卵巢肿瘤结构域蛋白酶1在脓毒症小鼠急性肺损伤中的作用:与TAK1-MAPK信号通路的关系

Role of deubiquitinase OTUD1 in acute lung injury in septic mice:relationship with TAK1-MAPK signaling pathway

目的评价卵巢肿瘤结构域蛋白酶1(OTUD1)在脓毒症小鼠急性肺损伤中的作用及其与转化生长因子β活化激酶1(TAK1)-丝裂原活化蛋白激酶(MAPK)信号通路的关系。方法雄性野生型和OTUD1基因敲除的C57BL/6N小鼠各20只,6~8周龄,体质量20~25 g。采用随机数字表法分别分为野生型假手术组(WT-Sham组)、野生型脓毒症组(WT-SEP组)、OTUD1基因敲除型假手术组(KO-Sham组)和OTUD1基因敲除SEP组(KO-SEP组),每组10只。采用盲肠结扎穿孔法制备脓毒症小鼠急性肺损伤模型。于术后24 h时处死小鼠,采集腹主动脉血样,取肺组织。采用血气分析仪行血气分析,计算氧合指数(OI),光镜下观察肺组织形态学并计算肺损伤评分,确定肺组织湿重/干重(W/D)比值,测定髓过氧化物酶(MPO)活性;采用ELISA法测定血浆TNF-α和IL-6浓度,Western blot法检测肺组织OTUD1、磷酸化(p-)TAK1、p38、c-Jun氨基末端激酶(JNK)和细胞外调节蛋白激酶(ERK)的表达。结果与WT-Sham组比较,WT-SEP组小鼠PaO_(2)和OI降低,肺损伤评分、W/D比值、MPO活性、血浆TNF-α和IL-6浓度升高,肺组织OTUD1、p-TAK1、p-p38、p-JNK和p-ERK表达上调(P<0.05);与WT-SEP组比较,KO-SEP组小鼠PaO_(2)和OI降低,肺损伤评分、W/D比值、MPO活性、血浆TNF-α和IL-6浓度升高,肺组织p-TAK1、p-p38、p-JNK和p-ERK表达上调(P<0.05)。结论OTUD1参与了脓毒症小鼠急性肺损伤的内源性保护机制,可能与抑制TAK1-MAPK信号通路激活,降低炎症反应有关。

Objective To evaluate the role of deubiquitinase OTUD1 in acute lung injury in septic mice and the relationship with transforming growth factor-beta activated kinase 1(TAK1)-mitogen-activated protein kinase(MAPK)signaling pathway.Methods Twenty male wild-type(WT)and 20 OTUD1 gene knockout(KO)C57BL/6N mice,aged 6-8 weeks,weighing 20-25 g,were divided into 4 groups(n=10 each)using a random number table method:wild-type+sham operation group(WT-Sham group),wild-type+sepsis group(WT-SEP group),OTUD1-KO+sham operation group(KO-Sham group)and OTUD1-KO+SEP group(KO-SEP group).The acute lung injury was induced by cecal ligation and perforation(CLP)in anesthetized septic mice.Mice were sacrificed at 24 h after operation,blood samples were collected from the abdominal aorta,and lung tissues were collected.Blood gas analysis was performed using the i-STAT blood gas analyzer,PaO_(2)and FiO_(2)were recorded,and the oxygenation index(OI)was calculated.The morphology of lung tissues was examined with a light microscope for evaluation of lung injury,and lung injury scores were calculated.The wet to dry lung weight(W/D)ratio was measured,and the activities of MPO were measured.The concentrations of TNF-αand IL-6 in plasma were detected by enzyme-linked immunosorbent assay,and the expression of OTUD1,phosphorylated TAK1(p-TAK1),phosphorylated p38(p-p38),phosphorylated c-Jun amino-terminal kinase(p-JNK)and phosphorylated extracellular signal-regulated kinase(p-ERK)was detected using Western blot.Results Compared with WT-Sham group,the PaO_(2)and OI were significantly decreased,the lung injury score,W/D ratio,MPO activity,and plasma TNF-αand IL-6 concentrations were increased,and the expression of OTUD1,p-TAK1,p-p38,p-JNK and p-ERK protein in lung tissues was up-regulated in WT-SEP group(P<0.05).Compared with WT-SEP group,the PaO_(2)and OI were significantly decreased,the lung injury score,W/D ratio,MPO activity,and plasma TNF-αand IL-6 concentrations were increased,and the expression of OTUD1,p-TAK1,p-p38,p-JNK and p-ERK protein in lung tissues was up-regulated in KO-SEP group(P<0.05).Conclusions OTUD1 is involved in the endogenous protective mechanism against acute lung injury in septic mice,which may be related to the inhibition of TAK1-MAPK signaling pathway activation and decreased inflammatory responses.