Remifentanil up‐regulates HIF1α expression to ameliorate hepatic ischaemia/reperfusion injury via the ZEB1/LIF axis

Abstract Ischaemia/reperfusion (I/R)‐induced hepatic injury is regarded as a main reason of hepatic failure after transplantation or lobectomy. The current study aimed to investigate how the opioid analgesic remifentanil treatment affects I/R‐induced hepatic injury and explore the possible mechanisms related to HIF1α. Initially, an I/R‐induced hepatic injury animal model was established in C57BL/6 mice, and an in vitro hypoxia‐reoxygenation model was constructed in NCTC‐1469 cells, followed by remifentanil treatment and HIF1α silencing treatment. The levels of blood glucose, lipids, alanine transaminase (ALT) and aspartate transaminase (AST) in mouse serum were measured using automatic chemistry analyser, while the viability and apoptosis of cells were detected using CCK8 assay and flow cytometry. Our results revealed that mice with I/R‐induced hepatic injury showed higher serum levels of blood glucose, lipids, ALT and AST and leukaemia inhibitory factor (LIF) expression, and lower HIF1α and ZEB1 expression (P < .05), which were reversed after remifentanil treatment (P < .05). Besides, HIF1α silencing increased the serum levels of blood glucose, lipids, ALT and AST (P < .05). Furthermore, hypoxia‐induced NCTC‐1469 cells exhibited decreased HIF1α and ZEB1 expression, reduced cell viability, as well as increased LIF expression and cell apoptosis (P < .05), which were reversed by remifentanil treatment (P < .05). Moreover, HIF1α silencing down‐regulated ZEB1 expression, decreased cell viability, and increased cell apoptosis (P < .05). ZEB1 was identified to bind to the promoter region of LIF and inhibit its expression. In summary, remifentanil protects against hepatic I/R injury through HIF1α and downstream effectors.

play important roles to relieve hepatic I/R injury. [6][7][8] In animal experiments, ischaemic pre-conditioning and low-temperature reperfusion alleviated hepatic I/R injury, but these preclinical findings are not yet translatable to clinical procedures. 9,10 Currently, there are few effective therapeutic strategies for protecting against I/R-induced hepatic injury. 2 Remifentanil (Ultiva™) is a selective μ-opioid receptor agonist, which is used clinically for analgesia and as part of general anaesthesia. [11][12][13] Remifentanil reportedly protects the heart from I/R injury via post-conditioning 14 and reduces hepatic I/R injury in rats. 15 However, the molecular mechanism by which remifentanil attenuates hepatic I/R injury is not fully understood. Daijo et al 16 showed that the expression of hypoxia-inducible factor 1α (HIF1α) was up-regulated by remifentanil. Guo et al 17 summarized current studies regarding the role of HIF1α in hepatic I/R injury, which indicated that stabilization of HIF1α can attenuate hepatic I/R injury. HIF1α belongs to the HIF transcription factor family, which participates in a variety of biological processes such as cell survival under hypoxic condition, glycolysis and angiogenesis. [18][19][20] Under hypoxia, HIF1α forms a heterodimer with HIF1β, which binds to hypoxia-responsive elements (HREs) in the promoter regions of downstream target genes. 21 In cortical ischaemia, up-regulation of Zinc finger E-box binding homeobox 1 (ZEB1) mRNA and protein was shown to be a protective response to ischaemia by neurons. 22 In glioblastoma, HIF1α could induce the expression of ZEB1, which is a member of zinc finger homeodomain transcription factor family and plays important roles in the epithelial-mesenchymal transition (EMT) of embryogenesis. 23 ZEB1 was also found to regulate EMT in epithelial cancers. 24 Edward et al 25 reported that ZEB1 binding sites were located within the leukaemia inhibitory factor (LIF) promoter region and could inhibit LIF expression in glioma cancer stem cells. LIF was first described in the 1990s as a factor for repressing the clonogenicity, while inducing the differentiation of monocytic leukaemia M1 cells in mice. 26 LIF functions by interacting with gp130, by which means intracellular signalling is transduced in neurons and oligodendrocytes, resulting in the increased expression of neuro-survival associated genes. 27 In this paper, we established an I/R-induced hepatic injury model in C57BL/6 mice, following established procedures 28,29 and hypoxia-induced cell line model in NCTC-1469 cells. By using a knockdown strategy, histological approaches and molecular methods, we further demonstrated that remifentanil ameliorates I/R-induced hepatic injury through the regulation of HIF1α and ZEB1/LIF axis. Our work deciphers the molecular mechanism of hepatic I/R injury, which could provide novel therapeutic strategy for the clinical application.

