Expression of nicotinamide adenine dinucleotide digestive enzyme CD38 in THP-1 cells treated with lipopolysaccharide / 中华围产医学杂志

ABSTRACT

Objective:To investigate the expression of nicotinamide adenine dinucleotide (NAD +) digestive enzyme CD38 in normal and endotoxin-tolerant human monocyte THP-1 cell lines treated with lipopolysaccharide (LPS). Methods:(1) Normal THP-1 cells The experiment and control group were treated with 100 ng/ml LPS for 1, 3, 6, 12 and 24 h or phosphate buffer for 24 h, respectively. Quantitative polymerase chain reaction (PCR) and Western blot were used to measure the expression of interleukin-6 (IL-6) and tumor necrosis factor (TNF) mRNA, CD38 mRNA and protein. (2) The induced endotoxin-tolerant THP-1 cells ①The model of endotoxin-tolerant cells was established firstly by treating the THP-1 cells with 100 ng/ml LPS for 24 h. THP-1 cells treated with phosphate buffer were set as blank group. After further stimulating the two groups with LPS (100 ng/ml) for 3 h, mRNA levels of IL-6 and TNF were measured by quantitative PCR to determine whether the modeling was successful or not. ②In addition, the expression of CD38 mRNA were detected by quantitative PCR before and 12 h after LPS stimulation, and the expression of CD38 protein of these two groups were detected by western blotting before and 1, 6 h after LPS stimulation. Two independent samples t-test and repeated measurement analysis of variance were used for statistical analysis. Results:(1) In normal THP-1 cells, the mRNA expression levels of IL-6 and TNF in the LPS-stimulated cells were significantly higher than those of the control at all time points. And a higher expression level of CD38 mRNA and protein was observed in LPS-stimulated cells from 3 to 24 h compared with the control (mRNA at 3 h 2.27±0.03 vs 1.00±0.18; protein at 3 h 1.47±0.14 vs 1.00±0.16, both P<0.05). (2) Endotoxin-tolerant THP-1 cells ①IL-6 and TNF mRNA levels in the model group were significantly lower than those in the blank group (both P<0.05), indicating that the endotoxin-tolerant THP-1 cell model was established successfully. ②Compared with the same points in the blank group, CD38 mRNA expression was upregulated in the model group before stimulating by LPS (14.18±1.19 vs 1.00±0.13, t=19.007) and 12 h after LPS stimulation (28.33±3.98 vs 7.61±0.88, t=8.803). Moreover, CD38 protein levels before stimulating by LPS (1.54±0.06 vs 1.00±0.10, t=7.796) and 1 h (1.59±0.09 vs 1.07±0.17, t=4.721), 6 h after LPS stimulation (2.48±0.09 vs 1.43±0.12, t=12.233) in the model group were all higher than those in the control group (all P<0.05). The intra-group comparison showed that in the model group the levels of CD38 mRNA at 12 h and CD38 protein at 6 h after LPS stimulation were significantly higher than those before (both P< 0.05). Conclusions:In both normal and endotoxin-tolerant THP-1 cells, LPS upregulates the expression of CD38, which is an NAD + digestive enzyme, and an indirect indicator of NAD + level in monocyte reinfection at the stage of immunosuppression. This study provides a preliminary reference for further investigation on the applicability of CD38 as a potential biological marker in the clinical diagnosis of neonatal sepsis.