2022 Volume 47 Issue 5 Pages 169-181
Dexmedetomidine (DEX) protects against acute stress-induced liver injury, but what’s less clear lies in the specific mechanism. To elucidate the specific mechanism underlying DEX on acute stress-induced liver injury, an in vivo model was constructed on rats with acute stress-induced liver injury by 15 min of exhaustive swimming and 3 hr of immobilization. DEX (30 μg/kg) or miR-34a-5p agomir was injected into model rats. Open field test was used to verify the establishment of the model. Liver injury was observed by hematoxylin-eosin (H&E) staining. Contents of norepinephrine (NE), alanine aminotransfease (ALT) and aspartate aminotransferase (AST) in serum of rats were detected by enzyme-linked immunosorbent assay (ELISA) and those of oxidative stress markers (reactive oxygen species (ROS), Malondialdehyde (MDA), Glutathione (GSH), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPX)) were measured using commercial kits. Apoptosis of hepatocytes was detected by Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Western blot was performed to detect the expressions of SOD2, COX-2, cytochrome C, Cleaved caspase 3, Bax, Bcl-2, P-JNK, JNK, P-p38, p38 and c-AMP, p-PKA and PKA in liver tissues. As a result, liver injury in model rat was alleviated by DEX. DEX attenuated the increase in the levels of NE, ALT, AST, MDA, ROS, apoptosis, SOD2, COX-2, Cytochrome C, cleaved caspase 3, Bax, and P-JNK, P-p38, c-AMP, P-PKA and miR-34a-5p, and the decrease in the levels of SOD, GPX, GSH and Bcl-2 in model rats. Furthermore, miR-34a-5p overexpression could partly reverse the effects of DEX. Collectively, DEX could alleviate acute stress-induced liver injury through ROS/JNK/p38 signaling pathway via downregulation of miR-34a-5p.
Stress is a state of the organism under the influence of internal and external stress or “stressor” (Mastorakos et al., 2005). As an important part of the organism’s protective mechanism, stress is a necessary response of all living organisms for better survival and development (Mastorakos and Pavlatou, 2005). However, due to the stress, a series of biochemical reactions in the body also destroys the stability of the internal environment of the body, leading to cell metabolism disorder, multi-organ failure and even death (Kim et al., 2016). Since the liver is an organ susceptible to stress (Wang et al., 2016), the damage of acute stress to liver could induce infection or shock, seriously endangering human health and life (Chen et al., 2017).
Dexmedetomidine (DEX), an effective alpha 2 adrenergic agonist (α-AR), is a nervous system drug with excellent sedation and anti-sympathetic effects (Carollo et al., 2008). It is primarily used for sedation in patients receiving initial intubation and ventilator during intensive care therapy (VanderWeide et al., 2016). In recent years, the research on its pharmacological action outside anesthesia and the underlying mechanism has become a hot spot in the field of medicine (Chen et al., 2018; Wang et al., 2019a; Zhang et al., 2014). Evidence has revealed that DEX has a variety of biological activities such as antioxidant stress response and anti-apoptosis (Acar et al., 2020; Ni et al., 2020; Wang et al., 2015, 2019b), but the specific molecular mechanism of its effect on acute stress-induced liver injury has not been expounded.
MicroRNAs (miRNAs) are a class of single-stranded, non-coding, endogenous small molecule ribonucleotides that are 18 to 25 nucleotides in length (Conrad et al., 2006). Although microRNAs were first discovered as RNAs without any role, recent studies have proved their participation in regulating assorted diseases, including cancer, cardiovascular disease, various injuries, etc. (Ali Syeda et al., 2020; Song et al., 2019; Sun et al., 2018). Additionally, the roles of miRNAs in the pathogenesis of liver injury have also attracted extensive attention. A previous study has proposed that DEX can improve propofol-induced hippocampal neuron damage in rats, and this process is related to the inhibition of miR-34a (Xing et al. 2020). Furthermore, inhibition of miR-34a-5p has a protective effect on diverse injuries (Chen et al. 2020; Wang et al., 2012). Thus, we speculated that the improvement of DEX in the acute stress-induced liver injury of rats may also be associated with the regulation of miR-34a-5p expression.
In our study, DEX was used to treat acute stress-induced liver injury in rats, and the specific molecular mechanisms of its antioxidant stress and anti-apoptosis effects were further investigated. This study is expected to provide a theoretical basis for DEX treatment of acute stress-induced liver injury.
