Study on the Mechanism of Trim47 in Acute Lung Injury via TAB1/I κB Inflammatory Signaling Pathway
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摘要:
目的 探讨Tripartite结构蛋白47（tripartite motif containing 47，TRIM47）对急性肺损伤急性肺损伤（acute lung injury，ALI）大鼠模型肺组织的影响以及对转化生长因子β激活激酶1（TGF-beta activated kinase 1，TAB1）/核因子κB抑制蛋白（inhibitor of NF-κB，IκB）调控的分子机制。 方法 构建SD大鼠ALI模型，模型组、空载体组（NC）组及si-TRIM47组诱导建立大鼠ALI模型，NC组与si-TRIM47组于建模后经尾静脉分别注射NC质粒及靶向TRIM47的siRNA干扰质粒（si-TRIM47）。干预1周后，通过苏木精-伊红（HE）染色观察各组肺组织病理学改变，ELISA检测外周血白细胞介素（Interleukin，IL）-1、IL-6、IL-10、IL-1β、肿瘤坏死因子（tumor necrosis factor，TNF-α）水平变化，qPCR测定肺组织中TAB1及IκB的mRNA相对表达量，Western blot分析肺组织中TAB1与IκB蛋白表达水平，同时检测NF-κB入核情况；CO-IP检测TRIM47和TAB1蛋白结合情况。 结果 在组织病理学观察中，与对照组相比，模型组和NC组的组织炎性细胞浸润程度升高；与模型组和NC组相比，si-TRIM47组的病理病变程度则相对较轻。ELISA检测结果显示，与对照组相比，模型组IL-1、IL-6、IL-1β、TNF-α升高（P < 0.01）。与模型组和NC组相比，si-TRIM47组大鼠的IL-1、IL-6、IL-1β、TNF-α降低，而IL-10升高（P < 0.01）；qPCR和Western blot结果表明，与对照组相比，模型组、NC组大鼠TAB1和核蛋白NF-κB升高、IκB表达水平降低（P < 0.01）；与模型组和NC组相比，si-TRIM47组TAB1降低和核蛋白NF-κB降低、IκB表达水平升高（P < 0.01）。此外，CO-IP实验显示，TRIM47促进TAB1蛋白表达。 结论 si-TRIM47可能通过抑制炎症因子释放及TAB1信号通路的激活，对大鼠ALI发挥保护作用。 Abstract:Objective To investigate the effect of tripartite motif containing 47 （TRIM47） on lung tissue of acute lung injury rat model and its effect on the molecular mechanism regulating transforming growth factor Transforming growth factor beta activated kinase 1 binding protein 1（TAB1） / inhibitor of NF-κB （IκB）. Methods The ALI model was constructed in SD rats, the model group, empty carrier （NC）group and si-TRIM47 group were induced to establish a rat ALI model. The NC group and si-TRIM47 group were injected with NC plasmid and siRNA interference plasmid targeting TRIM47 （si-TRIM47） via tail vein after modeling. One week after the intervention, pathological changes in the lung tissues of each group were observed via hematoxylin and eosin （HE） staining. Levels of peripheral blood interleukin （IL）-1, IL-6, IL-10, IL-1β, and Tumor Necrosis Factor （TNF-α）were detected by ELISA. The relative mRNA expression levels of TAB1 and IκB in lung tissues were measured using quantitative real-time PCR （qPCR）. Protein expression levels of TAB1 and IκB in lung tissues were analyzed by Western blot, while nuclear translocation of NF-κB was simultaneously detected. The protein interaction between TRIM47 and TAB1 was examined by co-immunoprecipitation （Co-IP）. Results In histopathological observations, compared with the control group, the model group and NC group exhibited increased inflammatory cell infiltration, whereas the si-TRIM47 group showed relatively milder pathological lesions compared to both the model and NC groups. ELISA results indicated that, compared with the control group, the model group had