A total of 108 patients with DM and 28 healthy individuals were admitted to the First Hospital of Dalian Medical University. Fasting venous blood was collected from diabetic patients and healthy people, centrifuged at 3.500 r/min for 10 min, and the serum/plasma samples were stored at – 80°C for subsequent assays. The criteria for inclusion were as follows: (1) meeting the diagnostic criteria for diabetic retinopathy of the Chinese Medical Association Diabetes Branch, (2) diagnosis confirmed by fundoscopy, and (3) no history of diabetes mellitus, hypertension, or related ocular diseases in the NC group. The exclusion criteria were as follows: (1) patients who had recently taken hormonal drugs or drugs that impair renal metabolism, (2) patients with a history of comorbid hypertension, (3) patients with a recent history of related ocular diseases, and (4) patients with a recent history of infection.

All experimental steps were performed according to the Institutional Guidelines for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Institutional Animal Care and Use Committee of the Experimental Animal Centre of Dalian Medical University. Male C57BL/6 mice, 6 weeks old, were obtained from the Experimental Animal Centre of Dalian Medical University and randomly divided into control and DM groups. The room temperature was 20 °C–25°C, the humidity was 55%–60%, and the mice were fed ad libitum with a 12-h day–night alternating environment for 1 week to allow adaptation. After this period, the control group was fed a basal diet, and the diabetic model group was fed a high-sugar, high-fat diet for 4 weeks, followed by a 12-h fasting period. The type 1 diabetic model group was injected intraperitoneally with streptozotocin (50 mg/kg) for 5 consecutive days (23), and the control group was injected with an equal volume of sodium citrate buffer. Fasting blood glucose was measured every other week after streptozotocin injection, and successful modeling was defined as a blood glucose level > 16.7 mmol/L along with insulin resistance (25, 26).

The human Müller cell line was provided by the School of Basic Medical Sciences, Sun Yat-sen University. It was cultured in DMEM (Gibco, USA) medium containing 10% fetal bovine serum (FBS; FBS-S500, NEWZERUM, New Zealand), penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37°C in 5% CO₂. The medium was changed every 1 to 2 days. Cells were washed with Phosphate-Buffered Saline (PBS) before the experiment. The soybean glycosides (MCE, HY-N0019) were dissolved in DMSO at 1 mg/mL and stored at 4°C.

The plasmids pIRES2-EGFP-Trx and pIRES2-EGFP-LacZ were stably transfected into Müller cells according to the Lipofectamine 2000 instructions. Müller cell lines stably expressing Trx and Lacz were thus established. The stably transfected cells were identified by quantitative real-time (qPCR) and Western blot following expansion in culture. ASK1 small-interfering RNA (siRNA) from Genepharma, China (sense 5′-GCAUGGUACCUCAAGUCUATT-3′, anti-sense 5′-UAGACUUGAGGUACCAUGCTT-3′) was used to downregulate ASK1 expression in Müller cells.

According to the manufacturer’s instructions, the IL-1β ELISA Kit (R&D, E20211101A) was used to detect the concentration of IL-1β in the serum of patients with diabetes. When the stop solution changed color from blue to yellow, the intensity was measured at 450 nm using a microplate reader. The concentration of IL-1β in the samples was calculated using a standard curve. The cytokines IL-1β, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) from the serum of diabetic mice, patients with diabetes, and the medium of Müller cells were detected by flow cytometry using the kit (Micron Biological Cytokine Detection Kit, 3060030064).

The cells were seeded into 96-well plates (Guangzhou Jet Bio-Filtration Co. Ltd., China) at a density of 5,000 cells per well, and the peripheral wells were filled with sterile 0.1 M PBS. The cells were cultured at 37 °C in a 5% CO₂ incubator and treated under different conditions. Subsequently, 10 μL of CCK8 reagent was added to each well, followed by incubation at 37 °C in 5% CO₂ for 2 h. Absorbance was measured at 450 nm using a microplate reader.

