miR-128-3p enhances the protective effect of dexmedetomidine on acute lung injury in septic mice by targeted inhibition of MAPK14

To investigate the role of miR-128-3p and MAPK14 in the dexmedetomidine treatment of acute lung injury in septic mice. SPF C57BL/6 mice were divided into 8 groups. The pathological changes and wet/dry weight ratio (W/D), PaO2, PaCO2, MDA, SOD and MPO levels in lung tissue and the serum levels of inflammation factors were observed. Dual luciferase reporter assay was used to detect the targeting relationship of miR-128-3p and MAPK14, and qPCR and WB were used to detect the expression of miR-128-3p and MAPK14. Compared with the Normal group, other groups had lower MDA, MPO, inflammatory factors levels and the expression level of MAPK14, while the content of SOD and the expression level of miR-128-3p was significantly decreased (all p < 0.05). Compared with the Model group, the contents of MDA, MPO, inflammatory factors in the DEX group and miR-128-3p mimic group were significantly decreased, and the content SOD was significantly increased, however, opposite results were occurred in oe-MAPK14 group (all p < 0.05). Compared with the DEX group, all the indicators in miR-128-3p mimic+DEX group showed significant improvement (all p < 0.05). Compared with the miR-128-3p mimic group, all the indicators were deteriorated in the miR-128-3p mimic+oe-MAPK14 group (all p < 0.05). The combination of DEX and oe-MAPK14 blocked the protective effect of dexmedetomidine on acute lung injury in septic mice. miR-128-3p can further enhance the protective effect of dexmedetomidine on acute lung injury in septic mice by targeting and inhibiting MAPK14 expression.


Background
Sepsis is the most common systemic clinical complication of severe trauma, burns and major surgery, which can deteriorate into multiple organ dysfunction syndrome, becoming the leading cause of death in these patients (Zhuo et al. 2019;Iwaki et al. 2019). Acute lung injury (ALI) is a common complication of sepsis as lung is particularly sensitive to sepsis damage (Peng et al. 2016). ALI is characterized by progressive hypoxemia, enhanced vascular permeability, edema, neutrophil infiltration and lung accumulation, which greatly increases patient morbidity and mortality (Mehaffey et al. 2017).
Therefore, the researches on the new targets for the treatments of sepsis are hotspot (Acosta-Herrera et al. 2015).
Dexmedetomidine (DEX), a α2-adrenergic receptor agonist, has an imidazole structure and sedative, analgesic and hemodynamic stabilization effects . A good anti-inflammatory effect of DEX has been discovered on important organs in mice with spinal cord injury, myocardial ischemia-reperfusion and sepsis, etc. . M o r e o v e r , D E X c a n i m p r o v e A L I , r e d u c e pathomorphological changes, inhibit oxidative stress damage, inflammatory response and apoptosis in lung epithelial cells by inhibiting TLR-4/NF-κB pathway (Meng et al. 2018a).
Increasing number of evidences have proved that miRNAs associated with certain inflammatory lung diseases Guo et al. 2014;Chen et al. 2017). miR-128-3p plays an important role in Dox-induced liver injury by targeting Sirt1 . However, whether miR-128-3p participates in the anti-inflammatory activity of dexmedetomidine in ALI remains unclear.
ALI is often mediated by a variety of intracellular signaling pathways, such as PI3K/Akt, c-Jun N-terminal protein kinase, mitogen-activated protein kinase, and p38 activation, which play a role in inflammatory responses, cell death, and alveolar epithelial cell damage in ALI (Yang et al. 2001;Padda et al. 2006). P38 mitogen-activated protein kinase (p38MAPK) is an important signal regulating cell proliferation and apoptosis. Its phosphorylated form can activate a variety of physiological processes, and MAPK14 is a member of the MAPK family (Feng et al. 2017). In this study, we found a targeting relationship between miR-128-3p and MAPK14 via bioinformatics prediction. We hypothesized that miR-128-3p may downregulate the expression of MAPK14, thereby inhibiting p38 signaling pathway to alleviate ALI in septic mice.
Therefore, we aimed to explore whether miR-128-3p will affect the p38MAKE pathway and play a role in acute lung injury in septic mice.
The sepsis model was established by cecal ligation and puncture. Briefly, the mice were fixed in table and anesthetized with 3% pentobarbital sodium (50 m/kg). After eyeball blood collection, the mice died of excessive blood loss, and lung tissue samples were retained. A 1 cm long surgical incision was made in the central part of the anterior abdominal cavity of the mice to separate out the cecum end, then the root of the cecum was ligated and punctured with a 4-gauge needle, and the contents in cecum were extruded out. Finally, the cecum and incision were sutured. Pre-warmed saline was injected postoperatively. Except for no ligation and puncture, the other steps in the Normal group were the same as those in the model group. Thirty-two animals died, so the success rate of model establishment was 70.90%, of which 70 were taken for the following experiments.
After the operation, mice intraperitoneally injected with DEX (12.5 μg/kg, GLPBIO, America) and miR-128-3p mimic, MAPK14 overexpression vectors (The NC, miR-128-3p and MAPK14 overexpression adenoviral vectors were constructed by GenePharma, Suzhou). After modeling and treatment, lung tissue and venous blood were taken from 5 mice in each group, and some lung tissues were fixed in 10% neutral formalin solution for 24 h, and was dehydrated by gradient alcohol, embedded in paraffin and sliced. This study was performed in The People's Hospital of Yinzhou, and experimental studies in accordance with the Basel declaration have been approved by the ethics committee of The People's Hospital of Yinzhou (No. D20181103).

