Dexmedetomidine alleviates pulmonary ischemia-reperfusion injury through modulating the miR-21-5p/Nr4a1 signaling pathway

1Department of Anesthesiology, The First Affiliated Hospital of Shantou University Medical College, Shantou City, Guangdong Province, 515041, China; 2Department of Sector 2 of Hepatic and Gallbladder Surgery, The First Affiliated Hospital of Shantou University Medical College, Shantou City, Guangdong Province, 515041, China; 3Department of Molecular Biology Laboratory, The First Affiliated Hospital of Shantou University Medical College, Shantou City, Guangdong Province, 515041, China; 4Department of Radiology, The First Affiliated Hospital of Shantou University Medical College, Shantou City, Guangdong Province, 515041, China; 5Department of paediatrics, The First Affiliated Hospital of Shantou University Medical College, Shantou City, Guangdong Province, 515041, China


INTRODUCTION
Pulmonary ischemia-reperfusion injury (PIRI) is a kind of aseptic acute pulmonary injury which is presented in many pathological conditions and medical situations, including pulmonary embolism (Clarke et al., 2002), hemorrhagic shock (Oda et al., 2002), trauma , pulmonary transplantation (Sugimoto et al., 2019) and open-heart surgery (Amour et al., 2019). This event is a common surgical complication that can lead to pulmonary dysfunction and reduce the survival rate of patients (den Hengst et al., 2010). Studies have shown that the pathogenesis of PIRI is complex, involving many pathophysiological processes and pathways, such as apoptosis (Wagner et al., 2006), autophagy , lymphocyte and macrophage infiltration (van der Kaaij et al., 2009) in oxidative stress injury , calcium overload  and inflammatory injury (Krishnadasan et al., 2002). Therefore, many pharmacological agents involved in these pathophysiological processes and pathways were studied to alleviate PIRI, for instance, dexmedetomidine (Dex) (Liang et al., 2019), pirfenidone (Saito et al., 2019) and dexamethasone (Zhou et al., 2012).
In particular, Dex is a highly potent and selective α2-adrenergic agonist which has multiple pharmacological functions, such as anxiolysis, sedation, antioxidant, anti-inflammatory and anti-apoptosis (Vora et al., 2015;Wang et al., 2015). Recent studies have suggested that Dex could alleviate ischemia-reperfusion injury of different organs (Yuan et al., 2017;Ren et al., 2018;Liu et al., 2019). Wang, et al., found that Dex may inhibit neuronal apoptosis via inhibition of hypoxia-inducible factor-1alpha to alleviate cerebral ischemia-reperfusion injury in rats . Ma, et al., indicated that Dex displayed renoprotection in renal ischemia-reperfusion injury, and acted as an anti-inflammatory effector to the parasympathetic nervous system activation (Ma et al., 2018). Though the previous studied have reported that Dex could reduce PIRI by its anti-apoptotic effects (Zhang et al., 2016;Kucukebe et al., 2017;Xue et al., 2019), the exact mechanisms underlying the effects of Dex on reducing PIRI remain to be further studied.
MicroRNAs (miRNAs) are non-coding RNAs of 22-25 nucleotides in length and play a negative role in post-transcriptional gene expression. In previous studies, it was shown that many microRNAs play an important role in ischemia-reperfusion injury Huang et al., 2019;. For example, miR-496 could reduce cerebral ischemia-reperfusion injury by inhibiting the BCL2L14 expression (Yao et al., 2019). And miR-219a-5p, an age-related miRNA, was reported to attenuate hepatic ischemia-reperfusion injury by inhibition of TP53BP2 in mice (Xiao et al., 2019). Importantly, miR-21-5p, acting as an anti-apoptotic agent, has been reported to mediate mesenchymal stromal cellsecreted exosomes to relieve ischemia-reperfusion injury in mice . However, the role of miR-21-5p in PIRI is still unclear.
