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Original article

Changes of sphingolipids profiles after ischemia-reperfusion injury in the rat liver

ZHAI, Shu-ting; LIU, Guang-yi; XUE, Fei; SUN, Gong-ping; LIANG, Liang; CHEN, Wei; XU, Guo-dong; LI, Jun-jian; YANG, Jun; LIANG, Ting-bo

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doi: 10.3760/cma.j.issn.0366-6999.2009.24.022
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Abstract

Hepatic ischemia-reperfusion (I/R) injury occurs in many clinical procedures, including liver transplantation, hepatic trauma, hemorrhagic shock, and liver resection. Necrotic and apoptotic liver cells appear during these procedures, but the molecular mechanisms are not well understood.1 Previous studies have demonstrated that numerous molecular events are associated with hepatocyte death, including tumor necrosis factor-α secretion, ceramide generation, and mitochondrial permeability transition pore.2,3 Tumor necrosis factor α (TNF-α) is a central mediator of hepatic I/R. Several pathways of TNF-α-related pro- and anti-apoptosis have been characterized in hepatocytes, but the role of sphingolipids (including ceramide) in TNF-α induced cell death is not completely understood.4

Sphingolipids have been considered as important building blocks of biological membranes with key structural functions but little relevance to cellular signaling. However, recent evidence indicated an important role of sphingolipids and their metabolites in intracellular and extracellular signaling pathways regulating cell functions such as proliferation, differentiation, adhesion, cell cycle arrest and cell death.5,6 Structurally, sphingolipids are composed of a long chain sphingoid base, an amide-linked fatty acid, and a polar head at the 1 position. Ceramide, N-acylated sphingosine, is in the center of sphingolipid metabolism and also serves as the structural backbone for other sphingolipids.7 On the contrary, ceramide can be generated by de novo synthesis, sphingomyelin hydrolysis, and recycling of sphingolipids. Ceramide can also be phosphorylated or utilized for synthesis of sphingomyelin and glycosphingolipids, or other bioactive sphingolipids, such as sphingosine or sphingosine-1-phosphate.8

Previous research has demonstrated that sphingolipids play a key role in death receptor-induced hepatocellular death, which contributes to the pathogenesis of liver diseases, such as steatohepatitis, hepatic I/R injury, viral hepatitis and hepatocarcinogenesis.8-11 However, sphingolipids have not been adequately researched in different liver diseases or pathological states, particularly due to the lack of high-throughput technologies for lipid analysis. Vast amounts of information have been generated regarding the various changes of lipids under different disease states after the emergence of “lipidomics”, in which several individual lipid components can be analyzed simultaneously by mass spectrometry (MS) technique.12,13 Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a widely used ionization method for proteins, has been successfully used in the determination and analysis of different sphingolipids in various tissues and disease states,14 such as changes of sphingolipids in patients with Gaucher disease15 and in brain tissues of rats with kindled seizures.16 Due to the close relationship between ceramide and other sphingolipids, such as sphingomyelin and gangliosides, it is necessary to study systemic changes in the liver after I/R in order to further interpret the relationship between sphingolipids and hepatic I/R injury.

METHODS

Animal model establishment and experimental design

All animal experiments were performed in accordance with the animal care guidelines of Zhejiang University, which conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (200-250 g; purchased from Shanghai Experimental Animal Institute, China) were housed in a standard animal facility under a 12-hour light-dark cycle. All animals had free access to water and food ad libitum.

We established a 70% liver I/R model as described previously.17 Rats were anesthetized by sodium pentobarbital (40 mg/kg, i.p). After laparotomy, the portal venous and hepatic arterial branches to the left and median hepatic lobes were isolated and occluded with a microvascular clamp. This avoided congestion of the gut, since the right-lobe inflow and outflow tract were still patent. Such a precaution is necessary, because rats do not tolerate total clamping of the portal vein. After 45 minutes of ischemia, the clamps were removed to allow reperfusion.