Dual luciferase reporter system assay
The targeting relationship and binding site of miR-128-3p and MAPK14 was analyzed via the biological prediction website (www.targetscan.org), when was next verified by the dual luciferase reporter system assay. The MAPK14 (PGL3-MAPK14wt) and mutants that bind to the miR-128-3p (PGL3-MAPK14mut) dual luciferase reporter vectors were separately constructed. The Rellina plasmids and the two reporter plasmids were co-transfected into HEK293T cells with the miR-128-3p plasmid and the NC plasmid, respectively. After 24 h of cell transfection, dual luciferase assays were performed according to the instruction of the dual luciferase reporter kit (Promega). Relative luciferase activity = firefly luciferase / Renilla luciferase (Meng et al. 2018a).

qRT-PCR
Trizol (Thermo Fisher Scientific, New York, USA) was used to extract total RNAs from lung tissue. The cDNA was synthesized by reverse transcription using TaqMan MicroRNA Assays Reverse Transcription Primer (Thermo scientific, USA). SYBR® Premix ExTaq™ II Kit (Xingzhi Biotechnology Co., Ltd., China) was used for quantitative PCR detection. The following components were added in sequence: 25 μL of SYBR Premix ExTaq™ II (2×), 2 μL of PCR upstream and downstream primers, ROXReferenceDye (50×) 1 μL, 4 μL DNA template, and 16 μL of ddH 2 O. Fluorescence quantitative PCR was performed in ABIPRISM® 7300 (model Prism® 7300, Shanghai Kunke Instrument Equipment Co., Ltd., China). The reaction conditions were: predenaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 60°C for 30 s, 32 cycles, extending at 72°C for 1 min. miR-128-3p with U6 as internal reference, and MAPK14 used GAPDH as internal reference. The relative expression amount of each gene of interest was calculated by 2 -ΔΔCt . Primer sequences are shown in Table 1.

