Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a)expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate,and stimulated with 1 μg/ml of LPS for 0 h,3 h,6 h and 12 h separately after 90 min.Cells were harvested and supernatant were collected 0 h,3 h,6 h and 12 h after incubation.The expressions of miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant of cells were assayed by enzyme-linked immunosorbent assay ( ELISA ).Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA.Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge ( 359.80 ±57.54) pg/ml (P ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.

Objective:To evaluate the role of brain-derived neurotrophic factor-antisense long-chain non-coding RNA (BDNF-AS) in amygdala in the development of neuropathic pain (NP) in rats.Methods:Healthy clean-grade male Sprague-Dawley rats, aged 2 months, weighing 200-260 g, were used to develop NP model via ligation of left L 5-6 spinal nerve, while control group was only subjected to the exposure of L 5-6 spinal nerve without ligation.This study was performed in two parts.Experiment Ⅰ Fifty-six rats were divided into 3 groups by the random number table method: sham operation group (Sham group, n=8), NP group ( n=24) and BDNF ( n=24). In BDNF group, exogenous BDNF was injected into bilateral amygdala at 1, 3, 6, 13 and 20 days after development of the model, with 100 pmol at each side.Eight rats were sacrificed at 7, 14 and 21 days after the model was developed in NP and BDNF groups and after the model was developed in Sham group, the brains were removed, and the amygdala was isolated for determination of the BDNF content (by enzyme-linked immunosorbent assay), the number of BDNF-positive cells (by immunohistochemistry), and expression of BDNF-AS (by real-time quantitative polymerase chain reaction). Experiment Ⅱ Thirty-two rats were divided into 4 groups ( n=8 each) using the random number table method: Sham operation group, NP group, BDNF group and siRNA group.At 1, 3, 6, 13 and 20 days after development of the model, exogenous BDNF 100 pmol and siRNA-BDNF-AS 50 nmol were injected into the amygdala at each side in BDNF group and siRNA group, respectively.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before development of the model (T 0) and at 4, 7, 14 and 21 days after development of the model (T 1-4). After the last behavioral test was completed, the rats were sacrificed, and the spinal cord tissues were collected to measure the contents of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α). Results:Experiment Ⅰ Compared with Sham group, the content of BDNF and the number of BDNF positive cells were significantly decreased, and the expression of BDNF-AS was up-regulated at each time point after development of the model in group NP ( P<0.05). Compared with NP group, the content of BDNF and the number of BDNF positive cells were significantly increased, and the expression of BDNF-AS was down-regulated at each time point after development of the model in group NP ( P<0.05). Experiment Ⅱ Compared with Sham group, MWT was significantly decreased and TWL was shortened at T 1-4, and the contents of IL-1β, IL-6 and TNF-α were increased in NP, BDNF and siRNA groups ( P<0.05). Compared with NP group, MWT was significantly increased and TWL was prolonged at T 1-4, and the contents of IL-1β, IL-6 and TNF-α were decreased in BDNF and siRNA groups ( P<0.05). Conclusions:The mechanism underlying the development of NP may be related to the up-regulation of BDNF-AS expression in amygdala, inhibition of BDNF synthesis and promotion of inflammatory responses in the spinal cord of rats.