Table 3_Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment.xlsx

BackgroundKnee osteoarthritis (KOA) is closely associated with an imbalance in macrophage M1/M2 polarization within its inflammatory microenvironment. Platelet-rich plasma (PRP) has demonstrated therapeutic efficacy in KOA. Moreover, microRNAs (miRNAs) also play a protective or destructive role in the pathogenesis of KOA. This study aims to elucidate the molecular mechanisms by which PRP-related miRNAs ameliorate the inflammatory microenvironment to alleviate KOA.
MethodsAn in vivo KOA rat model was established via intra-articular injection with monosodium iodoacetate (MIA). Safranin O-fast green staining and hematoxylin and eosin (HE) staining were used to assess cartilage degeneration and synovial inflammation, respectively. Macrophage phenotype was analyzed by immunohistochemistry (IHC) and immunofluorescence (IF). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to examine the expression of inflammatory cytokines. Chondrocyte anabolic and catabolic status was evaluated using IF and western blotting (WB). Bioinformatics analysis was employed to screen for differentially expressed miRNAs in PRP and Dual-luciferase reporter assay was conducted to verify that mRNA is a direct target for miRNA. Furthermore, we explored the biological functions of miRNA and mRNA by transfecting mimics and siRNA.
ResultsIn vitro, PRP inhibited M1-type macrophage polarization while promoting M2-type polarization, leading to suppressed pro-inflammatory cytokine release and enhanced anti-inflammatory cytokine release, collectively reducing cartilage degeneration. We identified microRNA let-7a-5p using bioinformatic approaches and subsequently investigated its molecular mechanisms. Similar to PRP, let-7a-5p was found to regulate macrophage polarization, the release of inflammatory cytokine, and cartilage degeneration. Furthermore, we identified and experimentally validated MAPK8 as a target gene of let-7a-5p.
ConclusionPRP reshapes macrophage polarization by regulating the let-7a-5p/MAPK8 axis, thereby improving the inflammatory microenvironment of KOA and providing a potential new therapeutic target for KOA management.

背景 膝骨关节炎（Knee osteoarthritis, KOA）与炎症微环境内巨噬细胞M1/M2极化失衡密切相关。富血小板血浆（Platelet-rich plasma, PRP）在膝骨关节炎治疗中已展现出明确疗效。此外，微小核糖核酸（microRNAs, miRNAs）在膝骨关节炎的发病机制中兼具保护与致病双重作用。本研究旨在阐明PRP相关微小核糖核酸通过调控炎症微环境以缓解膝骨关节炎的分子机制。
方法 本研究通过关节腔内注射碘乙酸单钠（monosodium iodoacetate, MIA）构建体内膝骨关节炎大鼠模型。分别采用番红O-固绿染色（Safranin O-fast green staining）与苏木精-伊红（hematoxylin and eosin, HE）染色评估软骨退变与滑膜炎症程度。通过免疫组织化学（immunohistochemistry, IHC）与免疫荧光（immunofluorescence, IF）技术分析巨噬细胞表型。采用逆转录定量聚合酶链反应（Reverse transcription-quantitative polymerase chain reaction, RT-qPCR）检测炎症细胞因子的表达水平。通过免疫荧光与蛋白质印迹（western blotting, WB）技术评估软骨细胞的合成与分解代谢状态。借助生物信息学分析筛选富血小板血浆中差异表达的微小核糖核酸，并通过双荧光素酶报告基因实验（Dual-luciferase reporter assay）验证mRNA与微小核糖核酸的直接靶向关系。此外，本研究通过转染微小核糖核酸模拟物与小干扰RNA（siRNA），探究目标微小核糖核酸及其靶mRNA的生物学功能。
结果 体外实验中，富血小板血浆可抑制巨噬细胞M1型极化并促进M2型极化，进而抑制促炎细胞因子释放、增强抗炎细胞因子分泌，最终减轻软骨退变程度。本研究通过生物信息学方法筛选得到微小核糖核酸let-7a-5p，并对其分子机制展开深入研究。与富血小板血浆的作用相似，let-7a-5p可调控巨噬细胞极化、炎症细胞因子释放以及软骨退变过程。此外，本研究筛选并通过实验验证了丝裂原活化蛋白激酶8（MAPK8）为let-7a-5p的靶基因。
结论 富血小板血浆通过调控let-7a-5p/MAPK8轴重塑巨噬细胞极化状态，进而改善膝骨关节炎的炎症微环境，为膝骨关节炎的临床诊疗提供了潜在的新型治疗靶点。