中性粒细胞颗粒蛋白对脂多糖诱导巨噬细胞生成一氧化氮的影响

Effects of neutrophilic granule protein on nitric oxide production in lipopolysaccharide-induced macrophages

目的探讨中性粒细胞颗粒蛋白(NGP)对脂多糖(LPS)诱导巨噬细胞生成一氧化氮(NO)的影响及其调控机制。方法体外培养NGP高表达小鼠巨噬细胞株RAW264.7细胞(NGP/RAW)、阴性对照空载体RAW264.7细胞(NC/RAW)、NGP敲除RAW264.7细胞(NGP KO/RAW)和野生型RAW264.7细胞(WT/RAW),取对数生长期细胞分别给予10 mg/L的LPS(LPS组)或磷酸盐缓冲液(PBS组)进行刺激。用Griess方法检测上清中NO含量;用定量反转录-聚合酶链反应(RT-qPCR)检测诱导型一氧化氮合酶(iNOS)的mRNA表达;用蛋白质免疫印迹试验(Western blotting)检测iNOS蛋白及磷酸化转录激活因子1(p-STAT1)蛋白表达。结果与PBS组相比,在LPS刺激后各时间点,4组细胞中iNOS mRNA表达和NO产生量均明显增加,iNOS mRNA表达于LPS刺激后12 h达峰值(2^(-ΔΔCt):NC/RAW细胞为38.45±1.34比1.00±0.00,NGP/RAW细胞为56.24±2.41比1.45±0.30,WT/RAW细胞为37.84±1.52比1.00±0.00,NGP KO/RAW细胞为5.47±0.62比0.98±0.40,均P<0.05),NO产生量于LPS刺激后24 h达峰值(μmol/L:NC/RAW细胞为24.15±1.26比0.15±0.04,NGP/RAW细胞为58.80±2.11比0.18±0.02,WT/RAW细胞为25.04±1.80比0.16±0.02,NGP KO/RAW细胞为2.42±0.38比0.12±0.03,均P<0.05)。给予LPS刺激后,NGP/RAW细胞内iNOS mRNA表达和NO生成量较NC/RAW细胞明显增加〔iNOS mRNA(2^(-ΔΔCt)):2 h为8.42±0.59比4.63±0.37,6 h为27.16±1.60比14.25±1.02,12 h为56.24±2.41比38.45±1.34;NO(μmol/L):6 h为4.12±0.25比2.23±0.17,12 h为16.50±1.52比6.35±0.39,24 h为58.80±2.11比24.15±1.26,均P<0.05〕;同时,p-STAT1和iNOS蛋白表达明显增强(p-STAT1/GAPDH:0 h为4.26±1.84比1.00±0.32,2 h为20.59±4.97比0.93±0.21,6 h为141.99±10.99比11.17±2.11;iNOS/GAPDH:0 h为1.27±0.86比1.00±0.22,2 h为7.94±1.94比2.01±0.92,6 h为24.24±4.88比3.72±1.11,均P<0.05),说明NGP可以通过促进转录激活因子1(STAT1)通路磷酸化来增加iNOS表达,进而使NO生成增多。LPS刺激后,NGP KO/RAW细胞内iNOS mRNA表达和NO生成量较WT/RAW细胞明显减少〔iNOS mRNA(2^(-ΔΔCt)):2 h为2.46±0.31比4.22±0.18,6 h为3.61±0.44比13.02±1.34,12 h为5.47±0.62比37.84±1.52;NO(μmol/L):6 h为1.22±0.19比2.01±0.12,12 h为1.60±0.44比5.15±0.62,24 h为2.42±0.38比25.04±1.80,均P<0.05〕。说明NGP敲除后iNOS活化减少,进而使NO生成减少。结论NGP可通过激活STAT1/iNOS通路正向调控活化巨噬细胞中NO的生成。

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^(-ΔΔCt):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^(-ΔΔCt)):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^(-ΔΔCt)):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.