and Erk after I/R injury compared to the basal level was attenuated by FTY720 treatment during the first 24 h after reperfusion (Figure 2B). Consistent with the results of normal livers, the mRNA levels of Egr-1, ET-1, iNOS and MIP-2 were also down-regulated by FTY720 during the first 24 h after reperfusion compared to those of the control groups (Figure 3B).

FTY720-activated Akt pathway together with heat shock proteins, anti-inflammatory cytokine in both normal and cirrhotic livers

Up-regulation of Phospho-Akt-ser473 was found after FTY720 treatment in primary hepatocytes isolated from normal rats. On the other hand, cleaved-caspase-3 was down-regulated by FTY720 (Figure 4A).

The Akt signaling pathway was compared between the rats with or without FTY720 treatment (Figure 4B, C). The phosphorylation of Akt was mainly found at ser473. The activation of Akt phosphorylation by FTY720 in normal liver was mainly found at 6 h and 24 h after reperfusion. Three targets of Akt, FKHR, GSK-3β and Bad, were phosphorylated and inactivated by phospho-Akt mainly in the FTY720 treatment group at 6 h and 24 h after reperfusion (Figure 4B).

In the cirrhotic liver, expression of phospho-Akt and its targets were only found at 6 h and 24 h after reperfusion (Figure 4C). Up-regulation of phospho-Akt and its targets after I/R injury compared to the basal level was mainly found in the FTY720 treatment group at 24 h after reperfusion.

Anti-inflammatory cytokine IL-10 was found with relatively higher mRNA (Figure 5A) and protein (213 [133–277] pg/mL vs. 151.6 [124–171] pg/mL, p = 0.025) levels in normal liver after FTY720 treatment at 24 h after reperfusion. Two heat shock proteins, HO-1 and Hsp70, were found with higher mRNA levels after reperfusion mainly in the cirrhotic liver group with FTY720 treatment (Figure 5B).

FTY720 up-regulated anti-apoptotic genes together with partial down-regulation of apoptotic genes

Anti-apoptotic protein-A20 was found with relatively higher mRNA levels in both normal and cirrhotic livers with FTY720 treatment (Figure 5A(d), B(d)). Over-expression of A20 occurred earlier (90 min after reperfusion) in normal livers than in cirrhotic livers (6 and 24 h after reperfusion).

There was no significant difference of cleaved caspase-3, -7, -9 between the rats with or without FTY720 treatment in normal liver during the first 24 h after reperfusion (Figure 6A). Down-regulation of cleaved caspase-7 was found in cirrhotic livers with FTY720 treatment, especially at 90 min and 6 h after reperfusion (Figure 6B). However, the changes of cleaved caspase-3, -9 after I/R injury were comparable between the rats with or without FTY720 treatment in the cirrhotic groups (Figure 6B).

FTY720 maintained liver architecture in both normal and cirrhotic livers

In the normal liver, hepatic lobular architecture was well preserved after FTY720 treatment at 6 h and 24 h after reperfusion (Figure 7A(a), A(b)). The hepatocytes and portal tracts showed normal morphological features. On the contrary, patchy necrosis was found around the portal tract in the control group at 6 h after reperfusion (Figure 7A(c)). Twenty-four hours after reperfusion, ballooning change of hepatocytes accompanied with focal necrosis was also found in the control group (Figure 7A(d)).

As for the cirrhotic liver, apart from the perivenular fibrosis induced by CCl₄, there was no obvious necrosis of hepatocytes at 6 h and 24 h after reperfusion in the FTY720 treatment group (Figure 7B(a), B(b)). Hepatic necrosis developed focally at 6 h after reperfusion in the control group (Figure 7B(c)) and deteriorated at 24 h after reperfusion (Figure 7B(d)).

The liver architecture was well maintained by FTY720 pre-treatment in the normal liver group. The hepatic ultrastructural features were significantly different between the cirrhotic livers with or without FTY720 treatment (Figure 8). The hepatic sinusoid was well maintained after FTY720 treatment (Figure 8a, b) compared to the total disruption of sinusoidal lining cells in the control group (Figure 8c). Degeneration of cytoplasm (Figure 8c) and tremendous mitochondria swelling (Figure 8d) were also found in the control group.

FTY720 attenuated apoptosis in both normal and cirrhotic livers

FTY720 prevented liver apoptosis remarkably in both normal and cirrhotic livers (Figure 9). The peak of apoptosis occurred at 6–24 h after reperfusion in the normal and cirrhotic liver groups. Only a few apoptotic liver cells were found in the FTY720 treatment group (Figure 9A(a), A(b); 9B(a), B(b)). Most of the apoptotic liver cells were found around the perivenular area in the control groups (Figure 9B(c), B(d)). Patchy necrosis was also found together with apoptotic nuclei at 24 h after reperfusion in normal livers without FTY720 treatment. Both massive apoptotic nuclei and focal necrosis were present in the control group with cirrhotic livers (Figure 9B(d)).