背景：类风湿关节炎（Rheumatoid Arthritis, RA）是以关节损伤为主要表现的自身免疫病。成纤维样滑膜细胞（Fibroblast-like synoviocytes, FLS）增生和炎症浸润是RA主要特征之一，也是骨及软骨破坏的主要诱因。目前，仍没有根治RA的治疗策略。因此，深入了解RA发病机制，寻找RA治疗有效药物具有重要临床意义。

近年来，对表观遗传修饰的深入了解为解析疾病机制提供了新视角。其中， N6-甲基腺苷（m⁶A）甲基化修饰极其重要，并且与RA进展密切相关。目前，关于m⁶A修饰酶在RA滑膜增殖和关节破坏中的作用尚缺乏全面的资料，需要进一步研究以阐明其在RA中的确切作用。此外，寻找靶向m⁶A修饰酶的小分子药物也有助于突破RA治疗困境。

基于以上问题，本论文主要包括以下四部分内容：

第一部分：m⁶A阅读蛋白IGF2BP3在RA中的表达及诊断意义

方法：采用Boruta、Rpart、LASSO、XGBoost和logistic回归五种不同的机器学习方法构建疾病诊断模型（RA与非RA）。并通过功能富集分析和单细胞数据分析，探讨核心m⁶A相关修饰酶参与的生物学功能。

结果：在五种诊断模型中均包含两个m⁶A相关修饰酶：IGF2BP3和YTHDC2。其中， IGF2BP3重要性最高。功能富集分析提示，IGF2BP3和YTHDC2参与细胞增殖等信号通路。单细胞数据分析表明RA患者的M1型巨噬细胞比例显著增加，并且IGF2BP3与CD86表达密切相关。

第二部分：IGF2BP3促进RA-FLS增殖和炎症活化的作用机制研究

方法：免疫组化检测RA滑膜组织中IGF2BP3的表达。对IGF2BP3进行过表达或者沉默，探究其对滑膜细胞生物学功能的影响。同时，通过m⁶A-RIP-seq和IGF2BP3-RIP-seq技术筛选IGF2BP3作用的靶点，进一步通过功能回复实验验证IGF2BP3调控滑膜细胞的作用机制。此外，基于IGF2BP3敲除鼠，我们构建关节炎模型，进一步探讨IGF2BP3对关节炎的影响。

结果：RA滑膜组织中IGF2BP3表达水平显著增加，并且与Vimentin共表达。并且，siIGFBP3抑制RA-FLS的细胞增殖，并促进自噬。此外，我们发现IGF2BP3识别RASGRF1 mRNA的m⁶A甲基化位点，并调控其稳定性，进而激活mTORC1，从而促进滑膜细胞增殖和促炎细胞因子表达。与WT关节炎小鼠相比，IGF2BP3^-/-关节炎小鼠踝关节的滑膜炎症，炎症细胞浸润和细胞增生明显减轻。

第三部分：IGF2BP3促进巨噬细胞向M1型极化的机制研究

方法： 免疫荧光检测RA滑膜组织中IGF2BP3与M1型巨噬细胞比例的关系。对IGF2BP3进行过表达或者沉默，探究其对巨噬细胞炎症激活的调控作用。RIP-qPCR检测IGF2BP3在巨噬细胞中与RASGRF1 mrna的结合能力。随后，功能回复实验进一步检测IGF2BP3通过调控巨噬细胞向M1型极化和ROS生成的作用机制。最后，在动物实验中，探讨IGF2BP3敲除对关节炎小鼠炎症状态的影响。

结果： IGF2BP3表达水平与M1型巨噬细胞比例呈正相关。此外，siIGF2BP3显著降低M1型巨噬细胞的比例和ROS的生成。在巨噬细胞中，IGF2BP3也以m⁶A依赖的方式调控RASGRF1 mRNA的稳定性，从而激活mTORC1-自噬轴，促进巨噬细胞向M1型极化。此外，与WT关节炎小鼠相比， IGF2BP3^-/-关节炎小鼠脾脏中M1型巨噬细胞比例降低，且滑膜中RASGRF1和iNOS表达下降。

