Autophagy lessens ischemic liver injury by reducing oxidative damage
- Kai Sun†1, 2,
- Xuqin Xie†1, 2,
- Yan Liu3,
- Zhipeng Han2,
- Xue Zhao1,
- Ning Cai1,
- Shanshan Zhang2,
- Jianrui Song2 and
- Lixin Wei1, 2Email author
© Sun et al.; licensee BioMed Central Ltd. 2013
Received: 26 January 2013
Accepted: 14 May 2013
Published: 10 June 2013
Hepatic ischemia/reperfusion is a multi-factorial process which causes liver injury. It is reported that ischemia alone is sufficient to induce liver injury. Nutrient deprivation is a crucial factor impacting ischemic injury of the liver. Therefore, we explored the role of autophagy in ischemia through using hepatic ischemia rat model in vivo and nutrient-free model in vitro.
We found that both ischemia in vivo and nutrient deprivation in vitro activated autophagy, inhibition of which aggravated ischemia- or nutrient deficiency-induced injury. In the nutrient-free condition, autophagy inhibition enhanced liver cell necrosis but not apoptosis by promoting reactive oxygen species (ROS) accumulation, and antioxidant NAC could reverse this trend. Inhibition of autophagy also resulted in the increase of the percentage of necrotic cell but not apoptotic cell in the ischemia-treated rat livers. Further studies showed that under nutrient deprivation, autophagy inhibition promoted mitochondrial ROS generation, which further aggravated mitochondria damage. These changes formed a “vicious cycle” that accelerated the process of cell necrosis. Autophagy inhibition also increased mitochondrial oxidative stress during hepatic ischemia, and antioxidant could suppress the aggravation of ischemia-induced liver damage in the co-treatment of autophagy inhibitor.
Taken together, our results suggested that autophagy suppressed ischemic liver injury by reducing ROS-induced necrosis. This finding will contribute to the development of the therapeutic strategy about the pre-treatment of liver surgery.
KeywordsLiver ischemia Nutrient deprivation Autophagy Reactive oxygen species Necrosis
Hepatic ischemia/reperfusion (I/R) is an important causing liver injury during liver surgery, especially in hepatic transplantation, hepatic resection, and trauma. I/R injury has a profound impact on the burden of liver diseases. However, how to improve liver function in the process of I/R is always a challenge due to incomplete understanding of the mechanism of I/R injury. Although the studies about I/R are nearly all focused on reperfusion, long time ischemia is also a crucial damage factor in liver injury. Understanding the mechanism of ischemia damage is important for reducing liver injury during surgery. Interruption of an organ’s blood flow subsequently leads to its lack of oxygen and nutrient supply, loss of ATP, and acidosis. Among the consequences, nutrient deprivation is a very important factor impacting liver ischemic injury . Macroautophagy (hereafter referred as autophagy) may play a crucial role in response to nutrient deprivation.
Autophagy is an evolutionary conserved process involved in degradation of long-lived proteins and excess or dysfunctional organelles . During the process of autophagy, cellular contents including organelles are sequestered in double-membrane vesicles called autophagosomes, then the autophagosomes fuse with lysosomes where hydrolysis or cargo occurs, supplying amino acids and macromolecular precursor for cells [2, 3]. Autophagy occurs at low levels under normal conditions and is important for the turnover of organelles [4, 5].
In recent years, various studies reported that autophagy could promote survival in response to ischemia. Wang, P. found that induction of autophagy contributed to the neuro-protection of nicotinamide phosphoribosyltransferase in cerebral ischemia . Hoshino, A. showed that p53-TIGAR axis attenuated mitophagy to exacerbate cardiac damage after ischemia . However, the mechanism by which autophagy protects cells from ischemia injury has not been clarified.
In our study we investigated the effect of autophagy on survival of hepatocytes in hepatic ischemia. We reported here that both ischemia in vivo and nutrient deprivation in vitro significantly induced autophagy. Inhibition of autophagy aggravated ischemia-induced liver injury and starvation-induced hepatocyte death. Notably, these increased cell death was mainly due to necrosis but not apoptosis. Further study showed that inhibition of autophagy aggravated starvation-induced reactive oxygen species (ROS) accumulation, especially mitochondrial ROS, which in turn led to further mitochondria damage. These excessive ROS contributed to hepatocyte necrosis. Meanwhile, autophagy inhibition also enhanced mitochondrial oxidative stress in the process of hepatic ischemia, thus resulted in aggravated liver injury, which could be significantly suppressed by antioxidant.