Briefly, proteins were obtained using cell extraction kits supplemented with protease and phosphatase inhibitors (KeyGEN BioTECH, KGP903, China), stored at 4 °C for 15 min, and then centrifuged at 15,000 rpm for 20 min to collect the supernatants. Protein concentrations in each sample were measured using a BCA quantitative kit. Equal amounts of protein from different groups were prepared for SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked for 2 h in a nonfat milk diluted with Tris-Buffered Saline with Tween 20 (TBST) (0.5% Tween-20) at room temperature and then incubated overnight at 4 °C with the following primary antibodies: Trx (1:1,000, 14999-1-AP, Proteintech, China), glial fibrillary acidic protein (GFAP) (1:1,000, 60190-1-Ig, Proteintech), Txnip (1:1,000, 18243-1-AP, Proteintech), caveolin-1 (1:1,000, 16447-1-AP, Proteintech), NLRP3 (1:1,000, WL0263, Wanleibio), cysteinyl aspartate-specific proteinase (caspase-1) (1:1,000, Solarbio, China), apoptosis-associated speck-like protein containing a CARD (ASC) (1:1,000, 10500-1-AP, Proteintech, China) LC3 (1:1,000, 14600-1-AP, Proteintech), P62 (1:1,000, A7758, Abclonal), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (10494-1-AP, Proteintech). The membranes were washed three times with 1 × TBST for 15 min. Subsequently, they were incubated with goat antirabbit IgG (1:2,000, AS014, Abclonal) or goat antimouse IgG (AS003, 1:2,000, Abclonal) for 1 h at room temperature and washed three times with 1 × TBST for 15 min. The membranes were then exposed to X-ray film using an enhanced chemiluminescence system. The intensity of the bands was measured using LabWorks 4.5.

According to the manufacturer’s instructions, total RNA was extracted from the cells after different treatments by using TRIzol (Takara, Japan), and cDNA was synthesized using the TransScript^® Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit. Real-time PCR was performed using the PerfectStart^® Uni RT&qPCR Kit. The conditions for cDNA synthesis from RNA were as follows: 37 °C for 15 min, 85 °C for 5 s, and 4°C indefinitely. GAPDH was used as the internal reference for PCR amplification. The primer sequences were used as follows: Trx Forward: 5′-GTAGTTGACTTCTCAGCCACGTG-3′ and Reverse: 5′-CTGACAGTCATCCACATCTACTTC-3′; Cav-1 Forward: 5′-GACAGGGCGTTAAAAGCAGG-3′ and Reverse: 5′-TAGCAGTTCCAAGCACCAGT-3′); and GAPDH Forward: 5′-GTCTCCTCTGACTTCAACAGCG-3′ and Reverse: 5′-ACCACCCTGTTGCTGTAGCCAA-3′. The qRT-PCR amplification conditions were as follows: 94 °C for 30 s, 94 °C for 5 s, 55 °C for 15 s, 72 °C for 10 s for 40 cycles, 95 °C for 60 s, 55 °C for 30 s, and 95 °C for 30 s. The data were analyzed using the 2(–Delta Delta C(T)) method.

The cells were infected with a tandem fluorescent monomeric Red Fluorescent Protein–Green Fluorescent Protein (mRFP-GFP)-tagged LC3 adenovirus (Beyotime, C3011, China). GFP and mCherry expression was observed using Keyence microscopy. Yellow spots (representing the merged GFP signal and RFP signals) indicate autophagosomes, while red spots (RFP signal alone) indicate autolysosomes. Autophagic flux was evaluated based on the GFP/mRFP color change.

The cells were treated with different conditions, collected, and fixed at 4 °C with glutaraldehyde, followed by fixation in 1% osmium tetroxide for 1 h. The samples underwent gradient ethanol dehydration, and the cells were embedded. Ultrathin sections were prepared and observed under a transmission electron microscope (JEM-2000EX*).

The cells were seeded and grown on glass coverslips and treated as indicated. After fixation with 4% paraformaldehyde for 30 min at room temperature, staining was performed, followed by three washes with PBS for 3 min each. Cells were permeabilized with 0.25% TritonX-100 for 5 min at room temperature and washed three times with PBS for 3 min each. Focal death was then detected using a one-step TdT-mediated dUTP nick-end labeling (TUNEL) kit (KGA7071) according to the manufacturer’s instructions. The number of positive cells was quantified under a microscope (Leica, Germany).

