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Isoflurane Preconditioning Alleviated Murine Liver Ischemia and Reperfusion Injury by Restoring AMPK/mTOR-Mediated Autophagy

Rao, Zhuqing MD*; Pan, Xiongxiong MD, PhD*; Zhang, Hui MD; Sun, Jie MD, PhD*; Li, Jingjin MD*; Lu, Ting MD*; Gao, Mei MD, PhD*; Liu, Siying MD*; Yu, Dan MD*; Ding, Zhengnian MD, PhD*

doi: 10.1213/ANE.0000000000002385
Pain and Analgesic Mechanisms: Original Laboratory Research Report
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BACKGROUND: Isoflurane has a pharmacological preconditioning effect against ischemia injury in the heart, kidney, and brain, but whether and how isoflurane preconditioning protects livers against ischemia and reperfusion (IR) injury is unclear.

METHODS: Mice were randomly divided into an isoflurane preconditioning (ISO) group and control group, receiving 1.5% isoflurane or carrier gas for 40 minutes, respectively (n = 8/group). A partial warm liver IR model was used, and liver injury was evaluated. Primary hepatocytes were pretreated with 1.5% isoflurane for 2 hours before the induction of cell death by hydrogen peroxide. Cell death and survival were evaluated with the lactate dehydrogenase and cell counting kit-8 assay. Autophagy and regulatory molecules in stressed livers and hepatocytes were analyzed by Western blot (n = 6/group). An autophagy inhibitor (3-methyladenine [3-MA]) and 5′ adenosine monophosphate-activated protein kinase (AMPK) inhibitor (dorsomorphin) were administered in vivo (n = 8/group) and in vitro (n = 6/group).

RESULTS: Compared to that observed in the control group, mice in the ISO group showed reduced liver injury (alanine aminotransferase [ALT] levels, control versus ISO group, 8285 ± 769 vs 4896 ± 917 U/L, P < .001) and enhanced hepatocellular antiapoptosis in livers after IR. Furthermore, liver autophagy was restored by ISO as indicated by elevated LC3B II protein levels accompanied with increased p62 degradation. The in vitro study of primary hepatocytes also found that ISO effectively attenuated hepatocyte cell death induced by hydrogen peroxide. In addition, 3-MA pretreatment showed no significant influence in the control group, but abrogated the protective role of ISO both in stressed livers (ALT levels, phosphate-buffered saline + ISO versus 3-MA + ISO group, 5081 ± 294 vs 8663 ± 607 U/L, P < .001) and in hepatocytes. Finally, signaling pathway analysis demonstrated that AMPK was activated by ISO. Pretreatment with an AMPK inhibitor also abrogated liver protection by ISO (ALT levels, phosphate-buffered saline + ISO versus dorsomorphin [DOR] + ISO group, 5081 ± 294 vs 8710 ± 500 U/L, P < .001), with no significant effect in control mice.

CONCLUSIONS: Our results indicate that isoflurane preconditioning attenuates liver IR injury via AMPK/mTOR-mediated hepatocellular autophagy restoration. Our findings provide a novel potential therapeutic strategy for managing liver IR injury.

From the Departments of *Anesthesiology and Liver Surgery, First Affiliated Hospital with Nanjing Medical University, Nanjing, China.

Accepted for publication June 26, 2017.

Funding: This study was supported by the National Natural Science Foundation of China (81370260).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Zhengnian Ding, MD, PhD, Department of Anesthesiology, First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China. Address e-mail to zhengnianding@njmu.edu.cn.

