Animals and model establishment
A total of 120 healthy SPF C57BL/6 mice (purchased from Zhejiang Experimental Animal Center), clean grade, body weight 35 ± 5 g, were used for experiment in this study. Mice were divided into 8 groups: Normal group (healthy mice without treatment), Model group (model mice), DEX group (model mice treated with dexmedetomidine), miR-128-3p mimic group (model mice treated with miR-128-3p overexpression vectors), oe-MAPK14 group (model mice treated with MAPK14 overexpression vectors), miR-128-3p mimic + oe-MAPK14 group (model mice treated with miR-128-3p and MAPK14 overexpression vectors), miR-128-3p mimic + DEX group (model mice treated with miR-128-3p overexpression vector and dexmedetomidine), DEX + oe-MAPK14 group (model group mice treated with MAPK14 overexpression vector and dexmedetomidine), with ten mice in each group.
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 hours, 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 [8].
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 ddH2O. Fluorescence quantitative PCR was performed in ABIPRISM® 7300 (model Prism® 7300, Shanghai Kunke Instrument Equipment Co., Ltd., China). The reaction conditions were: pre-denaturation 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.
Table 1
Gene | Sequence |
miR-128-3p | F: 5'-GGTC ACAGTGAACCGGTC-3' |
R: 5'-GTGCAGGGTCC GAGGT-3' |
MAPK14 | F: 5'-CTGACCGACGACCACGTTC-3' |
R: 5'-CTTCGTTCACAGCTAGGTTGC-3' |
U6 | F: 5'-CTCGCTTCGGCAGCACA-3' |
R: 5'-AACGCTTCACGAATTTGCGT-3' |
GAPDH | F: 5'-TTCAACGGCACAGTCAAGG-3' |
R: 5'-CACCAGTGGATGCAGGGAT-3' |
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 anti-mouse MAPK14 (ab31828, 1:500, Abcam, UK), GAPDH (ab22555, 1:2,000, 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 1:5,000) 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 (PaO2) and carbon dioxide partial pressure (PaCO2). 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 hours, 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 sclice was conventionally dehydrated, transparentized and sealed with neutral gum. Each sclice was observed under an optical microscope (XP-330, Shanghai Bingyu Optical Instrument Co., Ltd., Shanghai, China).
Activity detection of myeloperoxidase (MPO), mitochondrial superoxide dismutase (SOD) and malondialdehyde (MDA)
The lung tissue in size of 125 mm3 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 3,500 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: 69-21138, 69-22800, 69-25328, 69-40133; 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.