姜黄素减轻脓毒症小鼠急性肺损伤时SIRT1与铁死亡的关系

Relationship between SIRT1 and ferroptosis during curcumin-induced reduction of acute lung injury in a mouse model of sepsis

目的评价姜黄素减轻脓毒症小鼠急性肺损伤(ALI)时沉默信息调节因子1(SIRT1)与铁死亡的关系。方法SPF级雄性C57BL/6J小鼠152只,8周龄,体质量23~27 g,采用随机数字表法分为4组(n=38):假手术组(C组)、脓毒症组(S组)、姜黄素组(Cur组)和姜黄素+SIRT1抑制剂EX527组(CE组)。Cur组每日姜黄素200 mg/kg灌胃;CE组每日姜黄素200 mg/kg灌胃,并腹腔注射EX5275 mg/kg;C组和S组给予等量溶剂二甲基亚砜。各组连续给药5 d后采用盲肠结扎穿孔术制备脓毒症模型。每组随机取20只小鼠观察造模后7 d生存情况。造模后24 h时,收集支气管肺泡灌洗液(BALF),采用ELISA法检测TNF-α、IL-1β、IL-6和IL-18浓度;取肺组织,计算湿重/干重比值(W/D比值),HE染色后行肺损伤评分,采用比色法检测肺组织还原型谷胱甘肽(GSH)、MDA和铁含量,Western blot法检测肺组织SIRT1、谷胱甘肽过氧化物酶4(GPX4)、酯酰辅酶A合成酶长链家族成员4(ACSL4)的表达。结果与C组相比,S组造模后7 d生存率降低,BALF中TNF-α、IL-1β、IL-6及IL-18浓度、肺组织W/D比值及肺损伤评分升高,肺组织GSH含量降低,MDA和铁含量升高,SIRT1和GPX4表达下调,ACSL4表达上调(P<0.05);与S组相比,Cur组造模后7 d生存率升高,BALF中TNF-α、IL-1β、IL-6及IL-18浓度、肺组织W/D比值及肺损伤评分降低,肺组织GSH含量增加,MDA和铁含量降低,SIRT1和GPX4表达上调,ACSL4表达下调(P<0.05);与Cur组相比,CE组造模后7 d生存率降低,BALF中TNF-α、IL-1β、IL-6及IL-18浓度、肺组织W/D比值及肺损伤评分升高,肺组织GSH含量降低,MDA和铁含量升高,SIRT1和GPX4表达下调,ACSL4表达上调(P<0.05)。结论姜黄素减轻脓毒症小鼠ALI的机制可能与激活SIRT1,进而抑制铁死亡有关。

Objective To evaluate the relationship between silent information regulator 1(SIRT1)and ferroptosis during curcumin-induced reduction of acute lung injury in a mouse model of sepsis.Methods One hundred and fifty-two SPF-grade male C57BL/6J mice,aged 8 weeks,weighing 23-27 g,were divided into 4 groups(n=38 each)using a random number table method:sham operation group(C group),sepsis group(S group),curcumin group(Cur group)and curcumin plus SIRT1 inhibitor EX527 group(CE group).Curcumin 200 mg/kg was administered by intragastric gavage every day in Cur group.Curcumin 200 mg/kg was administered by intragastric gavage every day and EX5275 mg/kg was intraperitoneally injected in CE group.The equal volume of solvent dimethyl sulfoxide(DMSO)was given in C group and S group.Sepsis model was developed by cecal ligation and puncture(CLP)after 5 days of consecutive administration in anesthetized animals.Twenty mice in each group were randomly selected to observe the survival condition within 7 days after CLP.The bronchoalveolar lavage fluid(BALF)was collected at 24 h after developing the model to determine the concentrations of tumor necrosis factor-alpha(TNF-α),interleukin-1beta(IL-1β),IL-6 and IL-18(by enzyme-linked immunosorbent assay),and the lung tissues were obtained for microscopic examination of the pathological changes which were scored and for determination of wet-to-dry lung weight(W/D)ratio,contents of glutathione(GSH),malondialdehyde(MDA)and iron(by colorimetry),and expression of SIRT1,glutathione peroxidase 4(GPX4)and Acyl-CoA synthetase long chain family member 4(ACSL4)(by Western blot).Results Compared with C group,the 7-day survival rate after CLP was significantly decreased,the concentrations of TNF-α,IL-1β,IL-6 and IL-18 in BALF,W/D ratio and lung injury score were increased,the content of GSH in lung tissues was decreased,the contents of MDA and iron were increased,the expression of SIRT1 and GPX4 was down-regulated,and the expression of ACSL4 was up-regulated in S group(P<0.05).Compared with S group,the 7-day survival rate after CLP was significantly increased,the concentrations of TNF-α,IL-1β,IL-6 and IL-18 in BALF,W/D ratio and lung injury score were decreased,the content of GSH was increased,the contents of MDA and iron were decreased,the expression of SIRT1 and GPX4 was up-regulated,and the expression of ACSL4 was down-regulated in Cur group(P<0.05).Compared with Cur group,the 7-day survival rate after CLP was significantly decreased,the concentrations of TNF-α,IL-1β,IL-6 and IL-18 in BALF,W/D ratio and lung injury score were increased,the content of GSH was decreased,the contents of MDA and iron were increased,the expression of SIRT1 and GPX4 was down-regulated,and the expression of ACSL4 was up-regulated in CE group(P<0.05).Conclusions The mechanism by which curcumin attenuates acute lung injury may be related to activation of SIRT1 and further inhibition of ferroptosis in mice.