通过抑制铁死亡减轻脓毒症引起急性肺损伤的研究

Experimental Study of Reducing Sepsis-induced Acute Lung Injury by Inhibiting Ferroptosis

目的 观察盲肠结扎穿孔(cecal ligation and puncture,CLP)造成的急性肺损伤(acute lung injury,ALI)小鼠模型铁死亡水平及炎症指标;观察药物liproxstatin-1干预铁死亡后,小鼠铁死亡水平及炎症指标的变化。方法 选取24只C57BL/6雄性小鼠随机分为假手术组、CLP组、Lip-1组、CLP+Lip-1组,每组6只。假手术组和Lip-1组进行假手术,CLP组和CLP+Lip-1组进行CLP手术(盲肠结扎+穿刺术建立脓毒症动物模型);Lip-1组和CLP+Lip-1组术前腹腔注射liproxstatin-1(0.8 mg/kg),假手术组、CLP组注射同体积生理盐水;术后16 h处死小鼠,收集支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和肺组织。测定各组小鼠左肺叶肺组织湿/干重比(wet to dry weight ratio,W/D),苏木精-伊红(hematoxylin-eosin,HE)染色观察各组小鼠右上肺叶病理形态表现,检测BALF中的总蛋白浓度、肿瘤坏死因子α (tumour necrosis factor alpha,TNF-α)、白细胞介素6(interleukin 6,IL-6)表达情况。采用Western blot检测各组小鼠肺组织中谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、环氧合酶2(cyclooxygenase 2,COX-2)和长链脂酰辅酶A合成酶4(acetyl coenzyme A synthetase long-chain family 4,ACSL4)蛋白表达水平。采用免疫组织化学法检测小鼠肺组织丙二醛(malondialdehyde,MDA)、4-羟基壬烯醛(4-Hydroxynonenal,4-HNE)表达情况。采用试剂测定铁离子表达情况。结果 与假手术组比较,CLP组小鼠肺组织W/D明显升高(P<0.01);病理切片显示,CLP组支气管壁充血严重,存在明显炎性细胞浸润,肺泡壁充血严重,肺部存在明显纤维化;CLP组BALF中的总蛋白浓度、IL-6及TNF-α表达明显增加(P均<0.01),GPX4表达下降,ACSL4、COX-2、MDA、4-HNE表达,铁离子含量明显升高(P均<0.05)。与CLP组比较,CLP+Lip-1组肺组织W/D明显下降(P<0.01);病理切片显示,CLP+Lip-1组可见支气管壁充血与少量明显炎性细胞浸润,其中少数肺泡壁充血,肺部轻度纤维化,治疗效果显著;CLP+Lip-1组BALF中的总蛋白浓度、IL-6及TNF-α表达均明显降低(P均<0.01),GPX4表达升高,ACSL4、COX-2、MDA、4-HNE表达、铁离子含量明显下降(P均<0.05)。结论 铁死亡参与了脓毒症导致的ALI,通过抑制铁死亡,能对脓毒症导致的ALI起到一定的治疗作用。

Objective To observe the ferroptosis levels and inflammatory indexes in model of mice with acute lung injury(ALI) induced by cecal ligation and puncture(CLP);to observe changes of ferroptosis level and inflammatory markers in mice after liproxstatin-1 intervening ferroptosis.Methods A total of 24 male C57BL/6 mice were randomly divided into Sham operated group,CLP group,Lip-1 group and CLP+Lip-1 group,with 6 mice in each.The Sham operated group and Lip-1 group underwent sham surgery,the CLP group and CLP+Lip-1 group underwent CLP surgery(the animal model of sepsis was established by cecal ligation + puncture);the Lip-1 group and CLP+Lip-1 group were preoperatively injected intraperitoneally with liproxstatin-1(0.8 mg/kg),and the same volume of saline was injected into the Sham operated and CLP groups;mice were executed 16 h after surgery,and bronchoalveolar lavage fluid(BALF) and lung tissues were collected.Wet to dry weight ratio(W/D) of the left lung lobe of each mouse was measured,hematoxylin-eosin(HE) staining was performed to observe the pathological and morphological manifestations of the right upper lobe of the lungs of each group of mice,expressions of total protein concentration,tumour necrosis factor alpha(TNF-α) and interleukin 6(IL-6) in BALF were detected.The protein expressions of glutathione peroxidase 4(GPX4),cyclooxygenase 2(COX-2) and acetyl coenzyme A synthetase long-chain family 4(ACSL4) were detected in the lung tissues of mice in each group by Western blot.The expressions of malondialdehyde(MDA) and 4-Hydroxynonenal(4-HNE) in mice lung tissues were detected by immunohistochemistry.The expression of ferric ion was detected by reagant.Results Compared with Sham operated group,W/D of lung tissue in mice of CLP group statistically increased(P<0.01);pathological sections showed that bronchial wall of CLP group was severely congested,and there were obvious inflammatory cell infiltration,severe alveolar wall congestion,obvious lung fibrosis;expressions of total protein concentration,IL-6 and TNF-α in BALF of CLP group statistically increased(all P<0.01),the expression of GPX4 decreased,while the expressions of ACSL4,COX-2,MDA,4-HNE and content of iron ion significantly increased(all P<0.05).Compared with CLP group,W/D of lung tissues in CLP + Lip-1 group significantly decreased(P<0.01);the pathological sections showed that the CLP+Lip-1 group had bronchial wall congestion and small amount of obvious inflammatory cell infiltration,a few alveolar wall congestion,mild pulmonary fibrosis,and the therapeutic effects were remarkable;the expressions of total protein concentration,IL-6 and TNF-α in BALF in CLP+Lip-1 group significantly decreased(all P<0.01),the expression of GPX4 increased,while the expressions of ACSL4,COX-2,MDA,4-HNE and content of iron ion significantly decreased(all P<0.05).Conclusion Ferroptosis is involved in sepsis-induced ALI,and inhibition of ferroptosis can play a certain role in the treatment of sepsis-induced ALI.