| Animal treatment
A total of 108 male C57BL/6 mice (ageing 8-12 weeks and weighing 22 ± 2 g) were obtained from Experimental Animal Center of Xi'an Jiaotong University. The mice were randomly assigned into the following nine groups (12 mice in each group): normal, I/R, I/R + remifentanil, I/R + negative control (NC) for shRNA (sh-NC) (for sh-HIF1α), I/R + remifentanil +sh-NC (for sh-HIF1α), I/R + remifentanil +sh-HIF1α, I/R + sh-NC (for sh-ZEB1), I/R + remifentanil +sh-NC (for sh-ZEB1) and I/R + remifentanil +sh-ZEB1. Alternative mice used as back-ups for each group were not included in the statistical analysis. All mice were housed with a 12 hour/12 hour light-dark cycle at a temperature of 20-25°C, and 60% relative humidity. The mice were acclimated to their environment for 4 days before model establishment.
For hepatic I/R model establishment, the mice were anesthetized by intraperitoneal (i.p.) injection of 30 mg/kg of tiletamine/zolazepam solution supplemented with 10 mg/kg of xylazin. [29][30][31] After a midline laparotomy, the liver hilum was isolated carefully, followed by placement of a micro-vascular clamp at the first branch of the liver artery and portal vein supplying the left lateral and median lobes of the liver. The circulation of the caudal lobes remained intact to prevent congestion in the intestinal venous. The mice were placed on a heating pad, and saline soaked sterilized gauze was used to cover the peritoneum to prevent dehydration. Then, 90 minutes after hepatic ischaemia, the micro-vascular clamp was removed to allow the reperfusion, whereupon abdominal wall was closed with 6-10 nylon sutures. At 6 hours after reperfusion, whole blood was collected by retro-orbital puncture, and, upon euthanasia, liver samples were harvested for subsequent experiments. 29,32,33 The sham operation followed same procedure with omission of vascular occlusion. Before ischaemia, the control group was administrated with 0.9% saline i.p.
for three times for 1 minute at 5 minutes intervals. For remifentanil treatment, the hepatic I/R mice were injected (i.p.) with remifentanil (30 μg/kg in total; 091110, Yichang Renfu Pharmaceutical Industry) 3 times, and were then injected (i.p.) twice with 1 μL of lentivirus at a concentration of 5 × 10 8 TU/mL each time. [33][34][35] Animal use and experimental procedures were carried out in a protocol approved by the Experimental Animal Ethics Committee of Xi'an Jiaotong University. All experimental animals operating procedures were in line with the United States National Institutes of Health (NIH) laboratory animal care and usage guidelines.

| Liver function tests
The serum was isolated, and the levels of blood glucose, lipids, aspartate transaminase (AST), and alanine transaminase (ALT) were measure by an automatic chemistry analyser (Beckman). 36,37

| Haematoxylin and eosin (HE) staining
Mouse livers were dissected, fixed, dehydrated, embedded in paraffin and cut into 4 µm sections. The sections were dewaxed with xylene and rehydrated in graded ethanol as follows: xylene and then toluene for 5 minutes each, followed by 100% ethanol for 2 minutes, 95%, 80% and 75% ethanol for 1 minute each, and distilled H 2 O for 2 minutes.
Sections were then stained with haematoxylin for 5 minutes, washed with H 2 O, and rinsed with acid alcohol for 30 seconds, followed by immersion in water bath at 50°C for 5 minutes and eosin staining for 2 minutes. Finally, sections were dehydrated by 2 changes of 95% ethanol for 1 minute each, 2 changes of 100% ethanol for 1 minute each, xylene phenol (3:1) for 1 minute, and 2 changes of xylene for 1 minute of each and then sealed slide with mounting medium. Sections were then observed and photographed under an inverted microscope (XSP-8CA, Shanghai Optical Instrument Factory).

