S1PR3激动剂KRX-725促进细菌清除影响脓毒症小鼠预后

目的 通过合成特异性S1PR3激动剂KRX-725,探讨S1PR3参与细菌清除的功能及其分子机制.方法 20只健康清洁级雄性C57BL/6小鼠以随机数字法分为S1PR3特异性激动剂KRX-725治疗组和对照组.采用大肠杆菌(3×106)腹腔注射诱导脓毒症模型,治疗组小鼠于模型后立即腹腔注射KRX-725(10 mg/kg),对照组给予等体积的溶剂,比较两组小鼠48 h生存率(n=12).模型后24 h检测腹腔及血液细菌负荷(n=5),并取肺组织进行苏木精-伊红染色(HE染色)评估KRX-725治疗组及对照组的肺脏病理损伤程度(n=3).体外采用大肠杆菌刺激腹腔巨噬细胞(细胞∶细菌=1∶10),分别加入KRX-725及其对照溶剂,采用CM-H2 DCFDA探针标记细胞内活性氧,酶标仪检测检测不同时间点巨噬细胞活性氧水平.庆大霉素保护实验观察KRX-725对巨噬细胞的清除细菌能力的影响.生存率分析采用Log-rank test,组间数据比较采用独立样本t检验分析,P＜0.05为差异有统计学意义.结果 KRX-725治疗组脓毒症小鼠48 h生存率较对照组显著提高(P＜0.05).脓毒症模型后24 h,KRX-725治疗组较对照组血液及腹腔灌洗液中的细菌负荷显著降低(血液:t=3.17,P＜0.05;腹腔灌洗液:t=4.07,P＜0.01),同时KRX-725可显著降低脓毒症模型肺组织损伤程度(肺损伤评分:KRX-725组1.4 ±0.25,对照组2.4 ±0.25)(t=2.89,P＜0.05).体外实验,KRX-725治疗组较对照组可迅速上调大肠杆菌刺激后20 min及30 min细胞内活性氧的水平(20 min荧光强度:KRX-725组522.9±38.76,对照组385.9±15.90,P＜0.05;30min荧光强度:KRX-725组519.7 ±25.02,对照组384.5±15.28,P＜0.01).庆大霉素保护实验发现,KRX-725可显著降低细菌吞噬后3 h及6h巨噬细胞内存活的大肠杆菌的数量(3 h:KRX-725组286.5±98.35,对照组710.8 ±--107.8,P＜0.05;6 h:KRX-725组72.5±6.45,对照组205.8±66.76,P＜0.01).结论 体内应用KRX-725可改善脓毒症小鼠生存率,降低血液及腹腔细菌负荷,减轻肺脏损伤,从而改善脓毒症预后.体外KRX-725通过上调巨噬细胞活性氧的水平,提高巨噬细胞对清除细菌的能力.

S1PR3 agonist KRX-725 promotes bacterial clearance and affects the outcome of sepsis

Objective To study the role of KRX-725,a specific agonist of Sphingosin-1 phosphate receptor 3,S1PR3),in the function and mechanism of S1PR3 in respect of bacterial clearance.Methods Twenty healthy male C57BL/6 mice were randomly (random number) divided into two groups:KRX-725 group and control group.Septic mice model were established by intraperitoneal injection of E.coli (3 × 106),then KRX-725 (10 mg/kg) or the vehicle was administered intratracheally.Forty-eight-hour survival rate (n =12),bacterial colony numbers in peritoneal cavity and blood (n =5),and lung injury (n =3)were compared between two groups.In vitro,the peritoneal macrophages were stimulated by E.coli (cell∶E.coli=1∶10) with KRX-725 or the vehicle treatment.The reactive oxygen species (ROS) levels were detected by CM-H2DCFDA in macrophages.The bacteria clearance function of KRX-725 was observed by gentamicin protection test.Survival rates were analyzed with the Log-rank test.A 2-tailed student's t test was used to compare difference between two independent groups.Results Compared with the control group,the 48-hour survival rate of KRX-725 group was significantly higher (P ＜ 0.05).Bacterialload in the blood and the peritoneal lavage fluid (PLF) was greatly decreased in the KRX-725 group (blood:t =3.17,P ＜0.05;PLF:t =4.07,P ＜0.01).The lung tissues injury was also obviously reduced in the KRX-725 group of 24 hours after the injection of E.coli (lung injury score:KRX-725 group 1.4 ± 0.25;control group 2.4 ± 0.25) (t =2.89,P ＜ 0.05).In vitro,KRX-725 could up-regulate the ROS levels in macrophage at 20 min and 30 min after E.coli injected intra-peritoneally (20 min fluorescent intensity:KRX-725 group 522.9 ± 38.76,control group 385.9 ± 15.90,P ＜ 0.05;30 min fluorescent intensity:KRX-725 group 519.7 ±25.02,control group 384.5 ± 15.28,P ＜0.01).The bacterial load in the KRX-725 treated macrophage were significantly decreased at 3 h and 6 h after E.coli injected intra-peritoneally (3 h:KRX-725 group 286.5 ±98.35,control group 710.8 ± 107.8,P ＜0.05;6 h:KRX-725 group 72.5 ±6.45,control group 205.8 ±66.76,P ＜0.01).Conclusion In vivo,KRX-725 could improve the survival rate of septic mice,decrease the bacterial lioad in the blood and PLF,and reduce the lung injury.In vitro,KRX-725 could up-regulate the ROS level in macrophages and accelerate the bacterial clearance.