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Objective To explore the role of miR-199a-3p in osteoblast proliferation induced by fluid shear stress (FSS) and the potential molecular mechanism. Methods Osteoblast MC3T3-E1 was treated with 1. 2 Pa FSS with time gradients of 0, 15, 30, 45, 60, 75 and 90 min, respectively. MC3T3-E1 cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor. MC3T3-E1 cells were transfected with miR-199a-3p mimic and itsnegative control and then treated with 1. 2 Pa FSS for 45 min. The pc DNA NC, pc DNA-CABLES -1, si RNA NC and si RNA CABLES-1 were transfected into MC3T3-E1 cells. The pc DNA-CABLES-1 and mir-199a-3p mimic and SI NA-cables-1 and miR-199a-3p inhibitor were co-transfected, respectively. Cell activity was detected by CCK-8 assay. Real-time quantitative PCR (RT-qPCR) was used to detect expression levels of CABLES-1, miR-199a-3p, CDK 6, Cyclin D1 and PCNA. Luciferase reporting assay was used to detect targeting relationship between CABLES-1 and miR-199a-3p. Immunofluorescence was used to detect protein expression of CABLES-1.Western blot was used to detect protein expression of CABLES-1, CDK 6, PCNA and Cyclin D1. Results Mir- 199a-3p in MC3T3-E1 cells was significantly down-regulated by FSS. Over-expressed miR-199a-3p inhibitedosteoblast proliferation, and down-regulated miR-199a-3p expression promoted osteoblast proliferation. miR-199a- 3p could reverse the FSS-induced proliferation in osteoblasts. Dual luciferase assay showed that miR-199a-3p targeted to CABLES-1 and over-expressed miR-199a-3p inhibited expression of CBALES-1 protein. CABLES-1 could promote proliferation of osteoblasts. miR-199a-3p inhibited osteoblast proliferation induced by FSS through CABLES-1. Conclusions FSS-induced osteoblast proliferation can be realized by down-regulated miR-199a-3p expression via targeting CABLES-1. The findings in this study provide new direction for researches on mechanism of FSS-induced osteoblast proliferation, as well as new ideas for future research on clinical application of mechanical loading in the treatment of bone and joint diseases.
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Objective:To screen the time points of high survival rate and efferocytosis dysfunction of rat alveolar macrophages stimulated by cigarette smoke extract (CSE), establish an in vitro model of alveolar macrophage efferocytosis function, and study chronic respiratory diseases with chronic inflammatory reaction as the main pathological changes. Methods:① Time point screening experiment: rat alveolar macrophages (NR8383 cells) were cultured in vitro, and the cells in logarithmic growth phase were divided into blank control group (100 μL complete medium) and 5% CSE group (90 μL complete medium + 10 μL 100% CSE). Alma blue method was used to detect the effect of 5% CSE on the activity of NR8383 cells at 6, 12, 24 and 48 hours. ② Apoptosis induction experiment: rat type Ⅱ alveolar epithelial cells (RLE-6TN cells) were cultured in vitro as phagocytic target cells of NR8383 cells, and the cells in logarithmic growth phase were divided into blank control group and 10, 30 and 60 minutes groups after ultraviolet exposure (apoptosis was induced by 30 000 μJ/cm 2 ultraviolet irradiation for 15 minutes). Flow cytometry was used to detect the apoptosis rate of RLE-6TN cells cultured for 10, 30 and 60 minutes after ultraviolet exposure. ③ Cell efferocytosis experiment: NR8383 cells in logarithmic phase were divided into blank control group and 5% CSE group. Two hours before NR8383 cells were stimulated by CSE for 6, 12 and 24 hours, RLE-6TN cells were exposed to ultraviolet to induce apoptosis, and the RLE-6TN cell suspension was added to NR8383 cells (the ratio of RLE-6TN cells to NR8383 cells was 5∶1). Flow cytometry was used to detect the efferocytosis rate of NR8383 cells to RLE-6TN cells at different time points treated with 5% CSE. Results:① Compared with the blank control group, the activity of NR8383 cells significantly decreased after treatment with 5% CSE for 48 hours [cell reduction rate: (68.5±4.1)% vs. (73.6±2.3)%, P < 0.05]. However, there were no significant differences when the activities of NR8383 cells treated with 5% CSE for 6, 12 and 24 hours were compared with the blank control group, so these three time points were selected for the subsequent establishment of alveolar macrophage cell efferocytosis dysfunction in vitro model experiment. ② Compared with the blank control group, the apoptosis rate of RLE-6TN cells significantly increased at 10, 30 and 60 minutes after ultraviolet exposure [(66.87±8.63)%, (85.51±2.39)%, (96.13±2.74)% vs. (9.13±3.17)%, all P < 0.01] in a time-dependent manner. Considering that it taked about 50 minutes for RLE-6TN cells to be labeled with PKH26 membrane labeling probe, 10 minutes after ultraviolet exposure was selected to label RLE-6TN cells. ③ Compared with the blank control group, the efferocytosis function of NR8383 cells was significantly decreased after treatment with 5% CSE for 12 hours [cell efferocytosis rate: (33.64±1.30)% vs. (44.02±2.71)%, P < 0.01], but there was no significant effect on the efferocytosis function of NR8383 cells at 6 hours and 24 hours. Conclusions:CSE can induce alveolar macrophage cell efferocytosis dysfunction. Based on the test results of the effect of 5% CSE on NR8383 cell activity and cell efferocytosis function, 12 hours with high survival rate and weak efferocytosis effect of NR8383 cells can be selected as the in vitro model condition of alveolar macrophage cell efferocytosis dysfunction.
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As one of the top three causes of death in the world, chronic obstructive pulmonary disease (COPD) is a serious hazard to human health. Macrophages play an important role in COPD, and their efferocytosis function is essential for ending chronic inflammation of COPD. Efferocytosis damage of alveolar macrophages (AM) in patients with COPD causes the rising of bacterial infection and airway bacterial colonization risk in lungs, which is the main reason for the acute exacerbation and the rising of incidence rate and mortality rate in COPD. In recent years, the regulation of macrophage efferocytosis function in COPD has becoming a research hotspot. Progress on the role of macrophage efferocytosis function on COPD, and the breakthrough points of improving AM efferocytosis dysfunction by traditional Chinese medicine is reviewed, so as to provide new ideas for the prevention and treatment of COPD.