目的 通过生物信息学分析及动物实验探讨益气活血祛风除湿方（YHQCD）治疗自身免疫性葡萄膜炎 （AU）的作用机制。方法 （1）从 GEO 数据库下载 GSE66936 数据集，筛选健康者与 AU 患者的显著差异表达基 因（DEGs），通过 Metascape 数据库对显著 DEGs 进行基因本体论（GO）功能及京都基因与基因组百科全书 （KEGG）通路富集分析。（2）采用皮下注射含有光感受器间维生素 A 结合蛋白（IRBP）的乳糜剂诱导实验性 AU 模 型。将 60 只 Lewis 大鼠随机分为正常组、模型组、地塞米松（DXM，5 mg·kg⁻¹）组及 YHQCD 低（8.20 g·kg⁻¹）、 高（16.40 g·kg⁻¹）剂量组，每组 12 只。各给药组大鼠灌胃相应浓度的药物，正常组与模型组大鼠均给予等量生 理盐水灌胃，连续灌胃给药 12 d。采用裂隙灯显微镜及 HE 染色法检测大鼠眼前节炎症及眼组织病理学改变； ELISA 法检测血清炎症因子（IL-10、IL-17、TNF-α、IL-1β）水平；免疫荧光法和 Western Blot 法检测 PI3K/ AKT 信号通路相关蛋白及 T 细胞标志物视黄酸相关孤儿受体 γ（t RORγt）和叉头状转录因子 P3（FOXP3）蛋白表 达水平。结果 （1）生物信息学结果显示，共筛选出健康者与 AU 患者显著 DEGs 126 个，其中差异倍数最大的 10 个基因分别是 PHLDA1、GIMAP5、CST7、TNF、IKK、IFIT3、CDC27、ANXA1、EIF4E、EMP1；显著 DEGs 主要富集于炎症反应、T 细胞分化、PI3K/AKT 等信号通路。（2）正常组大鼠虹膜血管无扩张、水肿及炎症 细胞浸润，视网膜结构完整。与正常组比较，模型组大鼠重度虹膜粘连，虹膜有明显的扩张和充血，睫状体肿 胀，视网膜结构紊乱及出现大量炎性细胞浸润，前房有大量纤维蛋白渗出，瞳孔缩小。与模型组比较，各给药 组大鼠炎性细胞浸润、血管舒张、虹膜血管充血和水肿程度减轻，视网膜组织结构较完整，眼部炎症减少。 （3）与正常组比较，模型组大鼠眼前节临床评分、组织病理评分及血清中 TNF-α、IL-17 和 IL-1β 水平升高（P＜ 0.01），IL-10 水平降低（P＜0.01）。与模型组比较，各给药组大鼠眼前节临床评分、组织病理评分及血清中 TNF-α、IL-17 和 IL-1β 水平均降低（P＜0.01），IL-10 水平显著增加（P＜0.01）。（4）与正常组比较，模型组大 鼠眼组织中磷酸化蛋白（p-PI3K、p-AKT、p-mTOR）、RORγt 荧光强度增强及 p-PI3K/PI3K、p-AKT/AKT、 p-mTOR/mTOR、RORγt 蛋白表达水平均明显升高（P＜0.01），FOXP3 荧光强度减弱及其蛋白表达水平降低 （P＜0.01）；与模型组相比，各给药组大鼠眼组织中 p-PI3K、p-AKT、p-mTOR、RORγt 荧光强度减弱及 p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR、RORγt 蛋白表达水平均明显降低（P＜0.01），FOXP3 荧光强度增 强及蛋白表达水平升高（P＜0.01）。结论 YHQCD 具有较好的治疗 AU 的作用，其机制可能与抑制 PI3K/AKT 信号通路激活，减少炎性因子分泌，恢复 Th17/Treg 细胞平衡相关。