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Liver ischemia and reperfusion (IR) injury is a frequent complication for patients subjected to partial liver resection and liver transplantation. Direct hepatocellular injury caused by deprivation of oxygen and nutrition and subsequent intrahepatic inflammation are the 2 characteristic pathogeneses.1

Autophagy is constitutively active in most cells for development, differentiation, survival, and homeostasis.2 In the previous study, we have found that impaired autophagy contributed to bupivacaine-induced myotoxicity in mouse myoblasts.3 However, functions of autophagy in IR injury are highly divergent and controversial.4–6 In liver IR injury, autophagy mainly has a prosurvival function, allowing cells to cope with nutrient starvation and anoxia.5,7

Volatile anesthetics are often administered to patients undergoing general anesthesia. Previous studies have shown that isoflurane preconditioning protects against IR injury in organs including the kidney (animal study),8 heart (clinical study),9 and brain (animal study).10 Interestingly, autophagy is found to be activated by isoflurane preconditioning in cultured neuron cells11 or IR models,12 resulting in preservation of cell and organ function. However, the role of isoflurane preconditioning in liver IR injury and its underlying mechanism is not fully understood.

Using an in vivo partial warm liver IR model and in vitro primary hepatocyte injury model, we determined whether and how isoflurane preconditioning protects livers against IR injury.

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METHODS

Mice

Male wild-type C57BL/6 mice (8 weeks old) were purchased from the Laboratory of Animal Resources of Nanjing Medical University. Animals were housed under specific pathogen-free conditions with free access to tap water and food. All animals received humane care according to protocol (protocol number NMU08-092) approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

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Experimental Protocols

Mice were randomly divided into an isoflurane preconditioning (ISO) group or control (Ctrl.) group. Mice in ISO group were exposed to 1.5% isoflurane with 25% oxygen balanced with nitrogen for 0, 20, 40, 60, or 90 minutes, followed by 30 minutes of normal air for isoflurane washout before surgical ischemic procedures, while the control animals received the same gas without isoflurane.

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Model of Warm Liver IR Injury

A model of partial hepatic warm IR injury was used. In brief, after successful anesthesia with 10% chloral hydrate (0.3 g/kg intraperitoneally [IP]), mice were injected with heparin (100 mg/kg). An atraumatic clip was used to interrupt the arterial and portal venous blood supply to the cephalad lobes of the liver. The clip was removed to initiate liver reperfusion after 90 minutes of ischemia. All the mice were placed in a designed warm container (HTP-1500 Heat Therapy Pump; Adroit Medical Systems, Chicago, IL) to keep the temperature at 29°C. Mice were euthanized after 6 hours after reperfusion. Sham controls underwent the same procedure, but without vascular occlusion. In some experiments, 3-methyladenine (3-MA, 30 mg/kg IP at −1 hour; Sigma, Saint Louis, MO), dorsomorphin (20 mg/kg IP −1 hour; Tocris, Bristol, UK), or same volume of phosphate-buffered saline (PBS, control) were administered before isoflurane or control gas exposure. Carprofen (6 mg/kg) was administered intraperitoneally for analgesia in all groups before surgery.

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Serum Biochemical Measurements and Liver Histopathology

Mice were euthanized at 6 hours after reperfusion and blood and liver samples were collected. Serum alanine aminotransferase (ALT) levels were measured with an AU5400 automated chemical analyzer (Olympus, Tokyo, Japan). Part of the liver specimens were fixed in 10% buffered formalin and embedded in paraffin. Liver sections (4 µM) were stained with hematoxylin and eosin. The severity of liver IRI was graded blindly by one investigator using Suzuki’s criteria on a scale from 0 to 4. No necrosis, congestion/centrilobular ballooning is given a score of 0, whereas severe congestion and >60% lobular necrosis is given a score of 4.13

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Primary Hepatocyte Isolation and Isoflurane Preconditioning

Mouse livers were perfused in situ via the portal vein with Hank’s balanced salt solution, followed by 0.27% collagenase IV (Sigma). Percoll (40%, Sigma) was used for further hepatocyte isolation (centrifuged at 150g for 7 minutes). Williams E medium with hepatocyte plating/maintenance supplements was used for hepatocyte plating and culture. For hepatocyte isoflurane preconditioning experiments, hepatocytes were kept in an airtight, 37°C, humidified chamber, connected with an in-line calibrated agent-specific vaporizer to deliver 1.5% isoflurane (ISO group) or carrier gas (Ctrl. group) for 2 hours before cell death induction.