This study was approved by the Experimental Animal Committee of Zhejiang Chinese Medical University (ZJ202002013). All animal experiments were conducted in Zhejiang Chinese Medical University following the guidelines of the Chinese Council for Animal Care and Use. The experiment did utmost to minimize the pain and discomfort of the animals.
Animals and treatmentMiRNA agomir, a chemically-modified double-strand miRNA mimic (nucleotide fragment), has stable and long-lasting miRNA promotion effect. The agomir-NC is negative control of miR-34a-5p agomir. 48 male Wistar rats (200 ± 20 g, 6 weeks old) purchased from the Nanjing Institute of Model Zoology (Nanjing, China) were selected as subjects for this study. During the experiment, all rats were raised in a laboratory environment with temperature of 25°C and humidity of at least 40%. Prior to the experiment, the rats were randomly divided into six groups (n = 8): control group (no treatment); model group (the rats were subjected to 15 min of exhaustive swimming experiment and subsequently 3 hr of immobilization with a rat fixator to establish the model); model+DEX group (model rats were injected with 30 μg/kg DEX); DEX group (normal rats were injected with 30 μg/kg DEX); model+DEX+agomir-NC group (model rats were injected with 30 μg/kg DEX and miRNA agomir NC (miR4N0000001-4-5, Ribobio, Guangzhou, China) with 5 mg/kg); and model+DEX+agomir group (model rats were injected with 30 μg/kg DEX and miR-34a-5p agomir (miR40000815-4-5, Ribobio) with 5 mg/kg). The DEX administration was performed in rats through the intraperitoneal injection 24 hr before modeling. MiR-34a-5p agomir or miRNA agomir NC was injected into rats through the tail vein 24 hr with before modeling. All rats were fasted during modeling. After the modeling, the rats were removed from the fixator for open field experiment. After finishing open field test, the rats were anesthetized with etherand then killed by cervical dislocation, for collecting the blood. The liver samples of rats were removed completely by scissors and placed in a sterile culture dish. Finally, the liver samples were rinsed with sterile saline solution and weighed.
Open field testAfter the treatment, the immobility time, total distance, rearing number and crossing number of rats were measured by open field test in a quiet environment. The animal was placed in the center of the bottom of the box (a black wooden rectangular parallelepiped box (100 × 100 × 40 cm) without lid). Then, a camera was used to track and record performance of rats. After a 3-min observation, photography was stopped and the immobility time, total distance, rearing number and crossing number were recorded. The inside and bottom of the box were cleaned so that information left by the last animal (such as animal smell) would not affect the result of next test. The test was performed only once for each rat. Finally, the results of tests were analyzed using Super Maze Software (Shanghai Softmaze Information Technology Co. Ltd., Shanghai, China, http://softmaze.bioon.com.cn).
Hematoxylin-eosin (H&E) stainingThe liver tissues were fixed with 10% formalin and embedded in paraffin, followed by being sliced into 5-μm thick sections. Subsequently, xylene was used to dewax sections which were then successively rehydrated in anhydrous ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5 min. Then, the sections were transferred to distilled water for 1 min and stained with hematoxylin for 10 min, subsequent to which the sections were immersed in 1% hydrochloric acid for 30 sec and rinsed with tap water for 15 min, followed by re-staining with eosin (C0105, Beyotime Biotechnology, Shanghai, China) for 3 min. Following dyeing, the sections were dehydrated in 70% ethanol, 85% ethanol, 95% ethanol and anhydrous ethanol successively for 3 min each, and then transparentized by xylene for 10 min. Finally, the neutral gum was sealed, observed and photographed under a microscope (× 200) (POMEAS, Guangdong, China).
Enzyme-linked immunosorbent assay (ELISA)ELISA kit was applied to detect the contents of norepinephrine (NE) (E-EL-0047c, Elabscience, Wuhan, China, https://www.elabscience.cn), alanine aminotransfease (ALT) (E-EL-R1232km-1, Elabscience) and aspartate aminotransferase (AST) (E-EL-R0076c, Elabscience) in rat serum. The kit was taken out to stand for 30 min at room temperature. The standard group (6 concentrations), the blank group and the sample group to be tested were set. The samples were added as follows: 100 μL of the standard solution was added to the blank well in the standard group; 100 μL distilled water was added to the blank well. The remaining wells were filled with 100 μL of samples to be tested. 50 μL hydrocresol peroxide solution was added to each well of the standard group and the sample group to be tested. Then, the label board was sealed with sealing paper and incubated at 37°C for 1 hr. At the end of incubation, each well was filled with diluted detergent solution and stood for 15–30 sec. In addition, the label board was fully cleaned for 5 times and the paper was patted dry. 50 μL chromogenic solution A and 50 μL chromogenic solution B were added to each well, and reacted at 25–37°C in the dark for 15 min. Ultimately, 50 μL of termination fluid was added to terminate the reaction. OD value of each well at a wavelength of 450 nm was measured by Molecular Devices (Shanghai, China).