第四部分：雷公藤红素（Celastrol , CEL）靶向IGF2BP3缓解RA炎症进展的作用机制研究

方法： 通过分子对接，分子动力学模拟和SPR分析寻找与IGF2BP3结合的小分子化合物。然后，分子生物学实验检测CEL对滑膜细胞和巨噬细胞生物学功能的调控。并通过功能回复实验探讨CEL靶向IGF2BP3抑制滑膜细胞增殖和巨噬细胞炎症激活的分子机制。最后，基于IGF2BP3敲除鼠构建的关节炎模型，分析IGF2BP3在CEL缓解关节炎进展中发挥的作用。

结果： CEL与IGF2BP3稳定结合；并降低IGF2BP3表达。机制上，CEL通过抑制 IGF2BP3表达，从而减弱RASGRF1介导的mTORC1激活，进而抑制滑膜细胞增殖和巨噬细胞炎症激活。此外，与WT关节炎小鼠相比，IGF2BP3^-/-关节炎小鼠的关节肿胀、滑膜炎症和骨破坏明显改善；而CEL在IGF2BP3^-/-关节炎小鼠中未显示出进一步的缓解作用。

结论: m⁶A阅读蛋白IGF2BP3有希望作为RA诊断的重要指标，并且与RA-FLS细胞增殖和M1型巨噬细胞极化密切相关。机制上，IGF2BP3通过提高RASGRF1 mRNA的稳定性，激活mTORC1信号通路，从而加速S6K和ULK1磷酸化，促进滑膜细胞增殖并抑制细胞凋亡及自噬过程；此外， IGF2BP3也能通过RASGRF1介导的mTORC1-自噬轴促进巨噬细胞的ROS生成和M1型巨噬细胞极化。值得注意的是，CEL通过抑制IGF2BP3/RASGRF1/mTORC1信号通路减少关节微环境中的细胞增殖和炎症激活，从而缓解关节炎小鼠的关节损伤。

Background: Rheumatoid arthritis (RA) is an autoimmune disorder primarily characterized by joint damage. Fibroblast-like synoviocytes (FLS) hyperplasia and inflammatory infiltration are key features of RA, contributing significantly to bone and cartilage destruction. At present, there is no definitive cure for RA. Therefore, a comprehensive understanding of the pathogenesis of RA and the identification of effective therapeutic agents are of paramount clinical importance.

In recent years, advances in the study of epigenetic modifications have offered novel insights into disease mechanisms. Notably, N⁶-methyladenosine (m⁶A) methylation plays a crucial role in the progression of RA. However, current knowledge regarding the impact of m⁶A-modified enzymes on synovial hyperplasia and joint destruction in RA remains limited, necessitating further research to elucidate their precise roles. Additionally, exploring small molecule drugs targeting m⁶A-modified enzymes may provide new avenues to address the therapeutic challenges associated with RA.

Based on the above questions, this paper mainly includes the following three parts:

Part 1: Expression and diagnostic significance of m⁶A reading protein IGF2BP3 in RA

Methods: Five different machine learning methods, including Boruta, Rpart, LASSO, XGBoost, and logistic regression, were used to construct disease diagnosis models (RA vs. non-RA). Functional enrichment analysis and single-cell data analysis were also conducted to explore the biological functions involved in the core m⁶A related modifying enzymes.

Results: Two m⁶A related modifying enzymes, IGF2BP3 and YTHDC2, were included in all five diagnostic models, with IGF2BP3 being the most important. Functional enrichment analysis suggested that IGF2BP3 and YTHDC2 were involved in cell proliferation and other signaling pathways. Single-cell data analysis indicated that the proportion of M1 macrophages in RA patients was significantly increased, and IGF2BP3 was closely related to the expression of CD86.

Part 2: The mechanism of IGF2BP3 promoting the proliferation and inflammatory activation of RA-FLS

Methods: Immunohistochemistry was used to detect the expression of IGF2BP3 in RA synovial tissues. Overexpression or silencing of IGF2BP3 was performed to investigate its effects on the biological functions of RA-FLS. m⁶A-RIP-seq and IGF2BP3-RIP-seq techniques were used to screen the targets of IGF2BP3, and functional rescue experiments were conducted to verify the mechanism of IGF2BP3 in regulating RA-FLS. Additionally, an arthritis model was established based on IGF2BP3 knockout mice to further explore the impact of IGF2BP3 knockout on arthritis.