Western blot
Total protein in lung tissue was extracted using RIPA lysate containing PMSF (R0010, solarbio). The protein concentration was determined by BCA kit (thermo, USA). The protein sample was mixed with the loading buffer, boiled for 10 min. Then 50 μg of protein sample was electrophoresed at 70 V for 3 h and transferred onto a PVDF membrane (ISEQ00010, Millipore, Billerica, MA, USA) with constant flow 150 mA. The membrane was then blocked by 5% skim milk at 4°C for 2 h, washed with TBST, and incubated with anti-rabbit antimouse MAPK14 (ab31828, 1:500, Abcam, UK), GAPDH (ab22555, 1:2000, Abcam, UK) overnight at 4°C. After washing with TBST thrice, the membrane was incubated with HRP-labeled goat anti-rabbit IgG antibody (Beijing Zhongshan Biotechnology Co., Ltd., diluted 1:5000) for 2 h and wash TBST thrice. ECL fluorescence detection kit (Cat. No. BB-3501, Ameshame, UK) was used for color development and the membrane was photographed by Bio-Rad image analysis system (BIO-RAD, USA) and the results were analyzed by Image J software. The relative protein content = the gray value of the corresponding protein band / the gray value of the GAPDH protein band.
Blood gas analysis and lung tissue wet / dry weight ratio (W/D) The carotid artery blood was taken for blood gas analysis to observe arterial oxygen partial pressure (PaO 2 ) and carbon dioxide partial pressure (PaCO 2 ). The wet / dry weight ratio (W/D) was calculated to reflect the degree of edema of the lung. The left lung of the mouse was removed by thoracotomy, and the wet weight was weighed after clearing the lung surface by filter paper. After drying in an incubator at 80°C for 48 h, the sample in constant weight was weighted as the dry weight. Lung tissue wet / dry weight (W/D) = (lung wet weight / lung dry weight) * 100%.

HE staining
After modeling and treatment, some lung tissues were fixed in 10% neutral formalin solution for 24 h, and was dehydrated by gradient alcohol, embedded in paraffin and sliced. Then slice was treated with xylene transparent, hydrated by gradient alcohol and washed with distilled water for 1 min. Subsequently, the slice was stained with hematoxylin for 3 min, flushed with tap water, immersed in alcohol containing 0.5% hydrochloric acid for 10 s, stained with eosin dye solution for 5 min. Finally, the slice was conventionally dehydrated, transparentized and sealed with neutral gum. Each slice was observed under an optical microscope (XP-330, Shanghai Bingyu Optical Instrument Co., Ltd., Shanghai, China).
The lung tissue in size of 125 mm 3 was homogenized with 1 mL of PBS, centrifuged at 4°C, 12,000 xg for 10 min, and the supernatant was taken. The myeloperoxidase (MPO), which reflects the degree of neutrophil accumulation in lung tissue, was detected in strict accordance with the kit instructions (K744-100, Biovision, US). MDA and SOD in lung tissue were detected by MDA (A003-1-2) and SOD (A001-3-2) assay kits purchased from Nanjing Jiancheng Reagent Co., Ltd., respectively.

Enzyme-linked immunosorbent assay (ELISA)
Blood taken from mouse eyeballs were stand at room temperature for a while and at 4°C overnight, centrifuged at 3500 xg/min to collect serum and the samples were preserved at −80°C. The level of inflammatory factors was measured by according the ELISA kit instructions (kit numbers: 138,800,328,133; Wuhan Merck, China).

Statistical analysis
All data were processed by SPSS21.0 statistical software. The measurement data were expressed as mean ± standard deviation. One-Way ANOVA and Tukey post-Hoc test was used for comparison between groups. p < 0.05 indicates that the difference is statistically significant.

Results
Pathological changes of lung tissue in each group of mice HE staining was used to detect the pathological changes of lung tissue in mice (Fig. 1). Lung tissue of Normal group was in regular structure without obvious F: 5'-CTCGCTTCGGCAGCACA-3' R: 5'-AACGCTTCACGAATTTGCGT-3' GAPDH F: 5'-TTCAACGGCACAGTCAAGG-3' R: 5'-CACCAGTGGATGCAGGGAT-3' pathological damage. In Model group, oe-MAPK14 group, miR-128-3p mimic+oeMAPK14 group, there were different degrees of inflammatory cell infiltration in the alveoli and interstitial; there was effusion in the cavity and thickened alveolar septum, meanwhile, some alveoli were collapsed, atelectasis, and formed transparent membrane and alveolar structure was damaged. However, the lung tissue damage of the mice in the DEX group and the miR-128-3p mimic+DEX group was significantly improved compared with the above three groups. The degree of lung tissue damage in the DEX + oe-MAPK14 group was similar to that in the Model group.

