TLR2/TLR4在失血性休克引发的脾组织TIPE2和TLR2/TLR4信号通路分子表达上调中的作用

Roles of TLR2/TLR4 in hemorrhagic shock-induced up-regulation of TIPE2 and TLR2/TLR4 signaling molecules in spleen tissue

目的研究发现,肠淋巴液回流是导致失血性休克后免疫功能紊乱的重要因素。本研究应用TLR2^(-/-)和TLR4^(-/-)小鼠探讨失血性休克对脾组织TIPE2和TLR2/TLR4信号下游分子表达的影响。方法将不同(C57BL/6J、TLR2^(-/-)、TLR4^(-/-))来源雄性小鼠随机分为Sham组、Shock组、Shock+Drainage组,分别予以不同手术处理后,无菌获取各实验组小鼠的脾组织。应用RTPCR技术分别检测WT小鼠脾组织TIPE2、TLR2、TLR4、MyD88、TRIF、TRAF3、TRAF6的mRNA表达。应用Western blotting技术分别检测不同小鼠(C57BL/6J、TLR2^(-/-)、TLR4^(-/-))脾组织TIPE2、TLR2、TLR4、MyD88、TRIF、TRAF3、TRAF6的蛋白表达。结果与Sham组相比,失血性休克后脾脏组织TIPE2、TLR2/TLR4及其下游分子表达水平均上调,肠淋巴液引流显著降低了失血性休克导致的TIPE2、TLR2/TLR4及其下游分子的高表达。TLR2的缺失降低了Shock组脾组织TIPE2、TLR4、MyD88、TRAF6的蛋白表达。TLR4缺失降低了Shock组TIPE2、TLR2、MyD88、TRIF、TRAF6的蛋白表达。结论以TLR2、TLR4作为干预靶点,TIPE2可能通过负反馈机制调控TLR2/TLR4信号通路下游分子mRNA和蛋白的表达,有利于促进失血性休克后免疫平衡状态的恢复。

Tumor necrosis factor-α-induced protein 8-like molecule 2 is an important protein for maintaining immune dynamic homeostasis.Immune dysfunction is an important factor in the development of sepsis or septic shock after hemorrhagic shock.Recent studies have found that intestinal lymphatic reflux is an important factor contributing to immune dysfunction after hemorrhagic shock.In this study,TLR2^(-/-)and TLR4^(-/-)mice were used to investigate the effect of hemorrhagic shock on the expression of TIPE2 and TLR2/TLR4 signaling downstream molecules in splenic tissues.Male mice from different genotypes(C57BL/6J,TLR2^(-/-),TLR4^(-/-))were randomly divided into Sham,Shock and Shock+Drainage groups.The spleen tissues were obtained aseptically from each experimental group after different surgical treatments.RT-PCR was used to detect the mRNA expressions of TIPE2,TLR2,TLR4,MyD88,TRIF,TRAF3 and TRAF6 in splenic tissues of WT mice;Western blot was used to detect the protein expressions of TIPE2,TLR2,TLR4,MyD88,TRIF,TRAF3 and TRAF6 in splenic tissues of different mice(C57BL/6J,TLR2^(-/-),TLR4^(-/-)).Compared with the Sham group,the expression levels of TIPE2,TLR2/TLR4 and their downstream molecules were upregulated in splenic tissues after hemorrhagic shock.And the drainage of intestinal lymphatic significantly decreased the high expression of TIPE2,TLR2/TLR4 and their downstream molecules caused by hemorrhagic shock.Lack of TLR2 decreased the protein expressions of TIPE2,MyD88,TLR4 and TRAF6 in splenic tissues of Shock group,while Lack of TLR4 decreased the protein expression of TIPE2,TLR2,MyD88,TRIF,TRAF6 in Shock group.In conclusion,TLR2 or TLR4 acts as an intervention target,and TIPE2 may regulate the mRNA and protein expression of downstream molecules of TLR2/TLR4 signaling pathway through a negative feedback mechanism,which is beneficial to promote the restoration of immune balance after hemorrhagic shock.