Conbercept enhances the phagocytic activity of retinal Müller glia towards hard exudates in diabetic retinopathy

Conbercept enhances the phagocytic activity of retinal Müller glia towards hard exudates in diabetic retinopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Conbercept enhances the phagocytic activity of retinal Müller glia towards hard exudates in diabetic retinopathy Yingying Zhu, Shiyue Qin, Hai Xie, Yinping Liu, Xiaosa Li, Yanchun Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4405680/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Hard exudates (HEs) are one of the main factors affecting vision in patients with diabetic retinopathy (DR) and diabetic macular edema (DME). Anti-vascular endothelial growth factor (anti-VEGF) is the main therapy for DME, but its effect on Müller glia phagocytosis remains unclear. The aim of this study was to investigate the effects of conbercept on phagocytosis of HEs by Müller glia in DR and elucidate the underlying mechanism(s). Methods Twenty-one eyes from 17 patients diagnosed with DR or DME underwent optical coherence tomography (OCT) imaging at baseline and one week after each consecutive intravitreal conbercept injection (ICI), administered three times with an interval of over one month between each injection, to assess changes in HEs. The rat Müller cell line (rMC-l) was cultured under high glucose conditions to mimic a diabetic environment. Cells were treated with oxidized low-density lipoprotein (Ox-LDL) alone or in combination with conbercept. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8). The phagocytosis of Ox-LDL by rMC-l cells with or without conbercept was examined via immunofluorescence, flow cytometry, and Western blot. Gene expressions of several scavenger receptors and transporters involved in lipid metabolism, including RAGE, LOX-1, TLR-4, CD36, SR-AII, and ABCG-1, were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Results The area of the HEs exhibited minimal changes following the initial ICI, whereas a significant decrease in area was observed after three consecutive injections. The viability of rMC-l cells was obviously reduced at higher concentrations of conbercept (> 100 µg/mL). Under high glucose conditions, rMC-l cells phagocytosed Ox-LDL, particularly locolized around the nucleus, and conbercept further enhanced this phagocytosis. Ox-LDL treatment increased the expression of the receptors and transporters involved in phagocytosis and lipid metabolism, while conbercept treatment further enhanced their expressions, except for ABCG-1 which was decreased. Conclusion This study confirmed that Conbercept treatment can effectively reduce the area of HEs in DR and DME. This therapeutic effect may be attributed to the enhanced phagocytic capability of Müller glia towards HEs, which is mediated by the regulation of key lipid metabolism receptors and transport proteins. These findings provide novel mechanisms underlying the facilitation of HEs clearance in DR and DME by anti-VEGF therapy, thereby establishing a theoretical basis for future therapeutic strategies. Conbercept Müller glia oxidized low-density lipoprotein phagocytosis anti-VEGF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hard exudates (HEs) are lipid-rich deposits often observed in various retinal vascular diseases [ 1 ], including diabetic retinopathy (DR) and diabetic macular edema (DME), retinal vein occlusion (RVO), and etc. The standard treatment for above conditions is intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents. Recent clinical studies have suggested that anti-VEGF treatment not only reduces macular edema but also diminishes HEs [ 2 – 4 ], leading to speculation that these drugs might promote HEs absorption by modulation of retinal phagocytic cells. Conbercept, a recombinant fusion protein with potent anti-VEGF activity, has shown efficacy in reducing retinal vascular leakage and improving visual outcomes [ 5 ]. While its primary mechanism of action is inhibiting VEGF-mediated vascular permeability and neovascularization, recent studies have indicated that conbercept might also exert differential effects on retinal cells, such as enhancing the autophagy of retinal RF/6A cells [ 6 ]. Retinal Müller cells, the predominant glial cells in the retina, play a crucial role in maintaining retinal homeostasis [ 7 ]. Their cell bodies are primarily located in the Outer Plexiform Layer (OPL) and Inner Nuclear Layer (INL), which correspond to the sites where HEs typically accumulate as observed on OCT. As macrophage-like cells