Male Wistar rats weighing 180-220 g from the Animal Center of Dalian Medical University were used in this study, and maintained under standard laboratory conditions. All rats were fed standard laboratory food and water. Rats were housed in a barrier system at 25 °C with 12 h light-12 h dark cycles and acclimated for 1 wk before experimentation. All procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Dalian Medical University (Permit number: SCXK 2008-0002). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering.

Intestinal I/R was induced in rats according to a previously standardized method[16]. Briefly, after a midline laparotomy was performed, the superior mesenteric artery was gently isolated and occluded with an atraumatic microvascular clamp for 60 min and then followed by reperfusion for 240 min. When mesenteric pulsations ceased and the intestine became pale in color, occlusion was confirmed. And reperfusion was confirmed when the pusatile flow returned to the mesenteric artery and its branches. For sham operation, the superior mesenteric artery was only isolated without occlusion.

The rats were randomly divided into three groups (n = 8 each): (1) sham group; (2) I/R group; and (3) FO group. In the FO group, rats were pretreated intraperitoneally with 1 mL/d of FO emulsion (100 mL of FO emulsion contained 2.82 g of EPA and 3.09 g of DHA; Fresenius Kabi, Bad Homburg, Germany) for five consecutive days, and then surgery was performed as that in the I/R group. The same volume of normal saline was administered in the sham group and I/R group.

The dose of FO emulsion pretreatment was selected according to a previous study[16], and a pilot study has been performed. All animals were sacrificed at the end of reperfusion and tissue and blood samples were obtained for further analysis.

Intestinal and liver tissues were fixed in 10% formalin, embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin (H&E) for light microscopy. Scores of liver and intestine pathology were evaluated according to previous studies[7-9].

Measurement of serum aspartate aminotransferase, alanine aminotransferase, tumor necrosis factor-α, and liver malonaldehyde

Serum was obtained from blood samples by centrifugation (1000 g, 10 min, 4 °C) and stored at -80 °C for assessment. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured with an OLYMPUS AU1000 automatic analyzer (Aus Bio Laboratories Co., Ltd., Beijing, China). Serum tumor necrosis factor-α (TNF-α) was quantified using a rat enzyme-linked immunosorbent assay kit (BOSTER Bioengineering Co Ltd, Wuhan, China). Liver tissues were harvested and homogenized immediately on ice in five volumes of normal saline. Homogenates were centrifuged at 1200 g for 10 min. Liver malonaldehyde (MDA) concentration in the supernatant was determined using an assay kit (Nanjing Jiancheng Corp., Nanjing, China), according to the manufacturer’s recommendations. The activity of MDA is expressed in nmol/mg prot.

HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, CA, United States) supplemented with 10% fetal bovine serum. The cells were kept at 37 °C in a humidified atmosphere with 5% CO₂, 100 units/mL of penicillin, and 10 μg/mL of streptomycin. Media were collected and centrifuged 48 h after treatment with lipopolysaccharide (LPS; 10 μg/mL, Sigma-Aldrich, United States) in the presence or absence of FO (50 mmol/L EPA/50 mmol/L DHA each mixture) and TNF-α was measured with a specific ELISA kit (Jiancheng, Nanjing, China) according to the manufacturer’s protocol.

The doses of FO and LPS pretreatment were selected according to previous studies[16,18], and a pilot study has been performed.

Western blot analysis of p-AMPK, SIRT-1, LC3 II, Beclin-1, and P62 in liver tissue and HepG2 cells

For detecting p-AMPK, SIRT-1, LC3 II, Beclin-1, and P62 contents in liver tissue, 30 μg of protein was separated by 12% or 15% SDS-PAGE (Bio-Rad, Hercules, United States), and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, United States). The membranes were then incubated with primary anti-p-AMPK (Abcam Ltd., Cambridge, United Kingdom), anti-SIRT-1 (Abcam Ltd., Hong Kong, China), anti-LC3 I, anti-LC3 II, anti-P62, anti-Beclin-1, or anti-β-actin antibody (Santa Cruz Biotechnology, United States) in Tris buffered solution (TBS) containing 5% skimmed milk overnight at 4 °C. After washing three times in TBS with 0.1% Tween 20 (TTBS), the membranes were incubated with biotinylated secondary antibody diluted 1:1000 in PBST containing 5% skimmed milk for 2 h at 37 °C. After extensive washing with TTBS, the membranes were exposed to enhanced chemiluminescence-plus reagents (Beyotime Institute of Biotechnology, Hangzhou, China). Emitted light was documented with a BioSpectrum-410 multispectral imaging system with a Chemi HR camera 410 (UVP, Upland, CA, United States) and analyzed with Gel-Pro Analyzer Version 4.0 (Media Cybernetics, Bethesda, United States).

