A total of 40 specific pathogen free male Sprague-Dawley rats (n=40; Hebei Province Animal Research Center; age, 8 weeks; weight, 240-260 g) were used in the cuuremt study. Rats were housed in a temperature-controlled room (21±2˚C) with a relative humidity of 30-40% and a 12 h light/dark cycle. All rats had free access to water and food. Mouse monocyte macrophage RAW264.7 cells (American Type Culture Collection), ELISA kit for cytokines (R&D Systems, Inc. cat. no. ML-Elisa-1458), phosphate buffered saline (EMD Millipore), bicinchoninic acid (Beyotime Institute of Biotechnology), antibodies (all 1:1,000; all from Abcam), RNA reverse transcription kit (Roche Diagnostics GmbH), hematoxylin and eosin (H&E) staining reagent (Beyotime Institute of Biotechnology) and TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

In the present study, SD rats were divided into a sham group and a sepsis-induced lung injury (sepsis) group according to whether cecal ligation and puncture was performed. The RAW264.7 cells were stimulated with LPS and divided into the following groups, blank, LPS, LPS + control and LPS + mimic. The present study was approved by the Ethics Committee of the Fourth Central Hospital of Baoding City (Hebei, China).

Following anesthesia via intraperitoneal injection of pentobarbital sodium (dose, 40 mg/kg), rats underwent laparotomy via a linear abdominal incision. The cecum was punctured twice at different sites with an 18-gauge needle and gently pressed until the feces was squeezed out. The intestines were placed back in the abdomen and the abdominal incision was subjected to layered closure. Upon completion of surgery, saline (5 ml/100 g body weight) was injected subcutaneously into the rats in the sepsis and sham groups to replace the extracellular fluid that was isolated during peritonitis. The rats in the sham group underwent a sham surgery without ligation or puncture of the cecum.

Lung tissues and monocytes were homogenized in TRIzol^® reagent and total RNAs were extracted according to the manufacturer's protocol. Subsequently, 3 µg RNAs were reverse transcribed into complementary deoxyribose nucleic acids (cDNAs) at 37˚C for 1 h using the RNA reverse transcription kit. The PCR mixture contained 0.5 µl Taq polymerases, 1 µl of each primer and 2 µl of each cDNA sample, with a final volume of 20 µl. In all amplifications, three repeated wells were set and quantitative changes in mRNA expression were evaluated by qPCR. qPCR was subsequently performed using the SYBR-Green Master kit (Roche Diagnostics). Quantitative analysis was performed using the ABI 7500 fluorescence PCR amplification instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction system volume was 25 µl. The thermocycling conditions for qPCR were as follows: Pre-denaturation at 95˚C for 5 min, denaturation at 95˚ for 30 sec, annealing at 60˚ for 45 sec, extension at 72˚ for 3 min for 35 cycles, and then a final extension at 72˚C for 5 min. PCR products were stored at 4˚C. The following primer pairs were used for qPCR: GAPDH: Forward, 5'-ACAGCAACAGGGTGGTGGAC-3' and reverse, 5'-TTTGAGGGTGCAGCGAACTT-3'; TLR4: Forward, 5'-AAGGCATGGCATGGCTTACAC-3' and reverse, 5'-GGCCAATTTTGTCTCCACAGC-3'; NF-κB: Forward, 5'-CCCAAACCTTGGCATCCTG-3' and reverse, 5'-CCGAACAACACTCAAATCC-3'; miR-223: Forward, 5'-UGUCAGUUUGUCAAAUACCCCAAAA-3' and reverse, 5'-UGUCAGUUUGUCAAAUACCCCAUUU-3'. Finally, Cq values were processed using the 2^-∆∆Cq method (20), with GAPDH serving as the control.

Pneumonectomy was performed 24 h after the rat model was established in the two groups. The trachea was fixed at 25˚C with 100% ethanol for 48 h at a distance of 20 mm from the lung. Following fixation, the paraffin-embedded tissue sections were cut into slices (thickness, ~5 µm) and histologically stained with methylene blue at 25˚C for 5 min. The slices were stored at -65˚C overnight prior to the experiment. At the beginning of the experiment, the sections were deparaffinized in 65, 70 and 90% xylene tanks at 25˚C, dehydrated in ethanol with five gradually increasing concentrations (60, 75, 90, 95 and 98%), gently washed with deionized water four times and dried in the air. The tissues were subsequently spread on a sterile glass slide and placed above an alcohol lamp for 15-30 sec to dry. Eosin Y dye solution was added dropwise for 3 min for cell plasma staining. Following cytoplasm staining, the dye solution was diluted with distilled water to terminate the staining. The stained tissue samples were cleaned with ethanol and added to the methylene blue dye solution dropwise for cell nucleus staining. After 60 sec, staining was ceased by diluting the dye solution with deionized water. A light microscope (IX70; Olympus) was used to observe cells under five randomly selected fields of view. Following staining, the lung was independently evaluated and scored by two experts. For each rat, the following characteristics of three different lobes were examined: Interstitial edema, hemorrhage and neutrophil infiltration.

