Effects of neutrophilic granule protein on nitric oxide production in lipopolysaccharide-induced macrophages / 中华危重病急救医学

ABSTRACT

Objective:To explore the influences of neutrophilic granule protein (NGP) on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages and the regulatory mechanism.Methods:NGP highexpression RAW264.7 cells (NGP/RAW) and negative control empty vector cells (NC/RAW), NGP knockout RAW264.7 cells (NGP KO/RAW) and wild-type cells (WT/RAW) were cultured in vitro. Cells in logarithmic phase were stimulated with 10 mg/L LPS (LPS group) or phosphate buffered saline (PBS group) respectively. The content of NO in the supernatant was detected by Griess method. The mRNA expression of inducible nitric oxide synthase (iNOS) was detected by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expressions of iNOS and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) were detected by Western blotting.Results:Compared with PBS group, iNOS mRNA and NO expression were significantly increased at different time after LPS stimulation, the mRNA expression of iNOS peaked at 12 hours after LPS stimulation (2 -&Delta;&Delta;Ct 38.45±1.34 vs. 1.00±0.00 in NC/RAW cells, 56.24±2.41 vs. 1.45±0.30 in NGP/RAW cells, 37.84±1.52 vs. 1.00±0.00 in WT/RAW cells, 5.47±0.62 vs. 0.98±0.40 in NGP KO/RAW cells, all P < 0.05), and the production of NO peaked at 24 hours after LPS stimulation (μmol/L 24.15±1.26 vs. 0.15±0.04 in NC/RAW cells, 58.80±2.11 vs. 0.18±0.02 in NGP/RAW cells, 25.04±1.80 vs. 0.16±0.02 in WT/RAW cells, 2.42±0.38 vs. 0.12±0.03 in NGP KO/RAW cells, all P < 0.05). After being stimulated by LPS, the expression of iNOS mRNA and NO in NGP/RAW cells were increased significantly compared with NC/RAW cells [iNOS mRNA (2 -&Delta;&Delta;Ct) 8.42±0.59 vs. 4.63±0.37 at 2 hours, 27.16±1.60 vs. 14.25±1.02 at 6 hours, 56.24±2.41 vs. 38.45±1.34 at 12 hours; NO (μmol/L) 4.12±0.25 vs. 2.23±0.17 at 6 hours, 16.50±1.52 vs. 6.35±0.39 at 12 hours, 58.80±2.11 vs. 24.15±1.26 at 24 hours, all P < 0.05]. At the same time, the protein expressions of p-STAT1 and iNOS were also significantly enhanced (p-STAT1/GAPDH 4.26±1.84 vs. 1.00±0.32 at 0 hours, 20.59±4.97 vs. 0.93±0.21 at 2 hours, 141.99±10.99 vs. 11.17±2.11 at 6 hours; iNOS/GAPDH 1.27±0.86 vs. 1.00±0.22 at 0 hours, 7.94±1.94 vs. 2.01±0.92 at 2 hours, 24.24±4.88 vs. 3.72±1.11 at 6 hours, all P < 0.05), indicating that NGP might increase the expression of iNOS by promoting the phosphorylation of the signal transducer and activator of transcription 1 (STAT1) pathway, thereby increasing the production of NO. After being stimulated by LPS, the expression of iNOS mRNA and NO in NGP KO/RAW cells were significantly lower than that of WT/RAW cells [iNOS mRNA (2 -&Delta;&Delta;Ct) 2.46±0.31 vs. 4.22±0.18 at 2 hours, 3.61±0.44 vs. 13.02±1.34 at 6 hours, 5.47±0.62 vs. 37.84±1.52 at 12 hours; NO (μmol/L) 1.22±0.19 vs. 2.01±0.12 at 6 hours, 1.60±0.44 vs. 5.15±0.62 at 12 hours, 2.42±0.38 vs. 25.04±1.80 at 24 hours, all P < 0.05]. It showed that iNOS activation was reduced after NGP knockout, which in turn reduced NO production. Conclusion:NGP can positively regulate NO production in activated macrophages by activating the STAT1/iNOS pathway.