摘要目的 观察原花青素对盲肠结扎穿孔(CLP)诱导脓毒症小鼠的保护作用,探讨其作用机制.方法 将36只成年雄性C57BL/6小鼠按随机数表法随机分为Sham组、CLP组及治疗组.采用盲肠结扎穿孔建立脓毒症小鼠模型,Sham组仅做开腹处理,治疗组则在建模后腹腔注射原花青素(100 mg/kg).观察各组小鼠生存状态并记录72 h内死亡情况.另一批小鼠于建模后24h处死,收集外周血及肺组织.酶联免疫吸附试验(ELISA)检测外周血及肺组织中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6及IL-1β含量;苏木素-伊红(HE)染色检测肺组织病理变化;精密称量仪称取烘干前后肺组织质量并测定湿/干质量比值;Western blot法检测肺组织胞质及胞核中核因子-κB(NF-κB) p65的蛋白表达情况;ELISA法检测肺组织中NF-κB p65的DNA结合活性.结果 Sham组小鼠72 h生存率为100％,CLP组小鼠72 h生存率仅为20％,而治疗组小鼠72 h生存率为46.7％,差异有统计学意义(P =0.005).建模24 h后,与CLP组比较,治疗组小鼠外周血及肺组织中炎性因子TNF-α(214.000±16.310比426.300±22.980,F=111.300,P=0.000;593.300±39.040比936.500±55.360,F=123.600,P=0.000)、IL-6(1 020.000±72.150比1 741.000±83.710,F=148.400,P=0.000;1 107.000±68.530比1 830.000±99.190,F=140.500,P=0.000)及IL-1β(507.200±28.780比820.700±47.770,F=127.700,P=0.000;154.200±7.769比257.300±20.390,F=63.590,P=0.000)的含量均显著降低;肺组织中肺泡间隔增宽及大量炎细胞浸润等病理改变明显减轻;肺组织湿/干质量比值显著降低(3.966±0.255比5.116±0.261,F=16.500,P=0.000);肺组织胞质蛋白中NF-κB p65的表达明显增加(0.547±0.069比0.302±0.032,F=24.950,P=0.001),而胞核中NF-κB p65的蛋白表达显著减少(0.622±0.111比1.374±0.123,F=25.640,P=0.001);NF-κB p65的DNA结合活性也显著降低(1.948±0.155比2.884±0.188,F=41.330,P=0.000).结论 原花青素对CLP诱导脓毒症小鼠具有明显的保护作用,其机制可能与抑制NF-κB p65入核有关.

abstractsObjective To investigate the protective effect of proantho cyanidins (PCs) against cecal ligation and puncture (CLP)-induced acute lung injury in septic mice and its mechanism.Methods 36 adult male C57BL/6 mice were randomly divided into three groups,including Sham group,CLP group and treatment group.Each group has 12 mice,sepsis model was set up by CLP and Proantho Cyanidins (100 mg/kg) was injected intraperitoneally.The survival state and mortality by 72 h was observed.The peripheral blood and lung tissue were collected at 24 h after CLP.The protein expressions of tumor necrosis factor-α (TNF-α),interlukin (IL)-6 and IL-1β in peripheral blood and lung tissues were measured by enzyme linked immunosorbent assay (ELISA).The pathological changes in lung tissues were detected by hematoxylin and eosin (HE) staining.The wet/dry ratios of lung tissues were obtained by precision weighing instrument.The protein expressions of nuclear factor-κB (NF-κB) p65 in cytoplasm and nucleus were determined by Western blotting.The NF-κB p65 DNA binding activities were measured by ELISA.Results At 72 h after modeling,the survival rate of Sham group,CLP group and treatment group were 100％,20％ and 46.7％,respectively (P =0.005).At 24 h,compared with CLP group,the protein expressions of TNF-α (214.000 ± 16.310 vs.426.300 ± 22.980,F =111.300,P =0.000;593.300 ± 39.040 vs.936.500 ± 55.360,F=123.600,P=0.000),IL-6 (1020.000± 72.150 vs.1 741.000±83.710,F=148.400,P=0.000;1 107.000±68.530 vs.1 830.000±99.190,F=140.500,P =0.000) and IL-1 β (507.200 ± 28.780 vs.820.700 ± 47.770,F =127.700,P =0.000;154.200 ±7.769 vs.257.300 ± 20.390,F =63.590,P =0.000) in peripheral blood and lung tissues from treatment group were both significantly decreased.PCs treatment could remarkably ameliorate CLP-induced pathological changes,such as thickened alveolar septum and infiltration with inflammatory cells.The wet/dry ratios of lung tissues in treatment group was lower than that in CLP group (3.966 ± 0.255 vs.5.116 ±0.261,F =16.500,P =0.000).Compared with CLP group,the cytoplastic protein level of NF-κB p65 in treatment group was significantly increased (0.547 ± 0.069 vs.0.302 ± 0.032,F=24.950,P =0.001),while the nuclear protein level of NF-κB p65 was remarkably decreased (0.622 ±0.111 vs.1.374 ±0.123,F =25.640,P =0.001).The NF-κB p65 DNA binding activity in treatment group was significantly lower than that in CLP group (1.948 ±0.155 vs.2.884 ±0.188,F=41.330,P =0.000).Conclusion PCs had a protective effect against CLP-induced acute lung injury in septic mice,which may relate to the inhibition of NF-κB 065 nucleus translocation.