Total RNA was extracted from rat liver using Trizol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. Reverse transcription into cDNA was performed using a TaKaRa RNA polymerase chain reaction (PCR) kit Version 3.0 (TaKaRa, Dalian, China) for PCR analysis; primers are shown as follows: AMPK forward, 5’-G TGGTGTTATCCTGTATGCCCTTCT-3’ and reverse, 5’-CTGTTTAAACCATTCATGCTCTCGT-3’; SIRT-1 forward, 5’-AGCTGGGGTTTCTGTTTCCTGTGG-3’ and reverse, 5’-CGAACATGGCTTGAGGATCTGGGA-3’; β-actin forward, 5’-AGAGGGAAATCGTGCGTGAC-3’ and reverse, 5’-CAATAGTGATGACCTGGCCGT-3’. RT-PCR was performed with the SYBR Premix Ex Taq kit (Takara, Dalian, China) for fluorescence detection during amplification on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). PCR cycling was performed under the following conditions: initial denaturation at 95 °C for 30 s and 40 thermal cycles of 95 °C for 5 s, 60 °C for 34 s, and 72 °C for 30 s.

HepG2 cells were transfected with small interfering RNAs (siRNAs) directed against AMPK α1 and SIRT-1, with scrambled siRNAs used as controls. Both specific and control siRNAs were obtained from GenePharma (Shanghai, China), and transient siRNA transfection was performed as previously described. After siRNA transfection for 48 h, the cells were treated with FO for an additional 6 h. The cells were then collected for the protein and mRNA analyses.

Histopathological analysis of the liver in the I/R group showed apparent injury compared with the sham group, which was manifested as edema, hemorrhage, and neutrophil infiltration (Figure 1A). However, liver injury was significantly improved by pretreatment with FO emulsion, and the histopathological score was significantly reduced compared with the I/R group (Figure 1A, P < 0.01).

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Figure 1 Fish oil emulsion improves I/R induced liver and intestinal injury histopathologically. Pathologic changes of liver (A) and intestinal (B) tissues in different groups (mean ± SD, n = 8). ^bP < 0.01 vs sham group; ^dP < 0.01 vs I/R group.

Compared with the sham group, intestinal mucosal injury was also found in the I/R group with apparent hemorrhage and loss of lamina propria villi and glands (Figure 1B). Pretreatment with FO emulsion significantly reduced the intestinal damage and histopathology score (Figure 1B, P < 0.01).

Effect of FO emulsion on serum ALT, AST, TNF-α, and liver MDA levels

In the I/R group, there was a significant increase in serum ALT and AST levels compared with the sham group. However, pretreatment with FO significantly decreased serum levels of both ALT and AST compared with the I/R group (Figure 2A, P < 0.01). This indicated that FO emulsion was effective in improving liver function.

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Figure 2 Alanine aminotransferase and aspartate aminotransferase (A) as well as TNF-α and liver MDA levels (B) in different groups (mean ± SD, n = 8). ^bP < 0.01 vs sham group; ^dP < 0.01 vs I/R group.

Moreover, serum TNF-α and liver MDA increased significantly after intestinal I/R compared with the sham group (P < 0.01). Furthermore, FO emulsion pretreatment dramatically reduced proinflammatory cytokine levels (Figure 2B, P < 0.01).

Effect of FO emulsion on LC3 II, Beclin-1, and P62 expression in liver tissue after intestinal I/R

Western blot analysis was performed to examine the expression of LC3 II, Beclin-1, and P62 in liver tissue. Compared with the sham group, both LC3 II and Beclin-1 expression was markedly decreased but P62 expression increased in the I/R group. However, pretreatment with FO emulsion markedly increased the expression of both LC3 II and Beclin-1, and decreased the expression of P62 compared with the I/R group (Figure 3, P < 0.01), indicating that FO emulsion activated autophagy in intestinal I/R-induced liver injury.

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Figure 3 Western blot analysis of liver autophagy (LC3 II, Beclin-1, and P62) levels in different groups (mean ± SD, n = 8). ^bP < 0.01 vs sham group; ^dP < 0.01 vs I/R group.