In this study, the PIRI mouse model was established and mouse pulmonary vascular endothelial cells (MPVECs) were treated with hypoxia reoxygenation (H/R) to investigate the effect of Dex on pulmonary reperfusion injury. Also, TargetScan was used to predict biological targets of miR-21-5p, and Nr4a1 was identified. In addition, further rescue experiments proved that overexpression of Nr4a1, as a target of miR21-5p, could block the effect of Dex on H/R-treated MPVECs. Thus, the function and mechanism of Dex in PIRI was investigated in this study.

Drugs and reagents.
Dexmedetomidine hydrochloride (Dex) was purchased from Sigma Aldrich (St Louis, MO, USA). Dex was dissolved in normal saline at a 20 mg/mL concentration as a stock solution. The Nr4a1-WT or Nr4a1-MUT reporter plasmids were constructed by cloning the wild type or mutated miR-21-5p binding sequence into the pGL3 reporter plasmids (GeneScript, Nanjing, China). The Nr4a1 cDNA fragment was cloned into pcDNA3.1 (Life Technologies, Darmstadt, Germany) to construct the Nr4a1 overexpression vector. All of the constructed plasmids were verified by sequence analysis (not shown). In addition, the Nr4a1 overexpression vector was verified by qRT-PCR. The mimics of miR-21-5p, miR-21-5p inhibitor and negative control RNA were obtained from GenePharma (Shanghai, China). Other reagents involved in the study were acquired from a reputed vendor.
Cell lines. Mouse pulmonary vascular endothelial cells (MPVECs) (cat. no. BNCC340787) were purchased from the BeNa Culture Collection (BNCC). Human embryonic kidney cancer cells (HEK293) (cat. no. GNHu43) were purchased from the Cancer Institute of Chinese Academy of Medical Science. MPVECs and HEK293 cells were cultured at 37°C in Dulbecco's Modified Eagle's Medium (Hyclone, Logan, UT, USA) supplemented with 10% Fetal Bovine Serum (FBS) and 100 U/ml penicillin-streptomycin and were cultivated in a humidified atmosphere containing 5% CO 2 in the air.
Animals. This study was conducted at the First Affiliated Hospital of Shantou University Medical College with the consent from the Experimental Animals Ethics Committee of the First Affiliated Hospital of Shantou University Medical College. The permission number for animal study is SUMC2019-310. All procedures were performed according to the Guides for the Care and Use of Laboratory Animals.
Pulmonary ischemia-reperfusion injury model. Healthy female clean grade C57BL/6J mice (6 to 8-week-old) with body weight of 22-25g were involved to establish the PIRI mice model as previously described (Cao et al., 2015). Briefly, anesthesia was performed with air containing 1% isoflurane, through tracheostomy, and we then intubated a mouse with intravenous puncture needle and mechanically ventilated the mouse with an oxygen rich atmosphere by a small animal respirator. The mouse was placed on the right side, and the chest was cut through the 5th intercostal space through the left thoracic incision. After exposing the left pulmonary, we blocked the left pulmonary hilum with a non-invasive microvascular clip, resulting in pulmonary ischemia for 30 minutes. The microvascular clip was removed, and pulmonary blood perfusion was restored for 2 hours.
In this study, 24 mice were randomly divided into the Sham group, ischemia reperfusion (I/R) group, Sham+Dex group and ischemia reperfusion (I/R)+Dex group (n=6 in each group). Mechanical ventilation was performed without ischemia reperfusion in the Sham group. In the treatment group (Sham+Dex group and I/ R+Dex group), 20 μg/kg of Dex (100 μg /mL) was injected through the caudal vein.
Pulmonary tissue wet/dry (W/D) weight ratio. The W/D ratio was determined as previously described . After pulmonary blood flow reperfusion for 2 hours, the left pulmonary was taken and weighed immediately to obtain the wet weight of pulmonary tissue. Next, we dried the pulmonary in an oven at 60°C for 48 hours to measure the dry weight. The W/D ratio was used to assess the pulmonary water content.
