Activation of autophagy protects against cholestasis-induced hepatic injury
- Lu Gao†1,
- Gang Lv†2,
- Xianling Guo†1,
- Yingying Jing1,
- Zhipeng Han1,
- Shanshan Zhang1,
- Kai Sun3,
- Rong Li1,
- Yang Yang1 and
- Lixin Wei1, 3Email author
© Gao et al.; licensee BioMed Central Ltd. 2014
Received: 1 May 2014
Accepted: 1 August 2014
Published: 26 August 2014
Cholestasis is characterized by an abnormal accumulation of bile acids and causes hepatocellular injury. Recent studies show that autophagy is involved in the pathophysiology of many liver diseases. The potential role of autophagy in preventing cholestatic hepatotoxicity, however, has rarely been investigated. The aim of this study was to examine whether autophagy is involved in the cholestatic hepatotoxicity.
We found that bile duct ligation (BDL) led to cholestatic liver injury and hepatocytic autophagy activation in the mice. Suppression of autophagy with Chloroquine (CQ) increased liver injury and hepatocytes apoptosis; while activation of autophagy by rapamycin reduced cholestasis hepatotoxicity. In L02 normal liver cells, Glycochenodeoxycholate (GCDC) treatment would induce autophagy. Inhibition of autophagy by CQ could promote GCDC-induced cell apoptosis. In contrast, rapamycin treatment could protect against GCDC-induced cell death. Furthermore, autophagy contributed to the liver cells survival via modulation of reactive oxygen species (ROS).
These findings indicate that autophagy protects against cholestasis induced liver injury and hepatocyte apoptosis by eliminating ROS accumulation. Our data suggest that enhancement of autophagy may be a therapeutic strategy to mitigate cholestatic liver injury.
KeywordsAutophagy Bile acid Cholestasis Hepatocyte Reactive oxygen species
Cholestasis is a commonly clinical pathology of many diseases, which is characterized by an abnormal accumulation of bile acids. Many liver diseases have been demonstrated to have cholestatic pathophysiology, such as cholangiocarcinoma, bile duct stone, primary biliary cirrhosis, biliary atresia, and primary sclerosing cholangitis [1–3]. Initial studies suggested that the accumulation of hydrophobic bile acids in the liver contributes to cholestasis associated liver injury , and that Glycochenodeoxycholate (GCDC), the main toxic component of bile acid in patients, could induce necrosis in freshly isolated hepatocytes and primary cultured hepatocytes [5, 6], or induce apoptosis in isolated liver cell models [7, 8]. In recent years, the possible molecular mechanisms of liver damage induced by cholestasis, including oxidative stress, mitochondrial damage, and cell membrane disruption through their detergent action on lipid components have been found in bile acid-induced hepatocyte death. Despite this observation, the definitive mechanisms that underlie the hepatic injury during cholestatic liver diseases remain incompletely understood.
Autophagy is a catabolic process that enables cells to recycle amino acids and other intracellular nutrients to obtain energy . It is believed to be a mechanism for selective elimination of protein and dysfunctional organelles that are damaged under pathological conditions . Autophagy dysfunction is associated with various diseases, such as cancer, neurodegeneration, gastrointestinal disorders, microbial infection, and aging . The genes that regulate autophagy were first identified in yeasts. Among them, LC3, a marker of autophagosomes in mammalian cells, is activated and then relocalizes to intracellular vesicles when the lipid bilayer structure sequesters cytoplasm to form autophagosmes . In addition, p62 is a multifunctional protein that binds to LC3 and to ubiquitinated proteins, which mediates the recognition of protein aggregates for autophagic clearance . The accumulation of p62 marks the dysfunctional autophagy which is not enough to process the damaged proteins bound to p62 . In recent years, various studies reported that autophagy actively participates in liver physiology and pathogenesis [15, 16]. In liver, it was found that autophagy plays important roles in cytoprotection against multiple pathological insults, including liver steatosis, liver injury, dyslipidemia in alcoholic, and non-alcoholic fatty liver conditions [17–20]. However, whether autophagy plays roles in cholestasis is unclear.
In this study, we investigated whether the autophagy machinery could be activated in liver injury induced by cholestasis; we hypothesized that autophagy contributes to the cell survival via reactive oxygen species (ROS) modulation in hepatocytes.
Bile duct ligation promoted autophagy in the mouse liver
GCDC induced autophagy in L02 hepatocytes
Autophagy protects against BDL-induced liver injury
Activation of autophagy reduced GCDC-induced hepatocyte apoptosis
ROS contributes to cholestasis-induced liver injury
The major finding of this study is that autophagy is a survival mechanism for liver to protect against cholestasis-induced hepatocyte injury. Our results showed that blockade of autophagy by autophagic inhibitor-CQ exacerbated cholestasis-induced liver injury and hepatocytes death. In contrast, inducer of autophagy by rapamycin could partly inhibit the cholestasis hepatotoxicity. Moreover, modulation of ROS by autophagy could contribute to the cell survival during cholestasis.
