Dexmedetomidine alleviates hepatic ischaemia‐reperfusion injury via the PI3K/AKT/Nrf2‐NLRP3 pathway

Abstract Hepatic ischaemia‐reperfusion (I/R) injury constitutes a tough difficulty in liver surgery. Dexmedetomidine (Dex) plays a protective role in I/R injury. This study investigated protective mechanism of Dex in hepatic I/R injury. The human hepatocyte line L02 received hypoxia/reoxygenation (H/R) treatment to stimulate cell model of hepatic I/R. The levels of pyroptosis proteins and inflammatory factors were detected. Functional rescue experiments were performed to confirm the effects of miR‐494 and JUND on hepatic I/R injury. The levels of JUND, PI3K/p‐PI3K, AKT/p‐AKT, Nrf2, and NLRP3 activation were detected. The rat model of hepatic I/R injury was established to confirm the effect of Dex in vivo. Dex reduced pyroptosis and inflammation in H/R cells. Dex increased miR‐494 expression, and miR‐494 targeted JUND. miR‐494 inhibition or JUND upregulation reversed the protective effect of Dex. Dex repressed NLRP3 inflammasome by activating the PI3K/AKT/Nrf2 pathway. In vivo experiments confirmed the protective effect of Dex on hepatic I/R injury. Overall, Dex repressed NLRP3 inflammasome and alleviated hepatic I/R injury via the miR‐494/JUND/PI3K/AKT/Nrf2 axis.

critical for improving the prognosis of hepatic I/R injury, especially for those patients receiving surgery with prolonged ischaemia time or marginal liver transplantation.
The restoration of blood flow triggers tissue inflammation and ischaemic injury by activating multiprotein complex named inflammasome. 5 Inflammasome is implicated in the pathogenesis of hepatic I/R injury, which is accepted as a crucial contributor to hepatocyte injury. 5,6 After inflammasome activation, the affected tissues undergo apoptosis and another inflammation-associated cell death, namely pyroptosis. 5,7 Pyroptosis is a form of lytic programmed cell death initiated by inflammasomes. 8 Pyroptosis is viewed as a universal and natural immune mechanism in vertebrates, which causes inflammation in bacterial infection and various non-infectious diseases including types of hepatic injury. 9 Although there lacks direct evidence of the presence of pyroptosis in hepatic I/R injury, the inflammasome activation in hepatic I/R injury has been elucidated, indicating the involvement of pyroptosis in hepatic I/R injury. 10 Intriguingly, a previous study has unveiled that inhibition of pyroptosis ameliorates hepatic I/R injury and represses inflammatory reaction. 11 Activation of some critical survival pathways or inhibition of apoptotic/ pyroptotic pathways using drugs or small molecules contributes to alleviating hepatic I/R injury before or during liver surgery or transplantation. 4 Dexmedetomidine (Dex), a highly selective α 2 -adrenergic agonist, bears the potent properties of sedation and analgesia, which is extensively applied in critically ill and anaesthetic patients in clinical. 12 Accumulating evidences have elucidated that Dex protects lung, cerebrum and liver against I/R injury by suppressing proinflammatory signalling and reducing cell death. [13][14][15][16][17] For example, Dex exerts protective effect on hepatic I/R injury by repressing inflammatory response and oxidative stress in mice. 18 Also, the differentially expressed microRNAs (miRNAs) are verified to participate in the mechanisms of Dex action. 19 miRNA has been accepted as a novel target for treating hepatic I/R injury. 4

miR-494
represses hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis. 20 miR-494 attenuates hepatic I/R injury in a rat model. 21 Overexpression of miR-494 upregulated HIF-1α expression under hypoxia and exerts protective effects against hypoxia-induced apoptosis, which suggests that miR-494 functions as a therapeutic target for hepatic I/R injury. 22 Still, whether Dex can protect liver from I/R injury by targeting miR-494 remains unknown. This study investigated the specific protective mechanism of Dex in hepatic I/R injury, which shall confer novel insights into the management of hepatic I/R injury.

| Ethics statement
All the animal experiments were implemented on the guide for the care and use of laboratory animals and on minimized animal number and the least pains.

| Western blotting
Cells or tissues were treated with RIPA lysis buffer to extract the total protein, and the protein concentration was detected using the bicinchoninic acid kit (Pierce, Waltham, MA, USA). The protein sample was separated by 12% SDS-PAGE and transferred onto cellulose nitrate membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membranes were blocked with phosphate-buffered saline containing 5% skim milk for 2 h and incubated with the primary antibodies at 4°C overnight and then with the secondary antibodies for 2 h. The protein band was developed using enhanced chemiluminescence kit (Thermo Fisher Scientific) and quantified using ImageJ software. The primary antibodies were as