Rats were divided into two groups, control (sham operated group) and I/R group. In the control group, the rats underwent a laparotomy and isolation of the vessels, without occlusion of the portal venous and hepatic arterial branches to the left and median hepatic lobes. The rats were sacrificed at 0.5, 1, 3, 6, and 12 hours after reperfusion in each group. Five rats were assessed at each time point. The liver tissues were quickly separated, weighted, put into liquid nitrogen and stored at −70°C for further analysis.

Biochemical analysis and determination of TNF-α mRNA levels in the liver tissue

Blood samples taken at different time points, 0.5, 1, 3, 6, 12 hours after reperfusion, were obtained from different groups. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured using standard techniques with a serum analyzer (Hitachi, Japan).

TNF-α mRNA levels in the liver were determined using semiquantitative RT-PCR. Total RNA was isolated from liver tissue using the Trizol reagent (GIBCO BRL, USA). Primers were synthesized at the Shanghai Sangon Biological Engineering & Technology and Service Co. Ltd. (TNF-α, Sense: 5′-CCACGCTCTTCTGTCTACTG-3′, Antisense: 5′-GCTACGGGCTTGTCACTC-3′. β-actin, Sense: 5′-CCTAAGGCCAACCGTGAA-3′, Antisense: 5′-GGAGCCAGGGCAGTAATC-3′). 2 μl of RNA was reverse-transcribed to complementary DNA at 42°C for 90 minutes, and cDNA was then amplified. Thermal cycle conditions were 94°C for 45 seconds, 57°C for 45 seconds, and 72°C for 60 seconds, for a total of 30 cycles. The final cycle was followed by a 10-minute incubation at 72°C. Amplification of β-actin was performed with the same reaction program. The PCR product of 4 μl was electrophoresed on a 2.0% agarose gel. The results were detected with the Kodak Digital Science Image System (Kodak, USA).

Tissue preservation, lipids extraction and preparation of sphingolipid

Extraction of sphingolipids was conducted as described previously.16,18 First, rat liver tissues with the same wet mass were chopped and homogenized in 2:1 chloroform-methanol mixture (V/V) and diluted to 20-fold the volume of the original tissue sample volume using 2:1 chloroform/methanol (V/V). The extract was then mixed thoroughly with a 0.2 volume of water and the mixture was separated into two phases by centrifugation at 4770 × g for 15 minutes. Second, the lower phase was dried by vacuum centrifugation in a centrifugal evaporator (Speed-Vac,Thermo Savant, Holbrook, USA). Third, 500 μl of methanol containing 0.1 mol/L NaOH was added into each tube at 55°C for 1 hour to decompose glycerophospholipids. After neutralization with 100 μl of methanol containing 1 mol/L HCL, 500 μl of hexane and one drop of water were added to each sample. Fourth, the mixture was then centrifuged again at 4770 × g for 15 minutes and the lower phase was dried in a centrifugal evaporator after the upper phase was removed. The residue was mixed with 0.8 ml of theoretical lower phase (chloroform:methanol:water, 86:14:1, V/V) and 0.2 ml theoretical upper phase (chloroform:methanol:water, 3:48:47, V/V) for Folch partition, and the mixture was centrifuged at 4770 × g for 15 minutes. Finally, the upper phase was discarded to remove salts and the lower phase was evaporated in a centrifugal evaporator. The residue crude sphingolipid was stored at −70°C for MALDI-TOF-MS analysis.

MALDI-TOF MS analysis

For MALDI-TOF MS analysis, each sample was dissolved in 5 μl chloroform:methanol (2:1, V/V), followed by the addition of 5 μl matrix solution, ethylacetate containing 0.5 mol/L 2,5-dihydroxyl-benzoic acid (2, 5-DHB; Sigma) and 0.1% TFA, in a 0.5 ml Eppendorf tube. The tube was vortexed vigorously and then centrifuged in a microcentrifuge for 1 minute. One μl of mixture was directly added onto the sample plate and rapidly dried under a moderate warm stream of air in order to remove the organic solvent within seconds.