The eyeballs were fixed in Bouin’s fixative for 24 h, dehydrated through alcohol, and paraffin sections of the whole retina, including the optic disc, were prepared at a thickness of 5 μm and stained with hematoxylin–eosin (H&E). The total nuclear layer thickness of the retina was measured from the retinal pigment epithelium layer to the ganglion cell layer using Nikon software.

Paraffin sections were melted in an oven at 60 °C, then dewaxed and hydrated using xylene and alcohol. Antigen retrieval was performed by adding 0.1% trypsin dropwise at 37 °C. Sections were blocked with goat serum at room temperature for 30 min, followed by incubation with the GFAP antibody (1:200, 60190-1-Ig, Proteintech) diluted in PBS at 37 °C for 2 h. The fluorescein isothiocyanate-labeled secondary antibody was diluted 1:1,000 and incubated at room temperature for 1 h. Finally, the sections were mounted with an antifluorescence quenching solution and observed under a fluorescence microscope to capture pictures.

In this study, data were obtained from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/gds/). The single-cell RNA sequencing (scRNA-Seq) dataset GSE205123 was identified by searching study-related keywords such as “Müller cell”, “single-cell analysis”, “retina”, “diabetes”, etc. The data were divided into two groups: wild‐type mice (WT: BSK_WT; m/m) and the diabetic group (DB: BSK_DB; db/db). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed on the selected target genes (data source: https://sangerbox.com). Gene screening criteria were p_val_adj < 0.05, pct.1 > 0.5. Protein–Protein Internet (PPI) networks were drawn using the STRING database (data source: https://cn.string-db.org/).

The data are presented as the mean ± SD. The statistically significant differences between the two groups were analyzed using Student’s t-test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). All analyses were performed using GraphPad Prism software (Ver.8.0.2). A p-value < 0.05 was considered statistically significant (α = 0.05, Power = 0.8, Effect size = d = 0.5).

To investigate whether diabetes-induced inflammation produced by retinal Müller cells is related to glial cell activation, we examined inflammatory factors in human samples, mouse samples, and Müller cells, as well as the expression of GFAP in mouse and Müller cells, using ELISA and flow cytometry. As shown in Figures 1A–C, the levels of inflammatory factors TNF-α, IFN-γ, and IL-1β in the serum of diabetic patients and diabetic mice were significantly increased compared with the control group. In addition, the levels of TNF-α, IFN-γ, and IL-1β in Müller cells and their culture medium in vitro were also detected, with results shown in Figures 1J, K. Moreover, morphologic analysis of the retina using H&E staining revealed that total retinal thickness was reduced in diabetic mice compared with the control group (Figures 1D, E). Observation of GFAP expression in the retina using immunohistofluorescence staining indicated that GFAP expression was elevated in vivo and in vitro; its expression increased in the DM group compared with the control group (Figures 1F–I). These results suggest that Müller cells in a high-glucose environment lead to increased inflammatory factors and high GFAP expression, which may accelerate neurodegeneration by disrupting the vascular barrier, driving neovascularization, and ultimately causing irreversible vision damage.

