瓜氨酸预处理对脂多糖致小鼠急性肺损伤的作用机制研究

Investigation of the protective effects of citrulline pretreatment in lipopolysaccharide‑induced lung injury in mice

目的探讨瓜氨酸(citrulline,Cit)对脂多糖(lipopolysaccharide,LPS)导致的小鼠急性肺损伤(acute lung injury,ALI)是否具有保护作用。方法42只7~9周的C57雄性小鼠,进行2批实验,每批小鼠按完全随机分组法分为3组(每组7只):正常对照组(Con组)、LPS组和LPS+Cit组。首先对小鼠腹腔注射Cit 900 mg·kg^−1·d^−1(LPS+Cit组)或等容量生理盐水(Con组、LPS组)进行预处理7 d,第8天腹腔注射LPS 10 mg/kg(LPS组、LPS+Cit组)或等容量生理盐水(Con组),LPS干预后6 h麻醉插管检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中蛋白浓度和细胞数量,测定肺组织湿/干重比(wet/dry weight ratio,W/D),观察肺组织病理改变,Western blot法检测肺组织中核苷酸结合寡聚化结构域样受体蛋白3(NOD‑like receptor pyrin domain containing 3,NLRP3)炎性体、半胱氨酸蛋白酶‑1(cysteine aspartate‑specific protease‑1,caspase‑1)蛋白水平,ELISA法检测肺组织中IL‑1β、IL‑18水平。结果与Con组比较,LPS组BALF中蛋白浓度、细胞数量及肺组织W/D明显升高(P<0.05),NLRP3、caspase‑1蛋白水平及IL‑1β、IL‑18蛋白含量明显升高(P<0.05);与LPS组比较,LPS+Cit组BALF中蛋白浓度、细胞数量及肺组织W/D明显降低(P<0.05),NLRP3、caspase‑1蛋白水平及IL‑1β、IL‑18蛋白含量明显降低(P<0.05)。结论Cit可能通过抑制NLRP3炎性体激活,降低炎症因子释放,从而发挥对肺损伤的保护作用。

Objective This work investigates the effects of citrulline(Cit)on lipopolysaccharide(LPS)induced acute lung injury(ALI)and explores the possible underlying molecular mechanism in mice.Methods Forty‑two male C57 mice were randomly divided into 3 groups(n=7):control group(Con group),LPS group,LPS+Cit group.Cit(900 mg·kg−1·d−1)was injected intraperitoneally for 7 days before LPS(10 mg/kg)was administrated.After 6 h,the mice were anesthetized and bronchoalveolar lavage fluid(BALF)was collected for detecting the levels of protein and inflammatory cell counting.The wet/dry weight ratio(W/D)and pathological changes of lung tissue were examined.Lung tissues were homogenized to detect NOD‑like receptor pyrin domain containing 3(NLRP3)and cysteine aspartate‑specific protease‑1(caspase‑1)by Western blot.IL‑1βand IL‑18 were detected by enzyme‑linked immunosorbent assay(ELISA).Results Compared with the Con group,the cell count and protein levels in BALF and W/D ratio in the LPS group were markedly increased(P<0.05).In addition,the expression of NLRP3 and caspase‑1 cleavage were increased in the LPS group,as well as the secretion of IL‑1βand IL‑18(P<0.05).While compared with the LPS group,Cit attenuated ALI as shown in cell count and protein content levels in BALF and W/D(P<0.05).The expression of NLRP3 inflammasome and the subsequent caspase‑1 cleavage as well as the IL‑1βand IL‑18 secretion in the LPS+Cit group were decreased(P<0.05).Conclusions Our results indicate that Cit pretreatment could significantly attenuate LPS‑induced lung edema and the perfusion of inflammatory cells in the lung in mice,which might partly through inhibiting the NLRP3 inflammasome activation.