背景及目的:IgG4 相关性疾病(IgG4-Related Disease，IgG4-RD)是一种免疫 介导的纤维炎性疾病，常表现为血清中 IgG4 水平增高，受累组织中大量淋巴细胞、 浆细胞浸润及特异性纤维化(闭塞性脉管炎、席纹状纤维化)。早期生长应答基 因 1(Early Growth Response 1，EGR1)作为一种重要的转录因子，已被证实在多 种炎症及纤维化疾病中异常表达且发挥重要的致病作用，但其在 IgG4-RD 中所扮 演的角色仍不清楚。为明确这一问题，我们希望通过分析 IgG4-RD 患者颌下腺组 织(Submandibular Glands，SMGs)的单细胞转录组测序(single-cell RNA sequencing，scRNA-seq)结果中的 EGR1 表达情况以及构建 EGR1 异常表达的上皮 细胞模型，揭示 EGR1 在 IgG4-RD 导管上皮细胞中的异常表达情况及相关的致病 机制。

研究方法:本研究通过挖掘 IgG4-RD 患者组织 scRNA-seq 数据，探究 IgG4- RD 患者及对照组患者 SMGs 中各类细胞 EGR1 的表达情况。通过免疫印迹法 (Western Blot，WB)、免疫组化(Immunohistochemistry，IHC)、免疫荧光 (Immunofluorescence，IF)共定位的方法进行验证。并根据 IHC 结果与相对应患 者的临床特征及 Masson 染色结果进行相关性分析，进一步探究 IgG4-RD 患者 SMGs 中 EGR1 表达水平与疾病发生发展的关系。此外，为验证上皮细胞与免疫细 胞的作用关系，通过密度梯度离心法及磁珠分选收集 IgG4-RD 患者及健康对照者 (Healthy Control，HC)的各类免疫细胞，与人颌下腺鳞状上皮癌细胞(A253) 或人胰腺导管上皮细胞(hTERT-HPNE)共培养，通过 WB 检测各组上皮细胞中 EGR1 的表达情况，并通过 IF 进行验证，初步探索 IgG4-RD 患者上皮细胞 EGR1 异 常表达的免疫原因。

通过慢病毒转染过表达质粒或 siRNA 干扰片段构建 EGR1 过表达(EGR1- Overexpression，OE-EGR1)或 EGR1 敲低(EGR1-siRNA，si-EGR1)的颌下腺及 胰腺导管上皮细胞模型，流式细胞术(Flow cytometry，FCM)、WB、PCR 验证 各组细胞模型构建效果，采用 Transwell、细胞划痕、CCK8、克隆形成实验检测上 皮细胞模型的功能变化，并通过与胰腺成纤维细胞(Pancreatic stellate cell, PSCs Cat no.CM-H024)或 IgG4-RD 患者颌下腺成纤维细胞/对照组患者成纤维细胞共培 养，采用细胞划痕、CCK8 检测成纤维细胞功能改变及 PCR 检测胶原水平差异，证 实 EGR1 差异表达对上皮细胞功能及对成纤维细胞纤维化作用的影响。最后，将 OE-EGR1 及 si-EGR1 上皮细胞进行代谢组测序及全转录组测序分析，利用通路富 集分析、差异基因检测等生信分析探究上皮细胞中 EGR1 的调控通路及作用靶点。

研究结果:scRNA-seq 结果显示:与对照组患者(n=3)相比，IgG4-RD 患者 (n=3)的颌下腺上皮细胞(Submandibular Gland Epithelial Cells，SMGECs)中 EGR1 表达明显升高;IHC 染色结果显示: IgG4-RD 患者导管上皮细胞中 EGR1 阳 性面积比率显著升高;IF 结果显示:IgG4-RD 患者的 CD326 为标记的上皮细胞与 EGR1 共定位明显;IgG4-RD 患者 EGR1 阳性率与患者 IgG4 基线水平及 SMG 组织 纤维化程度呈正相关;SMGs 上皮细胞转录组学发现 IgG4-RD 患者上皮细胞中 EGR1 mRNA 水平明显升高。IgG4-RD 患者及 HC 的各免疫细胞与 A253/ hTERT- HPNE 细胞共培养结果显示:CD14+单核细胞能显著增加上皮细胞中 EGR1 表达水 平;SMGs 免疫荧光结果显示:IgG4-RD 患者的 M2 巨噬细胞与 EGR1 在上皮细胞 处共定位;另外，IgG4-RD 患者血清与 hTERT-HPNE 共培养也能显著刺激其高表 达 EGR1。

Transwell 及细胞划痕的结果表明:上皮细胞高表达 EGR1 后细胞迁移能力会 显著增强;CCK8 及克隆形成实验结果表明:上皮细胞高表达 EGR1 后细胞增殖能 力将明显减弱。上皮细胞与成纤维细胞共培养后，细胞划痕结果表明:上皮细胞 高表达 EGR1 后能显著增强成纤维细胞的迁移能力;CCK8 结果表明:上皮细胞高 表达 EGR1 后能显著增强成纤维细胞的增殖能力;PCR 结果进一步验证:a- SMA、 Vimentin、COlI，COlIV 等胶原相关基因的 mRNA 水平在 OE-EGR1 组共培养的成 纤维细胞中明显升高。

代谢组学结果证明，较 si-EGR1 组，OE-EGR1 中促炎、凋亡、纤维化、衰老 相关的代谢物明显上调。转录组学通路富集分析表明，OE-EGR1 中差异表达的基 因主要富集在与角化细胞和表皮细胞分化相关的通路，以及皮肤和表皮发育相关 的通路中。