To explore the relationship between Trx and ERS, autophagy, and focal death in Müller cells, bioinformatics was performed to examine their correlations. As shown in Supplementary Figures S1A, B, 11,781 cells were screened according to the Seurat criteria, of which 6,313 cells were from the WT group and 5,468 cells originated from the DB group. Based on retinal marker genes reported in the literature as screening criteria, cell annotation identified the following cell types with corresponding marker genes: cone cells (Opn1mw+), bipolar cells (Vsx2+, Pax6−), ganglion cells (Thy1+, Pax6+), Müller cell (Vsx2+, Pax6+, Rlbp1+), and Microglia Cell (Cx3cr1+), which were visualized using violin plots (Supplementary Figure S1A) and tSNE (Supplementary Figure S1B). Genes associated with ERS, cellular autophagy, and focal death-related enrichment pathways were selected, yielding 1,628 genes relevant to this study. The enriched 1,628 genes were screened using single-cell sequencing data, and five genes (DB group/WT group) related to this study were further identified: Txn1 (Trx-1), Hspa5 (GRP78), Cav-1, Map1lc3b (LC3II), and Txnip (Supplementary Figures S1C–G). The genes screened by single-cell sequencing were then combined with Trx, Txnip, ASK1, glucose-regulated protein 78 (GRP78), microtubule-associated protein1 light chain 3-II (LC3II), Sequestosome 1 (P62), NLRP3, etc., which are closely related to the subject study, for protein interaction network PPI mapping (data source https://cn.string-db.org/) (Supplementary Figure S1J). In vitro, Western blot for ERS-, autophagy-, and pyroptosis-related proteins showed that, compared with the control group, the high-glucose group exhibited significantly increased expression of the ERS proteins GRP78 (Supplementary Figures S1K, L), IRE1 (Supplementary Figures S1K, M), and C/EBP homologous protein (CHOP) (Supplementary Figures S1K, N); the autophagy proteins LC3II (Supplementary Figures S1O, Q) and P62 (Supplementary Figures S1O, R); and the pyroptosis-related proteins NLRP3 (Supplementary Figures S1S, V), caspase-1 (Supplementary Figures S1S, W), ASC (Supplementary Figures S1S, X), and Txnip (Supplementary Figures S1S, U). In contrast, the expression levels of Trx (Supplementary Figures S1S, T) and Cav-1 (Supplementary Figures S1O, P) were downregulated, which further validated the bioinformatics analysis.

To explore the effect of high glucose on Müller cell pyroptosis, cells were treated with high-glucose medium. To investigate the role of Trx in high-glucose-induced Müller cell pyroptosis, a Trx-overexpressing cell line and a parallel experimental group (Müller–Lacz cells) were used. As shown in Figures 2A–C, the expression of Trx was detected at both the gene and protein levels in Müller cells. The results of qPCR (Figure 2A) and Western blot analysis (Figures 2B, C) showed that Trx expression was increased after transfection compared with the control group. The optimal concentration and duration of high-glucose treatment in Müller–Lacz cells were determined using the CCK8 assay. The results showed that a significant decrease in Müller–Lacz cell viability occurred at a high-glucose concentration of 75 mM after 48 h (Figure 2D). The effect of high Trx expression on HG-induced Müller cell pyroptosis was analyzed, and the expression of Trx, Txnip, and pyroptosis-associated proteins was evaluated by Western blot. As shown in Figures 2F–K, the expression of Txnip (Figure 2H), NLRP3 (Figure 2I), caspase-1 (Figure 2J), and ASC (Figure 2K) was decreased, whereas the expression of Trx (Figure 2G) was increased in Müller–Trx cells compared with Müller–Lacz cells after high-glucose treatment. As shown in Figures 2E, L, TUNEL staining results demonstrated a significant decrease in the number of positively stained cells in Müller–Trx cells compared with Müller–Lacz cells after high-glucose treatment. These results suggest that changes in the expression of the redox system Trx/Txnip can be induced in response to high glucose, leading to the initiation of cellular pyroptosis, and that Trx overexpression can alleviate high-glucose-induced pyroptosis in Müller cells.

The effect of Trx on the presence of ERS and autophagy in high-glucose-induced Müller cells was investigated. In Figures 3A–D, ERS-related proteins and ASK1 were detected by Western blot. As shown in Figures 3E, F, to investigate the effect of Trx on high-glucose-induced autophagic flow in Müller cells, mCherry-GFP-LC3 was used to track the formation and degradation of autophagosomes. The results showed that the Müller–Lacz+HG group exhibited a significant increase in red and yellow spots compared with the Müller–Lacz group, and high Trx expression further increased the number of yellow spots in the cells. In addition, Müller cells treated with high glucose were collected, and the ultrastructure of intracellular autophagosomes was observed under a transmission electron microscope. The results showed that the number of autophagosomes in cells treated with high glucose was significantly higher than that in the group without high-glucose treatment, whereas the number of autophagosomes in Müller cells decreased after Trx overexpression (Figure 3G). Moreover, the gene expression of Trx-1 and Cav-1 was detected by qPCR (Figures 3H, I). As shown in Figures 3J–M, the expression of Cav-1 and the autophagy-related proteins LC3II and P62 was detected by Western blot. The results revealed that, in the Müller–Lacz+HG group compared with its control group, Cav-1 expression was significantly reduced, whereas LC3II and P62 expression was elevated. In contrast, in the Müller–Trx+HG group compared with the Müller–Lacz+HG group, the expression of Cav-1 and LC3II was elevated, while P62 expression was reduced. These results suggest that a high-glucose environment induces ERS and autophagy in Müller cells; however, Trx overexpression alleviates ERS and autophagy, possibly through ASK1- and Cav-1-mediated regulation of this process.