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cells or tissues using TRIzol reagent (Takara, Tokyo, Japan), and 500 ng total RNA was reverse transcribed into cDNA using PrimeScript RT Reagent kit (Takara). CFX 96 qPCR system (Bio-Rad) and SYBR RT-PCR kit (Takara) were used for RT-qPCR. The relative expression of miR-494 and JUND was calculated by 2 −ΔΔCT method, with GAPDH and U6 as the internal reference. Each sample was repeated 3 times independently. Primer sequences are shown in Table 1.  Dex was administered intravenously at the beginning of the operation at a loading dose of 3 μg/kg and then at a dose of 3 μg/ kg/min for the next 2 h. 24 The rats in the sham group received all operations except clamping the hepatic pedicle. After abdominal suture, the rats were allowed to recover with free access to food and water. After 24 h of reperfusion, rats were euthanized by intraperitoneal injection of 1% pentobarbital (800 mg/kg). The blood was extracted from left ventricle by a syringe, centrifuged at 2000 g and 4°C for 15 min and stored at −80°C. The rat liver tissues were collected and fixed in 4% paraformaldehyde. The rats were assigned into sham group (rats received all operations except clamping the hepatic pedicle), I/R group (rats received I/R treatment), I/R + DEX group (rats received I/R treatment and intravenous injection of Dex), I/R + DEX + antagomiR-NC group (after antagomiR-NC transfection, rats received I/R treatment and intravenous injection of Dex) and I/R + DEX + antago-miR-494 group (after antagomiR-494 transfection, rats received I/R treatment and intravenous injection of Dex). Each group had 12 rats, among which 6 rats were used for tissue section staining and 6 rats were used for protein or RNA extraction and detection.

| Haematoxylin and eosin (HE) staining
The liver tissues fixed in 4% paraformaldehyde for 24 h were em-

| Suzuki score
Suzuki score was used to evaluate liver tissue injury in rats, and the Suzuki score criteria are shown in Table 2 below.

| Immunohistochemical staining
The liver tissue sections were treated with 0.01 mol/L citric acid buffer

| Statistical analysis
Data were analysed and introduced using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Data are expressed as mean ± standard deviation. Kolmogorov-Smirnov test was used to test the normal distribution. The t test was adopted for comparison between two groups.
One-way analysis of variance (ANOVA) was employed for the comparisons among multiple groups, following Tukey's multiple comparisons test. The p value was obtained from a two-tailed test and the p < 0.05 meant the statistically significance.

| Dex protected H/R cells through miR-494
miRNA plays a vital role in hepatic I/R injury and may become a novel tool to diagnose and monitor hepatic I/R injury. 26 miR-494 can play a protective role in liver I/R injury. 21 Therefore, we specu-

| miR-494 targeted JUND
To investigate the potential mechanism of miR-494 in H/R cells, we

| Dex protected hepatic I/R injury in vivo
To was detected using RT-qPCR ( Figure 6A). The phosphorylation of PI3K/AKT/Nrf2 pathway-related proteins and the NLRP3 protein level were detected using Western blotting ( Figure 6E). The results were consistent with the above cell experiment results (p < 0.001).
Taken together, Dex exerted protective effect on hepatic I/R injury in rats.

F I G U R E 4 Upregulation of JUND reversed the protective effect of Dex on H/R cells. The effects of Dex on H/R cell pyroptosis and
inflammatory factors were observed after JUND was overexpressed in the model cells. A, JUND expression was detected using RT-qPCR. B, The pyroptotic proteins were detected using Western blotting. C, The contents of inflammatory factors were detected using ELISA. The cell experiment was repeated 3 times. Data were presented as mean ± standard deviation and analysed using one-way ANOVA, followed by Tukey's multiple comparison test, *** p < 0.001