Oxidative stress assayAccording to the instructions, the contents of reactive oxygen species (ROS), Malondialdehyde (MDA) and Glutathione (GSH) and the activities of Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPX) in liver homogenate were detected with the oxidative stress test kits for SOD (S0109, Beyotime Biotechnology), MDA (S0131S, Beyotime Biotechnology); GSH: (S0053, Beyotime Biotechnology), GPX (S0056, Beyotime Biotechnology), and ROS (S0033S, Beyotime Biotechnology)). The activity of SOD was measured by microplate spectrophotometry and the absorbance values of SOD were measured at 450 nm. The results of SOD activity were expressed as U/mg protein. The content of MDA was determined by TBA method and the absorbance value of MDA at 532 nm was determined by the spectrophotometer (D-8PC, Philes, Nanjing, China). The results of MDA content were expressed as nmol/ mg protein. The content of GSH was determined by DTNB method. The absorbance value of GSH at 412 nm was determined by spectrophotometer and the results of GSH content were expressed as μmol/g. The activity of GPX was determined by NADPH method. The absorbance values of GPX at 595 nm were determined by spectrophotometer and the results of GPX activity were expressed as U/mg. The ROS content was determined by DCF method, and the fluorescence was detected by an enzyme marker (488 nm excitation wavelength, 525 nm emission wavelength) (Molecular Devices).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assayApoptosis was detected using the TUNEL assay kit (C1091, Beyotime Biotechnology). The above tissue slices were placed in a dyeing cylinder. Then, the slices were washed twice with xylene (5 min for each time), twice with anhydrous ethanol (3 min for each time), once with 95% and 75% ethanol (3 min for each time), and with phosphate buffered saline (PBS) for 5 min. Following this, protease K solution of 20 μg/mL was added to hydrolyze the slices at room temperature for 15 min. Later, the slices were transferred to distilled water and rinsed for 4 times (2 min for each time). The excess fluid around the tissues on the slice was carefully removed with a filter paper, and 2 drops of TdT enzyme buffer were immediately added to the slices. Then, the slices were added with 54 μL TdT enzyme solution and placed in a wet box at 37°C for 1 hr. Subsequently, the slices were gently shaken every 10 min to stir the liquid slightly. Two drops of peroxidase-labeled anti-digoxin antibody were added directly to the slices, and the reaction was carried out in a wet box at room temperature for 30 min. Fresh 0.05% diaminobenzidine (DAB) solution was added directly to stain the tissue sections at room temperature for 5 min. After washing with PBS, the slices were re-dyed with methyl green at room temperature for 10 min. Ethanol was used for dehydration for 3 times, 2 min for each time, followed by being transparentized with xylene. After the slices were sealed and dried, the experimental results were observed and recorded under microscope (× 200) (POMEAS).
