单钠尿酸盐晶体诱导小鼠急性腹膜炎模型中线粒体DNA的表达及意义

Significance of mtDNA expression in acute peritonitis induced by monosodium urate in mice

目的 建立单钠尿酸盐（MSU）晶体诱导的小鼠急性腹膜炎模型,并探究炎症过程中线粒体DNA（mtDNA）的表达及意义.方法 64只C57BL16雄性小鼠采用随机数字法分为2组;PBS对照组（腹腔注射0.2 ml PBS）,MSU组（腹腔注射0.2 ml的15 mg/ml的MSU）,分别在2、4、6、8、12、16、20、24 h后,取小鼠外周血,PBS灌洗腹腔取腹腔灌洗液,并取腹膜组织荧光免疫染色.采用ELISA法检测血浆、腹腔灌洗液中的炎症因子IL-1β、IL-18水平,提取DNA,荧光定量实时PCR法测定各组血浆、腹腔灌洗液中mtDNA表达量.数据分析采用多因素方差分析.结果 MSU组的血浆、腹腔灌洗液中的炎症因子IL-1β[（27.0±2.0） pg/ml和（26.8±2.1） pg/ml]、IL-18水平在2、4h明显升高[（673±454） pg/ml和（752±495） pg/ml],高于其他时间点及对照组（F=22.778,P＜0.05;F=6.660,P＜0.05）;MSU组的血浆、腹腔灌洗液中mtDNA表达量4h开始升高,6h达峰值[（9.85±4.59）×106拷贝,（7.81±3.43）×106拷贝],8h下降,明显高于其他时间点及对照组（F=6.719,P＜0.05;F=1 1.181,P＜0.05）;荧光免疫染色发现MSU组给药12 h后开始形成中性粒细胞的聚集,并在24h形成大量中性粒细胞的聚集.结论 MSU诱导的小鼠急性腹膜炎炎症过程中,随着mtDNA的表达量升高,炎症反应缓解并最终形成中性粒细胞的聚集,mtDNA的表达可能是MSU导致炎症中的保护因素之一.

Objective To establish acute peritonitis induced by monosodium urate （MSU） of in mice and observe the significance of mitochondrial deoxyribonucleic acid （mtDNA） expression in the inflammatory processes.Methods The mouse models of acute peritonitis were made by intraperitoneal injection of MSU.Sixty-four male C57BL16 mice were randomly divided into the MSU group which were treated with 0.2 ml of 15 mg/ml MSU solution by i.p.injection and the control group which were treated with 0.2 ml of PBS.Respectively four mice from MSU group and four mice from control group were killed 2 hours, 4 hours, 6 hours, 8 hours 12 hour, 16 hours, 20 hours and 24 hours later and whole blood, peritoneal lavage and peritoneum were collected respectively.Four the mice from the MSU group and four mice from the control group were killed and whole blood, peritoneal lavage and peritoneum were collected.Immunoflourescence study of peritoneum tissues was performed.The levels of interleukin （IL）-1β, IL-18 in plasma and peritoneal lavage were examined by enzyme linked immunosorbent assay （ELISA）.DNA was extracted from blood and peritoneal lavage, and mtDNA level was detected by using real-time polymerase chain reaction （PCR）.The data was analyszied by multivariate analysis of variance.Results As compared with those killed at other time points from the MSU groups and the control group, the levels of IL-1β [（27.0±2.0） pg/ml vs （26.8±2.1） pg/ml], IL-18 [（673±454） pg/ml vs（752±495） pg/ml] in plasma and peritoneal lavage were increased progressively in those which were killed after i.p.injection of 2 hours and 4 hours from in the MSU group （F=22.778, P〈0.05;F=6.660, P〈0.05）.The mtDNA in plasma and peritoneal lavage of the mice began to be expressed 4 hours after i.p.injection 4 hours from in the MSU group.The peak level was detected in those i.p.injected MSU 6 hours later [（9.85±4.59）×106 copies, （7.81±3.43）×106 copies].Then 8 hours later the mtDNA began to slowly decreased.At these three time points, the mtDNA were all increased progressively than those at the other time points of the MSN group or at all time points of the control group （F=6.719, P〈0.05;F=11.181, P〈0.05）.By immunoflourescence study, there were neutrophil extracellular traps （NETs） were formed 12 hours later in the MSU group and aggregated NETs were found 24 hours later.Conclusion In the inflammatory processes of acute peritonitis induced by MSU of in mice, with the expression of mtDNA increasing, the inflammation is relieved, and aggregated NETs are formed in the end.Expression of mtDNA may be one for the protective factors of the inflammation induced by MSU.