To investigate whether overexpression of Trx regulates high-glucose-induced cellular pyroptosis through ASK-1-mediated ERS, the expression of GRP78 and CHOP was examined. As shown in Figures 4A–C, the expression of GRP78 and CHOP was significantly decreased in Müller–Trx cells after ASK1 siRNA transfection under high-glucose treatment. TUNEL staining was performed to assess the effect of ASK1 siRNA treatment on pyroptosis in Müller cells under high-glucose conditions. The results showed that the cellular pyroptosis rate of Müller–Trx–HG–siRNA was reduced compared with that in the Müller–Lacz–HG–siRNA group, suggesting that Trx overexpression alleviated high-glucose-induced Müller cell pyroptosis by regulating ASK-1 (Figure 4D). Moreover, the expression of Cav-1 and P62 was also examined, and the results are shown in Figures 4E–G.

To investigate the association between Trx overexpression and the regulation of high-glucose-induced cellular pyroptosis and autophagy, autophagic flux was examined. As shown in Figures 5A, B, changes in autophagic flux were detected using mCherry-GFP-LC3. Cells were pretreated with daidzein (Cav-1 inhibitor). Compared with the Müller–Trx+HG+daidzein group, the Müller–Lacz+HG+daidzein group showed an increased number of yellow spots and a decreased number of red spots. Furthermore, transmission electron microscopy (TEM) was used to observe the formation of autophagic vesicles in Müller cells. As shown in Figure 5F, the number of autophagosomes in Müller–Lacz cells was significantly increased compared with the control group after high-glucose treatment; however, the number of autophagosomes in Müller–Trx cells was decreased compared with that in Müller–Lacz cells. However, this process was reversed after daidzein treatment. To further investigate whether Trx-regulated Cav-1 affects autophagy, the expression of Cav-1 in the Müller–Trx+HG group treated with daidzein treatment was compared with that in the Müller–Trx+HG group and was found to be reduced. In addition, the expression of LC3 (Figures 5C, D) and P62 (Figures 5C, E) in the Müller–Trx+HG group treated with daidzein treatment was increased compared with that in the Müller–Trx+HG group. These results suggest that a high-glucose environment induces autophagy in Müller cells, whereas inhibition of Cav-1 exacerbates DR by promoting autophagosome accumulation and impaired autophagic lysosome degradation. This pathological process can be reversed by high Trx expression.

As shown in Figures 5H, K, pyroptosis was detected by TUNEL staining, and the number of positive cells was increased in Müller–Lacz cells compared with the control group after high-glucose treatment. This increase was inhibited in Müller–Trx cells. However, the number of positive cells was also increased in Müller–Trx cells after high-glucose treatment with daidzein. Moreover, Western blot results showed that the expression of Txnip (Figures 5G, J), NLRP3 (Figures 5G, L), caspase-1 (Figures 5G, M), and ASC (Figures 5G, N) was increased in the Müller–Lacz+HG group compared with the Müller–Lacz group, and expression was reduced in the Müller–Trx+HG group compared with the Müller–Lacz+HG group. This process was reversed after daidzein treatment. Trx expression of Trx was also detected (Figures 5G, I). These results indicate that Trx overexpression alleviates high-glucose-induced pyroptosis via Cav-1-mediated autophagy in Müller cells.