within the central nervous system, one of their physiological functions is phagocytosis, which is essential for preventing the accumulation of HEs. The phagocytic function of retinal microglia has been extensively documented [ 8 – 9 ], and there is growing interest among researchers regarding the phagocytic capacity of Müller glia in the retinal metabolism. Previous studies have shown that Müller glia can eliminate degenerated photoreceptors before microglia intervention, a process modulated by phosphatidylserine and Rac1 [ 10 ]. Furthermore, in the absence of microglia, Müller glia can engulf other apoptotic cells in the retina, underscoring their significant phagocytic role [ 11 ]. However, under diabetic conditions, this phagocytic function of Müller glia might be compromised, thereby contributing to the accumulation of HEs. Currently, there are no reports regarding the effect of conbercept on the phagocytosis of Müller glia as well as the related mechanism(s), and there is also a lack of in vitro studies to demonstrate its phagocytotic effect treated with or without conbercept. In this study, clinical samples from diabetic patients were utilized to investigate the impact of conbercept on retinal hard exudates. Additionally, we explored the phagocytosis of Ox-LDL by Müller glia in vitro treated with or without conbercept treatment under diabetic condition. The scavenger receptors and transporters involved in lipid metabolism were also explored. MATERIALS AND METHODS Patients Twenty-one eyes in 17 patients with DR or DME were diagnosed by comprehensive ophthalmologic examinations in the Department of Ophthalmology, Yijishan Hospital affiliated to Wannan Medical College, Wuhu, China, between January 2022 and January 2024. This study was approved by the Clinical Research Ethical Committee of Wannan Medical College Yijishan Hospital and adhered to the principles of the Declaration of Helsinki. All individual participants provided written informed consent. All patients underwent routine ophthalmic examinations before and after treatment, including best corrected visual acuity, intraocular pressure, slit lamp microscope and fundus examination. Patients were reexamined 1 week after each ICI. Inclusion criteria: (1) Diagnosis of diabetic retinopathy (with or without diabetic macular edema); (2) Presence of hyperreflective foci in the macular center with a size greater than 30 µm, exhibiting artifacts visible on fundus photography; (3) Diagnosis of type 2 diabetes by an endocrinologist. Exclusion criteria: (1) History of previous anti-VEGF treatment or other ocular surgeries; (2) Retinal laser therapy within the past six months; (3) Presence of any other eye diseases that may cause hard exudates in the macula; (4) Coexistence of eye conditions such as glaucoma, retinal detachment, and retinal vein occlusion that may lead to retinal pathology apart from diabetic retinopathy; (5) Inability to obtain clear images due to media opacities such as cataracts; (6) Unstable systemic condition or incomplete clinical data. Intravitreal injection of conbercept An experienced ophthalmologist performed all intravitreal injections aseptically at the temporal limbus through the eyeball’s pars plana. All patients received intravitreal injections of conbercept (10 mg/0.2 ml) using a 30-gauge needle. The interval between each injection was more than 1 month. A 1-week variation was allowed for every injection interval. OCT evaluation Retinal imaging was conducted using a Spectralis OCT system (Heidelberg Engineering GmbH, Germany) across a 2 mm×2 mm area centered on the fovea. HEs are defined as OCT-detected lesions with a size larger than 30 µm and the presence of artifacts, resembling the retinal pigment epithelium-Bruch's complex. These HEs are predominantly located in the inner retinal layers, spanning from the nerve fiber layer to the outer nuclear layer. The measurement of HEs was performed using ImageJ software (version 1.46r, Rawak Software Inc., Stuttgart, Germany). OCT scans were imported into ImageJ, converted to 8-bit, and then sharpened to enhance clarity. Subsequently, relevant areas were selected for measurement. Reagents and antibodies The Dil-Ox-LDL was purchased from Invitrogen (L34358; Carlsbad, CA, USA). The Ox-LDL and primary antibodies against anti-Ox-LDL were purchased from Yubo Biomed Technologies (Shanghai, China). The conbercept was purchased from Chengdu Kanghong Pharmaceutical Group Co., Ltd. (Sichuan, China). The DMEM High Glucose Medium (SH30021.01B) was purchased from HyClone (Logan, UT, USA). CY3 