All samples were analyzed using a Voyager-DE STR MALDI-TOF mass spectrometer (ABI Applied Biosystem, Framingham, USA) with a 337 nm N2 UV laser. The mass spectra of the samples were obtained in the positive ion mode. Mass/charge (m/z) ratios were measured in the reflector/delayed extraction mode with an accelerating voltage of 20 kV, a grid voltage of 67% and a delay time of 100 ns. C2-dihydroceramide (MW 343.6) was used to calibrate the instrument. The mass accuracy was 0.5. All lipid spectra were acquired using a low-mass gate at 400 Da. For each group, 5 spectra were determined from each animal. Only when a peak appeared in all of the 5 spectra with relatively stable intensity it was considered a candidate for analysis. All MS data were analyzed as described previously.16

Establishment of reference mass spectrum and relative quantification

For analyzing sphingolipids, we established a “reference mass spectrum” (RMS) method to deal with the problem of inter-cell variation, as well as the problem associated with the poor quantification ability of MALDI-TOF-MS. The basic principle is that only those peaks presented in all 5 mass spectra from the same group (one mass spectra from one animal) were chosen and placed in the RMS. By doing so, the interference of inter-group variation could be minimized. Since it is difficult to quantify the amount of each sphingolipid species using MALDI-TOF-MS, a relative quantification method was also created by comparing the ratio of different sphingolipid species instead of the absolute amount of each species. It was found in all the mass spectra obtained, peak 506.9, which corresponds to Cer(d18:1C11:0+K)+, always had the relative intensity of 100%, meaning it was the highest peak in the mass spectra. Thus, the relative intensities of other peaks were calculated by comparing to peak 506.9. The mean and standard deviation were then calculated of the relative intensity of each peak from control. Combining these data, a final RMS was established for the control group. All comparisons were then conducted using these RMS.

Histology

Liver specimens were fixed in 10% neutral-buffered formalin solution and embedded in paraffin. Sections were cut at 4 μm and stained with hematoxylin and eosin (H&E) for histopathology evaluation. Sinusoidal congestion, hepatocyte necrosis and ballooning degeneration were observed.

Statistical analysis

Statistical analysis was performed using Student's t test with SPSS13.0. Data are presented as mean ± standard deviation. A probability level of P <0.05 was considered statistically significant.

RESULTS

Serum ALT and AST Levels

Serum ALT and AST levels were significantly higher in comparison with the sham group (P <0.01) after I/R, peaking at the 6th hour (Figure 1). These changes indicated that liver damage after I/R was most serious at 6 hours after reperfusion.

Figure 1.
Figure 1.:
Serum ALT (A) and AST (B) levels from the different groups after hepatic I/R. Significantly increased serum concentrations of ALT and AST after reperfusion as compared with the control group. Data represent mean ± SD. * P <0.01 vs control group.

Histological assessment

No significant changes were observed in the control group by light microscopy. After reperfusion, significant periportal edema and cytoplasmic vacuolation were seen. At 6 hours after reperfusion, swelling of hepatocytes, structural derangement, necrosis, and inflammatory cell infiltration were easily found in the hepatic lobes (Figure 2).

Figure 2.
Figure 2.:
Hematoxylin and eosin-stained sections of the liver harvested from the control group (A, B) and 6 hours after reperfusion (C, D). The presence of a disruption of lobular architecture and neutrophils around the necrotic hepatocytes can be observed (Original magnifications: A, C×100; B, D×400).

TNF-α mRNA expression levels

From RT-PCR analysis, we found that TNF-α mRNA expression levels were significantly increased after reperfusion. As shown in Figure 3, TNF-α expression in the I/R group was elevated after reperfusion, with significant differences between the I/R and control groups (P <0.01).

Figure 3.
Figure 3.:
Densitometry of TNF-α mRNA levels normalized to β-actin at different time points after reperfusin (A: the control group; B: I/R group). Data represent the mean ± SD (C). * P <0.01 vs the control group.

