Objective:To explore the effects and mechanism of annexin A1 ( ANXA1)-overexpressing human adipose-derived mesenchymal stem cells (AMSCs) in the treatment of mice with acute respiratory distress syndrome (ARDS). Methods:The experimental study method was adopted. After the adult AMSCs were identified by flow cytometry, the 3 rd passage cells were selected for the follow-up experiments. According to the random number table (the same grouping method below), the cells were divided into ANXA1-overexpressing group transfected with plasmid containing RNA sequences of ANXA1 gene and no-load control group transfected with the corresponding no-load plasmid. The other cells were divided into ANXA1-knockdown group transfected with plasmid containing small interfering RNA sequences of ANXA1 gene and no-load control group transfected with the corresponding no-load plasmid. At post transfection hour (PTH) 72, the fluorescence expression was observed under a fluorescence microscope imaging system, and the protein and mRNA expressions of ANXA1 were detected by Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction respectively (with the sample numbers being 3). Fifty male C57BL/6J mice aged 6-8 weeks were divided into sham injury group, ARDS alone group, normal cell group, ANXA1-overexpressing group, and ANXA1-knockdown group, with 10 mice in each group. Mice in the last 4 groups were treated with endotoxin/lipopolysaccharide to make ARDS lung injury model, and mice in sham injury group were simulated to cause false injury. Immediately after injury, mice in sham injury group and ARDS alone group were injected with normal saline through the tail vein, while mice in normal cell group, ANXA1-overexpressing group, and ANXA1-knockdown group were injected with normal AMSCs, ANXA1-overexpressing AMSCs, and ANXA1-knockdown AMSCs, correspondingly. At post injection hour (PIH) 24, 5 mice in each group were selected, the Evans blue staining was performed to observe the gross staining of the right lung tissue, and the absorbance value of bronchoalveolar lavage fluid (BALF) supernatant of left lung was detected by microplate reader to evaluate the pulmonary vascular permeability. Three days after injection, the remaining 5 mice in each group were taken, the right lung tissue was collected for hematoxylin-eosin staining to observe the pathological changes and immunohistochemical staining to observe the CD11b and F4/80 positive macrophages, and the levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and IL-1β in BALF supernatant of left lung were determined by enzyme-linked immunosorbent assay. Data were statistically analyzed with paired sample t test, one-way analysis of variance, and least significant difference test. Results:At PTH 72, AMSCs in both ANXA1-overexpressing group and ANXA1-knockdown group expressed higher fluorescence intensity than AMSCs in corresponding no-load control group, respectively. At PTH 72, compared with those in corresponding no-load control group, the protein and mRNA expressions of ANXA1 in ANXA1-overexpressing group were significantly increased (wth t values of 249.80 and 6.56, respectively, P<0.05), while the protein and mRNA expressions of ANXA1 in ANXA1-knockdown group were significantly decreased (wth t values of 176.50 and 18.18, respectively, P<0.05). At PIH 24, compared with those in sham injury group (with the absorbance value of BALF supernatant being 0.041±0.009), the lung tissue of mice in ARDS alone group was obviously blue-stained and the absorbance value of BALF supernatant (0.126±0.022) was significantly increased ( P<0.05). Compared with those in ARDS alone group, the degree of blue-staining in lung tissue of mice was significantly reduced in normal cell group or ANXA1-overexpressing group, and the absorbance values of BALF supernatant (0.095±0.020 and 0.069±0.015) were significantly decreased ( P<0.05), but the degree of blue-staining in lung tissue and the absorbance value of BALF supernatant (0.109±0.016, P>0.05) of mice in ANXA1-knockdown group had no significant change. Compared with that in normal cell group, the absorbance value of BALF supernatant of mice in ANXA1-overexpressing group was significantly decreased ( P<0.05). Three days after injection, the lung tissue structure of mice in ARDS alone group was significantly damaged compared with that in sham injury group. Compared with those in ARDS alone group, hemorrhage, infiltration of inflammatory cells, alveolar collapse, and interstitial widening in the lung tissue of mice were significantly alleviated in normal cell group and ANXA1-overexpressing group, while no significant improvement of above-mentioned lung tissue manifestation was observed in ANXA1-knockdown group. Three days after injection, the numbers of CD11b and F4/80 positive macrophages in the lung tissue of mice in ARDS alone group were significantly increased compared with those in sham injury group. Compared with those in ARDS alone group, the numbers of CD11b and F4/80 positive macrophages in lung tissue of mice in normal cell group, ANXA1-overexpressing group, and ANXA1-knockdown group reduced, with the most significant reduction in ANXA1-overexpressing group. Three days after injection, compared with those in sham injury group, the levels of TNF-α, IL-6, and IL-1β in BALF supernatant of mice in ARDS alone group were significantly increased ( P<0.05). Compared with those in ARDS alone group, the levels of TNF-α, IL-6, and IL-1β in BALF supernatant of mice in normal cell group and ANXA1-overexpressing group, as well as the level of IL-1β in BALF supernatant of mice in ANXA1-knockdown group were significantly decreased ( P<0.05). Compared with that in normal cell group, the level of TNF-α in BALF supernatant of mice was significantly decreased in ANXA1-overexpressing group ( P<0.05) but significantly increased in ANXA1-knockdown group ( P<0.05). Conclusions:Overexpression of ANXA1 can optimize the efficacy of AMSCs in treating ARDS and enhance the effects of these cells in inhibiting inflammatory response and improving pulmonary vascular permeability, thereby alleviating lung injury of mice with ARDS.