goat anti-rabbit IgG (GB21303), FITC goat anti-mouse IgG (GB22301) and β-actin were purchased from Servicebio (Wuhan, China). The primers were purchased from Sangon Biotech (Shanghai, China). Rat Müller cell (rMC-l) culture Transformed rat retinal Müller cell line (rMC-l) was kindly supplied by Sarthy (Northwestern University, Chicago, IL, USA). The cells were cultured in high glucose (4.5 g/L) DMEM containing 10% fetal bovine serum (10099158; Gibco, Shanghai, China) and 1% penicillin/streptomycin (C0222; Beyotime Biotechnology) at 37°C with 5% CO₂ in a humidified incubator. Upon reaching ~ 80% confluence in a 10-cm dish, the cells were divided into three groups: vehicle control, Ox-LDL (10 µg/mL)-treated group, and Ox-LDL (10 µg/mL) + conbercept (100 µg/mL)-treated group. Two types of Ox-LDL were used in different experiments: 10 µg/mL Dil-Ox-LDL (ThermoFisher, USA) for immunofluorescence, flow cytometry and qRT-PCR, and 10 µg/mL Ox-LDL (Yubo, Shanghai, China) for Western blot. Cell viability assay Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; C0037; Beyotime Biotechnology). Cells were seeded in 96-well plates and treated with varying concentrations of conbercept (ranging from 10 to 500 µg/mL) for 24 hours. Post-treatment, 10 µL of CCK-8 solution was added to each well and incubated in the dark for 1 hour. Subsequently, the optical density was quantified using a microplate reader by measuring absorbance at 450 nm. Immunofluorescence of Ox-LDL in rMC-l cells rMC-l cells were incubated with Ox-LDL, either with or without conbercept, for 24 hours. The cells were then fixed in cold methanol for 30 minutes and blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour. Subsequently, the cells were first incubated with anti-Ox-LDL (1:100) and glial fibrillary acidic protein (GFAP) (1:500) overnight at 4℃. And then, after three 5-minute washes in PBS, incubated with the corresponding secondary antibody (1:1,000, anti-rabbit CY3; 1:1,000, anti-mouse FITC) for 2 hours at room temperature. Dil-Ox-LDL was used in accordance with the manufacture’s protocol. Then, the cells were further incubated with 4'6-diamidino-2-phenylindole (DAPI,100 ng/mL) for 5 minutes, followed by three washes (5 minutes each wash). Finally, the slides were visualized with a confocal microscope (LSM 800; Zeiss Microsystems, Germany). Protein extraction and Western blot The rMC-l cells were incubated for 24 hours with Ox-LDL, in conditions with and without conbercept supplementation. Cells were lysed in protein extraction radioimmunoprecipitation assay (RIPA; P0013B; Beyotime Biotechnology) buffer on ice. After 15-second ultrasonic treatment, the samples were placed on ice for 30 minutes before centrifugation. Protein concentrations were determined with bicinchoninic acid (BCA) Protein Assay Kit (A5586; Thermo Scientific, Shanghai, China). Equal amounts of protein were resolved on 12% SDS-polyacrylamide gels and subsequently transferred electrophoretically onto nitrocellulose membranes. The membranes were blocked in 5% PBS buffered nonfat milk at room temperature for 30 minutes, and then separately incubated with anti-Ox-LDL (1:1,000) or β-actin (1:2,000) overnight at 4℃. After being washed three times with 0.1% TBS-buffered Tween-20 (TBST), the membranes were incubated with the corresponding secondary antibodies (1:2,000, anti-rabbit) at room temperature for 2 hours, followed by washes with TBST (three times). Detection of the bands was done using Chemi Doc Touch System (Bio-Rad). Immunoblots were visualized by enhanced chemiluminescence (ECL) and analyzed using ImageJ software version 1.37 ( http://imagej.nih.gov/ij/ ). Samples were normalized to β-actin. Flow cytometry Dil-Ox-LDL, either with or without conbercept, was added to the medium and incubated for 4 hours. After three 5-minute washes in PBS, adherent rMC-l cells were collected, then digested with trypsin. The fluorescence of cells was analyzed with a flow cytometer (PE-A; Beckman CytoFLEX). Data were analyzed with FlowJo software (Tree Star Inc.). Quantitative real-time Polymerase Chain Reaction (qRT-PCR) The rMC-l cells were treated with Ox-LDL and Ox-LDL + conbercept for 24 hours. The mRNA expression levels were analyzed by qRT-PCR (CXF96, Bio-Rad) using SYBR Green (208054, QIAGEN, Germany) based gene expression. In brief, adherent rMC-l cells were lysed and the total RNA was extracted for cDNA synthesis (K16225, Thermo, USA). Levels of mRNA expressions were normalized by the intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data were expressed as a