| Cell counting kit-8 (CCK-8)
Cells were seeded in a 96-well plate with a density of 2 × 10 3 cell/well. A well containing only medium was used for normalization. After 24 hours of transfection, 10 μL of CCK8 was added to wells at 0, 24, 48, 72 and 96 hours, followed by incubation at 37°C for 4 hours. A microplate reader (Bio-Rad) was employed to measure the optical density (OD) at 450 nm. The ratio of OD experimental /OD control was calculated to depict the cell growth curve. Experiments were repeated 3 times.

| Flow cytometry
Cells in each group were digested with 0.25% trypsin at 2 days after transfection, followed by addition of RPMI-1640 medium with 10% foetal bovine serum (FBS) to stop the digestion. Cells were centrifuged at 1000 r/min for 5 minutes, and the supernatant was removed.
70% ethanol was used to fix cells to adjust the cell density to 1 × 10 6 cell/mL. Next, cells were stained with 10 mL of Annexin V-FITC/PI

| Real-time quantitative polymerase chain reaction (RT-qPCR)
The total RNA was extracted with Trizol (15596026, Invitrogen) and reverse-transcribed into cDNA using reverse transcription kit (RR047A, Takara) using 20 μL reaction system, 37°C, 15 minutes, 85°C, 5 seconds. Then, RT-qPCR was performed with the TaqMan MicroRNA Assay and TaqMan ® Universal PCR Master Mix using the following cycles: 95°C for 2 minutes, followed by 45 cycles at 95°C for 15 seconds and 60°C for 45 seconds. miRNA was reversetranscribed by miRNA First Strand cDNA Synthesis, which was subjected to qPCR using a SYBR Premix Ex Taq kit (RR420A, Takara) in a real-time PCR machine (ABI 7500, ABI). The qPCR system was set up as follows: SYBR Mix 9 μL, forward primer 0.5 μL, reverse primer 0.5 μL, cDNA 2 μL, RNase-free dH 2 O 8 μL; 95°C for 10 minutes, 95°C for 15 seconds and 60°C for 1 minute, repeated over 40 cycles.
Samples were loaded with 3 replicates. Primers were synthesized by Sangon Biotech Co., Ltd. (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal reference for mRNA and U6 was used for miRNA. Relative expression of target genes was calculated by the 2 −ΔΔCT method. ΔΔCT = Ct experiment − Ct control , ΔCT = Ct target − Ct reference . 40

| shRNA screening
Mice HIF1α and ZEB1 sequences were obtained from GenBank to design shRNA. BLAST was used to screen out non-specific siRNAs.
The 2 shRNA candidates (listed in Table S1) were inserted into the PsiRNA-neo vector. After validation by restriction enzyme digestion and sequencing, the constructs were designated as sh-HIF1α-1, sh-H1F1α-2, sh-ZEB1-1 and sh-ZEB1-2, all of which were transfected into HOSEpiC cells. Western blot analysis was used to measure the expression level of HIF1α and ZEB1, to select the most effective shRNA for the subsequent experiments.

| Statistical analysis
The data analysis was performed using SPSS 21.0 (SPSS Inc).
Quantitative data were presented as mean ± standard deviation.
Data analysis between 2 groups was analysed by unpaired t test.
Data of different groups were processed by single factor variance analysis with Tukey's post hoc test. P < .05 indicated a significant group difference.

| Identification of hepatic I/R injury mouse model
The serum levels of blood glucose, lipids, AST and ALT were measured after the establishment of the hepatic I/R injury mouse model.
The results showed significantly increased levels of all 4 indicators d (P < .05) ( Figure 1A). The evaluation of histopathology of liver was made after HE staining, which revealed the presence of disorganized hepatic cells, inflammatory cell infiltration, and ballooning degeneration hepatocytes in I/R-induced hepatic injury ( Figure 1B). These data suggested the successful establishment of the hepatic I/R injury mouse model.

| Remifentanil can ameliorate hepatic I/R injury
Based on a report that remifentanil can relieve hepatic I/R injury, 34 we investigated the underlying molecular mechanism by first es-  Figure 2D). These data indicated that remifentanil could ameliorate hepatic I/R injury.