Autophagy is an essential component occurs at low basal level in most cells to perform homeostasis functions such as protein and organelle tunerover, and be upregulated when response to some stressors. Activation of autophagy has been reported with liver injury induced by steatosis, alcoholic consumption, ischemia/reperfusion and toxic drugs [19, 25–27]. For example, in liver ischemia reperfusion injury, autophagy mainly has a prosurvival activity, allowing the cell for coping with nutrient starvation and anoxia [28, 29]. However, using animal models to study whether autophagy is involved in and its function has not been reported in this particular condition induced by cholestasis. We suspect that the enhancing autophagic capacity of the liver may adapt to the change of cholestatic environment and serve as an effective survival strategy to clearance of abnormal or damaged hepatic proteins and organelles. In our study, we found that autophagy was activated when liver cells explore to GCDC or BDL in vitro or in vivo at first. Then the result from the serum and cell morphology showed that treatment with CQ would exacerbated the hepatic injury by increasing apoptosis in cholestasis. Conversely, administration of the rapamycin significantly promoted the liver survival. Our data suggest that autophagy help hepatocyte overcoming cholestatic injury. Similarly, the protective effect of autophagy restores sevoflurane preconditioning lost by longer ischemic  and APAP induced hepatotoxicity . These findings indicate that autophagy activation in cholestatic injury directly contributes to the survival of liver cells.
How autophagy promotes cell survival to cholestasis environment, however, remains to be explored. Detection of apoptosis and necrosis are further conducted in liver tissue and hepatocytes. Apoptosis has been observed to occur with necrosis following BDL, but in vitro only apoptosis is to present the major cell death treated with GCDC. There may be two reasons for this phenomenon. First, actual bile salt concentration contacted in liver is not entirely consistently in vivo and in vitro. Second, the liver injury induced by cholestasis includes many factors, such as oxidative stress, mitochondrial damage, and cell membrane disruption and so on. Therefore, the different environment in vivo or in vitro results in different stimulation effects on bile acids effects. Our findings showed that there was no difference in necrosis but apotosis between the CQ and CQ + GCDC group. All of these results supported our hypothesis that pretreated with CQ exacerbated the hepatic injury due to increased apoptosis in cholestasis. Conversely, rapamycin administration would have the opposite effect, restoring the cell death. Previous report has been shown that autophagy, apoptosis and necrosis can overlap and autophagy delays the onset of both apoptotic and necrotic cell death in a model of ischemic cell death .
ROS was shown to act as a critical signal in the pathogenesis of bile acid-induced hepatocyte injury . In our study, we also found that cholestasis-induced autophagic activation, at least partially, via oxidative stress, which is critical for liver cells survival. We have observed a modest increase in cellular ROS activity caused by GCDC or BDL with model of cholestasis. Combination of CQ and GCDC or BDL resulted in a marked increase in ROS generation while surpression of ROS generation was shown when pretreated with rapamycin. More importantly, increased levels of ROS contributed to cell death and liver damage in cholestatic liver cells and tissues when autophagy was inhibited, but treatment with the antioxidant NAC antioxidant markedly reduced this phenonemon. Consisting with these reports, Ding ZB et al. found oxaliplatin-induced ROS generation is augmented by autophagy inhibition and has an important role in cell death . Sun K et al. stated that induction of autophagy reducing ROS-induced necrosis to suppressed ischemic liver injury . Our results support that autophagy is a cellular self-defense response to alleviate cholestasis-induced liver injury through modulating oxidative stress. However, current study has suggested that the mitophagy is the elimination of an important source of ROS . Thus, it is conceivable that autophagic degradation of damaged mitochondria is a part of the protection mechanism against cholestatic liver injury. Our work, however, does not clarify the specific mechanisms via which autophagy modulates ROS, especially the role of mitophagy in the cholestasis induced hepatocytes injury. Further studies are warranted in future.
In this study, we found that activation of autophagy can enhance the survival of liver injury induced by cholestasis. A proper autophagy capability in the liver may be crucial to reduce the detrimental effects of cholestasis, and that enhancement of autophagy may be a possible therapeutic strategy to mitigate the pathology associated with cholestasis liver disease.
Male C57BL/6 mice, 6–8 weeks old, weighting 20-25 g, were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China. All mice used in this study were housed in pathogen-free conditions, and all procedures were performed in accordance with guidelines established by the Chinese Academy of Sciences’ Committee on Animals. The animals received the laparotomy with bile duct ligaltion were performed as previously described in detail . In the control group, mice had undergone the surgical procedures but without BDL. Induction or suppression of autophagy was achieved by intraperitoneal administration of chloroquine (60 mg/kg), rapamycin (2 mg/kg) unless otherwise indicated in figure legends.
