目的：

糖尿病视网膜病变（Diabetic retinopathy，DR）是糖尿病常见微血管并发症之一，其早期病理特征表现为Müller细胞异常活化引发的视网膜神经血管损伤。过氧化物还原酶2（Peroxiredoxin 2，PRDX2）作为硫氧还蛋白家族成员，除维持氧化还原稳态外，还参与调控细胞增殖分化、血管重塑及炎症反应等生物过程，并在糖尿病肾病中展现出促炎作用。然而，PRDX2与DR的关联及其在Müller细胞胶质化中的具体作用尚待明确。本研究旨在探讨PRDX2与DR的相关性，并解析其在高葡萄糖诱导Müller细胞胶质化中的作用及潜在机制，进一步深化对DR致病机制的理解。

方法：

本研究包括临床-生信-基础实验，分两部分展开。第一部分，通过临床样本和生物信息学分析，探究PRDX2在DR患者血浆、玻璃体液和视网膜组织中的表达情况及其与DR的相关性。①建立病例对照队列（n=220），纳入173例2型糖尿病患者（无DR 51例、非增殖性DR 44例、增殖性DR 78例）及47例健康对照，采用ELISA检测血浆PRDX2及VEGFA水平，通过受试者工作特征曲线（ROC）及多因素logistic回归评估其诊断效能；②基于GEO（GSE53257）与ProteomeXchange（PXD036033）数据集，分析DR患者视网膜组织及玻璃体液中PRDX2表达情况；③采用ELISA验证性检测临床玻璃体样本（PDR患者9例 vs 非糖尿病患者6例）中PRDX2的表达水平。

第二部分通过体外细胞实验，探讨PRDX2在高葡萄糖诱导的Müller细胞胶质化中的作用及其调控机制。①基于人类蛋白质图谱（HPA）单细胞测序数据，分析PRDX2在视网膜细胞亚群的表达特征；②采用25 mM高葡萄糖诱导大鼠源Müller细胞（RMC-1）构建体外胶质化模型，并通过shRNA转染建立PRDX2稳定敲低细胞；③采用CCK8分析、Annexin V/PI凋亡检测、EdU增殖荧光染色、CyQUANT增殖分析、划痕-Transwell迁移实验，以及ATP及乳酸含量测定，全面评估细胞功能表型；④采用实时QPCR技术检测细胞外基质（COL3A1、SPP1、Integrin α5、Postn、DPT、FN1）、纤维化（TGF-β3/Smad3）、炎症（VEGFA、ICAM-1）和氧化应激（ALDH3A1、TXNIP、Trx1）相关基因的表达水平；⑤采用串联质谱同位素标记（TMT）定量蛋白质组学技术，筛选PRDX2基因敲低后RMC-1细胞中差异表达蛋白，构建PRDX2调控网络；⑥采用Western blot验证RhoA、ROCK1和RhoGDI1蛋白的表达变化。

结果：

血浆PRDX2与DR进展相关：血浆PRDX2水平随DR分期显著升高（Normal组 vs. NDR组 vs. NPDR组 vs. PDR组：128.6 pg/mL（IQR，46.27-228.5）vs 180.8 pg/mL [IQR，69.9-236.4] vs. 103.4 pg/mL [IQR，33.57-232.7] vs. 225.6 pg/mL [IQR，164.7-322.5]，P＜0.0001），与DR严重程度（r=0.267，P＜0.0001）和血浆VEGFA正相关（r=0.356，P＜0.0001）。多因素校正后，糖尿病患者高水平血浆PRDX2（＞192.0 pg/mL）显著增加NPDR（OR，2.379 [1.033-5.483]，P=0.042）和PDR（OR，3.423 [1.458-8.037]，P=0.005）的风险。血浆PRDX2对PDR的诊断效能（AUC=0.7376）显著优于VEGFA（AUC=0.6645），其截断值124.3 pg/mL时灵敏度达87.2%，特异性为59.09%；在调整年龄、高血压、VEGFA、FBG、Alb、A/G、GGT、SCr和Urea变量后，高水平血浆PRDX2（＞124.3 pg/mL）使PDR风险激增9.097倍（95%CI，[2.819-29.355]，P=0.0002）。