relative to the controls. The information for primers was provided in Table 1 . Table 1 Baseline characteristics of patients with DR or DME and HEs area comparisons after anti-VEGF treatment. Characteristics Baseline 1st ICI 2nd ICI 3rd ICI Eyes (No.) 21 - - - Patients (No.) 17 - - - Sex (male/female) 7/10 - - - Mean ages (years) 56.6 ± 9.2 - - - HEs area (mm²) 1.39 ± 1.41 1.38 ± 1.3 0.77 ± 0.9 0.45 ± 0.66 P value 0.938 0.021 0.002 ICI: intravitreal conbercept injection Table 2 The information of rat primers Gene Primers Nucleotide Sequence LOX-1 Forward 5’-CCACAAGACTGGATCTGGCAT-3’ Reverse 5’-AGATAGGCAATTCTCCCGACT-3’ CD36 Forward 5’-AGCTGCACCACATATCTACACA-3’ Reverse 5’-AGAATGGATCTTTGTAACCCCAC-3’ TNF-α Forward 5’-ATGGGCTGTACCTTATCTACTCC-3’ Reverse 5’-GAGGCTGACTTTCTCCTGGTA-3’ ABCG1 Forward 5’-TCGAATCTCGTGCCGTACCTG-3’ Reverse 5’-GCTGTTCTGATCACCGTACTCC-3’ SRA2 Forward 5’-CTTGGCACTGCTTCACGAAC-3’ Reverse 5’-TTCTTGTACCAGCAGTGCCAT-3’ TLR4 Forward 5’-ATCCCTGCATAGAGGTACTTCC-3’ Reverse 5’-ATCCAGCCACTGAAGTTGTGA-3’ GAPDH Forward 5’-GACATGCCGCCTGGAGAAAC-3’ Reverse 5’-AGCCCAGGATGCCCTTTAGT-3’ Statistical analysis Data were expressed as mean ± SD and analyzed using pairwise t-tests or one-way ANOVA. A P -value of < 0.05 was considered statistically significant. For data requiring statistical analysis, the experiments were repeated at least 3 times. Results Patient characteristics and the impact of Anti-VEGF therapy on reduction of HEs Twenty-one eyes from 17 patients with DR or DME were retrospectively reviewed in this study and baseline characteristics are presented in Table 1 . The participants included 10 females and 7 males, and the average age was 56.6 ± 9.2 years old. In Fig. 1 A, the OCT image of a 72-year-old female patient with DME exhibited a progressive reduction in macular edema size following an increasing number of conbercept injections. Notably, there was a concurrent decrease in HEs within the inner nuclear layer (INL), corresponding to the escalating frequency of conbercept injections. This observation suggests potential activation of Müller cells' phagocytic function, considering their anatomical localization within the INL. Statistically, there was no significant change in the area of HEs between baseline and the first follow-up, with measurements ranging from 1.39 mm² ± 1.41 mm² to 1.38 mm² ± 1.3 mm² ( P = 0.938). However, a significant reduction was observed at the second and third follow-ups, with the HEs area measured at 0.77 mm² ± 0.9 mm² ( P = 0.021) and 0.45 mm² ± 0.66 mm² ( P = 0.002), respectively (Fig. 1 C). rMC-l cells demonstrated phagocytosis of Ox-LDL Under high glucose conditions, confocal microscopy analysis revealed that Ox-LDL engulfment occurred in the cytoplasm of GFAP-labeled rMC-l cells, with close proximity to the nucleus (Fig. 2 A). Additionally, 2.5D reconstruction of Dil-Ox-LDL signals demonstrated their localization near the nuclei (Fig. 2 B), confirming the occurrence of phagocytosis. Conbercept decreased rMC-l cells viability in a dose-dependent manner The viability of rMC-l cells under high glucose conditions exhibited a dose-dependent response upon treatment with different concentrations of conbercept. As shown in Fig. 3 , the cell viability of rMC-l cells was observed to be 94.40% (10 µg/mL, n = 3, P > 0.05), 88.49% (50 µg/mL, n = 3, P < 0.05), 92.23% (100 µg/mL, n = 3, P < 0.05), 80.42% (300 µg/mL, n = 3, P < 0.01), and 70.76% (500 µg/mL, n = 3, P < 0.05) of the control, respectively, when treated with increasing concentrations of conbercept. Considering that conbercept exhibits a comparable and relatively modest (less than 12%) decline in rMC-l cell viability within the concentration range of 10–100 µg/mL, with a notable increase in inhibition to 20% at 300 µg/mL, it can be inferred that the concentration range of 0-100 µg/mL of conbercept is relatively safe for rMC-l cells. Consequently, a treatment concentration of 100 ug/mL was chosen for subsequent cellular experiments. Conbercept significantly enhanced the phagocytosis of Ox-LDL by rMC-l cells To further verify the increased uptake of Ox-LDL by rMC-l cells under high glucose conditions and explore the impact of conbercept, we employed immunofluorescence and western blot techniques to visualize and quantify the internalized amounts of Dil-Ox-LDL and Ox-LDL within the cells (Fig. 4 ). The analysis showed that conbercept treatment led to a 23% increase (n = 3, P < 0.05) in intracellular Dil-Ox-LDL levels by immunofluorescence, as well as a 30% increase (n = 3, P < 0.01) in Ox-LDL levels in rMC-l cells by western