Western blot assayRIPA lysis buffer (P0013B, Beyotime Biotechnology) (containing protease inhibitor (P1030, Beyotime Biotechnology) and phosphatase inhibitor (P1081, Beyotime Biotechnology)) was added to liver tissues and put into a homogenizer. After the tissues were ground into a homogenate, the whole tissue extracts were obtained after lysis at 4°C for 30 min, followed by centrifugation at 12000 rpm for 10 min at 4°C. BCA protein quantitative kit (P0012, Beyotime Biotechnology) was employed to determine the total protein concentration of each sample. After the measurement, 30 μg of the total protein was isolated by SDS/PAGE (sodium dodecyl sulfate/ polyacrylamide gel electrophoresis) (P0012A, Beyotime Biotechnology) and the marker (PR1910, Solarbio, Beijing, China) was used to mark the molecular weight of the protein. Then, protein was transferred to the polyvinylidene fluoride (PVDF) membrane (ISEQ00010/IPVH00010, MILLIPORE, Bedford, MA, USA). The membrane was blocked at room temperature for 1 hr with 5% skim milk. Then, Tris Buffered Saline with Tween 20 (TBST) was used to wash the excess skimmed milk. The membrane was incubated with the primary antibodies against SOD2 (#13141, 1:1000, CST), COX-2 (#12282, 1:1000, CST), P-JNK (ab131499, 1:500, Abcam, Cambridge, MA, USA), JNK (ab179461, 1:1000, Abcam), p38 (ab31828, 1:1000, Abcam), p-p38 (ab47363, 1:10000, Abcam,), Cytochrome C (ab133504, 1:5000, Abcam), Cleaved caspase 3 (ab49822, 1:500, Abcam), Bax (ab32503, 1:5000, Abcam), Bcl-2 (ab59348, 1:1000, Abcam), cAMP (ab76238, 1:1000, Abcam), p-PKA (#4781S, 1:1000, Cell Signaling, Danvers, MA, USA), PKA (#4782S, 1:1000, Cell Signaling, USA) and GAPDH (ab8245, 1:10000, Abcam) at 4°C overnight. On the next day, the primary antibody was removed, and the excess antibodies were washed with TBST. Then, the membrane was cultivated with the secondary antibody (ab6721, 1:10000, Abcam) at room temperature for 1 hr. After closure, the excess antibodies were also washed with TBST. The chemiluminescence solution (WBKLS0500, MILLIPORE) was added to enhance the visualization of the chemiluminescence advanced system (Bio-Rad, CA, USA). The gray value of band was analyzed by image J (1.8.0, National Institutes of Health, Bethesda, MD, USA).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)Total RNA was isolated from tissues and cells with miRcute miRNA Isolation Kit (DP501, TianGen, Beijing, China), followed by being reverse-transcribed to cDNA using One Step PrimeScript miRNA cDNA Synthesis Kit (D305A, TaKaRa, Kyoto, Japan). Afterwards, the cDNA was used as a template for qPCR, and the component of the reaction system was as follows: 2 µL cDNA, 10 µL 2 × SYBR Premix Ex Taq II (DRR081A, TaKaRa), 0.4 µL forward primer, 0.4 µL reverse primer and 7.2 µL ddH2O. Primers purchased from Sangon (Shanghai, China; Table 1) were mixed together, followed by reaction with Applied Biosystems 7900 system (Applied Biosystems Company, Foster, CA, USA) under reaction conditions of 95°C for 30 sec and 40 cycles of 95°C for 10 sec and 60°C for 30 sec. After the reaction, the relative expression of each target gene was calculated by 2−ΔΔCt method (Dalin et al., 2017). U6 served as an internal reference.
| Gene | Primer sequence | Species |
|---|---|---|
| miR-34a-5p | 5’-CCCACATTTCCTTCTTATCAACAG-3’ 5’- CCGACTCCACGACAC-3’ |
rat |
| U6 | 5’-CTCGCTTCGGCAGCACA-3’ 5’-AACGCTTCACGAATTTGCGT-3’ |
rat |
SPSS.19.0 (IBM, Armonk, NY, USA) was used for data analysis. The results were expressed as mean ± standard deviation (SD). Differences between multiple groups were compared using one-way analysis of variance (ANOVA). Image J was used to analyze the gray value of band. P < 0.05 was considered statistically significant.
Open field test is a common method to explore the autonomous behavior, exploratory behavior and tension of experimental animals in new and different environments, which is also usually used to verify the construction of stress models. We confirmed the successful construction of the acute stress-induced liver injury model by detecting immobility time, total distance, rearing number and crossing number. Compared with the control group, immobility time in the model group was significantly increased, while the total amount of distance, rearing number and crossing number were significantly decreased (Fig. 1A-D, p < 0.001). Those findings indicated that the acute stress-induced liver injury rat model of acute stress-induced liver injury was successfully constructed.
Open field experiment verified an acute stress rat model. (A): The immobility time was the amount of time that rats spent in the central lattice per unit time. (B): The total distance was the distance that rats moved. (C): The rearing number was obtained for the number of hind limbs standing. (D): The crossing number was the number of squares crossed by the forelimbs. * vs. control; *** P < 0.001.