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Induction of Hepatocyte Cell Death

Hydrogen peroxide (200 µM) was used to induce hepatocyte cell death as described previously.14 Cell culture medium was collected 24 hours after hydrogen peroxide treatment and then cell death and survival were detected using a lactate dehydrogenase (Sigma) and cell counting kit-8 assay (Dojindo, Kumamoto, Japan), respectively, according to the manufacturer’s instruction. In some experiments, 3-MA (5 mM) or dorsomorphin (2 µM) were added to the cell culture 1 hour before isoflurane preconditioning. For Western blot analysis, hepatocytes were collected 3 hours after hydrogen peroxide treatment.

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Western Blots

Liver tissue or cell lysate proteins were extracted and subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride nitrocellulose membrane. Primary antibodies against cleaved caspase 3, BCL-2, BCL-XL, LC3B, p62, phospho-5′ adenosine monophosphate-activated protein kinase-α (AMPKα) (Thr172), phospho-p70 S6K (Thr389), phospho-AKT (Thr308), phospho-ULK1 (Ser555), and β-actin (Cell Signaling Technology, Danvers, MA) were used and incubated overnight at 4°C. After 2 hours of incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (1:1000), Clarity Western ECL Substrate (Bio-Rad, Hercules, CA) was used for chemiluminescence development. Image J 1.47v software was used to quantify the Western blot bands.

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Quantitative Reverse Transcription-Polymerase Chain Reaction

Total RNA (2 μg) was reverse-transcribed to cDNA using a SuperScript III System (Invitrogen, Carlsbad, CA). Quantitative polymerase chain reaction was performed using the SYBR Green Master (Roche, Indianapolis, IN) as previously described.15

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Statistical Analysis

The results are shown as the mean ± SD. Multiple group comparisons were performed using 1-way analysis of variance followed by the Bonferroni post hoc test. All analyses were performed using Stata software (version 11.0). P values <.05 (2 tailed) were considered statistically significant. The sample size was calculated using Stata software with sampsi command. Based on the preliminary data (ALT levels in control group and ISO group after IR), mean 1 = 8000, mean 2 = 5000, SD = 1000, α = .05 (2 sided), and power = 0.9, the estimated required sample sizes were 3 mice/group.

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RESULTS

Isoflurane Preconditioning Attenuated Liver IR Injury

Figure 1.

Figure 1.

We first tested whether isoflurane preconditioning protected livers against IR injury in vivo. Groups of mice were subjected to different durations of isoflurane preconditioning (0, 20, 40, 60, and 90 minutes) before liver ischemia (90 minutes) followed by reperfusion (6 hours). Serum ALT was measured kinetically (Figure 1A). Interestingly, mouse serum ALT slightly decreased with 20 minutes of isoflurane preconditioning (ALT levels, 20- vs 0-minute group, 7479 ± 479 vs 9106 ± 618 U/L, n = 6, P < .001). The protective effects peaked with 40 minutes of isoflurane preconditioning (ALT levels, 40- vs 20-minute group, 5329 ± 230 vs 7479 ± 479, n = 6, P < .001). Thus, we chose 40 minutes of isoflurane preconditioning for further experiments. The protective role of isoflurane preconditioning (40 minutes) was further confirmed by lower levels of serum ALT (Figure 1B), better preserved liver architecture (Figure 1C), and lower Suzuki’s scores (Figure 1D).

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Isoflurane Preconditioning Enhanced Autophagy and Inhibited Hepatocellular Apoptosis in Livers After IR

Hepatocellular apoptosis in IR-stressed livers was evaluated. Antiapoptosis proteins, BCL-2 and BCL-XL, were measured by Western blot analysis (Figure 2A, B). Both BCL-2 and BCL-XL were activated by liver IR and were further enhanced by isoflurane preconditioning. Decreased protein levels of cleaved caspase 3 were also observed in the isoflurane preconditioning group. AKT signaling was inactivated by IR as indicated by decreased levels of AKT phosphorylation at Thr308. Furthermore, isoflurane precondition restored AKT activation after IR.