Hematoxylin and eosin (H&E) staining. The pulmonary tissues were dissected from the mice in different groups as described (Naderali et al., 2018). Briefly, the pulmonary tissues were fixed by dipping in 4% paraformaldehyde for 48 hours, then the tissues were embedded in paraffin and cut into 3-4 μm thick slices. Finally, hematoxylin and eosin staining (H&E) was performed to observe the pathological changes in pulmonary tissues under a microscope. Five fields of view were randomly selected under 200× magnifications and the number of alveolar cells containing more than two red blood cells or white blood cells was counted, and the alveolar damage index was calculated.
Measurements of MDA levels in pulmonary tissues. The pulmonary tissues were harvested and immediately homogenized on ice in 5 volumes of normal saline. Then, the supernatant was collected after homogenates' centrifugation at 1 200×g for 10 minutes. The MDA levels were measured in the supernatant according to the manufacturer's manual of a lipid peroxidation (MDA) Assay Kit (Biovision, San Francisco, USA).
Hypoxia reoxygenation (H/R) model. The H/R model was performed as previously described (Sauvant et al., 2009). Briefly, for hypoxia treatment, the MPVECs were cultured in an anaerobic chamber equilibrated with 5% CO 2 and 95% N 2 for 4 hours, and then maintained in a constant low oxygen concentration (<0.1%) chamber. After the hypoxia treatment, the MPVECs were moved back to a normal incubator with 5% CO 2 in the air. The treatment group was treated with different concentrations of Dex (1 μM, 5 μM, 10 μM or 20 μM).
Cell transfection. HEK293 and MPVECs were seeded at a density of 5×10 5 cells/well in 6-well culture plates (NEST, CA, USA). When the cell fusion rate reached 70%, the cells were transfected with miR-21-5p mimic (40 nM), inhibitor (55 nM) or Nr4a1 expression vector (1μg) and their corresponding controls using Lipofectamine 3000 (Invitrogen, CA, USA). After 4 hours, we discarded the transfection reagent and washed the cells with DMEM, then added fresh DMEM medium contain 10% FBS and different concentration of Dex (1 μM, 5 μM, 10 μM). 24 hours after transfection, the cells were harvested for further analysis.
Cell viability. To assay cell viability, the Cell Counting Kit-8 (CCK8) assay (Beyotime Biotechnology, Shanghai, China) was employed according to the manufacturer's manual. We seeded a total of 5×10 3 treated MPVECs in 96-well plates with 90 μL DMEM medium containing 10% FBS. Then, 10 μL of the CCK8 solution was added each well, and cells were incubated for 2 hours at 37°C at each indicated time point. Absorbance was then recorded at 450 nm using cytation 5 (BioTex VT, USA). Each experiment was repeated at least three times, and the mean of all measurements was calculated. The absorbance at each time point was used to plot the cell viability.
Apoptosis analysis. The Annexin V-FITC/PI apoptosis kit (eBioscience, UAS) was used to measure cell apoptosis according to the manufacturer's recommendations. Briefly, 24 hours after transfection, the MPVECs were harvested and washed with PBS at least two times. Then, the cells were resuspended in the binding cocktail containing Annexin V and PI, and kept in the dark for 15 min at room temperature. BD C6 flow cytometer (BD Biosciences, California, USA) was used to detect the stained cells. The results were analyzed by the Cell Quest software (BD Biosciences).
Luciferase reporter assay. MPVECs and 293T cells were co-transfected with 0.24 µg of Nr4a1-WT or Nr4a1-MUT reporter plasmids, together with 40 nM of miR-21-5p mimics or negative control oligoribonucleotides using Lipofectamine3000 (Invitrogen, USA) and following the manufacturer's instructions. In addition, 0.05 µg of Renilla luciferase expression plasmids were transfected into cells as a reference control, and 36 hours after transfection the cells were harvested to detect the firefly and renilla luciferase activities by dual luciferase reporter assays (Promega, E1910, WI, USA). The relative luciferase activity was calculated as a ratio of firefly and renilla luciferase activities.