H&E staining was used to observe the damage of rat liver tissue. As depicted in Fig. 2A, liver tissue was significantly damaged in the model rats compared to the normal rats. DEX alleviated liver damage from acute stress, but generated no damage to normal liver tissue. In order to further determine the effect of DEX on acute stress-induced liver injury, the contents of NE, ALT and AST in the serum of rats were detected by ELISA. In line with Fig. 2B-D, the levels of NE, ALT and AST in model rats were higher than those in normal rats, which, however, could be reduced by DEX in model rats (p < 0.001). All of the above implied that DEX could alleviate the liver injury caused by acute stress.
DEX relieved liver damage and oxidative stress caused by acute stress. (A): Liver injury was observed by H&E staining. (scale bar: 100 μm; magnification: × 200) (B-E): ELISA was used to detect the contents of NE, ALT and AST. (E-I): Commercial kit was used to detect oxidative stress-related indicators (SOD, GPX, GSH and ROS). * vs. control, # vs. Model; ## P < 0.01, *** or ### P < 0.001. DEX, Dexmedetomidine; ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin-eosin staining;
Oxidative stress is the main cause of liver injury in model rats. We detected MDA, SOD, GPX, GSH and ROS levels in the liver tissues of rats. The results showed that MDA and ROS levels were upregulated, while SOD, GPX and GSH levels were downregulated in the model group, compared with those in the control group (Fig. 2E-I, p < 0.01, p < 0.001). Meanwhile, the above changes of oxidative stress indexes were reversed in the DEX+Model group, while those in the DEX group shared almost the same oxidative stress index levels with the control group (Fig. 2E-I). Those signified that DEX could relieve the oxidative stress induced by acute stress.
DEX mitigated acute stress-induced cell apoptosis in liver tissueWe performed the TUNEL assay to detect the cell apoptosis of the liver. According to the results, the apoptosis of hepatocytes was enhanced in the model rats in contrast with the normal rats (Fig. 3A), but such enhancement could be offset by DEX. Also, Dex produced no effect on normal liver tissue (Fig. 3A). To affirm those results, further western blot analysis was performed on expressions of SOD2, COX-2, Cytochrome C, Cleaved caspase 3, Bax and Bcl-2. As evidenced by Fig. 3C-I, the expressions of SOD2, COX-2, Cytochrome C, Cleaved Caspase 3 and Bax were signally promoted (p < 0.05, p < 0.01, p < 0.001) and that of Bcl-2 was markedly reduced (p < 0.001) in the model rats as compared to the normal rats. In addition, we found that DEX could downregulate the expression of miR-34a-5p which was enhanced in model rats (Fig. 3B, p < 0.001).
DEX alleviated the apoptosis of rat hepatocytes induced by acute stress. (A): The TUNEL assay was used to observe apoptosis. (scale bar: 100 μm; magnification: ×200) (B): qRT-PCR determined the expression of miR-34a-5p. U6 served as an internal reference. (C-I): Western blot analysis of the expressions of SOD2, COX-2, Cytochrome C, Cleaved caspase 3, Bax and Bcl-2 in rat liver tissue. * vs. control, # vs. Model; # P < 0.05; ## P < 0.01; *** or ### P < 0.001. DEX, Dexmedetomidine; SOD2, superoxide dismutase 2; COX-2, Cyclooxygenase-2; Bax, Bcl-2 Associated X Protein; Bcl-2, B cell lymphoma/ leukemia-2.
To investigate whether acute stress caused changes in apoptosis-related pathways, we detected the expression levels of JNK, P-JNK, p38 and P-p38 in liver tissues by western blot. As illustrated by Fig. 4, the expressions of JNK and p38 in rat liver tissues showed slight changes between the model group and the control group, while those of P-JNK and P-p38 were promoted in the model group, when compared with the control group (Fig. 4A-G, p < 0.001). Expectedly, DEX reduced P-JNK and P-p38 expressions (Fig. 4A-G, p < 0.001) and did not affect the expressions of JNK, P38, P-JNK and P-p38.
DEX inhibited phosphorylation of JNK/p38 protein induced by acute stress. (A-G): Western blot analysis of the expressions of P-JNK, JNK, P-p38 and p38 in rat liver tissue. * vs. control, # vs. Model; *** or ### P < 0.001. DEX, Dexmedetomidine; JNK, c-Jun N-terminal kinase.
