牛膝醇提物通过滑膜液外泌体介导的m6A修饰抑制膝骨关节炎的分子机制研究

Research on the molecular mechanism by which achyranthes bidentata ethanol extract inhibits knee osteoarthritis via m6A modification mediated by synovial fluid exosomes

摘要:

目的 基于滑膜液外泌体介导的m6A修饰，探讨牛膝醇提物对膝骨关节炎（KOA）的影响。方法 利用改良Hulth法复制KOA大鼠模型，提取并鉴定滑膜液外泌体。通过逆转录聚合酶链反应（RT-PCR）等方法检测KOA大鼠滑膜液外泌体中m6A修饰的变化，识别差异m6A。构建滑膜炎症细胞模型，将实验分为对照组、IL-1β组、IL-1β + si-NC组、IL-1β + si-METTL3组，检测METTL3对滑膜细胞存活率及趋化因子分泌的影响。通过HE和番红固绿染色评估METTL3对KOA滑膜退变的组织学影响。使用Nanosight和RT-PCR检测牛膝醇提物对滑膜液外泌体的数量、活性及METTL3表达的影响。结果 KOA大鼠模型成功复制后，micro-CT显示对照组大鼠的膝关节表面光滑，关节间隙正常；而模型组大鼠的膝关节表面粗糙，关节间隙狭窄。通过Nanosight检测，外泌体颗粒丰度为76～80 nm，Western blotting检测结果显示，CD9和TSG101高表达，电子显微镜下外泌体为椭圆形，直径< 200 nm。m6A定量检测结果显示，KOA模型组m6A总量、METTL3表达均高于对照组（P <0.05），去甲基化酶表达均无明显变化（P >0.05）。METTL3沉默对滑膜细胞存活率的影响检测结果显示，与IL-1β组和IL-1β + si-NC组比较，IL-1β + si-METTL3组的细胞存活率升高（P <0.05）。检测METTL3沉默对滑膜细胞趋化因子SDF-1和MCP-1分泌的影响，结果表明，与IL-1β组和IL-1β + si-NC组比较，IL-1β + si-METTL3组SDF-1和MCP-1浓度均降低（P <0.05）。HE和番红固绿染色结果显示，对照组细胞层次分明，无明显炎症浸润；模型组和Scr-RNA组细胞排列紊乱，有血管增生和炎症浸润；Sh-METTL3组炎症浸润减少，血管增生缓解。牛膝醇提物含药血清刺激实验检测滑膜细胞存活率结果显示，与模型组比较，牛膝醇提物组滑膜细胞存活率升高（P <0.05）。与METTL3过表达组比较，METTL3过表达加牛膝醇提物组滑膜细胞存活率升高（P <0.05）。动物实验中，与对照组比较，牛膝醇提物组外泌体数量和乙酰胆碱酯酶活性均无明显变化（P >0.05），但模型组METTL3基因相对表达量高于对照组和牛膝醇提物组（P <0.05）。结论 KOA滑膜液外泌体中METTL3高表达，能够降低滑膜细胞存活率并促进趋化因子分泌。牛膝醇提物可能通过降低外泌体中METTL3的表达缓解KOA滑膜炎症。

Abstract:

Objective To explore the effects of Achyranthes bidentata ethanol extract on knee osteoarthritis (KOA) based on m6A modifications mediated by synovial fluid exosomes.Methods Establish a KOA rat model using a modified Hulth method, and extract and identify synovial fluid exosomes. Use RT-PCR and other methods to detect changes in m6A modifications in KOA synovial fluid exosomes and identify differential m6A. Construct a synovial cell inflammation model, dividing the experiment into: control group, IL-1β group, IL-1β + si-NC group, and IL-1β + si-METTL3 group, to assess the impact of METTL3 on synovial cell viability and chemokine secretion. Evaluate the histological impact of METTL3 on KOA synovial degeneration using HE staining and Safranin O-Fast Green staining. Use Nanosight and RT-PCR to examine the effects of Achyranthes bidentata ethanol extract on the quantity, activity, and METTL3 expression in synovial fluid exosomes.Results After successful model establishment, micro-CT showed that the knee joint surface in the control group was smooth with normal joint spacing, whereas the model group exhibited a rough joint surface and narrowed joint space. Nanosight detection showed that exosome particles ranged from 76 to 80 nanometers. Western Blot analysis indicated high expression of CD9 and TSG101, and electron microscopy observed that the exosomes were elliptical with diameters less than 200 nanometers. m6A quantification kit detection revealed that the total m6A level was elevated in the KOA model, with METTL3 expression significantly higher than that in the control group (P < 0.05), while the expression of demethylases showed no significant change (P > 0.05). The impact of METTL3 silencing on synovial cell viability was examined. Compared to the IL-1β group and the IL-1β+si-NC group, the IL-1β + si-METTL3 group exhibited increased cell viability, with statistically significant differences (P < 0.05). The effect of METTL3 silencing on the secretion of synovial cell chemokines SDF-1 and MCP-1 was also assessed. Results indicated that the concentrations of SDF-1 and MCP-1 were reduced in the IL-1β + si-METTL3 group compared to the IL-1β group and the IL-1β + si-NC group, with statistically significant differences (P < 0.05). HE and Safranin O-Fast Green staining showed that the normal group had distinct cell layers with no significant inflammatory infiltration; the model group and the Scr-RNA group displayed disorganized cell arrangements along with vascular proliferation and inflammatory infiltration; the Sh-METTL3 group and the Achyranthes bidentata extract group showed reduced inflammatory infiltration and alleviated vascular proliferation. In cell experiments using serum containing Achyranthes bidentata extract, synovial cell viability was assessed. Compared to the model group, the Achyranthes bidentata extract group showed increased synovial cell viability, with statistically significant differences (P < 0.05). Compared to the METTL3 overexpression group, the METTL3 overexpression plus Achyranthes bidentata extract group also exhibited increased synovial cell viability, with statistically significant differences (P < 0.05). At the animal level, compared to the control group, the Achyranthes bidentata extract gavage group showed no significant change in exosome quantity and ACHE activity (P > 0.05), but METTL3 expression levels were reduced, showing statistically significant differences (P < 0.05).Conclusion High expression of METTL3 in KOA synovial fluid exosomes can reduce synovial cell viability and promote chemokine secretion. Achyranthes bidentata ethanol extract may alleviate KOA synovial degeneration by reducing METTL3 expression in exosomes.