Quantitative real-time polymerase chain reaction (qRT-PCR). According to the manufacturer's instructions, total RNA from the pulmonary tissues and cultured cells were extracted by the TRIzol reagent (Invitrogen). Then, the RNAs were reverse transcribed into cDNA using the PrimeScript RT Reagent (TaKaRa, Japan). Finally, PCR was performed to detect the relative expression level of miRNA and mRNA. To normalize expression of miR-21-5p, U6 small nuclear RNA (sn-RNA) was used as an internal control, while GAPDH was used to normalize the expression of Nr4a1, and the 2-ΔΔ CT equation was used for calculating relative expression.
Western blot analysis. The pulmonary tissues and cultured cell samples were harvested in a lysis buffer, the samples were incubated on ice for 30 min and the super-natant was collected by centrifugation at 1.5×10 4 g for 5 min at 4°C. Then, we separated the total proteins (40 μg) on 12% SDS-PAGE and transferred the proteins onto PVDF membranes (Millipore) via a MiniGenie blotting system (Bio-Rad). Next, we blocked the membranes with TBST containing 1% skim milk powder for 1 hour and then incubated with rabbit monoclonal antibodies against murine Cleaved Caspase-9 (1:1 000; cat. no. 20750S; Cell Signaling Technology), Cleaved Caspase-3 (1:1 000; cat. no. 9579S; Cell Signaling Technology), GAPDH (1:1 000; cat. no. 5174S; Cell Signaling Technology), and rabbit polyclonal antibody NR4A1 (1:2 000; cat. no. PA5-27274; Invitrogen) at 4°C overnight. Next, the membranes were washed 3 times with TBST for 15 minutes, and then incubated with goat-anti-rabbit secondary antibodies (1:10 000; cat. no. 14708S; Cell Signaling Technology) for 1 hour at room temperature. Finally, the protein bands were visualized on the membranes with an enhanced chemiluminescence system (ECL) and Image J (National Institutes of Health, Bethesda, MD) was used to quantify the protein bands.
Statistical analysis. GraphPad Prism (Version 6.01 for Windows) statistical software was used to perform statistical analysis. Student's t-test was used to compare the significant differences between groups. Repeated measures ANOVA was used for intragroup analysis. Statistical significance was set at p<0.01.

Dex reduced the I/ R-induced pulmonary injury
To explore the function of Dex in I/R-induced pulmonary injury, mice were injected with Dex during I/R operation through the caudal vein. The W/D ratio of the pulmonary tissues was significantly higher in the I/R group when compared to the Sham group. The W/D ratio of the pulmonary tissues was significantly lower in the Sham+Dex group than in the I/R+Dex group. Importantly, as compared with the I/R group, the W/D ratio of the pulmonary tissues was decreased in the I/ R+Dex group (p<0.01) (Fig. 1A). The H&E staining analysis was performed to determine the histopathological changes, and the result revealed that the I/R operation caused more pulmonary injury as compared with the Sham group. In the presence of Dex, disruption of the histoarchitecture was attenuated in the I/R+Dex group when compared to the I/R group (p<0.01) (Fig. 1B). Measurement of MDA level in the pulmonary tissue could reflect the degradation of lipids caused by oxidative stress, which is associated with the changes in the structure and the permeability of pulmonary membrane. As shown in Fig. 1C, the MDA level in the I/R group was the highest, while Dex treatment (I/R+Dex) significantly reduced the MDA level when compared to the I/R group (p<0.01). These results demonstrated the protective role of Dex in the I/R-induced pulmonary injury.

Dex reduced the H/R-induced apoptosis of MPVECs
To further study the role of Dex in reducing the I/Rinduced pulmonary injury, H/R-treated MPVECs were administered with Dex. The CCK8 assay showed that the H/R treatment significantly reduced cell viability of MPVECs when compared to the control group. However, as compared to that in the H/R group, the Dex treatment (H/R+Dex) showed significantly increased vi-ability (p<0.01) ( Fig. 2A). In addition, Annexin V-FITC/ PI double staining using flow cytometry was performed to detect cell apoptosis. As shown in Fig. 2B, when compared to the control group, the apoptotic ratio was greatly increased in the H/R group (p<0.01), while the Dex treatment (H/R+Dex) reduced the apoptotic ratio (p<0.01). Levels of cell apoptosis-related proteins were detected by western blot analysis. Consistently, the protein levels of Cleaved Caspase-3 and Cleaved Caspase-9 in the H/R group were greatly increased as compared to those in the control group (p<0.01), and the Dex treatment (H/R+Dex) decreased expression levels of Cleaved Caspase-3 and Cleaved Caspase-9 (Fig. 2C). These findings indicated that Dex could reduce H/R-induced apoptosis of MPVECs.