Sphingolipids analysis of rat liver tissues

Three classes of sphingolipids, namely, ceramide, sphingomyelin and ceramide-monohexoside, were observed in all mass spectra. No differences were observed in type of intensity of sphingolipids in mass spectra of the control group. Peak 506.9 was assigned a relative intensity of 100%. The final RMS obtained is shown in Figures 4 A-F. Twenty-one major peaks were found, representatives of these three main classes of sphingolipids and were identified after careful comparison to published data and the sphingolipids database established by the data library (http://lipid.zju.edu.cn/). The ions characterized for special Cer and SM, as well as the values of their relative intensities, are listed in Table 1.

Figure 4.
Figure 4.:
Representative DE MALDI-TOF mass spectra of sphingolipids in liver tissues of rats in the control and I/R groups. A: control group; B-F: different time points after reperfusion, respectively (0.5, 1, 3, 6, 12 hours after reperfusion).
Table 1
Table 1:
The average relative intensity of major sphingolipids in rat liver of the control and I/R groups (mean±SD)

As shown in Figures 4 B and C, there were no obvious changes of sphingolipids at 0.5 hour and 1 hour after reperfusion compared with the control group. For ceramide, intensities of ions m/z 502.9 (Cer(d18:1C12:1+Na)+) and 511.0 (Cer (d18:1C14:0+H)+) increased; for sphingomyelin, ions m/z 641.5 (SM(d18:1C10:0+Na)+) appeared with low intensities, while 831.3 (SM(d18:1C24h:0+H)+) disappeared at both points. According to these changes, we found a decrease in total amounts of sphingomyelin (Table 2).

Table 2
Table 2:
Relative total amounts of sphingolipids classes at different time points after reperfusion

It was observed in Figure 4 D and E that significant changes of sphingolipids were observed at 3 hours after reperfusion in the I/R group compared with controls, particularly at the 6-hour time point (Figure 5). Also, there was a general increase in total sphingolipid amount, especially ceramide and sphingomyelin (Table 2). In detail, four new ceramide, ions m/z 537.8 (Cer(d18:1C16:0+H)+), 555.7 (Cer (d18:1C16:0+H2O+H)+), 567.7 (Cer(d18:1C17h:0+H)+) and 583.8 (Cer(d18:1C18:2+Na)+), and two novel sphingomyelin-ions m/z 683.5 (SM (d18:1C13:0+Na)+) and 731.4 (SM(d18:1C18:0+H)+), were observed. Meanwhile, intensity of most sphingomyelin forms reached the maximum at the 6-hour time point, such as 657.4 (SM(d18:1C10:0+K)+), 701.6 (SM(d18:1C16:1+H)+), 723.5 (SM (d18:1C16:1+Na)+), as well as ceramide-monohexoside 804.4 (CMH (d18:1C22:1+Na)+).

Figure 5.
Figure 5.:
Reference mass spectra of sphingolipids in the liver of rats in the control and I/R groups, marked A and B, with the individual m/z ions. The numbers of m/z ions correspond to different kinds of sphingolipids in Table 1. Different columns represent the intensity of each kind of sphingolipids in the control and I/R groups.

At the 12-hour point, the intensity of one ceramide, ions m/z 508.9 (Cer (d18:1C14:1+H)+), and one sphingomyelin, ions m/z 657.4 (SM(d18:1C10:0+K)+), decreased compared with controls, which was not consistent with the general changes in the amount of total sphingolipids (Table 2).

DISCUSSION

The results of the present study demonstrate that three main forms of sphingolipids accumulated in the liver after I/R, including ceramide, sphingomyelin and ceramide-monohexoside. Interestingly, these sphingolipids all had a “peak” at 6 hours after reperfusion, with most forms reaching their maximum at this time point.

