The role of transfer RNA (tRNA)-derived fragment (tRF) in various diseases has been established. However, the effect of tRF-3023b on inflammation remains unclear. Inflammation was imitated in RAW264.7 cells by adding Lipopolysaccharide (LPS). Cells were first divided into control, LPS, and LPS + Bulleyaconitine A (BLA) groups. The contents of TNF-α, IL-6, and MCP-1 were quantified using ELISA. The levels of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), and the phosphorylation of nuclear factor-kappa B (NF-κB)-P65 (p-P65) were detected by Western blotting. RNA sequencing was utilized to find differentially expressed tRFs (DE-tRFs) among three groups. The levels of various tRFs were checked by quantitative real-time PCR (qRT-PCR). Cell cycle and apoptosis were checked by flow cytometry. Dluciferase reporter assay was applied to predict and confirm the interaction between tRF-3023b and Cullin 4A (Cul4a), subsequently RNA pull-down followed by mass spectrometry analysis were conducted. BLA treatment decreased the contents of TNF-α, IL-6, MCP-1, and the expression levels of COX2, iNOS, p-P65. We found 6 DE-tRFs in LPS + BLA group compared to LPS group, tRF-3023b was high expression in control and BLA groups, and the lowest in LPS group. Cul4a was a direct target of tRF-3023b. tRF-3023b mimic affected the cell cycle distribution, promoted cells apoptosis, and suppressed the TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. The suppression of Cul4a affected the cell cycle distribution, resulted in an increase of cell apoptosis while a decrease of TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. Furthermore, Cul4a overexpression reversed the effect of tRF-3023b mimic. Cul4a knockdown reversed the effect of tRF-3023b inhibitor. Our study positions tRF-3023b as a compelling candidate, through its interaction with Cul4a, the underlying mechanism on inflammation maybe related to NF-κB pathway. The study provides a basis for exploring new therapeutic strategies for inflammation.

转移 RNA (tRNA) 衍生片段 (tRF) 在各种疾病中的作用已经确定。然而，tRF-3023b 对炎症的影响仍不清楚。通过添加脂多糖 (LPS) 在 RAW264.7 细胞中模拟炎症。首先将细胞分为对照组、LPS 组和 LPS + Bulleyaconitine A (BLA) 组。使用 ELISA 定量 TNF-α、IL-6 和 MCP-1 的含量。采用Western blotting检测环氧合酶2（COX2）、诱导型一氧化氮合酶（iNOS）水平以及核因子κB（NF-κB）-P65（p-P65）磷酸化水平。利用 RNA 测序来查找三组之间差异表达的 tRF (DE-tRF)。通过实时定量 PCR (qRT-PCR) 检查各种 tRF 的水平。通过流式细胞术检查细胞周期和凋亡。应用荧光素酶报告基因测定来预测和确认 tRF-3023b 和 Cullin 4A (Cul4a) 之间的相互作用，随后进行 RNA Pull-down 和质谱分析。BLA处理降低TNF-α、IL-6、MCP-1的含量以及COX2、iNOS、p-P65的表达水平。我们发现LPS+BLA组与LPS组相比有6个DE-tRF，tRF-3023b在对照组和BLA组中表达量较高，LPS组中表达量最低。Cul4a 是 tRF-3023b 的直接靶标。tRF-3023b 模拟物影响细胞周期分布，促进细胞凋亡，并抑制 TNF-α、IL-6、MCP-1、COX2、iNOS 和 p-P65。Cul4a的抑制影响细胞周期分布，导致细胞凋亡增加，同时TNF-α、IL-6、MCP-1、COX2、iNOS和p-P65减少。此外，Cul4a 过度表达逆转了 tRF-3023b 模拟物的作用。Cul4a 敲低可逆转 tRF-3023b 抑制剂的作用。我们的研究将 tRF-3023b 定位为一个引人注目的候选者，通过其与 Cul4a 的相互作用，炎症的潜在机制可能与 NF-κB 通路有关。该研究为探索新的炎症治疗策略提供了基础。