Chloroquine (CQ), Rapamycin, Glycochenodeoxycholate (GCDC) and N-acetyl-cysteine (NAC) were purchased from Sigma-Aldrich (St Louis, MO).
Transient transfection and identification of autophagy
GFP-tagged LC3 expression vector has been utilized to demonstrate the occurrence of autophagy and was detected using an inverted fluorescence microscope. L02 cells were seeded (1 × 103cells/well) in 96-well plates overnight and were transiently transfected with GFP-LC3 expressing plasmids using Fugene HD transfection reagent (Calbiochem, La Jolla, CA) according to the manufacturer’s instructions. After initial treatment, autophagy was detected by counting the number of GFP-LC3-positive dots per cell under fluorescence microscope (Olympus IX71).
Measurement of liver function
Two weeks after BDL, blood samples were collected from all mice. The plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and totalbilirubin (TBIL) levels were tested with a biochemical autoanalyzer (Fuji Medical System, Tokyo, Japan) according to the manufacturer’s instructions.
Western blot analysis
After special treatment, cells and tissues were lysed in RIPA lysis buffer (Beyotime) with 1 mM PMSF. Equal amount of protein was separated by SDS-PAGE and transferred to NC membrane. The membranes were washed, blocked, and incubated with specific primary anti-human antibodies against LC3 (Novus Biologicals, Littleton, CO) and p62/SQTM (Cell signaling Technology, Beverly, MA), Î²-actin antibody (Hangzhou HuaAn Biotech, Zhejiang, China), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Hangzhou HuaAn Biotech). Signals were visualized by chemiluminescent detection (Beyotime).
Histological examination and tunnel staining
Liver tissues were fixed in 4% paraformaldehyde, sectioned, and mounted on glass slides then stained with Meyer’s hematoxylin and eosin. Each sample was observed at a 200× magnification of microscopic field in 5 randomly selected areas. Tunnel staining (Calbiochem, La Jolla, CA) was used to assess the apoptosis level of paraffin-embedded fraction slides, according to the manufacturer’s instructions. The number of positive-apoptotic cells were counted and expressed to the total number of cells within 5 random fields (200×) of cells.
Electron microscopic analysis
At room temperature cells were fixed in 2.5% glutaraldehyde in PBS (pH = 7.4) for two hours, postfixed in 1% osmium tetroxide in water for one hour. After dehydrating in an ascending series of ethanol, the samples were then embedded in araldite. 50-60 nm sections were cut on a LKB-I ultramicrotome and picked up on copper grids, post-stained with uranyl acetate and lead citrate, observed in a Philips CM-120 TEM.
Flow cytometric analysis
Annexin V-fluorescein isothiocyannate (FITC) assay was used to measure cell death by flow cytometry according to the manufacturer’s instructions (Nanjing Keygen Biotech, China). Briefly, cells were collected together and resuspended in 300 µl 1 × binding buffer containing 5 µl of Annexin V and 5µof PI for 30 min at room temperature in the dark. After incubation, samples were analyzed by a BD FACSAria flow cytometer within one hour.
Measurement of intracellular ROS level
To examined the accumulation of reactive oxygen species (ROS), cells were incubated with 10 µM 2’, 7’-dichlorofluorescein diacetate (DCF-DA; Invitrogen) for 30 min at 37°C, respectively, followed by fluorescence microscopy.
All of the experiments were repeated at least three times. Quantitative data were expressed as mean ± SD. Significance between two groups was performed with the Student’s t test. P < 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 5.0 software.
- Annexin V-FITC:
Annexin V -fluoresceinisothiocyannate
Bile duct ligation
Green Fluorescent Protein
Hematoxylin and eosin
Microtubule-associated protein 1 light chain 3
Methyl thiazolyl tetrazolium
Polyacrylamide gel electrophoresis
Phenylmethyl sulfonyl fluoride
Reactive oxygen species
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
This work was supported by the Key Basic Research Project of China (Grant grant number 2012CBA01303, 2011CB966200, 2010CB945600, 2011CB965100), Key project of National Natural Science Foundation of China (Grant number 81030041), National Natural Science Foundation of China (Grant number 31171321, 81101622, 81372330), Special Funds for National key Sci-Tech Sepcial Project of China (Grant number 2012ZX10002-016, 2012ZX10002011-011), Shanghai Science and Technology Committee (Grant number 10ZR1439600, 11ZR1449500), Shanghai Municipal Health Bureau (Grant number XYQ2011044) and Science Fund for Creative Research Groups, NSFC, China (Grant number 81221061).
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