Hepatitis A virus cell membrane protein receptor 2 promotes endotoxin tolerance in mouse macrophages
10.3760/cma.j.cn121430-20201106-00705
- VernacularTitle:甲型肝炎病毒细胞膜蛋白受体2促进小鼠巨噬细胞内毒素耐受形成的研究
- Author:
Yao LIU
;
Yi LIU
;
Xiaohe ZHANG
;
Muhu CHEN
;
Yingchun HU
- From:
Chinese Critical Care Medicine
2021;33(4):472-477
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To screen out the potential key genes of endotoxin tolerance (ET), and to provide theoretical and experimental evidence for treatment and prognosis of sepsis.Methods:①Experiment 1 (gene chip and bioinformatics analysis): ET related data set GSE47783 was downloaded from the Gene Expression Omnibus (GEO). The data set was obtained from lipopolysaccharide (LPS) stimulated mouse macrophages to establish sepsis model (LPS group) and ET model (ET group). IDEP 0.92 software was used to screen differential expressed gene (DEG) between the two groups, analyze gene ontology (GO), and locate the main functions and signaling pathways of differential genes. The protein-protein interaction (PPI) network of DEG was constructed by the Search Tool for the Retrieval of Interacting Genes Database (STRING) to screen core genes hepatitis A virus cell membrane protein receptor 2 (HAVCR2) for following up validation study. ②Experiment 2 (reproduction of mouse macrophage RAW264.7 model): RAW264.7 cells were cultured in vitro, the ET model (ET group, cells were cultured with 10 μg/L LPS for 24 hours and then with 100 μg/L LPS for 4 hours) and sepsis model (LPS group, cells were cultured with 100 μg/L LPS for 4 hours) were reproduced by LPS stimulation. Phosphate buffer saline (PBS) group was given equal volume of solvent PBS for 4 hours. The mRNA and protein expressions of HAVCR2 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ③Experiment 3 (RAW264.7 cells transfected with HAVCR2 lentiviral vector): to further clarify whether HAVCR2 was involved in the formation of ET, after knockdown of HAVCR2 in RAW264.7 cells by lentiviral short hairpin RNA (shRNA) technology, the ET model (HAVCR2 --ET group) was constructed again, and the control group (ET group) without knockdown of HAVCR2 was set up. RT-qPCR method was used to detect the mRNA expressions of macrophage polarization key proteins [arginase 1 (ARG1), CD206, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide synthase 2 (NOS2)] in cells. Results:①Experiment 1: a total of 1 013 DEG were identified, compared with LPS group, 521 genes were up-regulated and 492 genes were down-regulated in ET group. The function of these DEG was to increase biosynthesis and reduce inflammatory reaction. Signal pathways were mainly enriched in Janus kinase/signal transducers and activators of transcription (JAK/STAT) , NOD like receptor, Toll-like receptor (TLR), TNF, hypoxia inducible factor-1 (HIF-1). The first up-regulated HAVCR2 in the ET group was selected as the target of the study. ②Experiment 2: the results of in vitro experiment showed that the mRNA expression of HAVCR2 after high-dose LPS stimulation was down-regulated as compared with PBS group, and the mRNA expression of HAVCR2 in ET group was significantly higher than that in LPS group (2 -ΔΔCT: 1.10±0.10 vs. 0.60±0.10, P < 0.05). The results of Western blotting were consistent with RT-qPCR results. ③Experiment 3: the mRNA expressions of ARG1 and CD206 in HAVCR2 --ET group were significantly lower than those in ET group [ARG1 mRNA (2 -ΔΔCT): 0.50±0.10 vs. 1.00±0.10, CD206 (2 -ΔΔCT): 0.73±0.10 vs. 1.00±0.10], and the mRNA expressions of TNF-α and IL-1β were significantly higher than those in ET group [TNF-α mRNA (2 -ΔΔCT): 2.20±0.10 vs. 1.00±0.10, IL-1β mRNA (2 -ΔΔCT): 9.00±0.10 vs. 1.00±0.10), with significant differences (all P < 0.05). There was no significant difference in the expression of NOS2 mRNA between the two groups. Conclusion:HAVCR2 is involved in the regulation of inflammatory factors downstream of sepsis and the formation of ET, which is expected to become a new therapeutic target of sepsis.