Objective To investigate the therapeutic mechanism of Yiqi Huoxue Qufeng Chushi Formula （YHQCD） in treating autoimmune uveitis （AU） through bioinformatics analysis and animal experiments. Methods （1） The GSE66936 dataset was downloaded from the GEO database to screen for significantly differentially expressed genes （DEGs） between healthy individuals and AU patients. Gene Ontology （GO） function and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses of the significant DEGs were performed using the Metascape database. （2）An experimental AU model was induced by subcutaneous injection of an emulsion containing interphotoreceptor retinoid-binding protein （IRBP）. Sixty Lewis rats were randomly divided into five groups （n=12 per group）：normal group，model group，dexamethasone （DXM，5 mg·kg-1 ） group，and low-dose （8.20 g·kg-1 ） and high-dose （16.40 g·kg-1 ） YHQCD groups. Rats in each treatment group received corresponding concentrations of the formula by gavage， while the normal and model groups received an equal volume of normal saline， once daily for 12 consecutive days. Slit-lamp microscopy and hematoxylin-eosin （HE） staining were used to assess anterior segment inflammation and histopathological changes in ocular tissues. ELISA was employed to detect the expression levels of serum inflammatory cytokines IL-10，IL-17，TNF-α，and IL-1β. Immunofluorescence and Western blot were used to detect the protein expression levels of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （AKT） signaling pathwayrelated molecules， as well as T cell markers retinoic acid-related orphan receptor γt （RORγt） and forkhead box transcription factor P3 （FOXP3）. Results （1） Bioinformatics analysis identified 126 significant DEGs between healthy individuals and AU patients. The top 10 genes with the largest fold changes were PHLDA1，GIMAP5，CST7， TNF， IKK， IFIT3， CDC27， ANXA1， EIF4E， and EMP1. These significant DEGs were primarily enriched in pathways related to inflammatory response，T cell differentiation，and the PI3K/AKT signaling pathway.（2） Rats in the normal group showed no iris vascular dilation， edema， or inflammatory cell infiltration， with intact retinal structure. Compared with the normal group，model group rats exhibited severe iris adhesion，marked iris dilation and hyperemia， ciliary body swelling， retinal structural disorganization with extensive inflammatory cell infiltration， significant fibrinous exudation in the anterior chamber， and miosis. Compared with the model group， rats in all treatment groups showed reduced inflammatory cell infiltration， vasodilation， iris vascular hyperemia and edema， relatively intact retinal tissue structure，and decreased ocular inflammation.（3） Compared with the normal group，the model group showed significantly increased clinical scores for the anterior segment， histopathological scores， and serum levels of TNF-α，IL-17，and IL-1β（P＜0.01），while serum IL-10 levels were significantly decreased （P＜ 0.01）. Compared with the model group， all treatment groups exhibited significantly decreased clinical scores， histopathological scores， and serum levels of TNF- α， IL-17， and IL-1β（P＜0.01）， along with significantly increased serum IL-10 levels （P＜0.01）.（4） Compared with the normal group，the model group showed significantly increased fluorescence intensity of phosphorylated proteins （p-PI3K， p-AKT， p-mTOR） and RORγt， as well as elevated protein expression levels of p-PI3K/PI3K， p-AKT/AKT， p-mTOR/mTOR， and RORγt in ocular tissues （P＜0.01），while FOXP3 fluorescence intensity and protein expression levels were significantly decreased （P＜0.01）. Compared with the model group， all treatment groups demonstrated significantly reduced fluorescence intensity of p-PI3K， p-AKT， p-mTOR， and RORγt， decreased protein expression levels of p-PI3K/PI3K， p-AKT/AKT，p-mTOR/mTOR，and RORγt （P＜0.01），along with increased FOXP3 fluorescence intensity and protein expression levels （P＜0.01）. Conclusion YHQCD demonstrates a significant therapeutic effect on AU，and its mechanism may be associated with inhibiting the activation of the PI3K/AKT signaling pathway， reducing the secretion of inflammatory cytokines，and restoring the Th17/Treg cell balance.

R285.5

国家自然科学基金项目（82374429）；刘良院士工作站指导项目（22YS003）；湖南省重点研发计划项目（2024JK2122）；湖南省卫生 健康高层次人才项目（20240304116）；中华中医药学会青年人才托举工程项目（2024-QNRC2-B25）。