Increasing evidence has shown that sphingolipids, essential components of membrane rafts, have emerged as lipid intermediates that play a significant role in signal transduction pathways of stress response and cell death.10,19 In particular, ceramide has attracted considerable attention in hepatocyte death. Ceramide can directly activate mitochondrial permeability transition pore (MPTP), which is a common mechanism of different types of cell death including necrotic and apoptotic death of hepatocytes, or indirectly through JNK activation and subsequent events.2,10,19 Indeed, many different stimuli, such as death ligands (e.g. TNF), bacterial and viral infections, bile acids, and chemotherapy generation or upregulation of ceramide are involved. TNF-α is a central mediator in hepatic response to I/R. Accumulation of TNF-α could cause activation of the “sphingomyelin cycle”, the key event consisting of enzymatic cleavage of sphingomyelin, one of the most abundant sphingolipids in membranes, and formation of ceramide.5,6 The main apoptotic effects of TNF-α are mediated by the tumor necrosis factor receptor-1 (TNF-R1), associated with lipid rafts, with functional domains transducing unique intracellular signals by interacting with different intracellular adaptor proteins.9,10,20 Consistent with the role of ASMase in TNF-α-mediated hepatocellular death, ASMase ablation by siRNAs in vivo has been shown to improve survival during total hepatic I/R in mice by preservation of mitochondrial function.19 Thus, ceramide generated from ASMase plays a key role in I/R-induced liver damage. Despite the definite role of each type of ceramide is still uncomplete, Osawa et al21 reported that endogenous C16-ceramide is elevated during TNF-α-induced apoptosis in both rat and mouse primary hepatocytes in vitro. Thus, based on our results, we hypothesized that different kinds of ceramide might participate in I/R-induced liver damage.

Sphingomyelin, the major phospholipid class located in the cellular membranes of hepatocytes, is broken down during I/R, generating intracellular ceramide and sphingosine, the former being instrumental for ischemic injury. It was noted that sphingomyelin levels increased after reperfusion, even with several new forms appearing. Due to the increase in both ceramide and sphingomyelin, we suggest that other metabolism pathways may be activated besides the sphingomyelin cycle, such as the de novo pathway. The capacity for de novo ceramide biosynthesis is widespread among cell types and tissues. De novo ceramide synthesis is necessary for many cellular functions as inferred from the detrimental consequences of its inhibition.22,23 The enzyme serine palmitoyl transferase (SPT), the rate limiting enzyme in ceramide biosynthesis, has been documented to play a crucial role in ceramide generation, which is involved in mitochondrial damage resulting in downstream activation of caspases and apoptosis.24,25 Unlike the “sphingomyelin cycle” pathway, the de novo pathway contributes to the slow but sustained generation of ceramide. At the late phase (>6 hours after reperfusion) of hepatic I/R injury,26 significant changes in sphingomyelin and ceramide were observed. Thus, SPT may play a crucial role in the lipidomic change profile of sphingolipids after I/R.

There are also problems faced by the lipidomic study. For example, the assignments for sphingolipid molecule solely based on the mass determined by MALDI-TOF-MS may not be accurate, and only by further MALDI-TOF-MS analysis could the definite molecular structures be assigned. Still, in the present study, we have analyzed the changes of sphingolipids in rats liver after warm I/R using DE MALDI-TOF-MS in rats. Due to matrix noise in the low mass range of MALDI-TOF-MS spectra, only those relatively abundant sphingolipids could be compared.16 In spite of the limitations of our current methodology, this is the first report on the role of sphingolipids in rat liver tissues after I/R injury. Our data shows three main spingolipids, ceramide, sphingomyelin and ceramide-monohexoside, are related to hepatic I/R injury. The changes in liver sphingolipids profiles in the rat and other documents27 demonstrate that the elevation of the sphingolipids is the key factor responsible for hepatic I/R injury, which provide a new perspective in understanding the mechanism(s) of hepatic I/R injury.

There are many other “rheostats” present in sphingolipid metabolism, some of which may have the opposite effect in regulating cell function and determining the fate of cells, such as Cer vs S-1-P,28 Cer vs ceramide-1-phosphate,29 etc. Some of the “rheostats” may participate in the increase in sphingolipids after reperfusion, while the modulation of the “rheostats” may have positive effects in protecting cell damage, since they have opposite roles in regulating hepatocyte death.8,10,28,29 Much more work must be done to make clear the speculation.

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Keywords:

lipidomic; sphingolipids; liver; ischemia-reperfusion; matrix-assisted laser desorption ionization time-of-flight mass spectrometry

© 2009 Chinese Medical Association