Figure 2.

Figure 2.

The protective roles of autophagy in liver IR injury have been shown in previous studies.16,17 We investigated whether liver autophagy activities were enhanced by isoflurane preconditioning. Autophagy markers, LC3B I/II and p62, were measured by Western blot (Figure 2A, B). Clearly, liver IR increased LC3B II protein levels. As LC3B II levels alone could not reflect autophagic flux, we measured p62 protein degradation to further determine autophagic flux in the groups. Surprisingly, p62 protein levels were slightly increased by liver IR, indicating that IR impaired autophagic flux in livers. Interestingly, isoflurane preconditioning not only enhanced the increase in LC3B II but also significantly deceased p62 protein levels in livers after IR. Furthermore, compared to that in the control group, markedly elevated levels of phosphorylated AMPKα at threonine 172 were detected in IR-stressed livers in the isoflurane preconditioning group. mTOR, a key negative regulator of autophagy, was decreased by isoflurane preconditioning, as indicated by the phosphorylated protein levels of p70 S6K. Interestingly, phosphorylation of ULK1 at Ser555 was also enhanced by isoflurane preconditioning. These results indicated that while IR impaired autophagy flux in livers, isoflurane preconditioning effectively restored autophagy flux in IR-stressed livers. AMPK may be an important regulator of autophagy activation by isoflurane preconditioning in ischemic livers.

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AMPK/mTOR-Dependent Activation of Autophagy by Isoflurane Preconditioning in Primary Hepatocytes

Figure 3.

Figure 3.

To address the role of isoflurane preconditioning in the regulation of hepatic autophagy and the underlying molecular mechanisms, we conducted primary hepatocyte cell culture in vitro. Primary hepatocytes were isolated and pretreated with isoflurane or vehicle gas before hydrogen peroxide stimulation. Consistent with the results in ischemic livers in vivo, Western blot analysis of harvested primary hepatocytes also showed that isoflurane preconditioning decreased protein levels of cleaved caspase 3, accompanied with increased protein levels of antiapoptosis BCL-2 and BCL-XL (Figure 3A, B). Isoflurane preconditioning also restored hepatocyte autophagic flux, as indicated by increased LC3B II and decreased p62 protein levels. To study whether autophagy regulation by isoflurane preconditioning was dependent on AMPK activation, dorsomorphin, a specific AMPK inhibitor, was used to block AMPK activation in hepatocytes (Figure 3A, B). AMPK phosphorylation was well inhibited by dorsomorphin as confirmed by Western blot. Dorsomorphin treatment showed no significant effects on hepatocellular apoptosis and autophagy in the control group, as indicated by the unchanged protein levels of cleaved caspase 3, BCL-2, BCL-XL, LC3BII, p62, p-p70 S6K, and p-ULK1 (Ser555). However, AMPK inhibition abrogated the effects of isoflurane preconditioning in antiapoptosis and autophagy enhancement. Increases in the protein levels of BCL-2, BCL-XL, LC3B II, and p-ULK1 (Ser555) were abolished after AMPK blocking. At the same time, the protein levels of cleaved caspase 3, p62, and p-p70 S6K increased with AMPK inhibition in isoflurane-preconditioned hepatocytes. Thus, our results showed that isoflurane preconditioning could promote antiapoptosis and autophagy in primary hepatocytes, dependent on AMPK activation.

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Critical Roles of the AMPK/mTOR-Mediated Autophagy Activation by Isoflurane Preconditioning in Stressed Hepatocytes and Livers

Functions of autophagy activation by isoflurane preconditioning were further determined in vitro. Primary hepatocytes were isolated from mouse livers, and hydrogen peroxide was added to the cell culture medium to induce cell death. Cell death and survival were detected by lactate dehydrogenase and cell counting kit-8 assay, respectively. As shown in Figure 4A, B, hepatocytes with isoflurane preconditioning had lower levels of cytotoxicity and higher levels of cell viability. 3-MA, a specific autophagy inhibitor, was used to determine the role of autophagy activation by isoflurane preconditioning in the protection of hepatocyte cell death. Clearly, autophagy inhibition by 3-MA showed no significant effect on hepatocyte cell death and survival in the control group. In contrast, 3-MA abrogated the protective role of isoflurane preconditioning in stressed hepatocytes. The role of AMPK activation in the regulation of autophagy activation was studied as well. AMPK inhibition by dorsomorphin also abrogated the protection of hepatocyte cell death by isoflurane preconditioning.