Cervical dislocation (following anesthesia with intraperitoneal injection of pentobarbital sodium at a dose of 40 mg/kg) was used as the method of euthanasia. The tissues surrounding the lungs were dissected and stored in equal parts at -80˚C and were thawed prior to use. The RIPA (radioimmunoprecipitation assay) protein lysate (Beyotime Institute of Biotechnology) was mixed with the tissues evenly, swirled three times for 15 sec each time and centrifuged at 13,500 x g/min at 4˚C for 40 min. Total protein concentration was calculated by bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Institute of Biotechnology). The protein concentration was obtained according to 450 mm absorbance and the samples were boiled for denaturation. Subsequently, the proteins were separated via SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore). Then non-specific antigen sites were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at 25˚C for 1 h and incubated with TLR4 and NF-κB antibodies (1:1,000; cat. no. ab32536) and GAPDH (1:1,000; cat. no. ab8245) at 4˚C for 14 h. Finally, secondary antibodies (HRP-conjugated goat anti-rabbit IgG; 1:5,000; cat. no. ab6721) were added at 25˚C for 1 h. Immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (Amersham; Cytiva). The gray value was analyzed using ImageJ software (version 1.38; National Institutes of Health).

RAW264.7 cells were evenly spread in a six-well plate with a total volume of 1 ml. LPS was added after 12-16 h of culture at 37˚C and, after 24 h of stimulation, the stimulated inflammatory cells were selected for seeding into 24-well plates at 1x10⁵ cells/well. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform the transfection. The cells were transfected with miR-223 mimic (20 µm) and miR-223 control (20 µm) when the confluency reached 70-80%. The sequences were as follows (Dojindo Molecular Technologies, Inc.): miR-223 mimic: Forward, 5'-CCUACGGAGUUACCAACCUGGC-3' and reverse, 5'-AAGACUGGCCAGCAUUAUAGAC-3'; miR-223 control: Forward 5'- GCCAGGACGUUCGAGACGUCAG-3' and reverse, 5'-GCAGCUGCGACGUUACCUUAGA-3'. The transfection reagent was added into the cells for 12 h of culturing at 37˚C and then replaced with a normal culture medium to observe the cell state after 48 h.

The supernatant from the blank, LPS, LPS + control and LPS + mimic groups was collected for analysis. Bronchoalveolar lavage (BAL) fluid was collected from the blank, LPS, LPS + control and LPS + mimic groups. The diluted standard substance was defrosted and diluted according to the 50% ratio gradient. Then 90 µl of standard substance and horseradish peroxidase (HRP)-labeled working solution were added to the standard well. ELISA plates (cat. no. 201303; Lianyungang Jinma Biotech Co., Ltd.) were coated with 100 µl/well of the diluted antigen for 1 h at 37˚C, after which plates were washed three times with PBS containing 0.05% Tween 20 and blocked with 200 µl 1% bovine serum albumin and 0.05% Tween 20 diluted in PBS for 2 h at 37˚C. The plate was cleaned five times and 90 µl HRP reaction substrate was added to each well in the dark. After 20 min, the enzymatic reaction was terminated, the optical density value was detected and the experimental data were recorded.

Experimental results were obtained and analyzed using Statistical Product and Service Solutions 17.0 software (SPSS, Inc.). Differences between two groups were analyzed by using a Student's t-test. Comparisons between three groups was performed using one-way ANOVA followed by the Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Compared with that in the sham group, the expression levels of miR-223 and NF-κB and TLR4 proteins were significantly higher than in the sepsis group (P<0.05), indicating that miR-223 is associated with the TLR/NF-κB signaling pathway (Fig. 1).

Compared with the sham group, the sepsis group demonstrated significantly higher IL-6 and IL-1β content in the BAL fluid, indicating a strong inflammatory response in the sepsis group (P<0.05; Fig. 2).

H&E staining and microscopic observation of the lung tissue from the sham group indicated alveoli with a normal structure and few pathological changes. In the sepsis group, the alveoli were observed to be surrounded by a large number of neutrophils, the mesenchyme was swollen, part of the alveolar wall exhibited fibrosis and the alveolar wall was thickened (Fig. 3), suggesting that the inflammatory response of cells is enhanced following sepsis.

Transfection was verified to be successful (Fig. 1B). Following LPS stimulation, the expression levels of miR-223 and TLR4 and NF-κB proteins in the cells of the LPS group were significantly increased when compared with the blank group (P<0.05). When compared with the other three groups, miR-223 expression in the LPS + mimic group was the highest (Fig. 4A). The protein expression levels of TLR4 and NF-κB in the LPS + mimic group were significantly reduced when compared with the LPS group and the LPS + control group (P<0.05) (Fig. 4B and C). qPCR revealed that the mRNA expression levels of TLR4 and NF-κB in the LPS + mimic group were significantly higher when compared with those in the blank group, but they were significantly decreased in the LPS + mimic group when compared with the LPS group and the LPS + control group (P<0.01; Fig. 4D). The results indicate that the inflammatory responses weaken after miR-223 is elevated and the anti-inflammatory effect of miR-223 is related to the inhibition of the TLR4/NF-κB pathway.

Following LPS stimulation, the content of IL-6 and IL-1β in the cell supernatant increased significantly. Compared with that in the LPS group and the LPS + control group, the content of IL-6 and IL-1β in the supernatant of the LPS + mimic group was significantly decreased (P<0.05; Fig. 5), indicating that miR-223 inhibits the expression of IL-6 and plays an anti-inflammatory role.