Autophagy protects liver from ischemic injury in the rats
Then we examined the impact of autophagy inhibition on ischemia-induced liver injury. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), two classical markers of liver injury, were prominently increased in the ischemia-treated rats as compared to those of normal rats. CQ injection before ischemia led to two fold or more increase in the levels of ALT and AST, but CQ treatment alone had no obvious influence on the levels of ALT and AST (Figure 1E and F). The hematoxylin and eosin (H&E) staining also revealed that ischemia group had significant liver injury such as loosing hepatocyte cords and ischemia + CQ group had further marked morphological alterations in the livers, including unclear structure of hepatic lobules, disarranged hepatocyte cords, narrowed hepatic sinusoids and swollen hepatocytes (Figure 1G). These data demonstrated that inhibition of autophagy significantly increased liver injury during ischemia.
Autophagy protected liver cells from cell necrosis induced by nutrient deprivation
Taken together, these data suggested that autophagy protected liver cells against nutrient deprivation-induced cell necrosis.
Autophagy protected liver cells from nutrient deprivation-induced necrosis by eliminating ROS-generating mitochondria in vitro
To examine whether autophagy played its protective role mainly through eliminating oxidative stress in the nutrient-free condition, we used antioxidant N-acetyl-L-cysteine (NAC) to treat Chang liver cells. Examination of cell viability showed that NAC prominently promoted the survival of Chang liver cells in the EBSS, EBSS + CQ and EBSS + 3-MA group (Figure 5C). Hoechst 33342/PI staining assay also suggested that NAC significantly attenuated cell necrosis in the EBSS and EBSS + CQ groups. More importantly, the level of cell necrosis had no obvious difference between EBSS, EBSS + CQ and EBSS + 3-MA group after NAC treatment (Figure 5D). Notably, NAC treatment had no influence on the apoptotic level of cells in the EBSS, EBSS + CQ and EBSS + 3-MA groups (Figure 5E). These results demonstrated that autophagy protected Chang liver cells from nutrient deprivation-induced cell necrosis by eliminating excessive ROS.
ROS lead to DNA oxidative damage, which could be assayed by 8-Hydroxy-2’deoxy Guanosine (8-OHdG) staining. Massive immunoreactivity of 8-OHdG was found in EBSS-treated cells, and much stronger fluorescence intensity was observed after co-treatment with CQ or 3-MA (Figure 6C). Most notably, fluorescence staining for 8-OHdG mostly localized in cell nuclei in the EBSS group, but a large part of 8-OHdG localized in cytoplasm in the cells of the EBSS + CQ group. Since only mitochondrial DNA (mtDNA) localizes in cytoplasm but not nucleus, the increased oxidative damaged DNAs were very likely to be mtDNA. Damaged mtDNA is also an indicator of mitochondria damage . Therefore, these results suggested that in the nutrient-free condition, autophagy reduced the generation of mitochondrial ROS and thus prevented further mitochondria damage which otherwise would produce more ROS in the cells. Therefore, when autophagy is inhibited, there might have a loop that would increase mitochondria damage and ROS accumulation, finally very likely leading to the increase of cell necrosis under nutrient deprivation.
Antioxidant protects autophagy inhibition induced liver injury under ischemia in vivo
Pathological structure analysis showed that NAC treatment prominently reduced the hepatic structural damage in the ischemia and ischemia + CQ groups. And there was no obvious histological difference between the ischemia + NAC and ischemia + CQ + NAC groups (Figure 8E). Consistent with these results, pretreatment with NAC significantly reduced the elevated levels of serum ALT and AST in the ischemia + CQ group (Figure 8F and G). These results clearly indicated that autophagy played its protective role by suppressing ischemia-induced mitochondrial ROS accumulation.