MiR-34a-5p agomir was used to enhance the expression of miR-34a-5p in cells, the success of which has been demonstrated by the detection results of qRT-PCR (Fig. 5A, p < 0.001). Next, oxidative stress levels and apoptotic protein expressions in cells treated with DEX and/or miR-34a-5p agomir were detected. According to Fig. 5B-F, DEX reduced the level of oxidative stress in model rats, while miR-34a-5p overexpression partially reversed the effect of DEX (p < 0.01, p < 0.001). Additionally, the results of western blot detection regarding the expressions of apoptosis-related proteins were delineated in Fig. 5G-M. The DEX-alleviated apoptosis of hepatocytes in model rats was also reversed by miR-34-5p overexpression (p < 0.001). In the light of the results of western blot, the expressions of p-JNK and P-p38 were dwindled by DEX, and such effect was also reversed by miR-34a-5p overexpression (Fig. 6, p < 0.001).
MiR-34a-5p partially reversed the effects of DEX on oxidative stress and apoptosis of model rats. (A): qRT-PCR detected the transfection efficiency of miR-34a-5p agomir. U6 served as an internal reference. (B-F): Commercial kit was used to detect oxidative stress-related indicators (SOD, GPX, GSH and ROS). (G-M): Western blot analysis of the expressions of SOD2, COX-2, Cytochrome C, Cleaved caspase 3, Bax and Bcl-2 in rat liver tissue. * vs. Model, # vs. Model+DEX+MC; # p < 0.05, ## p < 0.01, *** or ### P < 0.001. DEX, Dexmedetomidine. M, miR-34a-5p agomir; MC, agomir-NC (negative control of miR-34a-5p agomir).
MiR-34a-5p partially reversed the effect of DEX on JNK/p38 signal pathway of model rats. (A-G): Western blot analysis of the expressions of P-JNK, JNK, P-p38 and p38 in rat liver tissue. * vs. Model, # vs. Model+DEX+MC; *** or ### P < 0.001. DEX, Dexmedetomidine; JNK, c-Jun N-terminal kinase. M, miR-34a-5p agomir; MC, agomir-NC (negative control of miR-34a-5p agomir).
As shown in Supplementary Fig. 1, DEX significantly increased the expressions of adrenergic receptor signaling genes (cAMP and p-PKA) at protein level, as well as p-PKA/PKA level in model rats (p < 0.001). However, the effect of DEX was abrogated by miR-34a-5p overexpression (p < 0.05).
Stress can be divided into acute stress and chronic stress, which is mainly determined by the time that the stress source exerts an effect on the organism and the intensity of the stimulus (Calcia et al., 2016). In general, transient chronic stress will be gradually adapted by the body without serious consequences (Shi and Wu, 2020). However, acute stress can destroy the stability of human internal environment, and seriously cause damage to the body and even death (Liu et al., 2016). The present study revealed that DEX attenuated acute stress-induced liver injury, apoptosis and oxidative stress in rats via regulating the miR-34a-5p/ROS/JNK/p38 signaling pathway, and involved with cyclic adenosine monophosphate (cAMP) pathway. The data indicated that DEX provides marked protection against acute stress-induced liver injury in rats.
DEX is a 2-adrenergic receptor agonist, which has been proved to have many biological activities such as anti-oxidative stress reaction and anti-apoptosis (Sha et al., 2019b; Sun et al., 2017). In the research of Pilz et al. (2000) oxidative stress will lead to ROS accumulation in the body or cells, thereby causing oxidative damage. ROS reacts with double bonds of polyunsaturated fatty acids (PUFAs) to produce lipid hydrogen peroxide. MDA is formed as a secondary product during lipid peroxidation of PUFAs. Furthermore, SOD, GPX and GSH are also factors related to oxidation (Braidy et al., 2019; Strycharz-Dudziak et al., 2019). By detecting these indicators related to oxidative stress, we disclosed that DEX relieved liver injury induced by acute stress through antioxidant stress response. Then, we further examined the effect of DEX on the apoptosis of hepatocytes. In addition to detecting the alleviating effect of DEX on cell apoptosis through TUNEL assay, we also unveiled that DEX changed the expressions of apoptosis-related proteins such as SOD2, COX-2, Cytochrome C, Cleaved Caspase 3 and Bax in hepatocytes (Ong et al., 2006; Zhang et al., 2019), thus reducing the apoptotic ability of cells. DEX may protect the liver against IR injury in rats by the attenuation of oxidative stress (Kucuk et al., 2014; Sahin et al., 2013; Tüfek et al., 2013). Lim et al. (2021) reported DEX suppresses hepatic ischemia/reperfusion (IR) injury in rats by the attenuation of inflammatory response and the inhibition of apoptosis. Sha et al. (2019a) demonstrated that DEX attenuates lipopolysaccharide (LPS)-induced liver oxidative stress and cell apoptosis in rats. In this study, we found that DEX protected the liver against acute stress-induced injury in rats by reducing oxidative stress, apoptosis. Similar report also showed that DEX has a protective effect on acute stress-induced liver injury by reducing inflammation and apoptosis, involved with MKP-1, and NF-κB pathway (Sha et al., 2019a).