Dex upregulated miR-21-5p level and decreased Nr4a1 expression in H/R-treated MPVCs
Previous reports have described that miR-21-5p participated in the alleviation of I/R-induced injury in mouse . TargetScan database was used and predicted that Nr4a1 was a putative target for miR-21-5p (Fig. 4A). qRT-PCR and western blot analysis were performed to detect the expression levels of miR-21-5p and Nr4a1 in H/R-induced MPVECs at different times (3 h, 6 h, 12 h and 24 h). The results showed that in the H/R-treated MPVECs, miR-21-5p was downregulated in a time-dependent manner (Fig. 3A). In contrast, Nr4a1 was upregulated in the H/R-treated MPVECs in a time-dependent manner (Fig. 3B). However, in the H/R-treated MPVECs, miR-21-5p was dose-dependently upregulated and Nr4a1 was downregulated by the Dex treatement (all p<0.01) ( Fig. 3C and Fig. 3D). Taken together, Dex could upregulate the expression of miR-21-5p and downregulate the expression of Nr4a1 in H/R-treated MPVCs.

Dex reduced H/R-induced apoptosis of MPVECs cells by inhibiting Nr4a1 expression
To confirm the mechanism of Dex on reducing H/Rinduced apoptosis in MPVECs, we constructed an Nr4a1 expression vector and transfected it into MPVECs. The result of qRT-PCR showed that the relative Nr4a1 mRNA level was highly upregulated as compared to the control group (Fig. 5A). Then, the Nr4a1 expression vector was transfected into H/R induced MPVECs that were treated with Dex. The result of CCK8 assay demonstrated that overexpression of Nr4a1 (H/R+Dex+ Nr4a1) remarkably reduced the cell viability in comparison with the vector group (H/R+Dex+vector) (p<0.01) (Fig. 5B). Also, the data obtained by Annexin V-FITC/ PI double staining using flow cytometry indicated that the apoptotic ratio was greatly increased by overexpression of Nr4a1 (H/R+Dex+Nr4a1) as compared with the vector group (H/R+Dex+vector) in H/R-induced MPVECs that treated with Dex (p<0.01) (Fig. 5C). The results from western blot analysis indicated that the protein levels of Cleaved Caspase-3 and Cleaved Caspase-9 were upregulated by overexpression of Nr4a1(H/ R+Dex+Nr4a1) when compared to the vector group (H/R+Dex+vector) in H/R induced MPVECs that were treated with Dex (p<0.01) (Fig. 5D). These results indicated that DEX exerts anti-apoptotic effects via upregulation of miR-21-5p and subsequent downregulation of Nr4a1.