Results: The expression level of IGF2BP3 was significantly increased in RA synovial tissues and co-expressed with Vimentin. Moreover, siIGFBP3 inhibited the proliferation of RA-FLS and promoted autophagy. Furthermore, we found that IGF2BP3 recognized the m⁶A methylation site of RASGRF1 mRNA and regulated its stability, thereby activating mTORC1 to promote the proliferation of synoviocytes and the expression of inflammatory factors. Compared with WT arthritis mice, the synovial inflammation, inflammatory cell infiltration, and cell proliferation in the ankle joints of IGF2BP3^-/- arthritis mice were significantly reduced.

Part 3: The mechanism of IGF2BP3 promoting the polarization of macrophages to type M1

Methods: Immunofluorescence was used to detect the relationship between IGF2BP3 expression and the proportion of M1 macrophages in RA synovial tissues. Overexpression or silencing of IGF2BP3 was performed to investigate its regulatory effect on the inflammatory activation of macrophages. RIP-qPCR was used to detect the binding ability of IGF2BP3 to RASGRF1 mRNA in macrophages. Subsequently, functional rescue experiments were conducted to further explore the mechanism of IGF2BP3 in regulating the polarization of macrophages to type M1 and ROS generation. Finally, in animal experiments, the impact of IGF2BP3 knockout on the inflammatory state of arthritis mice was investigated.

Results: The expression level of IGF2BP3 was positively correlated with the proportion of M1 macrophages. Additionally, siIGF2BP3 significantly reduced the proportion of M1 macrophages and ROS generation. In macrophages, IGF2BP3 also regulated the stability of RASGRF1 mRNA in an m⁶A-dependent manner, thereby activating the mTORC1-autophagy axis to promote the polarization of macrophages to type M1. Moreover, compared with WT arthritis mice, the proportion of M1 macrophages in the spleen of IGF2BP3^-/- arthritis mice was decreased, and the expression of RASGRF1 and iNOS in the synovium was reduced.

Part IV: The mechanism of Celastrol (CEL) targeting IGF2BP3 to alleviate the inflammatory progression of RA

Methods: Small molecule compounds binding to IGF2BP3 were identified through molecular docking, molecular dynamics simulation, and SPR analysis. Then, molecular biology experiments were conducted to detect the regulation of CEL on the biological functions of RA-FLS and macrophages. The molecular mechanism of CEL targeting IGF2BP3 to inhibit RA-FLS proliferation and macrophage inflammatory activation was explored through functional rescue experiments. Finally, the role of IGF2BP3 in CEL alleviating the progression of arthritis was analyzed based on the arthritis model constructed in IGF2BP3 knockout mice.

Results: CEL stably binds to IGF2BP3 and reduces its expression. Mechanistically, CEL inhibits IGF2BP3 expression, thereby weakening RASGRF1-mediated mTORC1 activation, and subsequently inhibiting RA-FLS proliferation and macrophage inflammatory activation. Additionally, compared with WT arthritis mice, IGF2BP3^-/- arthritis mice showed significant improvement in joint swelling, synovial inflammation, and bone destruction; however, CEL did not show further alleviation effects in IGF2BP3^-/- arthritis mice.

Conclusions: The m⁶A reader protein IGF2BP3 is a promising biomarker for RA diagnosis and is closely related to RA-FLS proliferation and the polarization of macrophages to type M1. Mechanistically, IGF2BP3 activates the mTORC1 signaling pathway by enhancing RASGRF1 mRNA stability, to accelerate S6K and ULK1 phosphorylation, thereby promoting RA-FLS proliferation and inhibiting apoptosis and autophagy. In addition, IGF2BP3 also promotes ROS generation and the polarization of macrophages to type M1 through the RASGRF1-mediated mTORC1-autophagy axis. More importantly, CEL alleviates joint damage in arthritis mice by inhibiting the IGF2BP3/RASGRF1/mTORC1 signal pathway to reduce cell proliferation and inflammatory activation in the joint microenvironment.