Figure 4.

Figure 4.

Figure 5.

Figure 5.

Finally, we tested whether autophagy was involved in the protective role of isoflurane preconditioning in liver IR injury in vivo. 3-MA was administered to inhibit autophagy before the onset of liver ischemia, and liver IR injury was evaluated at 6 hours after reperfusion (Figure 5A–C). Liver injuries in the control group were not influenced by 3-MA treatment. In contrast, autophagy inhibition by 3-MA abrogated the protection of isoflurane preconditioning in IR-stressed livers, as evidenced by higher serum ALT levels (ALT levels, PBS + ISO versus 3-MA + ISO group, 5081 ± 294 vs 8663 ± 607 U/L, n = 8, P <.001), and worse preserved liver architecture than that in the control group (hematoxylin and eosin staining and Suzuki scores). Consistent with our observation in the primary hepatocyte injury model, AMPK inhibition by dorsomorphin pretreatment showed no significant effects on liver injury in the control group but increased liver injury in the isoflurane preconditioning group (ALT levels, PBS + ISO versus DOR + ISO group, 5081 ± 294 vs 8710 ± 500 U/L, n = 8, P < .001). These results indicated that AMPK-mediated autophagy activation played a critical role in the protection of hepatocyte cell death and liver IR injury by isoflurane preconditioning.

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DISCUSSION

Our study demonstrated critical protective effects of volatile anesthetic preconditioning in liver IR injury. Isoflurane preconditioning significantly reduced hepatic injury by promoting autophagy activation via the AMPK/mTORC1 signaling pathway accompanied with enhanced hepatocellular antiapoptosis in livers after IR.

Two distinctive stages of liver IR injury have been defined. Oxygen and nutrition depletion during the ischemia stage could result in direct liver parenchyma cell injury, while secondary inflammation and oxidative stress during the reperfusion stage exacerbate the severity of liver IR injury. IR induces an overgeneration of reactive oxygen species, leading to various proinflammatory mediators, endoplasmic reticulum stress, and, ultimately, cell death.18 Hepatocellular injury or death not only represents hepatic IR injury but also triggers intrahepatic inflammatory immune activation by release of damage-associated molecular patterns in turn. Thus, interventions to reduce hepatocellular injury may have potential protective roles in liver IR injury. In the present study, isoflurane preconditioning significantly increased hepatocellular antiapoptosis and decreased intrahepatic inflammation, as indicated by decreased transcription levels of anti-inflammatory tumor necrosis factor-α and interleukin (IL)-6 but increased levels of anti-inflammatory IL-10 (Figure 1E).

Autophagy and apoptosis are both well-controlled adaptive processes in cells under normal physiological conditions or pathological stress and can be triggered by similar stimuli. Autophagy has long been recognized as a critical pathway in the regulation of cell death and survival. Clearance of misfolded proteins and the recycling of cellular proteins and glycogen for ATP generation are the important mechanism by which autophagy maintains cell viability. Proteins involved in autophagy including Beclin-1 and Atgs play an important role in the regulation of apoptosis. Genetic inhibition of Beclin-1 shifted protective autophagy to apoptosis by increasing proapoptosis caspase-8 enzymatic activity19 or decreasing antiapoptosis Bcl-2 activation.20 However, studies have also shown that genetic depletion of Atg5 and Atg3 resulted in suppression of caspase-8 activation and apoptosis.21 Our in vivo and in vitro study using an autophagy inhibitor showed that autophagy inhibition resulted in increased cleaved caspase 3 and decreased activation of antiapoptosis factors, Bcl-2 and Bcl-XL, indicating a negative regulation of apoptosis by autophagy.

