MicroRNA-141-3p attenuates oxidative stress-induced hepatic ischemia reperfusion injury via Keap1/Nrf2 pathway

Hepatic ischemia reperfusion injury (IRI) is a major factor affecting the prognosis of liver transplantation through a series of severe cell death and inflammatory responses. However, the potential role of miR-141-3p in hepatic IRI is currently unknown. We collected the serum of liver transplantation patients to study the relationship between miR-141-3p and liver injury. A mouse hepatic IRI model was established to measure hepatic dysfunction and cell apoptosis. MiR-141-3p mimic and inhibitor were transfected into hepatocytes to explore the characteristics of hypoxia/reoxygenation (H/R), a classical hepatic IRI in vitro model. We found that miR-141-3p levels were negatively correlated with alanine aminotransferase (ALT)/aspartate aminotransferase (AST) in liver transplantation patients. The results demonstrated that miR-141-3p was decreased in mouse liver tissue after hepatic IRI in mice and in hepatocytes after H/R. Overexpression of miR-141-3p directly decreased Kelch-like ECH-associated protein 1 (Keap1) levels and attenuated cell apoptosis in vivo and in vitro, while inhibition of miR-141-3p facilitated apoptosis. Further experiments revealed that overexpression of miR-141-3p also attenuated oxidative stress-induced damage in hepatocytes under H/R conditions. Our results indicate that miR-141-3p plays a major role in hepatic IRI through the Keap1 signaling pathway, and the present study suggests that miR-141-3p might have a protective effect on hepatic IRI to some extent.


Introduction
Liver transplantation is an effective treatment for end-stage liver disease [1]. The liver is susceptible to hypoxia due to being highly dependent on oxygen. Hepatic ischemia reperfusion injury (IRI) is a common consequence of liver transplantation and liver resection. Hepatic IRI includes two stages: ischemic injury and inflammatory-regulated reperfusion. A series of events occur during hepatic IRI, including reactive oxygen species (ROS) generation, peroxidation of deoxyribonucleic acid (DNA) and proteins, which can cause a series of cascading reactions, such as inflammation, cell death, and hepatic failure [2,3]. As previously reported, some treatment measures for hepatic IRI have been studied, such as ischemic preconditioning, surgical interventions, targeted therapy, and gene therapy [4,5]. However, these strategies are controversial due to their poor validity.
MiR-141 belongs to the miR-200 family. It has been reported that miR-141 strongly influences nonalcoholic fatty liver disease (NAFLD) development by negatively regulating expression of the sirtuin 1 (SIRT1) gene and protein while at the same time ameliorating liver function [15]. MiR-141 has also been proposed as a molecular biomarker of various hepatic disorders [16]. As previously reported, miR-141 suppresses Keap1 levels, which activate the Nrf2-dependent pathway and confer 5-FU resistance in hepatocellular carcinoma (HCC) [17]. However, the underlying function of miR-141 in hepatic IRI is not clear.
In this study, we demonstrated that miR-141-3p expression is decreased in liver transplantation patients after surgery and hepatocytes under hypoxia/reoxygenation (H/R) conditions. MiR-141-3p overexpression protects against liver IRI. Moreover, Keap1 might be a target of miR-141-3p during mice hepatic IRI and hepatocyte H/R stress. This study provides new ideas for the development of novel treatment strategies for hepatic IRI.

Serum collection
Whole blood from 27 liver transplantation patients was collected preoperatively, 4 h after reperfusion, and on days 1, 2, and 3 after surgery. Blood was collected allowed to stand and clot at room temperature (RT). Then the tube was centrifuged at 3000 g for 15 min at 4 °C, and stored at − 80 °C. The serum samples were used to detect the level of miR-141-3p, Interleukin-6 (IL-6) and interleukin-1β (IL-1β). All procedures were approved and were performed with the patient's informed consent, and this research was approved by the human ethics committee of the First Affiliated Hospital of Chongqing Medical University.