elevated levels of IL-1, IL-6, IL-1β, and TNF-α （P < 0.01）; in contrast, compared with the model group and NC group, the si-TRIM47 group demonstrated reduced levels of IL-1, IL-6, IL-1β, and TNF-α but increased IL-10 （P < 0.01） group and NC group, the si-TRIM47 group demonstrated reduced levels of IL-1, IL-6, IL-1β, and TNF-α but increased IL-10 （P < 0.01）. Furthermore, qPCR and Western blot results revealed that, compared with the control group, TAB1 and nuclear NF-κB protein levels were elevated while IκB expression was markedly reduced in the model and NC groups（P < 0.01）; conversely, the si-TRIM47 group exhibited decreased TAB1 and nuclear NF-κB protein alongside increased IκB expression compared to the model and NC groups （P < 0.01）. Additionally, the CO-IP assay confirmed that TRIM47 promoted TAB1 protein expression. Conclusion si-TRIM47 may exert a protective effect against ALI in rats by inhibiting the release of inflammatory factors and the activation of the TAB1 signaling pathway. -
Key words:
- Si-trim47 /
- Acute lung injury /
- Inflammatory factors /
- TAB1 signaling path
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图 1 si-TRIM47干预对大鼠ALI病理学的影响（200×）（n = 3）
Figure 1. Effect of si-TRIM47 intervention on rat ALI pathology （200×） （n = 3）
图 2 各组大鼠IL-1、IL-6、IL-10炎症因子表达比较（$\bar x \pm s $，n = 15）
aP < 0.01 vs Control组；bP < 0.01 vs Model组；cP < 0.01 vs NC组。
Figure 2. Comparison of inflammatory factor of IL-1、IL-6、IL-10 expressions among different groups of （$\bar x \pm s $，n = 15）
图 3 si-TRIM47对ALI大鼠TAB1和IκB基因mRNA表达的影响 （$\bar x \pm s $，n = 3）
aP < 0.01 vs Control组；bP < 0.01 vs Model组；cP < 0.01 vs NC组。
Figure 3. Effect of si-TRIM47 on the mRNA expression of TAB1 and IκB genes in ALI rats （$\bar x \pm s $，n = 3）
图 4 si-TRIM47对ALI大鼠TAB1和IκB蛋白表达的影响 （$\bar x \pm s $，n = 3）
A：TAB1和IKB蛋白条带图；B：TAB1和IKB蛋白统计图；aP < 0.01 vs Control组；bP < 0.01 vs Model组；cP < 0.01 vs NC组。
Figure 4. Effect of si-TRIM47 on the protein expression of TAB1 and IκB in ALI rats （$\bar x \pm s $，n = 3）
图 5 si-TRIM47靶向抑制TAB1抑制NF-κB入核（$\bar x \pm s $，n = 3）
A：TAB1和TRIM47 CO-IP图；B：NF-κB入核蛋白条带图和统计图；aP < 0.01 vs Control组；bP < 0.01 vs Model组；cP < 0.01 vs NC组。
Figure 5. si-TRIM47 targets and inhibits TAB1 to suppress NF-κB nuclear translocation （$\bar x \pm s $，n = 3）
表 1 qPCR检测中不同基因的引物序列
Table 1. Primer sequences of different genes in qPCR detection
基因名称 引物序列（5'-3'） 引物长度（bp） TAB1 Forward：5′- CGACGCGTTGGCGGCGCAGAGGAGGAGCTTGC -3′ 23 Reverse：5′- ACGCGTCGAC TACCCTGGGGTCAGGCTGCCCAGGA -3' IκB Forward：5′- TGAAAAACTGGATGTCCCTGTATG -3′ 22 Reverse：5′- GGCCCATTTCCCGCCCCCTGGCAT -3′ GAPDH Forward: 5'- GTACGACTCACTATAGGGA -3′
Reverse: 5'- AGGTCCACCACCCTGTTGCTGT -3′20 
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