| DISCUSS ION
The liver is highly dependent on oxygen supply and vulnerable to hypoxia; and the adverse consequences of hepatic I/R injury remain a tough issue in clinical practice. 33 Dex is an anaesthetic adjuvant that can reduce inflammatory reaction during the perioperative period, suggesting that it may become a novel therapeutic approach to alleviate I/R injury. 34 The pathogenesis of hepatic I/R injury includes the occurrence of oxidative stress and the release of pro-inflammatory factors, and Ma XG, et al. found that Dex reduced H/R-induced elevation of ROS and MDA levels. 35 Meanwhile, Zhou H, et al. found that Dex pretreatment can promote the activation of macrophage M2 and inhibit the activation of hepatic inflammatory innate immunity in a PPARγ/STAT3 dependent manner. 36 In this study, we found the Dex regulates the activation of NLRP3 inflammasomes by regulating miR-494/JUND, which further confirmed that Dex protects hepatic I/R by inhibiting inflammation in the liver.
Hepatic I/R induces inflammation and oxidative stress, resulting in the injury of liver and distant organs. 37 Dex alleviates inflammatory response and shows protective effect in various animal models of I/R injury. 13 The liver enzymes ALT and AST are commonly utilized to evaluate hepatic injury; and α-GST serves as a sensitive marker of hepatic I/R injury. 38 Our results demonstrated that Dex treatment notably reduced the levels of α-GST, IL-6, TNFα, ALT and AST in serum, suggesting that Dex exerted protective effects on hepatic I/R injury clinically. Dex mitigates hepatic I/R injury, and its protective mechanism may be concerned with reducing biochemical factors AST and ALT and inflammatory cytokines and enhancing Bcl-2, thereby attenuating inflammatory response and suppressing apoptosis in mice. 18,24 Our study confirmed the protective role of Dex in hepatic I/R injury.
To observe the effect of Dex on cell model, we simulated hepatic I/R injury in human liver cell line L02 through H/R treatment.
Inflammasome activation has been recently described as a promising therapeutic target of hepatic I/R injury. 39 Typical inflammasomes, including NLRP3, recruit caspase-1 under a series of microbial stimulation and endogenous risk signals, and then caspase-1 induces a form of lytic programmed cell death called pyroptosis. 40 It is well established that inflammasome activation is responsible for pyroptosis. 41 Pyroptosis is featured by the activation of caspase-1, which eventually results in the cleavage of GSDMD and the secretion of IL-1β and IL-18. 37 GSDMD is a genetic substrate of inflammatory caspases, which is implicated in pyroptosis and IL-1β release. 25 ASC represents a scaffold protein that is essential to recruit effector enzyme pro-caspase-1 into NLRP3 inflammasome. 42  and AKT in each group. A, Nrf2, PI3K, p-PI3K, AKT, p-AKT and NLRP3 levels were detected using Western blotting. The cell experiment was repeated 3 times. Data were presented as mean ± standard deviation and analysed using one-way ANOVA, followed by Tukey's multiple comparison test, *** p < 0.001 F I G U R E 6 Dex protected hepatic I/R injury in vivo. The rat model of hepatic I/R was established. A, miR-494 and JUND expression was detected using RT-qPCR. B, The levels of ALT and AST in serum of rats were detected using ELISA. C, The rat liver sections were stained with HE staining. D, The rat liver sections were stained with immunohistochemical staining. E, The changes of JUND, p-AKT, AKT, Nrf2 and NLRP3 were detected using Western blotting. N = 6. Data were presented as mean ± standard deviation and analysed using one-way ANOVA, followed by Tukey's multiple comparison test, *** p < 0.001 from HMGB1-induced cell injury. 45 Consistently, Dex ameliorates myocardial I/R injury in rats and represses H/R-induced pyroptosis in cardiomyocytes by reducing miR-29b. 46 These results indicated that Dex protected the cell model of I/R injury by suppressing pyroptosis.
Then, we investigated the molecular mechanism of Dex protecting liver from I/R injury. Aberrant miRNA expression is implicated in the pathogenesis of I/R injury, because they modulate the cell participants and humoral factors related to I/R injury. 26 miR-494 is differentially expressed in cerebral I/R injury in rats 47 and shows protective effect on myocardial I/R injury. 48  Nrf2 activation is a potent intervention that can protect hepatic I/R injury during and after surgery. 56 In this study, H/R treatment inhibited the phosphorylation of PI3K and AKT and the level of Nrf2, while Dex reversed these trends and activated the signalling pathway. The inhibition of oxidative stress and apoptosis observed in Dex-treated mice with I/R injury may be attributed to the enhanced Nrf2. 18 Excessive activation of NLRP3 facilitates myocardial, cerebral and hepatic I/R injury. 57 The crosstalk between Nrf2 and inflammasome has been unveiled, and Nrf2 activation represses NLRP3 inflammasome and inflammation, 31,58 which is consistent with the results of Western blot in our study. NLRP3 silencing exerts protective effects on hepatic I/R injury in mice mainly by downregulating caspase-1 activation and reducing IL-1β and IL-18 secretion. 59 Briefly, Dex repressed NLRP3 inflammasome by activating the PI3K/ AKT/Nrf2 pathway. Moreover, in vivo experiments verified that Dex reduced the positive expression of NLRP3, and Dex had a protective effect on hepatic I/R injury in rats.
To sum up, Dex upregulated miR-494 expression to inhibit JUND and activated the PI3K/AKT/Nrf2 pathway, thereby repressing NLRP3 inflammasome and alleviating hepatic I/R injury. This study simply revealed that Dex inhibited inflammation through miR-494 and its target gene to protect liver from I/R injury, but the specific mechanism of Nrf2 in NLRP3 inflammasome failed to be deeply studied. Nrf2 activation is tightly related to its entry and exit from the nucleus, and the regulation mechanism of out-and inside nucleus of Nrf2 in NLRP3 remains to be studied. In the future, we will further study on the mechanism of Nrf2 in regulating NLRP3, which may provide novel insights into the clinical treatment of hepatic I/R injury.

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

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data generated or analysed during this study are included in this published article.