结论:IgG4-RD 患者 SMGECs 高表达 EGR1，促进上皮细胞炎症、凋亡、EMT 过程发生以及成纤维细胞的增殖及活化， 主动参 与 IgG4-RD 有望为后续的研究及治疗 提供新的思路及治疗靶点。

Background and Purpose: IgG4-Related Disease (IgG4-RD) is an immunologically mediated fibroinflammatory disorder characterized by increased serum IgG4 levels, infiltration of numerous lymphocytes and plasma cells in affected tissues, and specific fibrosis, such as storiform fibrosis and obliterans phlebitis. Early Growth Response 1 (EGR1), as an important transcription factor, has been shown to be abnormally expressed and play a significant pathogenic role in various inflammatory and fibrotic diseases, but its role in IgG4-RD remains unclear. To clarify this issue, we aim to analyze the expression of EGR1 in the single-cell sequencing (scRNA-seq) results of submandibular gland (SMGs) tissues from IgG4-RD patients，and construct epithelial cells with abnormal EGR1 expression to reveal the abnormal expression of EGR1 in ductal epithelial cells of IgG4- RD and its related pathogenic mechanisms.

Methods: This study explores the expression of EGR1 in various cells of the SMGs from IgG4-RD patients and control patients through scRNA-seq data. Validation was performed using Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) co-localization methods. Subsequently, we analyzed the correlation between abnormal EGR1 expression in the SMGs of IgG4-RD patients and the progression of disease based on the IHC results, corresponding clinical inflammation indicators of the patients, and Masson staining results. Immune cells from IgG4-RD patients and healthy controls (HC) were collected using density gradient centrifugation and magnetic bead sorting，and co-cultured with human submandibular gland carcinoma cells (A253) or human pancreatic ductal epithelial cells (hTERT-HPNE). WB was used to detect EGR1 expression and validate by IF, which to preliminarily explore the immune causes of abnormal EGR1 expression in epithelial cells of IgG4-RD patients.

EGR1 overexpression (OE-EGR1) or EGR1 knockdown (EGR1-siRNA, si-EGR1) models of submandibular gland and pancreatic ductal epithelial cells were constructed using lentiviral transfection. Flow cytometry, WB, and PCR were used to verify the construction effects of each cell model. Functional changes in epithelial cell models were tested using Transwell, wound healing assay, CCK8, and colony formation experiments. The effects of EGR1 abnormal expression on epithelial cell function and the fibrotic action of fibroblasts were confirmed by co-culturing with pancreatic stellate cells (PSCs Catno.CM-H024) or fibroblasts from the SMGs of IgG4-RD patients/control group patients, using wound healing assay, CCK8, and PCR to detect changes in fibroblast function and collagen levels.

Whole transcriptome sequencing and metabolomics analysis were performed on OE- EGR1group and si-EGR1 group, using pathway enrichment analysis, differential gene detection and bioinformatics analyses to explore the regulatory pathways and target genes of EGR1 in epithelial cells.

Results: The scRNA-seq results showed that compared with control group patients (n=3), EGR1 expression in the epithelial cells of the submandibular glands (SMGECs) from IgG4-RD patients (n=3) was significantly increased. IHC results showed that the positive area ratio of EGR1 in the ductal epithelial cells of IgG4-RD patients was significantly increased. IF results showed that co-localization of EGR1 with CD326- marked ductal epithelial cells was significantly increased in IgG4-RD patients. The positive rate of EGR1 in IgG4-RD patients was positively correlated with IgG4 basic level and the degree of fibrosis in the SMGs. Transcriptomics of SMGECs revealed a significant increase in EGR1 mRNA levels in IgG4-RD patients. The results of co-culture of various immune cells from IgG4-RD patients and HC with A253/hTERT-HPNE cells showed that CD14+ monocytes could significantly increase EGR1 expression levels in epithelial cells. IF results of SMGs tissue from IgG4-RD patients showed that M2 macrophages in IgG4- RD patients co-localized with EGR1 in epithelial cells. Additionally, serum from IgG4- RD patients co-cultured with epithelial cells could significantly stimulate hTERT-HPNE to highly express EGR1.

The results of Transwell and wound healing assay indicated that epithelial cells with high EGR1 expression significantly enhanced their migration ability; CCK8 and colony formation experiment results indicated that epithelial cells with high EGR1 expression significantly reduced their proliferation ability. After co-culture with fibroblasts, the wound healing assay results showed that epithelial cells with high EGR1 expression significantly enhanced the migration ability of fibroblasts; CCK8 results indicated that epithelial cells with high EGR1 expression significantly enhanced the proliferation ability of fibroblasts; PCR results further verified that the mRNA levels of collagen-related genes such as Vimentin, a-SMA, COlI, COlIV, were significantly increased in fibroblasts co- cultured with the OE-EGR1 group.

Metabolomic results have demonstrated that compared to the si-EGR1 group, there is a significant upregulation of metabolites associated with pro-inflammatory, apoptotic, fibrotic, and senescence pathways in the OE-EGR1 group. Transcriptomic pathway enrichment analysis indicates that differentially expressed genes in OE-EGR1 are primarily enriched in pathways related to keratinocyte and epidermal cell differentiation, as well as skin development and epidermis development.

Conclusion:Patients with IgG4-RD exhibit high expression of EGR1 in SMGECs, which promotes inflammation, apoptosis, and epithelial-mesenchymal transition processes in epithelial cells, as well as proliferation and activation of fibroblasts. This indicates that abnormal expression of EGR1 actively participates in the inflammatory and fibrotic processes of IgG4-RD disease and cellular damage, potentially providing new insights and therapeutic targets for subsequent research and treatment.