Using a series of morphological and biochemical assays, we found that autophagy was activated in the process of ischemia, and moderated ischemia-induced liver injury. In order to explore the underlying mechanism, the major damage factor of ischemia, nutrient deprivation, was mimicked through EBSS treatment in Chang liver cells. Inhibition of starvation-induced autophagy significantly increased cell necrosis but not apoptosis in vitro. Further studies showed that autophagy inhibition aggravated starvation-induced ROS accumulation, especially mitochondrial ROS, and mitochondria damage. The increase of starvation-induced cell necrosis and ischemia-induced liver damage, both of which resulted from autophagy inhibition, could be reduced by antioxidant NAC.
Our results showed mitochondria of cells deprived of nutrition were disrupted, and produced abundant ROS (Figure 6A, B and Figure 7G). ROS can induce oxidative damage of organisms, macromolecules, including DNA, lipids and proteins [16–18]. Excessive ROS could result in cell necrosis . Part of the damaged mitochondria were normally sequestered and degraded through autophagy, which helped cells to escape from cell death . But when autophagy was inhibited, this process was hampered and thereby leading to damaged mitochondria accumulation, subsequently to more ROS production, and ultimately to more cell necrosis. It was a “vicious cycle” in which initial ROS-induced mitochondria damage enhanced ROS production that, in turn, led to further mitochondrial damage and eventually massive hepatocytes death.
On the basis of the results above, we concluded that during the process of ischemia, autophagy generally was induced to decrease cell necrosis and liver injury mainly through suppressing ROS accumulation, especially produced by mitochondria. However, patients usually had underlying diseases including metabolic syndrome, diabetes, hypertension, and advanced age. Many of these conditions have been shown to interfere with autophagy . And various studies showed that livers with impaired autophagy were vulnerable to hepatic I/R [21, 22]. Wang JH et al. found that livers of older patients had significantly less reparative capacity following I/R injury, which occurred during these operations. Immunoblot, autophagic flux, genetic, and imaging analyses all showed that autophagy inhibition increased the sensitivity of liver to I/R injury. Atg4B overexpression blocked the mitochondrial permeability transition and decreased cell death induced by I/R in old patients . In addition, another recent study showed that autophagic proteolysis was inhibited in steatotic liver, due to impairment of autophagosome acidification and cathepsin expression. Using a murine model, Takeshi Suzuki et al. provided evidence that the steatotic liver was vulnerable to hepatic I/R . And in the study of Ramalho FS et al. steatotic livers showed impaired regenerative response and reduced tolerance to hepatic injury compared with non-steatotic livers . All evidences above showed that inhibition of autophagy was correlated with high sensitivity of liver to injury. However, these studies did not investigate that which stage of I/R, ischemia or reperfusion, is the main stage in which autophagy exerts protective effect. Or maybe autophagy protects liver in the whole process of I/R? In this report, we showed that autophagy, at least, was an important protector in the process of ischemia, although the exact role of autophagy in reperfusion needs further investigation.
Restoration or enhancement of autophagy may ameliorate the damage to liver function in the process of ischemia, especially to livers with low level of autophagy. Further work about this will provide more applicable therapeutic strategy about the pretreatment of liver surgery.
Materials and methods
Animals and experimental design
Male Sprague–Dawley rats (10–12 week-old, weighing 220–250 g) were obtained from the Shanghai Experimental Center, Chinese Science Academy, Shanghai, and were maintained at an animal facility under pathogen-free conditions. The animals were housed in a temperature and humidity controlled environment with a 12 h light/12 h dark cycle. All animals received humane care according to animal protocols approved by the Second Military Medical University Animal Care Committee.
48 rats were randomly divided into eight equal groups, including sham, ischemia, sham + CQ, sham + NAC, sham + CQ + NAC, ischemia + CQ, ischemia + NAC, ischemia + CQ + NAC groups. All animals were fasting overnight before operation. CQ and NAC (both from Sigma-Aldrich, St Louis, MO) were used as autophagy inhibitor and antioxidant, respectively. CQ (60 mg/kg) and NAC (150 mg/kg) were given by intraperitoneal injection to rats 2 hours before sham or ischemia operation.
