DAPK1在急性肺损伤小鼠肺组织中的表达及其作用

doi:10.3877/cma.j.issn.1674-6902.2017.02.008

目的

探讨死亡相关蛋白激酶1(DAPK1)在急性肺损伤小鼠肺组织中的表达及在炎症失控中的作用。

方法

将30只雄性C57小鼠按随机数字表法分为5组：正常对照组、LPS诱导急性肺损伤3、6、12、24 h组。应用2 mg/kg DAPK1抑制剂TC-DAPK 6预处理小鼠后，再用10 mg/kg LPS诱导小鼠肺损伤。HE染色光镜观察小鼠肺组织病理改变；免疫组化检测肺组织中DAPK1的表达和分布；应用RT-PCR和Western blot检测肺组织DAPK1和NF-κB p65的表达；ELISA检测血清炎症因子TNF-α、IL-6水平变化；Kaplan-Meier生存分析对各组小鼠存活时间进行分析。

结果

LPS致伤组肺组织可见明显病理损伤改变且随时间进展加重，而DAPK1蛋白在正常对照组小鼠肺组织仅少量表达，在急性肺损伤小鼠肺组织中表达随时间进展明显增加；与正常对照组相比，LPS组肺组织DAPK1 mRNA蛋白表达水平均明显增高(P<0.05)，血浆炎症因子TNF-α、IL-6均明显升高(P<0.05)。抑制小鼠肺DAPK1表达可明显降低LPS诱导的肺组织中DAPK1和NF-κB p65蛋白水平、血清炎症因子TNF-α、IL-6的水平(P<0.05)。此外，抑制DAPK1可延长LPS致急性肺损伤小鼠的生存期(P<0.01)。

结论

DAPK1通过调控NF-κB炎症通路参与了LPS诱导的小鼠急性肺损伤，其可作为潜在的治疗靶点。

关键词: 死亡相关蛋白激酶1, 急性肺损伤, 核因子κB

Objective

To investigate the expression of death associated protein kinase 1(DAPK1) in lung tissues of mouths with LPS induced acute lung injury(ALI) and its function in ALI.

Methods

Thirty C57 male mice were randomly divided into 5 groups: control group, LPS induced acute lung injury group for 3 h, 6 h, 12 h and 24 h group. Histological changes of the lungs were observed by HE staining; The expression and distribution of DAPK1 in the lung tissues was detected by western blot, qPCR and Immunohistochemistry. The serum levels of TNF-α and IL-6 were detected by ELISA. Six mice were pretreated with 2 mg/kg TC-DAPK 6, a selectivity DAPK1 inhibitor, and then 10 mg/kg LPS was used to induce mice acute lung injury by intraperitoneal injection. The expression of DAPK1 and NF-κB p65 was evaluated using western blot and qPCR, and ELISA was used to analyze the level of serum TNF-α and IL-6. Kaplan-Meier analysis was evaluated the lifespan of these mice.

Results

The HE stain showed that pathological of lung tissue damage became severe along with time. Immunohistochemistry demonstrated that. The area of DAPK1 protein expression was more large with time increased in LPS group, compared to control group (P<0.05). The level of serum TNF-α and IL-6 was significantly higher than control group (P<0.05). The expression of DAPK1 and NF-κB p65 and serum TNF-α and IL-6 level was significantly decreased after pretreated with TC-DAPK 6 and stimulated with LPS, compared to LPS alone group (P<0.05). Moreover, Kaplan-Meier analysis showed the lifespan of mice by pretreated with TC-DAPK 6 was longer than LPS alone group (P<0.01).

Conclusion

DAPK1 involved in LPS induced mice acute lung injury by modulation NF-κB p65 and the production and released of inflammation mediator. It might be a potential target for ALI/ARDS treatment.

Key words: Death associated protein kinase 1, Acute lung injury, Nuclear factor-κB

图1 LPS致大鼠急性肺损伤；注：A.HE染色观察LPS处理小鼠不同时间点肺组织的损伤(×20)；①对照组；② LPS处理3 h；③LPS处理6 h；④LPS处理12 h；⑤ LPS处理24 h；B. ELISA检测血清TNF-α、IL-6水平；*，与control组比较，P<0.05; ^，与LPS处理3 h组比较，P<0.05; #，与LPS处理6 h组比较，P<0.05

图2 LPS致小鼠急性肺损伤各个时相点DAPK1的表达及定位。A.免疫组化染色观察LPS处理小鼠不同时间点对肺组织中DAPK蛋白的定位和表达(×20)；注：①对照组；②LPS处理3 h；③LPS处理6 h；④ LPS处理12 h；⑤ LPS处理24 h；B. Western blot检测LPS致小鼠急性肺损伤各个时相点DAPK蛋白的表达情况；C. qPCR检测LPS致小鼠急性肺损伤各个时相点DAPK mRNA的表达情况；*与control组比较P<0.05; ^与LPS处理3 h组比较，P<0.05；#与LPS处理6 h组比较，P<0.05；&与LPS处理12 h组比较，P<0.05

图3 TC-DAPK 6抑制LPS诱导的肺损伤，下调DAPK1及NF－κB p65的表达；注：A: HE染色观察TC-DAPK 6预处理后，LPS诱导小鼠急性肺损伤(×20). 1:单独LPS组；2：TC-DAPK 6＋LPS组；B：qPCR检测DAPK1的表达水平。*与control组比较P<0.05; ^与LPS组比较P<0.05；C：Western blot检测DAPK1和NF-κB p65蛋白的表达情况

图4 TC-DAPK 6下调LPS诱导的小鼠血清炎症因子血清IL-6、TNF-α水平并延长小鼠生存期；注：A和B，ELISA检测血清TNF-α、IL-6水平。*与control组比较，P<0.05; ^与LPS处理组比较，P<0.05；C：Kaplan-Meier生存分析小鼠生存时间。*与LPS组比较，P<0.05

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Expression and function of DAPK1 in lung tissues of mouse with acute lung injury

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Expression and function of DAPK1 in lung tissues of mouse with acute lung injury