Enzyme-linked immunosorbent assay (ELISA)
Serum samples were collected from patients as described above. IL-6 (4A Biotech, China) and IL-1β (NeoBioscience, China) concentrations in the serum were determined by ELISA kits according to the instructions.

Serum levels of aminotransferase
Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are indexes of liver injury. ALT and AST assay kits (Nanjing Jiancheng, China) and microplate readers (Biotek, USA) were used to measure mouse serum ALT and AST levels.

Construction of the hepatic ischemia/reperfusion (I/R) injury model
C57BL/6 J mice (male, 18-20 g, 6-8 months old) were purchased from Chongqing Medical University Experimental Animal Center (Chongqing, China). Animals were maintained in a specific pathogen-free (SPF) setting with 12 h light/12 h dark conditions. All animal experiments were approved by the institutional animal care and use committee.
A warm partial (70%) liver I/R injury model was established as previously described [18]. After anesthesia with the sodium pentobarbital (intraperitoneal injection, 50 mg/ kg), we opened the abdomen and clamped the left liver and middle liver artery. The clamp was removed after 60 min of ischemia and then reperfusion was performed (reperfusion time: 0 h, 1 h, 3 h, 6 h, 9 h) (n = 5 in each time point). The control group was the sham operation group (n = 5). Mice were euthanized with an overdose of sodium pentobarbital (100 mg/kg intravenous). Subsequently, the liver tissues and 1 ml venous blood were harvested for subsequent experiments.

Cell culture
The human liver LO2 cell line was purchased from Zhong Qiao Xin.
Zhou Biotechnology Co., Ltd (Shanghai, China). Cells were incubated in RPMI 1640 basic medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Biological Industries, Israel). For the H/R model, cellular hypoxia was induced by incubation in serum-free medium and culturing cells in a tri-gas incubator (Thermo, MA, USA) with 94% N 2 , 1% O 2 , and 5% CO 2 . Cells were exposed to hypoxia for 2 h, 4 h, 8 h, 12 h and 24 h followed by reoxygenation for 6 h.

Hematoxylin and eosin (HE) staining
After the left lobe of the liver was obtained, part of the liver tissue was fixed in 10% buffered formalin. After deparaffinization and rehydration, hematoxylin/eosin staining and mounting were performed and sections were observed at 200× magnification.

Western blot analysis
Total protein and nuclear protein were extracted primarily using RIPA and nuclear protein extraction kit (Beyotime, Shanghai, China). Protein concentration was measured by BCA (Beyotime, Shanghai, China). After separation in gels, blocking was performed for 15 min using quick western blocking solution. After overnight incubation with primary antibodies, proteins were incubated with secondary antibody (1:10,000 diluent) for 1.5 h. We visualized the proteins using Fusion FX7.

Measurement of intracellular ROS accumulation
DCFH-DA fluorescence dye was used to examine intracellular ROS accumulation. Briefly, LO2 cells were seeded in plates and treated with the miRNA-141-3p mimic and inhibitor. After incubation, DCFH-DA was added to serumfree culture medium, which was added to the cells and then incubated for 15 min in the dark at 37 °C. For flow cytometry analysis, dichlorofluorescein (DCF) fluorescence intensity was measured using a BD FACSAria II flow cytometer (USA). For confocal laser scanning microscopy (CLSM), after DCFH-DA staining, cells were incubated with Hoechst for 15 min at 37 °C, and ROS levels were observed under CLSM (ZESS, Germany) at 488 nm by comparing the fluorescence intensity (green signal).

Statistical analysis
Data are expressed as the mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance (ANOVA). Correlation analysis was performed using Spearman's rank correlation method. SPSS 18.0 software (SPSS, Chicago, IL, USA) was used to perform statistical analyses, and P < 0.05 was considered statistically significant.