Rats were anaesthetized by sodium phenobarbital at a dose of 30 mg/kg. A complete midline incision was made. Hepatoduodenal ligament was separated after entry into the belly. The hepatic pedicle including hepatic artery and portal vein, which supplies the left and median liver lobes (70% of liver mass), was occluded with a microvascular clamp for 90 min . Sham-operated rats were only subjected to anesthesia without ischemia operation. Then livers and bloods of rats were immediately collected without reperfusion.
Serum ALT and AST were analyzed using a Fuji DRICHEM 55500 V (Fuji Medical System, Tokyo, Japan) according to the manufacturer’s instructions. The levels of MDA and T-AOC were measured using the assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions.
Human liver cell line Chang liver was maintained in RMPI1640 medium (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (GIBCO), 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator under 95% air and 5% CO2 at 37°C.
Nutrient deprivation and drug treatments of cells
To obtain nutrient-free condition, hepatocytes were washed three times with phosphate buffered saline (PBS) and incubated in EBSS (Sigma-Aldrich, E2888) for indicated time at 37°C. CQ, 3-MA (Sigma-Aldrich) and NAC were used at 10 μM, 5 mM and 1 mM for indicated time, respectively.
Transient transfection and identification of autophagy
GFP-tagged LC3 expression vector was utilized to demonstrate the occurrence of autophagy. Chang liver cells were seeded (7 × 103 cells/well) in 96-well plates and cultured overnight, then GFP-LC3 expression plasmids were transiently transfected into the cells using Fugene HD transfection reagent (Roche, Basle, Switzerland), according to the manufacturer’s instructions. The cells were subjected to the indicated treatments 24 h after transfection. At the end of the treatments, the puncta were observed under a fluorescent microscope (Olympus IX71, Olympus Optical Co. Ltd, Tokyo, Japan). A minimum of 200 cells per sample was counted in triplicate for each experiment.
Gene silencing with lentivirus-delivered shRNA
shRNA candidate target sequence to Atg5 is 5’-CCTTTCATTCAGAAGCTGTTT-3’. Scrambled (SCR) shRNA sequence, which was used as a negative control, is 5’-TTCTCCGAACGTGTCACGT-3’. The oligonucleotides encoding the Atg5-shRNA or SCR-shRNA sequence were inserted into the GFP express vector pGCL-GFP (Shanghai GeneChem, shanghai, china). The recombinant virus was packaged using Lentivector Expression Systems (Shanghai GeneChem). Chang liver cells were infected. After 3 days, GFP-positive cells were counted under fluorescence microscope. Atg5 expression after shRNA infection was revealed by western blot analysis at 4th day.
The measurement of viable cell mass was performed with CCK8 (Dojindo Laboratories Co., Kumamoto, Japan). Cells (7 × 103 cells/well) were seeded in 96-well plates and cultured overnight, and then were treated as indicated. As soon as the treatments were completed, 10 μl solution of CCK8 was added to each well. These plates were continuously incubated for 1 h in a humidified CO2 incubator at 37°C. Finally, the absorbance of sample was measured on a microplate reader ELX800 (BIO-TEK Instruments, Inc, Winooski, VT) at 490 nm.
Cell death analysis
The percentages of apoptotic or necrotic cells were assessed by Apoptosis and Necrosis Assay Kit (Beyotime, Haimen, Jiangsu, China). After incubation, cells were stained with Hoechst 33342 and PI and then examined by fluorescence microscopy. The apoptotic cell showed a high Hoechst 33342 staining and a low PI staining while its nucleus was condensed or fragmented. The PI strong positive and Hoechst weak positive cells were regarded as necrotic ones. In four microscopic fields containing 200 cells, the number of viable cells, necrotic cells and apoptotic cells was counted [26, 27].
Histological analysis, immunohistofluorescence and tunel staining
Collected livers were fixed with 10% neutral buffered formalin and embedded in paraffin. All paraffin-embedded sections were stained with H&E for conventional morphological evaluation. The primary immunohistofluorescence antibody is HGMB1 (Abcam, Cambridge, UK). Tunel staining (Calbiochem, La Jolla, CA) was used to assess the apoptosis level of paraffin-embedded fraction slides, according to the manufacturer’s instructions.