blot. Additionally, flow cytometry analysis confirmed a significant 21% enhancement (n = 3, P < 0.01) in Dil-Ox-LDL uptake with conbercept treatment (Fig. 5 ). These results indicated that conbercept could effectively enhance the phagocytotic activity of rMC-l cells towards Ox-LDL under diabetic conditions. Conbercept upregulated the expression of scavenger receptors and cholesterol efflux transporters in Ox-LDL-induced rMC-l cells qRT-PCR analysis revealed that treatment with Ox-LDL upregulated the expressions of several receptors and transporters involved in lipid metabolism, including RAGE (receptor for advanced glycation end products), LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), TLR-4 (toll-like receptor 4), CD36 (cluster of differentiation 36), SR-AⅡ (scavenger receptor class A, type II), and ABCG-1 (ATP binding cassette subfamily G member 1). Notably, the addition of conbercept to Ox-LDL-treated cells further amplified the expressions of these genes, except for ABCG-1 which exhibited a decrease. This suggests a differential regulatory effect of conbercept on genes associated with lipid metabolism and phagocytosis (Fig. 6 ). Additionally, our study results indicate that there is a correlation between the expression levels of RAGE and SR-AⅡ, CD36, as well as between TLR-4 and LOX-1, CD36 genes in rMC-l cells (Fig. 7 ). DISCUSSION Our results revealed a novel mechanism of action of conbercept in the high glucose environment, offering innovative insights for the resolution of HEs in DR or DME. Clinical studies have demonstrated that retinal photocoagulation therapy, intravitreal injection of steroid hormones or anti-VEGF therapy can effectively alleviate HEs, and the mechanism involves the regulation of microvascular leakage, lipid accumulation and inflammatory response [ 12 – 14 ]. The results of our study are consistent with previous findings, demonstrating a significant reduction in the area of HEs within the macular region following ICI. This reduction was particularly prominent at the INL, which corresponds to the location of Müller soma, indicating the activation of phagocytic activity of Müller by conbercept (Fig. 1 ). Subsequent in vitro investigations confirmed the phagocytic effect of Müller glia on Ox-LDL, which can be enhanced by conbercept (Fig. 2 – 5 ), thereby providing a more comprehensive understanding of the mechanism underlying reduction in HEs following anti-VEGF treatment and offering significant clinical implications. HEs formation results from increased vascular permeability due to lesions in the microvessels in the retina. Normally, endothelial cells of retinal blood vessels form tight junctions that prevent plasma components from entering retinal tissue [ 15 ]. But in DR or DME, impaired vascular barrier function leads to leakage of lipids and proteins from the blood into the parenchymal tissue of the retina. These leakages usually accumulate between the OPL and the INL of the retina, because the anatomy and vascular supply of this region make it a reservoir for leakages. Specifically, the OPL is the synaptic region of neurons with a rich microvascular network, while the INL contains the nucleus of retinal cells, and the structural characteristics of these layers lead to the easy deposition of exudates to form HEs here. Previous studies have demonstrated the phagocytic functions of both Müller glia and microglia [ 16 – 17 ]. Müller cells are more likely to be directly involved in HEs clearance due to their anatomic location. The cell body of Müller glia is situated in the INL, with its fibers extending from the inner limiting membrane to the outer nuclear layer. This unique cellular morphology not only provides structural support for maintaining retinal homeostasis [ 18 ], but also facilitates direct contact between Müller glia and the region where HEs typically accumulate, specifically between the OPL and INL. Therefore, it is reasonable to speculate that Müller glia, owing to their strategic location and functional role, are likely to be the primary mediators of exudative material clearance in the retina when HEs accumulate. Our study further substantiated the phagocytic function of Müller glia, particularly for lipid products (Fig. 2 ), a finding that may have positive implications for improving retinal lipid metabolism and alleviating hard exudate accumulation, among others. The upregulation of scavenger receptors such as RAGE, SR-AⅡ, and CD36 in Müller glia in response to conbercept treatment implies a direct influence on the cells' lipid handling capabilities (Fig. 6 ), crucial for mediating the clearance of Ox-LDL, a key factor in the formation of HEs. Indeed, hyperglycemia has been reported to induce the upregulation of RAGE, leading to RAGE signaling and subsequent pro-inflammatory responses by retinal Müller glia, both in vivo and in vitro upon exposure to high glucose [ 19 ]. Additionally, advanced glycation end-products (AGEs) stimulate the uptake of Ox-LDL into macrophages through the Cdk5-CD36 pathway via RAGE-mediated oxidative stress [ 20 – 22 ]. Findings suggest that SR-AII could directly prevent the activation of the RAGE-MAPK-NF-κB signaling pathway and diminish the secretion of proinflammatory cytokines in microglia [ 23 ]. Our study revealed a positive correlation between the gene expression levels of RAGE with CD36 (Fig. 7 ), suggesting that conbercept potentially enhanced the uptake of Ox-LDL into rMC-l cells by promoting the CD36 pathways, which are induced by the AGEs-RAGE axis. The increased expression of SR-AⅡ by conbercept can reduce the effects of inflammation activation caused by RAGE. This upregulation is pivotal, as scavenger receptors, particularly CD36 and SR-AⅡ, are known to be the principal receptors for binding and uptake of Ox-LDL in macrophages, exhibiting specific recognition and binding capabilities for Ox-LDL that facilitate lipid engulfment, intracellular degradation, and metabolism [ 24 ]. This process typically involves endosome formation and cytoskeletal modifications, where SR-AⅡ functions to dampen lipid-related inflammatory responses, and CD36 actively participates in intracellular lipid transport and metabolism [ 25 – 26 ]. By enhancing the phagocytotic capacity of Müller glia through these pathways, conbercept may help mitigate the buildup of lipid-rich deposits, a hallmark of diabetic retinal diseases like DR, and contribute to the reduction of lipid deposition and plaque formation. TLR-4 is mainly recognized for detecting bacterial lipopolysaccharides (LPS) and is vital in regulating metabolism, especially in lipid metabolism related to diseases like atherosclerosis and diabetes [ 27 ]. Once activated, TLR-4 affects lipid uptake in different cells by altering the expression of genes related to lipid metabolism and transport. Additionally, TLR-4 activation can enhance the phagocytic activity of BV2 cells [ 28 ]. These receptors, central to the innate immune system, demonstrate intricate interactions, i.e., TLR-4 influences the expression or activity of other lipid receptors such as LOX-1 and CD36, thereby impacting cellular lipid uptake. Specifically, LOX-1 serves as a receptor for Ox-LDL, recognizing and binding it to facilitate cellular uptake, intracellular degradation, and metabolism, thereby reducing extracellular lipid accumulation [ 29 – 30 ]. Concurrently, the synergistic effect between LOX-1 and TLR-4 mutually enhanced their activity [ 31 ], while TLR-4 further augmented the uptake of Ox-LDL by macrophages via CD36 upregulation and initiates inflammatory signaling pathways [ 32 ]. The observed modulation of LOX-1, CD36, and TLR-4 expression by conbercept suggests a multifaceted impact on the retinal immune response (Fig. 6 ). Our findings underscore a significant correlation between TLR-4 and the expression of LOX-1 and CD36 (Fig. 7 ), underlining the complexity of conbercept's role in modulating lipid metabolism and the immune response in the context of DR and DME, potentially contributing to the reduction of chronic inflammation associated with these conditions. Ox-LDL typically enters lysosomes and is hydrolyzed into free cholesterol and fatty acids by neutral cholesteryl ester hydrolases (CEH), with subsequent metabolism occurring via ABCA1 and ABCG1 pathways [ 33 ]. The downregulation of ABCG1 expression may be associated with the cellular endeavor to maintain cholesterol homeostasis, potentially serving as a mechanism for attenuating cholesterol efflux in response to heightened phagocytosis of Ox-LDL. The observed downregulation of ABCG-1 suggests a strategic shift in the Müller glia' lipid metabolism towards enhanced phagocytic clearance of Ox-LDL, which could serve as a protective mechanism against oxidative damage and inflammation. This shift aligns with the broader role of Müller glia in retinal homeostasis and supports the hypothesis that conbercept's therapeutic benefits may include the modulation of lipid metabolism, enhancing cellular resilience against the diabetic milieu. The dual role of conbercept in both inhibiting pathological angiogenesis and modulating Müller glia function