MiR-141-3p is correlated with the recovery of liver function in the serum of patients with liver transplantation
To examine the significance of miR-141-3p in liver transplantation, we first collected serum from 27 liver transplantation patients at the following time points: preoperation, 4 h after reperfusion, and on postoperative days 1, 2 and 3. Basic patient characteristics are summarized in Table 1, and the laboratory data of patients are summarized in Table 2  and Supplementary Table 1. Next, we assessed miR-141-3p expression. We found that expression of miR-141-3p 4 h after perfusion was lower than that at preoperation, and over time, miR-141-3p increased (Fig. 1a). In contrast, AST and ALT levels, which reflect liver function, were increased 4 h after perfusion compared to preoperation and decreased gradually from days 1 to 3 (Fig. 1b). As shown in Fig. 1c, IL-1β and IL-6 were increased after liver transplantation and gradually decreased after postoperative day 2. Moreover, we analyzed the correlation between expression of miR-141-3p and ALT/AST levels. We found a significant negative correlation between miR-141-3p expression and ALT/AST levels (Fig. 1d, e). Expression of miR-141-3p was strongly correlated with ALT/AST levels 4 h after perfusion (r > 0.80, P < 0.001). These data all suggest that miR-141-3p is negatively correlated with the recovery of liver function.

MiR-141-3p is downregulated in liver tissue in the hepatic IRI model
To determine expression of miR-141-3p in the hepatic IRI model, we established a mouse hepatic IRI model. We found that levels of mouse serum ALT or AST in the IRI groups were significantly increased compared to the sham group (Fig. 2a). Levels of both serum ALT and AST gradually increased with reperfusion time. HE analysis revealed large areas of hepatocyte necrosis in hepatic IRI group mice compared to sham group mice (Fig. 2b). MiR-141-3p expression  was decreased in the IRI groups compared to the sham group (Fig. 2c). We chose 6 h as our reperfusion time with consideration of the expression between miR-141-3p and ALT/ AST levels. These data demonstrate that the miR-141-3p levels are decreased in the hepatic IRI model.

MiR-141-3p inhibits IRI-triggered hepatic dysfunction and cell apoptosis in vivo
To further establish the influence of miR-141-3p on hepatic IRI, mice were transfected with lentivirus. We found that miR-141-3p expression in liver tissues was significantly decreased in both the model and vector control groups, while it was increased in the miR-141-3p treatment group (Fig. 3a). HE analysis revealed large areas of hepatocyte necrosis in the model group mice compared to the sham group, and the degree of necrosis in the miR-141-3p treatment group was less than that in the model and vector control groups (3b-c). Furthermore, Bcl-2 expression in the miR-141-3p group was higher than in the vector control group, and Bax and cleaved-caspase3 expression in the miR-141-3p group was lower than in the vector control group (Fig. 3d). Finally, western blot analyses showed that Keap1 expression in the miR-141-3p group was lower than in the vector control group (Fig. 3e). Therefore, miR-141-3p ameliorates IR-triggered hepatic dysfunction and apoptosis.

MiR-141-3p inhibits the generation of intracellular ROS in H/R-induced injury
We tested ROS generation mediated by miR-141-3p in LO2 cells. Intracellular ROS levels were measured using the DCFH-DA fluorescence method after treatment.
Flow cytometry data showed that ROS induction in the H/R + miR-141-3p mimic group was decreased compared to the NC group and was increased in the H/R + miR-141-3p inhibitor group (Fig. 5a). Meanwhile, we measured intracellular ROS with CLSM after DCFH-DA and Hoechst staining. Our data revealed that ROS levels were decreased in the H/R + miR-141-3p mimic group (Fig. 5b). This indicates that miR-141-3p also inhibits ROS generation in LO2 cells.