Western blot analysis
Whole cell lysates were subjected to SDS–PAGE. The blots were incubated with desired primary antibodies, which included anti-LC3 (Novus Biologicals. Littleton, CO), anti-p62, anti-cleaved caspase3, anti-cleaved caspase7 and anti-Atg5 (all from Cell Signaling Technology, Beverly, MA), and then with anti-rabbit IgG peroxidase conjugated secondary antibody (Hangzhou HuaAn Biotech, Hangzhou, Zhejiang, China) and chemiluminescent substrates. Hybridization with anti-GAPDH (Hangzhou HuaAn Biotech) was used to confirm equal protein loading.
Mitochondria isolation from liver
The mitochondria of rat livers were prepared using Tissue Mitochondria Isolation Kit (Beyotime), according to the manufacturer’s instructions.
Measurement of intracellular ROS level and mitochondrial superoxide level
Cells were incubated with 10 μM 2’, 7’-dichlorofluorescein diacetate (DCF-DA) for 20 min at 37°C to assess intracellular ROS level. After washing twice in PBS, positively stained cells were observed under fluorescent microscope and quantified with Image J software (US National Institutes of Health, Bethesda, MD), or were analyzed at an excitation wavelength of 480 nm and an emission wavelength of 525 nm by BD FACScan flowcytometry (BD Biosciences, San Jose, CA).
To examine accumulation of mitochondrial superoxide, cells were incubated with 2.5 μM MitoSOX Red mitochondrial superoxide indicator (Invitrogen) for 10 min, and then were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. Cell nuclei were labeled using DAPI (1 mg/ml) for 4 min. Fluorescent images were captured using a fluorescence microscope. Quantification of MitoSOX Red fluorescence was also analyzed with excitation/emission at 510/580 nm using a FACScan flowcytometry (BD Biosciences).
Freshly prepared frozen liver sections were incubated with 2 μM MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen) for 30 min at 37°C. Then they were observed by fluorescence microscopy and quantified with Image J software.
Mitochondrial membrane potential examination
Mitochondrial membrane potential of chang liver cell was measured by the incorporation of a cationic fluorescent dye Rhodamine 123 (Rho123, 5 μg/ml, Sigma). After the indicated treatment periods, the cells were stained with Rho123 and incubated for 15 min at 37°C in the dark. The fluorescence intensity of cells was observed under fluorescence microscopy.
Freshly prepared mitochondrial suspensions (0.5 mg protein/ml) of rat livers were incubated with 2 mM Rho123 for 30 min 37°C in the dark and then washed and suspended in PBS. Samples were analyzed immediately with excitation/emission at 488/530 nm using a FACScan flowcytometry.
Cells (4 × 104 cells/well) were seeded in 24-well plates and cultured overnight. At the end of the designated treatments, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After incubation for 1 hour in blocking buffer (10% goat serum in PBS), the cells was incubated with anti-8-OHdG (Abcam) antibody at 4°C overnight. On the following day, cells were washed twice with PBS and incubated with second antibody at room temperature for 45 min. After washing with PBS, cell nuclei were stained using DAPI (1 mg/ml) for 4 min. Then cells were observed under fluorescent microscope.
Data were presented as mean ± SEM. Differences were analyzed by the Student t test and one-way ANOVA. A p value of less than 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA).
Reactive oxygen species
Total anti-oxidation competence
Hematoxylin and eosin
Earle’s balanced salt solution
Microtubule-associated protein 1 light chain 3
High power field.
This project was supported by Key Basic Research Project of China (Grant NO. 2011CB966203, 2010CB945600, 2012CBA01303); Key project of National Natural Science Foundation of China (Grant NO. 81030041); National Natural Science Foundation of China (Grant NO. 31171321, 81101622, 81201584); Special Funds for National key Sci-Tech Special Project of China (Grant NO.2012ZX10002-016, 2012ZX10002011-011); Shanghai Science and Technology Committee (Grant NO. 11ZR1449500, 12431900802, 12ZR1454200) and Science Fund for Creative Research Groups, NSFC, China (Grant NO. 81221061).
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