Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Rd, Atlanta, GA 30322, USA.
J Neuroinflammation. 2011 May 8;8:42. doi: 10.1186/1742-2094-8-42.
Traumatic brain injury (TBI) causes acute inflammatory responses that result in an enduring cascade of secondary neuronal loss and behavioral impairments. It has been reported that progesterone (PROG) can inhibit the increase of some inflammatory cytokines and inflammation-related factors induced by TBI. Toll-like receptors (TLRs) play a critical role in the induction and regulation of immune/inflammatory responses. Therefore, in the present study, we examined the genomic profiles of TLR-mediated pathways in traumatically injured brain and PROG's effects on these genes.
Bilateral cortical impact injury to the medial frontal cortex was induced in C57BL/6J mice. PROG was injected (i.p., 16 mg/kg body weight) at 1 and 6 h after surgery. Twenty-four hours post-surgery, mice were killed and peri-contusional brain tissue was harvested for genomic detection and protein measurement. RT-PCR arrays were used to measure the mRNA of 84 genes in TLR-mediated pathways. Western blot, ELISA and immunohistochemistry were used to confirm the protein expression of genes of interest.
We found that 2 TLRs (TLR1 and 2), 5 adaptor/interacting proteins (CD14, MD-1, HSPA1a, PGRP and Ticam2) and 13 target genes (Ccl2, Csf3, IL1a, IL1b, IL1r1, IL6, IL-10, TNFa, Tnfrsf1a, Cebpb, Clec4e, Ptgs2 and Cxcl10) were significantly up-regulated after injury. Administration of PROG significantly down-regulated three of the 13 increased target genes after TBI (Ccl-2, IL-1b and Cxcl-10), but did not inhibit the expression of any of the detected TLRs and adaptor/interacting proteins. Rather, PROG up-regulated the expression of one TLR (TLR9), 5 adaptor/interacting proteins, 5 effectors and 10 downstream target genes. We confirmed that Ccl-2, Cxcl-10, TLR2 and TLR9 proteins were expressed in brain tissue, a finding consistent with our observations of mRNA expression.
The results demonstrate that TBI can increase gene expression in TLR-mediated pathways. PROG does not down-regulate the increased TLRs or their adaptor proteins in traumatically injured brain. Reduction of the observed inflammatory cytokines by PROG does not appear to be the result of inhibiting TLRs or their adaptors in the acute stage of TBI.
创伤性脑损伤（TBI）会引起急性炎症反应，导致继发性神经元丢失和行为障碍的级联反应持续存在。据报道，孕酮（PROG）可以抑制 TBI 引起的一些炎症细胞因子和炎症相关因子的增加。Toll 样受体（TLRs）在诱导和调节免疫/炎症反应中起着关键作用。因此，在本研究中，我们检测了 TLR 介导的通路在创伤性脑损伤中的基因组谱和 PROG 对这些基因的影响。
在 C57BL/6J 小鼠的内侧额皮质诱导双侧皮质冲击损伤。手术后 1 小时和 6 小时腹腔内注射 PROG（16mg/kg 体重）。手术后 24 小时，处死小鼠，采集损伤区周围脑组织进行基因组检测和蛋白测量。RT-PCR 阵列用于测量 TLR 介导的通路中 84 个基因的 mRNA。Western blot、ELISA 和免疫组织化学用于确认感兴趣基因的蛋白表达。
我们发现，2 种 TLR（TLR1 和 TLR2）、5 种衔接蛋白/相互作用蛋白（CD14、MD-1、HSPA1a、PGRP 和 Ticam2）和 13 个靶基因（Ccl2、Csf3、IL1a、IL1b、IL1r1、IL6、IL-10、TNFa、Tnfrsf1a、Cebpb、Clec4e、Ptgs2 和 Cxcl10）在损伤后显著上调。PROG 给药后，TBI 后上调的 13 个靶基因中有 3 个（Ccl-2、IL-1b 和 Cxcl-10）显著下调，但不抑制任何检测到的 TLR 和衔接蛋白/相互作用蛋白的表达。相反，PROG 上调了 1 种 TLR（TLR9）、5 种衔接蛋白/相互作用蛋白、5 种效应物和 10 个下游靶基因。我们证实 Ccl-2、Cxcl-10、TLR2 和 TLR9 蛋白在脑组织中表达，这与我们观察到的 mRNA 表达一致。
结果表明，TBI 可增加 TLR 介导通路中的基因表达。PROG 不会下调创伤性脑损伤中增加的 TLR 或其衔接蛋白。PROG 减少观察到的炎症细胞因子似乎不是在 TBI 的急性期抑制 TLR 或其衔接蛋白的结果。
