Role of 14-3-3σgene in the regulation of endotoxin/lipopolysaccharide-induced inflammatory responses in human pulmonary epithelial cells
10.3760/cma.j.cn501120-20191203-00450
- VernacularTitle:14－3－3σ基因在调控内毒素/脂多糖诱导的人肺上皮细胞炎症反应中的作用
- Author:
Chunxia GAN
1
;
Mingzhuo LIU
;
Xincheng LIAO
;
Zhonghua FU
;
Xiaoping ZENG
;
Hongmei WANG
;
Guanghua GUO
Author Information
1. 南昌大学第一附属医院烧伤科 330006
- Keywords:
Endotoxins;
Lipopolysaccharides;
Inflammation;
14-3-3σ;
Bax/B-cell lymphoma-2;
Pulmonary epithelial cell
- From:
Chinese Journal of Burns
2020;36(4):260-266
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the mechanism of 14-3-3σgene in regulating inflammatory response of human pulmonary epithelial cells induced by endotoxin/lipopolysaccharide (LPS).Methods:(1) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group and PCMV6-14-3-3σgroup using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid, and cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid. The protein expression of 14-3-3σin cell was detected by Western blotting at 48 hours after transfection. (2) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group, PCMV6-14-3-3σgroup, PCMV6-14-3-3σ+ LPS group, and LPS group using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid for 42 hours. Cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in PCMV6-14-3-3σ+ LPS group were stimulated with 1 μg/mL LPS (the same final mass concentration below) for 6 hours after being transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in LPS group were stimulated by LPS for 6 hours. The protein expressions of Bax and B-cell lymphoma-2 (Bcl-2) were detected by Western blotting, and the ratio of Bax to Bcl-2 was calculated. Apoptotic rate was detected by flow cytometry. The mRNA expressions of tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1β) in cells were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction technique. Content of TNF-α and IL-1β in cell culture supernatant was detected by enzyme-linked immunosorbent assay. Data were statistically analyzed with t test, one-way analysis of variance, and least significant difference test. Results:(1) At 48 hours after transfection, the protein expression of 14-3-3σin cells of PCMV6-14-3-3σgroup (1.05±0.03) was significantly higher than that in control group (0.78±0.04, t=5.41, P<0.01). (2) Compared with those in control group, the ratio of Bax to Bcl-2, apoptotic rate, mRNA expressions of TNF-α and IL-1β, and content of TNF-α and IL-1β in cell supernatant in PCMV6-14-3-3σgroup showed no significant difference ( P>0.05); the above-mentioned indexes of cells in LPS group were significantly higher or increased ( P<0.01). Compared with those in LPS group, the above-mentioned indexes of cells in PCMV6-14-3-3σ+ LPS group were significantly lower or decreased ( P<0.01). Conclusions:14-3-3σis a key factor in regulating apoptosis. It can alleviate the LPS-induced inflammatory responses by regulating the ratio of apoptotic regulators Bax to Bcl-2 and inhibiting apoptosis of human pulmonary epithelial cells.