Blood gas analysis and W/D of each group of mice
The W/D of the lung tissue, PaO 2 and PaCO 2 of each group are shown in Fig. 2. Compared with the Normal group, W/D, PaCO 2 was significantly higher and PaO 2 was significantly lower in the other groups (all p < 0.05). Compared with the Model group, W/D, PaCO 2 , and PaO 2 showed no significant differences in miR-128-3p mimic+oe-MAPK14 group, in addition, W/ D and PaCO 2 were significantly decreased and PaO 2 was significantly increased in DEX group, miR-128-3p mimic group, miR-128-in 3p mimic+ DEX group, while opposite results occurred in oe-MAPK14 group (p < 0.05). Compared with the miR-128-3p mimic group, the W/D and PaCO 2 decreased and PaO 2 increased in the miR-128-3p mimic+DEX group, but the miR-128-3p mimic+ oe-MAPK14 group has opposite results (both p < 0.05). Compared with the DEX group, the W/D and PaCO 2 increased and PaO 2 decreased in the DEX + oe-MAPK14 group (all p < 0.05).

Serum levels of inflammatory factors in mice
The serum levels of inflammatory factors in each group were detected by ELISA (Fig. 3). Compared with the Normal group, the serum levels of inflammatory factors (interleukin (IL)-8, IL-17, IL-6 and tumor necrosis factor (TNF)-α) were significantly higher in the other groups (p < 0.05). Compared with the Model group, there was no significant difference in serum inflammatory factors in miR-128-3p mimic+oe-MAPK14 group, which were significantly lower in DEX group, miR-128-3p mimic group, miR-128-3p mimic+ DEX group (p < 0.05), but the oe-MAPK14 group had opposite results (p < 0.05). Compared with miR-128-3p mimic group, the serum levels of inflammatory factors were significantly less in miR-128-3p mimic+DEX group, but opposite results occurred in miR-128-3p mimic+oe-MAPK14 group (p < 0.05). Compared with the DEX group, the serum levels of IL-1β, TNF-α and IL-6 in the DEX + oe-MAPK14 group were significantly increased (all p < 0.05).

MPO, SOD and MDA contents in lung tissue of mice in each group
The results of MPO, SOD and MDA in lung tissue of each group showed in Fig. 4. Compared with the Normal group, the SOD content was significantly lower in the other groups, while the MPO and MDA contents were significantly higher (p < 0.05). C, the content of MPO, SOD and MDA in miR-128-3p mimic+oe-MAPK14 group was not statistically different (p > 0.05). SOD content increased and MPO and MDA contents decreased in miR-128-3p mimic group and DEX group when compared with the Model group (p < 0.05). The    The biological prediction website (http://www.microrna.org/ microrna/home.do) predicted that miR-128-3p and MAPK14 have specific binding sites (Fig. 5a). The dual luciferase report system assay showed (Fig. 5b) that the luciferase activity of the Wt-MAPK14 and miR-128-3p mimic transfected group was significantly lower than that in the Wt-MAPK14 and NC mimic group (p < 0.05). However, the luciferase activity of the group transfected with Mut-MAPK14 and miR-128-3p mimic or Mut-MAPK14 and NC mimic group showed no significant difference (p > 0.05). Therefore, miR-128-3p could target and negatively regulate MAPK14 gene expression.
To investigate how miR-128-3p protects mouse acute lung injury through p38 signaling pathway, we detected the gene expression of miR-128-3p and MAPK14 by qRT-PCR and Western blot (Fig. 5 c-e). Compared with the Normal group, miR-128-3p down-regulated and MAPK14 upregulated in the other groups (all p < 0.05). Compared with the Model group, the expression of MAPK14 showed no significant difference in miR-128-3p mimic+oe-MAPK14 group (p > 0.05); however, the expression levels of MAPK14 and p-MAPK14 were significantly decreased in miR-128-3p mimic group and DEX group, which increased in oe-MAPK14 group (p < 0.05). Compared with miR-128-3p mimic group, the expression levels of MAPK14 and p-MAPK14 significantly decreased in miR-128-3p mimic+DEX group, but increased in miR-128-3p mimic+oe-MAPK14 group (p < 0.05); MAPK14 expression level was significantly elevated in the oe-MAPK14 group and the miR-128-3p mimic+oe-MAPK14 group (all p < 0.05). miR-128-3p expression level was significantly increased in the miR-128-3p mimic group, the miR-128-3p mimic+DEX group, and the miR-128-3p mimic+oe-MAPK14 group (all p < 0.05). Compared with the DEX group, the expression levels of MAPK14 and p-MAPK14 in the DEX + oe-MAPK14 group were significantly increased (p < 0.05).

