Effect of tumor necrosis factor-α on the expression of tight junction proteins ZO-1 and Claudin-4 in rat alveolar epithelial cells
10.3760/cma.j.issn.1671-0282.2019.09.007
- VernacularTitle: 肿瘤坏死因子-α对大鼠肺泡上皮细胞紧密连接蛋白ZO-1、Claudin-4表达的影响
- Author:
Guocui SHI
1
;
Shenmao MA
2
;
Xigang MA
3
Author Information
1. Department of Respiratory Medicine, Cangzhou People’s Hospital, Cangzhou 061000, China
2. Resident Standardized Training Base, General Hospital of Ningxia Medical University, Yinchuan 750004, China
3. Department of Critical Care Medicine, General Hospital of Ningxia Medical University, Yinchuan 750004, China
- Publication Type:Journal Article
- Keywords:
Tumor necrosis factor-α
;
Tight junction proteins;
Acute lung injury;
Pulmonary epithelial barrier;
Apoptosis
- From:
Chinese Journal of Emergency Medicine
2019;28(9):1093-1099
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of TNF-α on the expressions of tight junction protein ZO-1 and Claudin-4 in rat alveolar epithelial typeⅡ cells (AEC-Ⅱ).
Methods:Rat AEC-Ⅱ cells were cultured in vitro and divided into the control group, and TNF-α 24 h, 48 h, 72 h groups. The control group was cultured with DME F12 medium containing fetal bovine serum, and the TNF-α groups were intervened by TNF-α with a concentration of 10 ng/mL for 24 h, 48 h, and 72 h, respectively. After co-culture, the transmission electron microscopy was used to identify and observe the ultrastructural changes of rat AEC-Ⅱ cells. The cell inhibition rate was determined by MTT assay, and the cell apoptosis rate was measured by flow cytometry. The expression and distribution of tight junction protein ZO-1 and Claudin-4 in each group were observed by immunofluorescence method. The transcriptional levels of ZO-1 and Claudin-4 were detected by real-time fluorescent quantitative PCR. The expressions of ZO-1 and Claudin-4 were detected by Western blot. One-way analysis of variance (ANOVA) was used for comparisons among groups and SNK-q test was used for pairwise comparisons between groups. The non-parametric test of rank transformation was used when homogeneity of variance were not met. The value of P<0.05 was considered significantly different.
Results:Transmission electron microscopy showed that the AEC-Ⅱ cells in the control group obtained characteristic lamellar bodies of different sizes. The lamellar bodies in each TNF-α group were gradually emptied and apoptotic cells were observed under the visual field. The cell inhibition rate and early apoptosis rate of each TNF-α group were significantly higher than those of the control group (all P<0.05). Confocal microscopy showed that ZO-1 was linearly distributed along the cell membrane, and Claudin-4 was scattered along the cell membrane. In the TNF-α groups, the fluorescence intensity of ZO-1 was weakened and the continuity was broken as well as the linear structure. The fluorescence intensity and density of Claudin-4 were decreased in the TNF-α groups. Besides, the transcription and expression of ZO-1 in the TNF-α 24 h group were lower than those in the control group, but there was no significant difference (P>0.05). However, the mRNA level of ZO-1 in the TNF-α 48 h group (0.28±0.06) and 72 h group (0.13±0.07) were significantly lower than those in the control group. And their protein levels of TNF-α 48 h group (0.44±0.09) and of TNF-α 72 h group (0.2±0.01) were lower than that of the control group (0.69±0.12). Compared with the control group, the transcription level (24 h: 0.16±0.03; 48 h: 0.04±0.01; 72 h: 0.01±0.00 vs 1.00±0.00) and expression (24 h: 0.49±0.08; 48 h: 0.34±0.05; 72 h: 0.04±0.01 vs 0.96±0.13) of Claudin-4 in each TNF-α group were significantly decreased, and showed a decreasing trend with time (all P<0.05).
Conclusions:TNF-α can damage the pulmonary epithelial barrier by damaging alveolar epithelial typeⅡ cells and down-regulating the expression and distribution of ZO-1 and Claudin-4.