Western blot and RT-PCR analyses were performed to determine AMPK and SIRT-1 protein and mRNA contents in the liver. After intestinal I/R, the ratio of p-AMPK/AMPK protein and AMPK mRNA in the liver was dramatically decreased compared with the sham group (Figure 4A, P < 0.01). Similarly, both SIRT-1 protein and mRNA expression in the liver were dramatically decreased compared with the sham group (Figure 4B, P < 0.01). Nevertheless, not only the ratio of p-AMPK/AMPK protein and AMPK mRNA, but also the SIRT-1 protein and mRNA expression in the liver were dramatically increased by pretreatment with FO emulsion compared with the I/R group (Figure 4A and B, P < 0.01, P < 0.05).

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Figure 4 Western blot analysis (A) of liver p-AMPKα1/AMPKα1 and qRT-PCR analysis (B) of liver AMPK expression in different groups (mean ± SD, n = 8). ^bP < 0.01 vs sham group; ^cP < 0.05, ^dP < 0.01 vs I/R group.

To investigate the role of the AMPK/SIRT-1/autophagy pathway affected by FO emulsion in intestinal I/R-induced liver injury, HepG2 cells were employed and challenged with LPS to mimic the circumstance in vivo. Western blot analysis was then performed to examine the AMPK, SIRT-1, and Beclin-1 expression in LPS-stimulated HepG2 cells. Further, TNF-α and MDA were determined using experimental kits according to the manufacturer’s protocols. Compared with the con group (control + LPS), the expression of p-AMPK, SIRT-1, and Beclin-1 was significantly increased in the FO group (FO + LPS) (Figure 5A, P < 0.05). This was associated with a significant decrease of TNF-α and MDA levels (Figure 5B, P < 0.01). However, p-AMPK, SIRT-1, and Beclin-1 expression was decreased significantly in the FO + siA (FO + AMPK siRNA + LPS) group compared with the FO group. Moreover, reduced SIRT-1 and Beclin-1 expression was observed in the FO + siS (FO + siRNA of SIRT-1 + LPS) group compared with the FO group (Figure 5A, P < 0.05). This was associated with a significant increase of TNF-α and MDA levels in HepG2 cells, although there was no change of p-AMPK (Figure 5B, P < 0.05). Thus, FO emulsion may ameliorate liver injury induced by intestinal I/R through the AMPK/SIRT-1/autophagy pathway.

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Figure 5 p-AMPK, AMPK, SIRT-1, Beclin-1, TNF-α, and MDA expression in HepG2 cells challenged with LPS, LPS + FO, LPS + FO + siAMPK, or LPS + FO + siSIRT-1 (mean ± SD, n = 3). ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05 vs FO.

The liver is the most vulnerable organ after intestinal ischemia/reperfusion (I/R) because the liver and intestine share the anatomical common pathway such as coupled vasculature. The mechanism is obscure and has multiple overlapping pathways. Increased evidence indicates that the AMPK/SIRT-1/autophagy pathway may be involved in this process. Fish oil (FO) may regulate the AMPK/SIRT-1/autophagy pathway to affect liver injury induced by intestinal I/R.

Understanding and regulating AMPK/SIRT-1/autophagy pathway using a safe and effective substance like FO will be an important area of future research.

This study aimed to provide evidence that FO can attenuate intestinal I/R induced liver injury by inducing autophagy through the AMPK/SIRT-1 pathway. This will provide a therapeutic target for future clinical applications of FO to prevent intestinal I/R associated liver disease.

This research was performed using Wistar rats challenged by intestinal I/R and HepG2 cells stimulated with LPS to mimic the in vivo pathogenesis. Further, RNA interference has been employed, which laid a foundation for the future gene knockout animal study.

Intestinal I/R induced apparent liver injury, including histopathological injury and liver dysfunction, and this was associated with increased TNF-α and MDA, and decreased AMPK/SIRT-1/autophagy molecular function. FO emulsion restored the balance of the factors and alleviated liver injury. The similar results were observed in HepG2 cells stimulated with LPS.

Intestinal I/R induced liver injury is associated with decreased AMPK/SIRT-1/autophagy (LC3 II, Beclin-1, and P62 expression) molecular function. FO emulsion restored the beneficial factors and alleviated liver injury. Similar results were observed in HepG2 cells. We present a novel theory here that FO can prevent intestinal I/R associated liver disease via the AMPK/SIRT-1/autophagy pathway. This may be a potential novel target for patients. We may provide the first evidence of AMPK/SIRT-1/autophagy regulated by FO in liver injury induced by intestinal I/R. Our hypothesis was confirmed using rat models and HepG2 cells treated with LPS and RNA interfere to mimic the conditions in vivo. Thus, FO and FO-related products may have novel clinical applications to prevent intestinal I/R associated liver disease in future.

This study has some limitations and further studies should be performed using gene knockout animals and clinical application should be concentrated.