The JNK/p38 signaling pathway is considered to be a pivotal pathway involved in initiating apoptosis (Xia et al., 1995). Sha et al. (2019a) put forward that MKP-1 upregulation may attenuate inflammation and apoptosis in acute stress-induced liver injury by inhibiting NF-κB pathway and phosphorylation of JNK and P38. The formation of ROS is closely related to the activation of the stress-activated kinases, JNK and p38 (Wang et al., 2018), Previous reports have pointed out that oxidative stress in the liver could change the JNK/p38 signaling pathway and ultimately promote the apoptosis of hepatocytes (Mendelson et al., 1996; Sun et al., 2016). Intriguingly, DEX upregulates the expression of MKP-1 (Sha et al., 2019a). In addition, it has been reported that DEX ameliorates acute stress-induced kidney injury by attenuating oxidative stress and apoptosis via blocking the ROS/JNK signaling pathway (Chen et al., 2018). To further comprehend the mechanism of DEX in acute stress-induced liver injury, we examined the effect of DEX on JNK/p38 signaling pathway. Consistently, in our study, it was proved that DEX could attenuate the liver injury caused by acute stress through the JNK/P38 signaling pathway.
Except the above results, we were also pleasantly surprised to find that DEX could downregulate the expression of miR-34a-5p in the liver tissues of model rats, but could not alter that in the normal liver tissues. We know from a previous study that miR-34a-5p has been shown to play a vital role in assorted injury mechanisms (Wang et al., 2012). Similarly, our study unraveled that miR-34a-5p overexpression could partially reverse DEX-alleviated oxidative stress and cell apoptosis. Furthermore, we also discovered that miR-34a-5p overexpression could partly reverse the regulatory effects of DEX on JNK/P38 signaling pathway. Additionally, since DEX is alpha 2-adrenergic receptor agonist, and cAMP level can be inhibited by the alpha 2-receptor antagonist (Osborne, 1991), the cAMP signaling has been also detected. cAMP, a key second messenger molecule, modulates various cellular functions including lipid inflammation and injury. The elevated intracellular cAMP level promotes the activation of PKA (Wang et al., 2019c; You et al., 2019; Zhou et al., 2019), and the cAMP-PKA pathway involved in liver injury (Ji et al., 2012, 2013; Yoshikawa et al., 2020). DEX also regulates cAMP-PKA signaling pathway in rats with rats with postoperative cognitive dysfunction (Zhu et al., 2019). The present study revealed that miR-34a-5p overexpression could partly reverse the regulatory effects of DEX on cAMP-PKA signaling pathway. These findings also indicated that DEX may act as an agonist of alpha 2 adrenergic receptor that exerted the protective effect on acute stress-induced liver injury.
The current study is not without limitation. Acute hypothalamic-pituitary-adrenal (HPA) function after a stressor is greater in adult female rodents than that in males, which is attributed largely to regulation by the gonadal hormones testosterone and estradiol (Heck and Handa, 2019). Furthermore, Luft et al. (2019) reported that males could be more vulnerable to the short-term effects of acute stress in Balb/c mice. Thus, only male rats were used in this research to avoid the underlying confounding effects of estrous cycle and sex hormones of female rats. This context may lead to sex bias and constrain the generalizability of the findings in females.
In summary, DEX exerts its antioxidant stress and anti-apoptosis abilities through miR-34a-5p/ROS/JNK/p38 signaling pathway, and may act as an agonist of alpha 2 adrenergic receptor. Our study revealed the molecular mechanism of DEX in relieving acute stress-induced liver injury, looking forward to laying a theoretical foundation for the treatment of acute stress-induced liver injury.
This work was supported by the Zhejiang Medical and Health Science and Technology Platform Project [2018KY001]; the Clinical Research Fund Project of Zhejiang Medical Association [2018ZYC-A02].
Conflict of interestThe authors declare that there is no conflict of interest.
