Supplementary Material for: Silencing of TRIM44 Inhibits Inflammation and Alleviates Traumatic Brain Injury in Rats by Downregulating TLR4-NF-κB Signaling

Background: Neuroinflammation subsequent to traumatic brain injury (TBI) is important for the recovery of patients and is associated with neurodegenerative changes post-TBI. The tripartite motif containing 44 (TRIM44) protein is an E3 ligase involved in the regulation of immune function with no previously known link to TBI. This study explores the connection between TRIM44 and TBI. Methods: After induction of TBI in rats by control cortex injury, TRIM44 expressions were determined with quantitative real-time reverse transcription polymerase chain reaction and Western blot, and Toll-like receptor 4 (TLR4)-NF-κB signaling was examined by the expression of TLR4, p65 phosphorylation, and the specific NF-κB transcription activity. The effects of TRIM44 knockdown on inflammation, neurological function, and TLR4-NF-κB signaling in TBI rats were revealed by the detection of proinflammatory cytokines and TLR4-NF-κB signaling molecules, modified neurological severity score, brain water content, and Evans blue permeability. Results: We found that TRIM44 expression was significantly increased following TBI induction along with TLR4-NF-κB activation. Silencing of TRIM44 suppressed proinflammatory cytokine production, improved neurological outcomes, alleviated brain edema, and inhibited TLR4-NF-κB signaling in TBI rats. Conclusion: Our findings suggest that suppressing TRIM44 or modulation of relevant pathways may be a therapeutic strategy for TBI.研究背景：创伤性脑损伤（traumatic brain injury, TBI）继发的神经炎症对患者康复至关重要，且与创伤性脑损伤后发生的神经退行性病变密切相关。含三结构域蛋白44（tripartite motif containing 44, TRIM44）是一种参与免疫功能调控的E3泛素连接酶，此前尚未发现其与创伤性脑损伤存在关联。本研究旨在探讨TRIM44与创伤性脑损伤之间的联系。 研究方法：采用控制性皮质损伤法构建大鼠创伤性脑损伤模型，通过实时定量反转录聚合酶链反应（quantitative real-time reverse transcription polymerase chain reaction）与蛋白质印迹（Western blot）检测TRIM44的表达水平，并通过检测Toll样受体4（Toll-like receptor 4, TLR4）的表达、p65磷酸化水平以及特异性核因子κB（NF-κB）转录活性，分析TLR4-NF-κB信号通路的激活情况。通过检测促炎细胞因子与TLR4-NF-κB信号通路分子、改良神经功能缺损评分（modified neurological severity score）、脑含水量以及伊文思蓝（Evans blue）通透性，明确TRIM44敲低对创伤性脑损伤大鼠炎症反应、神经功能以及TLR4-NF-κB信号通路的影响。 研究结果：本研究发现，创伤性脑损伤造模后，TRIM44的表达水平显著升高，同时伴随TLR4-NF-κB信号通路的激活。敲低TRIM44可抑制创伤性脑损伤大鼠的促炎细胞因子生成，改善神经功能预后，减轻脑水肿，并抑制TLR4-NF-κB信号通路的活化。 研究结论：本研究结果表明，抑制TRIM44表达或调控相关信号通路，有望成为创伤性脑损伤的潜在治疗策略。