【摘要】 探索芍药舒筋片在干预膝骨关节炎（KOA）进展过程中是否通过METTL3介导的m⁶A甲基化调控miR-140的表达发挥治疗作用。体外培养ATDC5细胞，使用15%的双乙酰基赖氨酸进行KOA细胞建模，随机分为11组。与芍药舒筋片相关的组别加入10μmol/L的芍药舒筋片溶液进行培养，与METTL3相关的组别使用lipofectamine^TM 2000进行转染，与miR-140相关的组别加入miR-140 mimic。qRT-PCR评估每组ATDC5细胞中miR-140的水平，MeRIP-qRT-PCR检测各组ATDC5细胞的m⁶A甲基化修饰水平，CCK8实验、划痕试验和β-半乳糖苷酶染色评估软骨细胞的增殖、迁移能力和衰老程度。之后，将SD大鼠随机分为6组：对照组、假手术组、KOA组、芍药舒筋片组、芍药舒筋片+KOA+shMETTL3-NC组和芍药舒筋片+KOA+shMETTL3组。KOA组构建KOA大鼠模型，芍药舒筋片组给予芍药舒筋片灌胃治疗，shMETTL3组在治疗的基础上给予相应的慢病毒注射。qRT-PCR评估各组大鼠软骨组织中的miR-140水平，并通过MeRIP qRT-PCR检测各组大鼠软骨组织中m⁶A甲基化修饰水平，番红O-固绿染色观察各组大鼠软骨组织的形态变化。结果显示：模型组的m⁶A甲基化修饰水平、miR-140的表达水平、细胞增殖和迁移能力显著降低，细胞衰老程度增加（P<0.05）;而加入芍药舒筋片治疗的组别能够显著提高细胞中的m⁶A甲基化修饰水平，并增加miR-140水平、细胞增殖及迁移能力，并抑制细胞衰老（P<0.05）。当METTL3被沉默时，芍药舒筋片的治疗作用被阻断，相关组的m⁶A甲基化修饰水平、miR-140的表达水平、细胞增殖和迁移能力显著降低，细胞衰老程度增加（P<0.05）;而在METTL3被沉默的基础上加入miR-140 mimic的组别的细胞中的m⁶A甲基化修饰水平得到提升，细胞增殖及迁移能力加强，并抑制细胞衰老（P<0.05）,miR-140的高表达逆转了METTL3沉默时带来的对治疗效果的阻断。在大鼠KOA模型中，miR-140和m⁶A甲基化修饰水平显著降低，软骨组织也被破坏。在芍药舒筋片的治疗下，miR-140和m⁶A甲基化修饰水平升高，软骨组织损伤恢复，在这个基础上沉默METTL3时，上述的治疗效果发生了逆转。芍药舒筋片可以通过METTL3介导的m⁶A甲基化修饰水平来调节miR-140的表达水平，修复损伤的KOA大鼠软骨细胞，起到保护作用。更多还原

【Abstract】 Osteoarthritis（OA） is a prevalent degenerative joint disease that significantly impairs the quality of life of middle-aged and elderly individuals and imposes a substantial financial burden on both patients and society.Due to their notable biological pro-perties, Shaoyao Shujin tablets have garnered increasing attention for their potential in OA treatment.This study aimed to investigate whether Shaoyao Shujin tablets could exert therapeutic effects on knee osteoarthritis（KOA） progression by regulating miR-140 through METTL3-mediated m⁶A methylation.An in vitro model of inflammation was established by treating the chondroprogenitor cell line（ATDC5） with 15% diacetyllysine.ATDC5 cells were then treated with a 10 μmol/L solution of Shaoyao Shujin tablets.To explore the role of METTL3 and miR-140,cells were transfected with Lipofectamine^TM 2000 for METTL3-related groups, and miR-140 mimics were added for miR-140-related groups.Chondrocyte proliferation, migration, and senescence were evaluated using the CCK8 assay, scratch test, and β-galactosidase staining, respectively.For in vivo experiments, SD rats subjected to anterior cruciate ligament transection（ACLT） surgery were treated with Shaoyao Shujin tablets via gastric lavage.Additionally, lentivirus-mediated intra-articular injections were administered with or without small interfering RNA（shRNA） targeting METTL3 or miR-140 mimics.Cartilage damage was assessed through histological analysis.Quantitative real-time PCR（qRT-PCR） was used to evaluate miR-140 expression levels, while MeRIP-qRT-PCR detected m⁶A methylation modification levels.In vitro analyses demonstrated that Shaoyao Shujin tablets enhanced chondrocyte proliferation and migration while reducing cellular senescence in 15% diacetyllysine-treated ATDC5 cells.These protective effects were shown to be mediated by METTL3.Further investigation revealed that Shaoyao Shujin tablets reduced m⁶A methylation levels by influencing miR-140’s targeted interaction with METTL3,leading to inhibition of OA progression.The in vivo results further validated these findings, demonstrating that Shaoyao Shujin tablets effectively increased the expression of miR-140 and METTL3,thereby slowing osteoarthritis（OA） progression and reducing osteophyte formation in ACLT mice.Notably, silencing METTL3 reversed these therapeutic effects, highlighting the critical role of METTL3 in mediating the observed outcomes.Furthermore, overexpression of miR-140 significantly mitigated the effects of METTL3 silencing, suggesting a potential interplay between miR-140 and METTL3 in regulating OA progression.Shaoyao Shujin tablets exert protective effects on chondrocytes in KOA by regulating miR-140 expression through METTL3-mediated m⁶A methylation modification.These findings highlight the therapeutic potential of Shaoyao Shujin tablets in mitigating KOA progression and restoring chondrocyte integrity.更多还原