DISCUSSION
PIRI is a pathological phenomenon in which tissue damage is aggravated and even irreversible damage occurs after blood flow restores in the ischemic tissues and organs (Mariscal et al., 2018). In recent years, various studies have shown that the pathogenesis of PIRI involves many pathophysiological processes and pathways, including autophagy, autoimmune oxidative stress injury and apoptosis, etc. (Zhang et al., 2003;Zhang et al., 2013;Denlinger 2015;Laubach & Sharma 2016). In order to study the mechanism of PIRI and explore the therapeutic effect of medicines, animal models of PIRI were established (Xu et al., 2018;Gu et al., 2019). In these models, the W/D weight ratio and MDA were used as indirect markers of pulmonary injury. On account of that, wet pulmonary weight can indicate the pulmonary tissue damage resulting from parenchymal inflammation or immune system disorders, such as alveolar hemorrhage, congestion and pulmonary edema (Thomsen et al., 2008). As an end product of lipid peroxidation, MDA was reported to be an important precursor of oxidative injury (Aydin et al., 2009). Meanwhile, the histopathological analysis of pulmonary tissues from a mice model was also performed in respect to the rates of vascular congestion, inflammation, and thrombosis (Jacobson et al., 2005). Since the pulmonary tissue consists of various kinds of cells, it is difficult to precisely analyze the apoptotic effect of medicines in vivo. The H/R-induced MPVECs in vitro model was also established to study the apoptotic effect of medicines Liu et al., 2014). The study presented here is based on the PIRI mouse model and H/Rinduced apoptosis of MPVECs is used as an in vitro model. In the PIRI mouse model, the W/D ratio of the pulmonary tissues was decreased, more pulmonary injury was observed, and the MDA level was increased as compared to the Sham group. These results suggested a successful construction of the PIRI mouse model. The following analysis showed that these investigations in the PIRI mouse model were reversed by Dex treatment.
Dex has been recently found to alleviate the ischemia and reperfusion induced injury in different organs Si et al., 2018;. In our present study, Dex was found to reduce PIRI by reducing the MDA level, decreasing W/D ratio of the pulmonary tissues and attenu- ate pulmonary histoarchitecture injury. In addition, Dex also reduced the H/R-induced apoptosis of MPVECs, and downregulated protein levels of Cleaved Caspase-3 and Cleaved Caspase-9. Liang, et al., reported that Dex treatment could attenuate PIRI by activating the PI3K/ Akt signaling pathway at the transcriptional level (Liang et al., 2019). We found that Dex upregulated expression of miR-21-5p and downregulated expression of Nr4a1 in H/R-induced MPVCs.
Increasing evidences have reported that miRNAs play important roles in various biological processes, such as cell metabolism, proliferation and apoptosis (Diaz et al., 2017;Sun et al., 2019;Wu et al., 2019). Among them, miR-21-5p has been widely demonstrated to be dysregulated in pulmonary tumors Zhong et al., 2019). Caruso, et al., described that miR-21 level was reduced in pulmonary injury induced by hypoxia challenge (Caruso et al., 2010). Li, et al., demonstrated that miR-21-5p could mediate mesenchymal stromal cellsecreted exosomes to alleviate ischemia/reperfusion injury in mouse . Our study indicated that miR-21-5p was downregulated in the H/R induced MPVECs in a time-dependent manner. In addition, expression of miR-21-5p could be upregulated by Dex in a dose-dependent manner.
The orphan nuclear receptor 4A1 (Nr4a1), belonging to the steroid nuclear hormone receptor superfamily, has been shown to play a role in complex pathways of cell survival and apoptosis (Li et al., 2006). The study of Godoi, et al., reported that Nr4a1 mediated cell apoptosis and autophagy via binding to the anti-apoptotic B cell lymphoma gene 2 family proteins (Godoi et al., 2016). Our study indicated that the expression of Nr4a1 was upregulated in H/R induced MPVECs in a time-dependent manner. And Dex could downregulate the expression of Nr4a1 in a dose-dependent manner. The research of Yan, et al., showed that miR-21 inhibited NR4A1 expression in the human endometrial stromal cells dagger . In the present study, bioinformatics analysis identified Nr4a1 as a putative target for miR-21-5p, and this was confirmed by the luciferase assay. Also, knockdown of miR-21-5p could upregulate the expression of Nr4a1, and overexpression of Nr4a1 reversed the protective effect of Dex on attenuating MPVECs injury induced by H/R. Taken together, these results indicated that Dex inhibited Nr4a1 expression by upregulation of miR-21-5p to reduce the apoptosis of MPVECs.
In conclusion, our study indicated a remarkable biological role of Dex in the I/R-induced pulmonary injury. This study demonstrated that Dex could attenuate I/Rinduced pulmonary injury by regulating Nr4a1 expression via miR-21-5p, which thus increased cell viability and decreased cell apoptosis. Our study provides a theoretical evidence for clinical use of Dex in the therapy of pulmonary injury.