| Remifentanil alleviates hepatic I/R Injury by up-regulating the expression HIF1α
Since remifentanil reportedly promotes the expression of HIF1α, 16 we investigated the effects of remifentanil treatment in HIF1α ex- The CCK8 assay showed that cell proliferation was significantly increased in the remifentanil + I/R + sh-NC cells compared to I/R + sh-NC, whereas flow cytometry showed that apoptosis was significantly decreased (both P < .05). However, the cell proliferation was significantly decreased while apoptosis was significantly increased in remifentanil + I/R + sh-H1Fiα compared to remifentanil + I/R + sh-NC (P < .05) ( Figure 3H,I). In

| Remifentanil promotes ZEB1 and inhibits LIF expression by up-regulating HIF1α expression
Since HIF1α could promote ZEB1 expression, 41 we treated hypoxia-induced NCTC-1469 cell with remifentanil and silenced HIF1α. RT-qPCR and Western blot analysis showed that ZEB1 expression was considerably increased while LIF expression was considerably reduced in the remifentanil + I/R + sh-NC group compared to I/R + sh-NC (P < .05).
Compared to remifentanil + I/R + sh-NC treatment, the expression of ZEB1 was significantly lower while the expression of LIF was significantly higher with remifentanil + I/R + sh-HIF1α treatment (P < .05) ( Figure 4A,B). RT-qPCR and Western blot analysis showed that, in comparison to the I/R + sh-NC group, ZEB1 expression was considerably up-regulated while LIF expression was considerably down-regulated in

| Remifentanil ameliorates hepatic I/R injury by regulating ZEB1/LIF axis
Previous data demonstrated that remifentanil could promote ZEB1 and inhibit LIF expression by regulating the expression of HIF1α. To study this in detail, we designed 2 different shRNAs to knockdown ZEB1 in the hypoxia-induced NCTC-1469 cell line. Western blot analysis demonstrated that ZEB1 expression in sh-ZEB1-1 and sh-ZEB1-2 cells was significantly down-regulated compared to sh-NC (P < .05), where sh-ZEB1-2 showed better silencing efficiency, and was thus chosen for the subsequent experiments ( Figure 5A). Next, RT-qPCR and Western blot analysis results displayed that the expression of HIF1α and ZEB1 was notably increased while that of LIF was notably decreased in the remifentanil + I/R + sh-NC group compared to I/R + sh-NC group (P < .05). Compared to the remifentanil + I/R + sh-NC group, HIF1α expression was unchanged (P > .05) but ZEB1 expression was notably decreased (P < .05) and LIF expression was notably increased in the remifentanil + I/R + sh-ZEB1 group ( Figure 5B,C). CCK8 analysis of cell viability ( Figure 5D) and flow cytometry for apoptosis ( Figure 5E) showed that the cell viability was remarkably increased (P < .05) and apoptosis was remarkably decreased (P < .05) in the remifentanil + I/R + sh-NC group compared to I/R + sh-NC group. Compared to remifentanil + I/R + sh-NC group, cell viability was remarkably decreased (P < .05) and apoptosis was remarkably increased (P < .05) in the remifentanil + I/R + sh-ZEB1 group.   Figure 5I). In summary, these data indicated that remifentanil could ameliorate the hepatic I/R injury by regulating the ZEB1/LIF axis.

| D ISCUSS I ON
Hepatic I/R injury after transplantation or surgery is a major reason of liver failure. In recent years, remifentanil has gained increasing attention as a protective agent against I/R-induced injury in liver, intestine, uterus, heart and other organs. 5         HIF1α readily interacts with HIF1β to form a heterodimeric transcriptional complex. 46,47 Others have reported that HIF1α expression was up-regulated by remifentanil. 16 In addition, HIF1α has been investigated to protect mice from hepatic ischaemic damage. 48  In summary, our data demonstrate that remifentanil can ameliorate I/R-induced hepatic injury by regulating the ZEB1/LIF axis through up-regulation of HIF1α ( Figure 6). For the future clinical applications, our work offers potential therapeutic targets such as by inhibiting LIF expression for the treatment of hepatic I/R injury.
However, we note some limitations of our study, which call for further investigations. First, we established an in vitro I/R injury model in NCTC-1469 cells via hypoxia-reoxygenation, which may not completely mimic the pathophysiological process of hepatic I/R injury.
Besides, the mouse model of hepatic I/R injury was developed according to previous reported studies, including the selection of time of hepatic ischaemia and reperfusion, 29,32 but there is no accepted standard for these parameters. Therefore, in our future investigations, we shall explore further influence of durations of ischaemia and reperfusion.

ACK N OWLED G EM ENTS
We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data not shared.