PRDX2在DR视网膜微环境中特异性高表达：DR患者视网膜组织（GSE53257，P＜0.05）和玻璃体液（PXD036033，P＜0.0001；临床样本验证，PDR组中位数126.8 pg/mL vs Non-diabetic组中位数97.79 pg/mL，P=0.026）中PRDX2表达较非糖尿病患者显著上调。

PRDX2在视网膜Müller细胞中高表达：人类视网膜单细胞测序生信分析显示PRDX2基因在Müller细胞中高表达（平均表达丰度451.4 nTPM），仅次于视锥细胞（平均表达丰度574.3 nTPM）。

高糖诱导Müller细胞胶质化并上调PRDX2表达：高葡萄糖（25 mM）诱导RMC-1细胞活力增加，凋亡减少，增殖加快，迁移增强，乳酸含量增加，促进ECM重塑（COL3A1、SPP1、Integrin α5、Postn、TGF-β3、Smad3基因表达上调）、纤维化（TGF-β3、Smad3基因表达上调）、炎症和氧化应激（ICAM-1、ALDH3A1 、TXNIP基因表达上调），同时显著性上调PRDX2基因/蛋白表达（P＜0.05）。

PRDX2基因敲低抑制高糖诱导的Müller细胞胶质化：敲低PRDX2显著性降低高糖环境下RMC-1细胞活力、增殖率及存活率（P＜0.05），使高糖诱导细胞表型恢复至近生理状态。

PRDX2-RhoGDI1-RhoA/ROCK信号轴机制：TMT标记定量蛋白组学分析联合WB验证显示，高糖刺激上调RMC-1细胞PRDX2、RhoA和ROCK1蛋白表达；而PRDX2基因敲低抑制巢蛋白（Nestin）、RhoA和ROCK1表达，并使RhoGDI1（RhoA GTP酶负调控因子）表达量提升2倍以上（P＜0.05）。

结论：

本研究首次探究了PRDX2与DR的潜在关联及其在调控Müller细胞功能与存活中的关键作用，结果发现，DR患者血浆、视网膜组织及玻璃体液中PRDX2表达较非糖尿病人群显著升高，且血浆PRDX2水平与DR严重程度呈正相关，高水平PRDX2是DR进展的重要风险因子。机制层面，PRDX2通过RhoGDI1-RhoA/ROCK信号轴驱动Müller细胞胶质化。高糖诱导可显著促进Müller细胞内PRDX2表达，激活RhoA/ROCK1信号，而敲低PRDX2基因则能上调RhoGDI1，从而抑制高糖诱导的RhoA/ROCK1信号活化，有效缓解Müller细胞反应性胶质样变。本研究拓宽了PRDX2在DR领域的功能机制空白，并为开发靶向神经胶质保护的DR精准诊疗提供了新靶点和理论依据。

Objective:

Diabetic retinopathy (DR), a principal microvascular complication of diabetes mellitus, is characterized by early-stage pathological alterations mediated through Müller cell gliosis and consequent neurovascular impairment. As a pivotal thioredoxin peroxidase, PRDX2 serves dual functions in redox homeostasis maintenance and modulation of critical cellular processes including proliferation dynamics, vascular architecture remodeling, and inflammatory signaling cascades. Paradoxically, while PRDX2 has demonstrated pro-inflammatory regulatory roles in diabetic nephropathy, its pathophysiological involvement in DR pathogenesis - particularly its mechanistic influence on Müller cell gliosis under hyperglycemic conditions - remains elusive. This study aims to: (1) elucidate the spatiotemporal expression profile of PRDX2 in DR progression, and (2) investigate its regulatory mechanisms in high glucose-induced Müller cell activation through integrated molecular and functional analyses. Our findings provide novel insights into the regulatory nature of PRDX2 in diabetic microvascular complications and advance the understanding of redox-mediated mechanisms underlying DR development.

Methods:

This study integrates two complementary approaches: clinical-bioinformatics analysis and in vitro cellular experiments. (1) Clinical-Bioinformatics Analysis: We established a case-control cohort (n=220) comprising 173 type 2 diabetic patients (51 non-DR, 44 NPDR, 78 PDR) and 47 healthy controls. Plasma PRDX2 and VEGFA levels were quantified using ELISA, with diagnostic efficacy assessed through ROC curves and multivariate logistic regression. We analyzed GEO (GSE53257) and ProteomeXchange (PXD036033) datasets to evaluate PRDX2 expression in DR retinal tissues and vitreous humor. Clinical validation was conducted via ELISA on vitreous samples (9 PDR vs. 6 non-diabetic controls). (2) In Vitro Studies: Single-cell RNA sequencing data from the Human Protein Atlas (HPA) were utilized to determine PRDX2 expression in retinal cell subpopulations. A high glucose (25 mM)-induced Müller cell (RMC-1) gliosis model was developed. PRDX2-knockdown cells were created through shRNA transfection. Functional assays included CCK-8 viability, Annexin V/PI apoptosis, EdU proliferation, CyQUANT proliferation, scratch-Transwell migration, and ATP/lactate quantification. QPCR analyzed gene expression related to extracellular matrix (COL3A1, SPP1, Integrin α5, Postn, DPT, FN1), fibrosis (TGF-β3/Smad3), inflammation (VEGFA, ICAM-1), and oxidative stress (ALDH3A1, TXNIP, Trx1). Tandem Mass Tag (TMT)-based proteomics identified differentially expressed proteins in PRDX2-knockdown cells. Western blot validated RhoA, ROCK1, and RhoGDI1 protein levels.

Results:

Plasma PRDX2 Correlates with DR Progression: Plasma PRDX2 levels increased significantly with DR severity (P<0.0001), showing strong positive correlations with DR staging (r=0.267) and plasma VEGFA (r=0.356) (both P<0.0001). Multivariate analysis revealed high PRDX2 (>192.0 pg/mL) as an independent risk factor for NPDR (OR=2.38, 95% CI: 1.03–5.48) and PDR (OR=3.42, 1.46–8.04). Notably, PRDX2 outperformed VEGFA in diagnostic accuracy for PDR (AUC=0.758 vs. 0.665), with an optimal cutoff of 124.3 pg/mL achieving 87.2% sensitivity and 59.09% specificity. Adjusted PRDX2 levels (>124.3 pg/mL) elevated PDR risk by 9.097-fold (95% CI: 2.82–29.36, P=0.0002).

PRDX2 Overexpression in DR Microenvironment: PRDX2 was significantly upregulated in DR retinal tissues (GSE53257, P<0.05) and vitreous humor (PXD036033, P<0.0001; clinical validation: PDR median=126.8 pg/mL vs. Non-diabetic=97.8 pg/mL, P=0.026).

Müller Cell-Specific PRDX2 Expression: Single-cell analysis showed PRDX2 highly expressed in Müller cells (451.4 nTPM), second only to cone photoreceptors (574.3 nTPM).

High Glucose Induces Gliosis and PRDX2 Upregulation: High glucose (25 mM) enhanced RMC-1 viability, proliferation, migration, and lactate production, while upregulating ECM remodeling (COL3A1, SPP1, Integrin α5, Postn), fibrosis (TGF-β3/Smad3), inflammation (ICAM-1), oxidative stress (ALDH3A1, TXNIP), and PRDX2 expression (P<0.05).

PRDX2 Knockdown Attenuates Gliosis: PRDX2 knockdown significantly reduced cell viability, proliferation rate, and survival rate under high glucose conditions (P < 0.05), restoring the high glucose-induced phenotype of RMC-1 cells to near-physiological states.

PRDX2-RhoGDI1-RhoA/ROCK Axis: Proteomics and Western blot confirmed that PRDX2 knockdown upregulated RhoGDI1 (>2-fold) while downregulating Nestin, RhoA, and ROCK1 (P<0.05), thereby inhibiting RhoA/ROCK signaling.

Conclusion:

This study establishes the previously unrecognized association between PRDX2 and DR, revealing its critical regulatory role in Müller cell homeostasis. Our findings demonstrate that elevated PRDX2 levels in plasma, retinal tissue, and vitreous humor exhibit a strong correlation with DR progression, positioning it as both a promising diagnostic biomarker and therapeutic target. Mechanistically, we identified a PRDX2-driven RhoGDI1-RhoA/ROCK signaling axis orchestrating Müller cell gliosis. High-glucose exposure induced PRDX2 overexpression and subsequent RhoA/ROCK1 activation, whereas PRDX2 knockdown promoted RhoGDI1 expression and suppressed hyperglycemia-induced RhoA/ROCK1 signaling, effectively attenuating reactive Müller cell gliosis. This mechanistic delineation bridges a fundamental knowledge gap in diabetic neurogliopathy, providing a potential biomarker for Müller cell-targeted precision interventions in DR therapeutics.