Moreover, recent studies have demonstrated that autophagy plays an important role in inflammation and immunity in various disease models, including cancer, infection, and inflammatory disorders.22–24 In an acute toxic liver injury model, the macrophage-specific knockout of autophagy gene Atg5 promoted the inflammasome-dependent IL-1β production, leading to increased liver injury.25 Macrophage autophagy also protected against liver fibrosis by limiting IL1α and IL1β secretion.26 In contrast, increased autophagy was found in CD4+ T cells from rheumatoid arthritis patients and in vitro activated CD4+ T cells. Atg5 knockout in T cells resulted in impaired T-cell activation and proliferation.27 In our partial warm liver ischemia reperfusion model, we found that intrahepatic inflammation was decreased after isoflurane preconditioning. However, we focused on autophagy regulation by isoflurane preconditioning in liver parenchymal cells but not in nonparenchymal cells such as macrophages, T cells, and neutrophils. Whether and how isoflurane preconditioning functions in regulating these immune cells still remains to be further determined.

Controversial results have been found regarding the effect of autophagy in IR injury in various organs including the kidney,28 heart,29 and liver.7 Autophagy was induced in response to renal IR injury both in vivo and in vitro.30,31 Autophagy deficiency in Atg5 or Atg7 knockout mice aggravated kidney proximal tubule cell apoptosis and renal IR injury. In heart models, both activated and impaired autophagy have been found.32–34 Furthermore, autophagy may be protective during ischemia but detrimental during reperfusion.35 In the liver IR model, autophagy was regulated by IR depending on the ischemia degree. Transient (30 minutes) ischemia triggered hepatic autophagic flux, but, in contrast, prolonged (90 minutes) ischemia resulted in impaired autophagic flux.36 Prolonged ischemia could substantially reduce expression of autophagic machinery proteins such as ATG7 and Beclin-1, which in turn impairs autophagic function.37 Activation of autophagy by mTOR inhibitors effectively protected livers against IR injury.16 However, the opposite effects were found in steatotic liver IR by using inhibitors of autophagy.38,39 In a cold ischemia and orthotopic transplantation model, autophagy suppression reduced both liver damage and mortality of rats.40 Thus, autophagy may have distinct effects in IR injury depending on the severity of ischemia, the phase of IR, and the degree and cell type of autophagy activation. Recent studies have shown that LC3B II alone does not reflect the activity of autophagy because increased protein levels of LC3B II could also be caused by impaired autophagic flux. Thus, we combined the measurement of LC3B II levels with that of p62, which binds to LC3 and is selectively degraded through autophagy, to determine autophagic flux.3,41 Consistent with the findings of other studies,36,37 IR-stressed livers and H2O2-stressed hepatocytes both demonstrated increased LC3B II levels but unchanged p62 levels, indicating impaired autophagic flux. Isoflurane preconditioning in vivo and in vitro effectively restored autophagic flux as indicated by increased LC3B II levels accompanied with enhanced degradation of p62. Autophagy inhibition by 3-MA abrogated the protective role of isoflurane preconditioning both in the liver IR injury and in the hepatocellular injury model, which confirmed the critical role of autophagy in the protection of hepatic injury.

The beneficial effects of AMPK/mTOR signaling have been observed in ischemic livers. AMPK activation led to ATP preservation and reduced lactate accumulation during prolonged ischemia. Preconditioning activated AMPK and inhibition of AMPK abolished the protective role of preconditioning in liver IR injury.42 mTOR inhibition by rapamycin inhibited endoplasmic reticulum stress and enhanced autophagy in hepatocytes, leading to less hepatocellular cell injury.43,44