Objective:To explore the epidemiological characteristics and treatment outcomes of patients with inhalation injuries combined with total burn area less than 30% total body surface area (TBSA).Methods:A retrospective observational study was performed on medical records of 266 patients with inhalation injuries combined with total burn area less than 30%TBSA who were admitted to the First Affiliated Hospital of Naval Medical University from January 2008 to December 2016 and met the inclusion criteria. The following statistical data of the patients were collected, including gender, age, injury site, injurious factors of inhalation injury, degree of inhalation injury, combined total burn area, tracheotomy, time of tracheotomy, mechanical ventilation, whether stayed in intensive care unit (ICU) or not, microbial culture results of bronchoalveolar lavage fluid, length of hospital stay, length of ICU stay, mechanical ventilation days, and respiratory tract infections. Single factor and multivariate linear regression analysis were used to screen out the risk factors impacting the length of hospital stay, length of ICU stay, and mechanical ventilation days of patients. Single factor and multivariate logistic regression analysis were used to screen out the risk factors impacting respiratory tract infections of patients.Results:The 266 patients included 190 males and 76 females, with the majority age of above or equal to 21 years and below 65 years (217 patients). The major injury site was confined space. The major factor causing inhalation injury was hot air. Mild and moderate inhalation injuries were more common in patients. The combined total burn area was 9.00% (3.25%, 18.00%) TBSA. In 111 patients who had tracheotomy, most of them received the procedures before being admitted to the First Affiliated Hospital of Naval Medical University. The length of hospital stay of patients was 27 (10, 55) days. The length of ICU stay of 160 patients who were hospitalized in ICU was 15.5 (6.0, 40.0) days. The mechanical ventilation days of 109 patients who were conducted with mechanical ventilation were 6.0 (1.3, 11.5) days. A total of 119 patients were diagnosed with respiratory tract infections, with 548 strains including 35 types of pathogens isolated, mainly Gram-negative bacteria. Single factor linear regression analysis showed that age, injurious factors of inhalation injury, combined total burn area, degree of inhalation injury (moderate and severe), tracheotomy, mechanical ventilation, and respiratory tract infections were the factors impacting the length of hospital stay of patients ( β=-0.198, -0.224, 0.021, 0.127, 0.164, -0.298, 0.357, 0.447, 95% confidence interval (CI)=-0.397--0.001, -0.395--0.053, 0.015-0.028, 0.009-0.263, 0.008-0.319, -0.419--0.176, 0.242-0.471, 0.340-0.555, P<0.1). Multivariate linear regression analysis showed that with mechanical ventilation and respiratory tract infections were the independent risk factors impacting the length of hospital stay of patients ( β=0.146, 0.383, 95% CI=0.022-0.271, 0.261-0.506, P<0.05 or P<0.01). Single factor linear regression analysis showed that injurious factors of inhalation injury, combined total burn area, degree of inhalation injury (moderate and severe), tracheotomy (no tracheotomy and prophylactic tracheotomy), mechanical ventilation, and respiratory tract infections were the factors impacting the length of ICU stay of patients ( β=0.225, 0.008, 0.237, 0.203, -0.408, -0.334, 0.309, 0.523, 95% CI=0.053-0.502, 0.006-0.010, -0.018-0.457, -0.022-0.428, -0.575--0.241, -0.687--0.018, 0.132-0.486, 0.369-0.678, P<0.1). Multivariate linear regression analysis showed that with respiratory tract infections was the independent risk factor impacting the length of ICU stay of patients ( β=0.440, 95% CI=0.278-0.601, P<0.01). Single factor linear regression analysis showed that injury site, injurious factors of inhalation injury (smoke and chemical gas), combined total burn area, degree of inhalation injury (moderate and severe), tracheotomy (no tracheotomy and prophylactic tracheotomy), and respiratory tract infections were the factors impacting mechanical ventilation days of patients ( β=-0.300, 0.545, 0.163, 0.005, 0.487, 0.799, -0.791, -0.736, 0.300, 95% CI=-0.565--0.034, 0.145-0.946, 0.051-1.188, 0.001-0.009, 0.127-0.847, 0.436-1.162, -1.075--0.508, -1.243--0.229, 0.005-0.605, P<0.1). Multivariate linear regression analysis showed that smoke inhalation, severe inhalation injury, and respiratory tract infections were the independent risk factors impacting mechanical ventilation days of patients ( β=0.210, 0.495, 0.263, 95% CI=0.138-0.560, 0.143-0.848, 0.007-0.519, P<0.05 or P<0.01). Single factor logistic regression analysis showed that age, injury site, combined total burn area (10%-19%TBSA and 20%-29%TBSA), degree of inhalation injury (moderate and severe), tracheotomy (prophylactic tracheotomy and no tracheotomy), and mechanical ventilation were the factors impacting respiratory tract infections of patients (odds ratio=1.079, 0.815, 1.400, 1.331, 1.803, 1.958, 0.990, 0.320, 3.094, 95% CI=0.840-1.362, 0.641-1.044, 1.122-1.526, 1.028-1.661, 1.344-2.405, 1.460-2.612, 0.744-1.320, 0.241-0.424, 2.331-4.090, P<0.1). Multivariate logistic regression analysis showed that with mechanical ventilation was the independent risk factor impacting respiratory tract infections of patients (odds ratio=4.300, 95% CI=2.152-8.624, P<0.01). Conclusions:The patients with inhalation injuries combined with total burn area less than 30%TBSA are mainly young and middle-aged males. Smoke inhalation, degree of inhalation injury, with mechanical ventilation and respiratory tract infections are the factors that affect the outcomes of patients with inhalation injuries combined with total burn area less than 30%TBSA. Additionally, prophylactic tracheotomy shows its potential value in reducing respiratory tract infections in patients with moderate or severe inhalation injuries.