脂氧素A4早期干预对脓毒症小鼠的保护作用

Protective effect of early intervention with lipoxin A4 on septic mice

目的 探讨在脓毒症早期进行脂氧素A4(LXA4)干预对脓毒症小鼠的影响。方法 将6~8周龄健康雄性Balb/c小鼠随机分为假手术组、脓毒症组、脓毒症造模1h后干预组和脓毒症造模6h后干预组,每组8只小鼠。采用盲肠结扎穿孔术制造脓毒症模型,造模后干预组于术后1h或6h给予LXA40.01μg/g体重;术后24h摘眼球取血,收集腹腔灌洗液,取肝、肺组织。稀释涂布平板法计数全血、腹腔灌洗液细菌菌落;CBA法检测血清肿瘤坏死因子α(TNF-α)、IL-6、单核细胞趋化蛋白1(MCP-1)水平,ELISA法检测血清高迁移率族蛋白1(HGMB1)水平;流式细胞仪检测腹腔灌洗液巨噬细胞及中性粒细胞水平;肝肺组织行石蜡切片、苏木精-伊红染色,观察病理损伤。结果 与假手术组相比,脓毒症组腹腔灌洗液巨噬细胞比例减少、中性粒细胞比例增加(P<0.05);血清IL-6、TNF-α、MCP-1、HMGB1水平增高(P<0.05);肝组织可见较多空泡样变性、肝细胞肿胀并有炎性细胞浸润;肺组织可见毛细血管充血、肺间隔增厚、炎性细胞浸润、部分组织结构破坏。与脓毒症组相比,1h干预组及6h干预组腹腔灌洗液巨噬细胞比例增加(P<0.05),中性粒细胞比例及腹腔灌洗液细菌载荷量无明显差异(P>0.05),全血中细菌载荷量降低(P<0.05);血清IL-6、TNF-α、MCP-1、HMGB1水平降低(P<0.05);肝、肺组织损伤及炎性细胞浸润程度减轻。与6h干预组相比,1h干预组血清HMGB1水平降低(P<0.05),其余指标在两组间比较差异无统计学意义(P>0.05)。结论 LXA4早期干预减轻了脓毒症小鼠的肝、肺组织损伤,可能与其降低血清IL-6、TNF-α、MCP-1、HMGB1水平有关;同时减少了脓毒症小鼠全血中细菌的播散程度,可能与增加腹腔巨噬细胞比例有关。

Objective To study the effect of early intervention with lipoxin A4 (LXA4) on septic mice. Methods Healthy male Balb/c mice aged 6-8 weeks were randomly divided into sham-operation group, sepsis group, 1-hour intervention group (intervention at 1 hour after sepsis), and 6-hour intervention group (intervention at 6 hours after sepsis)(n=8 each). A sepsis model was prepared by cecal ligation and puncture. The intervention groups received LXA4 at 0.01 μg/g body weight 1 or 6 hours after the model was established. Blood was taken from eyeballs at 24 hours after operation. Peritoneal lavage fluid and liver and lung tissue samples were collected. The bacterial colonies of whole blood and peritoneal lavage fluid were counted by dilution plating. The serum levels of tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) were determined by cytometric bead array. The serum level of high mobility group box-1 (HGMB1) was determined using ELISA. The percentages of macrophages and neutrophils in peritoneal lavage fluid were determined by flow cytometry. Paraffin sectioning and hematoxylin-eosin staining were performed for the liver and lung tissue samples to observe pathological damage. Results Compared with the sham-operation group, the sepsis group had a significantly decreased percentage of macrophages and a significantly increased percentage of neutrophils in peritoneal lavage fluid (P<0.05), as well as significantly increased serum levels of IL-6, TNF-α, MCP-1, and HMGB1 (P<0.05);in addition, the sepsis group showed more vacuolar degeneration, hepatocyte swelling, and inflammatory cell infiltration in liver tissue, and more capillary congestion, pulmonary septal thickening, inflammatory cell infiltration, and partial tissue destruction in lung tissue. Compared with the sepsis group, the 1-hour and 6-hour intervention groups had a significantly increased percentage of macrophages in peritoneal lavage fluid (P<0.05) and significantly reduced bacterial load in whole blood (P<0.05), serum levels of IL-6, TNF-α, MCP-1, and HMGB1 (P<0.05), and degree of liver and lung tissue damage and inflammatory cell infiltration, but there was no significant difference in the percentage of neutrophils and bacterial load in peritoneal lavage fluid (P>0.05). Compared with the 6-hour intervention group, the 1-hour intervention group had a significantly decreased serum level of HMGB1 (P<0.05), but there was no significant difference in other indicators between the two groups (P>0.05). Conclusions Early intervention with LXA4 may attenuate liver and lung injuries in septic mice, which may be explained by the decrease in serum levels of IL-6, TNF-α, MCP-1, and HMGB1, and it also may reduce the bacterial dissemination in the whole blood of septic mice, which may be explained by the increase in the percentage of peritoneal macrophages.