Discussion
IRI is a condition in which ischemic organs experience restored blood flow, which leads to more serious damage, further inducing organ dysfunction. Hepatic IRI often occurs in hypovolemic shock, hepatic resections and liver transplantation [19]. In the current study, we measured the function of miR-141-3p in hepatic IRI. Our results showed that Keap1 signaling might be the signaling pathway  ern blot after H/R. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group. e MiR-141-3p levels were analyzed after treatment with miR-141-3p mimic and inhibitor. f Western blot analysis of Bax, cleaved-caspase3 and Bcl-2 expression. g Western blot analysis of Keap1, Nrf2, HO-1 and NQO1. n = 6 for each group *P < 0.05, **P < 0.01, ***P < 0.001 compared to the NC group through which miR-141-3p attenuates oxidative damage and apoptosis in vivo and in vitro. Some miRNAs have been reported to participate in modulating apoptosis and oncogenesis [20]. A previous study indicated that ischemia preconditioning attenuates hepatic post-ischemia tumor necrosis factor release from Kupffer cells, reducing liver injury following hepatic IRI and that the effect of preconditioning is mediated by nitric oxide [21]. MiR-141 belongs to the miR-200 family, and the miR-200 family is upregulated early after ischemic preconditioning and is neuroprotective primarily by downregulating proly1 hydroxylase 2 levels [22]. Therefore, we speculated that miR-141-3p overexpression exerts protective effects similar to ischemic preconditioning in hepatic IRI.
In the present study, our vision shifts onto the expression profile and local action of miR-141-3p throughout the whole stage of hepatic IRI. It is worth mentioning that in the early stage of liver transplantation, differential expression of miR-141-3p in the serum is meaningful in the present study, as it was negatively correlated with ALT/ AST levels. As clinical prognosis data require long-term follow-up, prognostic analysis was not performed in this study, but relevant analyses will appear in our future clinical studies. Consistent with the serum results, we also found that miR-141-3p levels were decreased after hepatic IRI or hepatocyte H/R both in vivo and in vitro.
Reports have shown that miR-141 also participates in modulating oxidative stress-induced apoptosis. MiR-141 attenuates ultraviolet (UV)-induced damage through Nrf2 stabilization and activation in retinal pigment epithelium cells [23]. MiR-141-3p interacts with Chromodomain-helicase-DNA-binding protein 8 (CHD8), which plays critical roles in cardiomyocyte apoptosis induced by H/R [24]. The Keap1-Nrf2 complex may ameliorate hepatic IRI in orthotopic liver transplantations, as Keap1 negatively regulates Nrf2, and Keap1-Nrf2 regulates protein kinase B (Akt) activation, which is beneficial to cell survival [25]. There is evidence that depletion of Nrf2 increases susceptibility to liver injury [26], indicating that Nrf2 plays a major role in the hepatic protective pathway. HO-1 is a stress-induced isoform that attenuates oxidative damage, and previous b Confocal laser scanning microscopy analysis of intracellular ROS (200 × magnification). Scale bar = 30 μm. **P < 0.01 compared to the NC group studies have reported that HO-1 protects against liver, neurological, renal, and intestinal IRI [27,28].
These studies have shown that miR-141-3p regulates oxidative stress by targeting Keap1. In our study, it was confirmed that miR-141-3p alleviates oxidative stress damage in the liver through Keap1. In addition, a previous report showed that miR-141 attenuates myocardial IRI via antithetical regulation of intercellular adhesion molecule-1(ICAM-1) and inflammatory cells [29]. This report indicates that miR-141 also ameliorates hepatic IRI through other pathways, but this hypothesis requires further investigation.
In conclusion, in this study, we combined clinical and basic experimental research methods and found that miR-141-3p ameliorates hepatic IRI both in vivo and in vitro. The potential mechanism of this protection is related to the inhibition of Keap1 and resulting reduced degradation of Nrf2, which attenuated oxidative stress-induced damage and apoptosis. The study suggests that miR-141-3p might be a potential test index molecule and therapeutic target in hepatic IRI.