Complicated signaling pathways regulating autophagy have been defined. In IR models, AMPK/mTORC1 pathways have been widely studied since they are sensitive to nutrition and energy status, which are dramatically changed during IR. mTORC1 is an important negative regulator of autophagy. Meanwhile, AMPK represses mTORC1 activity, leading to autophagy activation. In an in vitro IR injury model of renal proximal tubular cells, AMPK knockdown by small hairpin RNA significantly increased mTORC1 phosphorylation and decreased autophagy induction, leading to enhanced cell apoptosis. In contrast, an mTOR inhibitor promoted autophagy and attenuated cell injury.45 In a kidney IR model, decreased AMPK activity accompanied with increased mTORC1 activity was detected. AMPK activation by its agonist restored autophagy activation, resulting in attenuated cellular stress markers.46 The ULK1 complex plays an important role in initiating autophagosome formation. Phosphorylation of ULK1 by AMPK at Ser555 is required for starvation-induced autophagy.47 Here, we also found decreased AMPK levels in the hepatocyte injury model, and isoflurane preconditioning restored AMPK activity and autophagic flux with decreased mTORC1 activity and increased ULK1 phosphorylation at Ser555. AMPK was activated by liver IR in a previous study.36 In our in vivo IR model, AMPK levels were slightly but not significantly elevated by IR. Interestingly, AMPK levels were significantly increased by IR in the ISO group, accompanied with decreased mTORC1 activation. AMPK inhibition by its inhibitor dorsomorphin abrogated the inactivation of mTORC1 and upregulation of autophagy by isoflurane and subsequently the protection of hepatic injury both in vitro and in vivo.

Protective roles of heme oxygenase 1 (HO-1) signaling have been reported in liver IR injury.48 Lv et al49 found that isoflurane attenuated IR injury by increasing the HO-1 expression and activity in a rat IR model. In the present study, we did not analyze the HO-1 signaling but focused on the role of isoflurane preconditioning in regulation of hepatocellular autophagy. However, the HO-1 signaling may also have functions in regulating IR injury in our mouse model. Interestingly, recent studies have reported the regulation of autophagy by HO-1. Obesity impaired hepatic autophagy activity and decreased hepatic HO-1 expression. Induction of HO-1 by ischemic preconditioning restored autophagy activity, resulting in alleviated liver IR injury.17

A number of limitations in this study should be addressed. No cell type–specific roles of isoflurane preconditioning were evaluated in vivo. As discussed above, isoflurane preconditioning could also have effects in other cells such as macrophages and neutrophils in the liver. In addition, we used the pharmacological inhibitors to inhibit autophagy and AMPK activation for mechanism analysis both in vivo and in vitro. No genetic modification was applied. Hepatocyte-specific ATG5 or ATG7 knockout mice may be applied to further confirm our current findings.

In conclusion, our study demonstrated that isoflurane preconditioning protects livers against IR injury. Isoflurane preconditioning promotes hepatocyte cell survival and inhibits hepatocellular death/apoptosis by restoring AMPK/mTOR-mediated autophagy in hepatocytes. These findings suggest that isoflurane preconditioning is a novel preventive or therapeutic strategy to protect against liver IR injury and may be promising in the setting of transplantation.

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DISCLOSURES

Name: Zhuqing Rao, MD.

Contribution: This author helped design experiments, perform in vitro and in vivo experiments, and also write the article.

Name: Xiongxiong Pan, MD, PhD.

Contribution: This author helped conduct the study and analyze the data.

Name: Hui Zhang, MD.

Contribution: This author helped to do the statistical analysis.

Name: Jie Sun, MD, PhD.

Contribution: This author helped conduct the study and analyze the data.

Name: Jingjin Li, MD.

Contribution: This author helped conduct the study and analyze the data.

Name: Ting Lu, MD.

Contribution: This author helped conduct the study and analyze the data.

Name: Mei Gao, MD, PhD.

Contribution: This author helped conduct the study and analyze the data.

Name: Siying Liu, MD.

Contribution: This author helped conduct the study and analyze the data.

Name: Dan Yu, MD.

Contribution: This author helped conduct the study and analyze the data.

Name: Zhengnian Ding, MD, PhD.

Contribution: This author helped design and supervise the study.

This manuscript was handled by: Jianren Mao, MD, PhD.

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