二烯丙基硫化物对百草枯中毒大鼠的抗炎机制

目的 探讨二烯丙基硫化物(DAS)对急性百草枯(PQ)中毒大鼠肺泡巨噬细胞(AM)中核转录因子-κB (NF-κB)蛋白的影响.方法 将45只Wistar雄性大鼠按随机数字表法分为生理盐水对照组(NS)、PQ中毒模型组及DAS治疗组,每组15只.将百草枯溶液(70 mg/kg)一次性灌胃制备中毒模型;对照组灌胃等量生理盐水.制模后分别经腹腔注射DAS 100 mg/kg(DAS治疗组)、等量生理盐水(模型组和对照组),定时每日1次,共14 d.各组分别于制模后3、7、14d随机各组处死5只大鼠.根据不同时间将各组动物用3.6％水合氯醛腹腔注射麻醉,解剖胸腔暴露肺,行支气管肺泡灌洗,收集灌洗液测定肺泡灌洗液中蛋白含量并分离培养肺泡巨噬细胞(AM);用免疫细胞化学方法测定AM中NF-κB的表达.结果 ①大鼠原代巨噬细胞的分离培养:采用差速贴壁的方法分离纯化肺泡巨噬细胞,30 min可以见到较多的巨噬细胞贴壁.②支气管肺泡灌洗液蛋白含量测定:NS组和PQ组比较,支气管肺泡灌洗液中蛋白含量3 d(261.6±17.16) μg/mL vs.(673.4±151.9) μg/mL;7 d(265.6±18.37) μg/mL vs.(581.3 ±134.58)μg/mL;14 d(253.8 ±11.43) μg/mL vs.(589.07 ±33.85) μg/mL,均P＜0.05.PQ组和DAS组比较支气管肺泡灌洗液中蛋白含量3d (673.4±151.9) μg/mL vs.(342.9±39.03) μg/mL;7d(581.3±134.58) μg/mL vs.(383.7 ±7.37) μg/mL,P＜0.05;14 d(589.07±33.85) μg/mL vs.(282.9±15.59) μg/mL,P＜0.05.③免疫细胞化学结果NS组和DAS组肺泡巨噬细胞质有极少量NF-κB p65阳性表达;PQ组细胞核中有较多NF-κB p65阳性表达;DAS治疗组中NF-κB p65阳性表达较模型组明显减少,吸光度积分值NS组与PQ组比较,PQ组与DAS组比较,差异均有统计学意义,P＜0.05.结论 DAS能下调PQ中毒大鼠肺泡巨噬细胞NF-κB的达,减少支气管肺泡灌洗液中蛋白量,故DAS具有一定抑制炎症介质释放,减少炎性渗出的作用.

The protective effects of diallyl sulfide on acute lung injury in rats with paraquot poisoning

Objective To investigate the expression of nuclear factor-κB in alveolar macrophages of paraquat-induced rats and the effect of diallyl sulfide on it.Methods Forty five male wistar rats were randomly divided into three groups,namely control group,model group,and DAS treatment group (n =15 in each).The model of paraquat poisoning was reproduced by single does of 70 mg/kg given by intra-gastric administration,while the equal volume of normal saline (NS) was given to the rats in control group instead.The dose of 100 mg/kg of DAS was given to rats by intra-peritoneal injection in DAS treatment group.The equal volume of NS was given to the rats by intra-peritoneal injection in model group and control group instead.The rats of model group and DSA treatment group were exposed to paraquat once a day for 14 days.Five rats in each group were sacrificed at 3,7,14 days.Alveolar maerophages were harvested by bronchalveolar lavage (BAL).The protein content of BAL fluid were examined.The exprossion of NF-κB was measured with immunocytochemistry technique.Results Alveolar macrophage cultures were carried out by using differential adherence of isolated and purified alveolar macrophages,and after 30 minutes culture,more adherent macrophages can be seen.Compared with model group,the protein content of BAL fluid at dfferent intervals in the control group were obviously lower,especially on the 3 rd day (261.6 ± 17.16) μg/mL vs.(673.4 ± 151.9) μg/mL;7 d (265.6 ± 18.37) μg/mL vs.(581.3 ± 134.58) μg/mL;14 d (253.8 ± 11.43) μg/mL vs.(589.07 ± 33.85) μg/mL,P ＜ 0.05.Comparisons of protein content in BLA fluid between PQ group and DAS treatment group were on the 3 rd day (673.4 ± 151.9) μg/mL vs.(342.9 ±39.03) μg/mL;on the 7 th day (581.3 ± 134.58) μg/mL vs.(383.7 ±7.37) μg/mL,P＜0.05;on the 14 th day (589.07±33.85) μg/mL vs.(282.9±15.59) μg/mL,P＜0.05.The immunocytochemistry analysis revealed minimal NF-κBp65 expression in the cell cytoplasm in the control group,while high NF-κBp65 expression was found in nuclear in the model group.Minimal NF-κBp65 expression was found in the cell cytoplasm in the DAS treatment group,and integral A value was significantly lower in the DAS treatment groups than that in the model group (P ＜ 0.05).Conclusions Treatment with an intra-peritoneal injection of DAS is capable of attenuating the extent of PQ-induced ALI in rats by lowering BLA fluid protein content,inhibiting the expression of NF-κB in alveolar maerophages.