Discussion
As an invasive disease with a high mortality, sepsis is common in the intensive care unit (Qin et al. 2019;Cheng et al. 2019). Studies have proved that cytokine bursts caused by uncontrolled inflammatory responses could induce severe damage in tissue and organ, even causing death (Mo et al. 2018). The lung is the easiest infection target in sepsis, which also plays a critical role in the secretion and release of inflammatory mediators (Dong et al. 2018). Acute lung injury (ALI) is caused by excessive inflammatory response in the lungs. ALI is characterized by respiratory distress, accompanying with diffuse endothelial and epithelial damage, inflammatory cell infiltration as well as pro-inflammatory cytokines release (Cadirci et al. 2019;Yu and Li 2019). Although many attempts have been made to determine new treatment strategies and treatment options for ALI, there are no effective treatments available for clinical use (Yao and Sun 2019). Therefore, there is an urgent need to develop new ALI treatment strategies and explore possible mechanisms for improving the survival rate of ALI patients.
Dexmedetomidine is a α2-adrenergic drug used in clinic, which can be used as an anti-oxidative drug before anesthesia, reducing the concentration of cytokines in kidney tissue and can also reduce lung damage caused by lipopolysaccharide, ischemia-reperfusion and ventilation in animal models (Fu et al. 2017;Gu et al. 2011, Meng et al. 2018b). Dexmedetomidine has also been shown to reduce oxidative stress and apoptotic lesions in lung tissue (Xie et al. 2015). In addition, it can reduce lung tissue fibrosis in rats after acute lung injury (Zhang et al. 2015). The expression of miR-128-3p is frequently observed in a variety of human diseases, including myocardial failure, diabetes, etc. (Cao et al. 2019;Wang et al. 2019). It has been reported that the p38 MAPK signaling pathway is involved in the ALI inflammatory response and mediates the production of many cytokines, including IL-1β, TNF-α and IL-6 (Xiong et al. 2016;. Many anti-inflammatory drugs act by targeting p38 MAPK (Bode et al. 2012).
In our experiment, we evaluated the protective effect of dexmedetomidine on sepsis-induced lung injury and we found that after the injection of Dex, the inflammatory response and oxidative stress damage were alleviated. We also found that MAPK14 expression was up-regulated in the lung tissue of sepsis mice and overexpression of MAPK14 resulted in massive release of inflammatory factors and abnormal oxidative stress damage, which indicates that MAPK14 may be involved in the occurrence and development of acute lung injury in sepsis mice. To further explore the molecular regulation mechanism, we confirmed the target relationship between miR-128-3p and MAPK14. In addition, the expression of miR-128-3p was down-regulated in the lung tissue of sepsis mice. We speculate that miR-128-3p may affect the lung injury of sepsis mice by regulating MAPK14. We also found that both miR-128-3p overexpression can inhibit inflammatory factor release and oxidative stress damage, enhance the therapeutic effect of DEX, while overexpression of MAPK14 can reverse the protective effect dexmedetomidine. in lung tissues of each group. Compared with Normal group, p < 0.05; compared with Model group, # p < 0.05; compared with DEX group, % p < 0.05; compared with miR-128-3p mimic group, & p < 0.05; compared with oe-MAPK14 group, $ p < 0.05; compared with miR-128-3p mimic+oe-MAPK14 group, @ p < 0.05; compared with miR-128-3p mim-ic+DEX group, Δ p < 0.05. DEX, dexmedetomidine. UTR, untranslated region Therefore, we conclude that overexpression of miR-128-3p in septic mice can target and inhibit P38 signaling pathway and improve the protective effect of dexmedetomidine on acute lung injury in septic mice.
In summary, this study demonstrates that miR-128-3p mediates the P38 signaling pathway by targeting the MAPK14 gene, thereby inhibiting inflammatory factor release and oxidative stress damage. The pathogenesis of acute lung injury was further elucidated in this study, which laid a theoretical foundation for the treatment of acute lung injury induced by sepsis in clinic. To further confirm the above results, we need further research to explore how dexmedetomidine specifically acts on the P38 signaling pathway.