highlights its potential as a more comprehensive therapeutic agent for DR and DME. These findings suggest that the therapeutic effects of conbercept might not be limited to VEGF inhibition but could also include modulation of cellular metabolic and immune responses, which are crucial in the pathogenesis of diabetic retinal diseases. Considering the complex interplay of angiogenesis, inflammation, and lipid metabolism in the progression of DR and DME, our study underscores the need for a holistic approach in treatment strategies. Conbercept's ability to simultaneously influence various pathways offers a promising therapeutic avenue, potentially addressing multiple aspects of retinal pathology. Further investigation into this relationship can potentially lead to more effective and comprehensive treatment approaches for retinal vascular diseases associated with HEs. This study has several limitations. First, for the expression levels of scavenger receptors, only mRNA levels were detected, which need to be confirmed by their protein expressions. Furthermore, we did not investigate whether conbercept exerts its influence on these molecules through direct regulation or indirectly via antagonism of its own targets. Additionally, the primary mediator of Müller-cell phagocytosis among VEGFA, VEGFB, or PlGF targeted by conbercept was not explored in this study. In conclusion, conbercept treatment enhanced the phagocytotic effect of retinal Müller glia under diabetic conditions, and this effect might be mediated by a series of key phagocytic receptors (Fig. 8 ). This study broadens our understanding of conbercept's role in retinal health, particularly in the context of diabetes-induced retinal changes. The enhanced phagocytotic activity of Müller glia and the modulation of key receptors involved in lipid metabolism and innate immunity by conbercept could be critical in developing more effective treatment strategies for retinal diseases like DR and DME. Future research should aim to further elucidate the molecular mechanisms underpinning these effects and explore the long-term outcomes of such modulation on retinal health and disease progression. Declarations Funding Declaration This work was supported by the National Natural Science Foundation of China, No. 82171062 (to JFZ), No. 82301222 (to CYZ) and No. 32201244 (to XSL) Ethics Statement This study was approved by the Clinical Research Ethical Committee of Wannan Medical College Yijishan Hospital and adhered to the principles of the Declaration of Helsinki. All individual participants provided written informed consent. Conflicts of Interest The authors declare no conflict of interest. Author Contribution Y.Z. wrote the manuscript. S.Q. contributed to the interpretation of these results for the manuscript.H.X. and Y.L. supervised the project.X.L. and Y.Z. provided essential theoretical insights and critically revised the manuscript for important intellectual content.C.Z. and J.Z. conducted a thorough review and final approval of the manuscript prior to submission.All authors reviewed the manuscript. Availability of Data and Materials All data generated or analyzed during this study are included in this published article and its supplementary information files. References Yanko L, Ungar H, Michaelson IC. The exudative lesions in diabetic retinopathy with special regard to the hard exudate. Acta Ophthalmol (Copenh). 1974;52(1):150-160. Domalpally A, Ip MS, Ehrlich JS. Effects of intravitreal ranibizumab on retinal hard exudate in diabetic macular edema: findings from the RIDE and RISE phase III clinical trials. Ophthalmology. 2015 Apr;122(4):779-786. Tao Y, Jiang P, Zhao Y, Song L, Ma Y, Li Y, Wang H. 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Ehlers JP, Yeh S, Maguire MG, Smith JR, Mruthyunjaya P, Jain N, Kim LA, Weng CY, Flaxel CJ, Schoenberger SD, Kim SJ. Intravitreal Pharmacotherapies for Diabetic Macular Edema: A Report by the American Academy of Ophthalmology. Ophthalmology. 2022 Jan;129(1):88-99. O'Leary F, Campbell M. The blood-retina barrier in health and disease. FEBS J. 2023;290(4):878-891. doi:10.1111/febs.16330 Fan W, Huang W, Chen J, Li N, Mao L, Hou S. Retinal microglia: Functions and diseases. Immunology. 2022;166(3):268-286. doi:10.1111/imm.13479 Bejarano-Escobar R, Sánchez-Calderón H, Otero-Arenas J, Martín-Partido G, Francisco-Morcillo J. Müller glia and phagocytosis of cell debris in retinal tissue. J Anat. 2017;231(4):471-483. Reichenbach A, Bringmann A. Glia of the human retina. Glia. 2020;68(4):768-796. Zong H, Ward M, Madden A, et al. Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE). Diabetologia. 2010;53(12):2656-2666. Terasaki M, Shibata K, Mori Y, et al. SMTP-44D Inhibits Atherosclerotic Plaque Formation in Apolipoprotein-E Null Mice Partly by Suppressing the AGEs-RAGE Axis. Int J Mol Sci. 2023;24(7):6505. Published 2023 Mar 30. Yashima H, Terasaki M, Sotokawauchi A, et al. AGE-RAGE Axis Stimulates Oxidized LDL Uptake into Macrophages through Cyclin-Dependent Kinase 5-CD36 Pathway via Oxidative Stress Generation. Int J Mol Sci. 2020;21(23):9263. Published 2020 Dec 4. Xanthis A, Hatzitolios A, Fidani S, Befani C, Giannakoulas G, Koliakos G. Receptor of advanced glycation end products (RAGE) positively regulates CD36 expression and reactive oxygen species production in human monocytes in diabetes. Angiology. 2009;60(6):772-779. Ma K, Xu Y, Wang C, et al. A cross talk between class A scavenger receptor and receptor for advanced glycation end-products contributes to diabetic retinopathy. Am J Physiol Endocrinol Metab. 2014;307(12):E1153-E1165. Yu XH, Fu YC, Zhang DW, Yin K, Tang CK. Foam cells in atherosclerosis. Clin Chim Acta. 2013;424:245-252. Lin CS, Lin FY, Ho LJ, et al. PKCδ signalling regulates SR-A and CD36 expression and foam cell formation. Cardiovasc Res. 2012;95(3):346-355. Collot-Teixeira S, Martin J, McDermott-Roe C, Poston R, McGregor JL. CD36 and macrophages in atherosclerosis. Cardiovasc Res. 2007;75(3):468-477. Xu XH, Shah PK, Faure E, et al. Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation. 2001;104(25):3103-3108. Kim DC, Kim SH, Jeong MW, Baek NI, Kim KT. Effect of rottlerin, a PKC-delta inhibitor, on TLR-4-dependent activation of murine microglia. Biochem Biophys Res Commun. 2005;337(1):110-115. Kattoor AJ, Goel A, Mehta JL. LOX-1: Regulation, Signaling and Its Role in Atherosclerosis. Antioxidants (Basel). 2019;8(7):218. Published 2019 Jul 11. Liu H, Li Y, Lin N, et al. Interleukin-1β Promotes Ox-LDL Uptake by Human Glomerular Mesangial Cells via LOX-1. Int J Med Sci. 2020;17(8):1056-1061. Published 2020 Apr 27. Ding Z, Liu S, Wang X, et al. Lectin-like oxidized low-density lipoprotein receptor-1 regulates autophagy and Toll-like receptor 4 in the brain of hypertensive mice. J Hypertens. 2015;33(3):525-533. Sun Z , Yuan W , Li L ,et al.Macrophage CD36 and TLR4 Cooperation Promotes Foam Cell Formation and VSMC Migration and Proliferation Under Circadian Oscillations[J].Journal of Cardiovascular Translational Research, 2022, 15(5):985-997. Tiwari RL, Singh V, Barthwal MK. Macrophages: an elusive yet emerging therapeutic target of atherosclerosis. Med Res Rev. 2008;28(4):483-544. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4405680","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":302715027,"identity":"2ecde2b8-7095-439e-b351-541583a1f693","order_by":0,"name":"Yingying Zhu","email":"","orcid":"","institution":"the First Affiliated Hospital of Wannan Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Zhu","suffix":""},{"id":302715028,"identity":"1f7aeae0-1354-4eb9-a035-112bb7b5eb3d","order_by":1,"name":"Shiyue Qin","email":"","orcid":"","institution":"Taizhou People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shiyue","middleName":"","lastName":"Qin","suffix":""},{"id":302715029,"identity":"307c1309-753a-4a9e-a3ef-9cb9c1faaf93","order_by":2,"name":"Hai Xie","email":"","orcid":"","institution":"Shanghai General Hospital, Shanghai First People’s Hospital), Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"Xie","suffix":""},{"id":302715030,"identity":"914591d7-2111-4488-a828-e8e97984ad43","order_by":3,"name":"Yinping Liu","email":"","orcid":"","institution":"the First Affiliated Hospital of Wannan Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yinping","middleName":"","lastName":"Liu","suffix":""},{"id":302715031,"identity":"d9129a60-d3b1-483a-970a-0a8dc82e3243","order_by":4,"name":"Xiaosa Li","email":"","orcid":"","institution":"Shanghai General Hospital, Shanghai First People’s Hospital), Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaosa","middleName":"","lastName":"Li","suffix":""},{"id":302715032,"identity":"ce0e36d6-e630-4d5e-95e1-b40bdab44cbb","order_by":5,"name":"Yanchun Zhang","email":"","orcid":"","institution":"Shanxi Eye Hospital, Xi'an People's Hospital (Xi'an Fourth Hospital), Affiliated People's Hospital of Northwest University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanchun","middleName":"","lastName":"Zhang","suffix":""},{"id":302715033,"identity":"eec9b690-a0a1-4140-a7ce-afdd70d2d83f","order_by":6,"name":"Chaoyang Zhang","email":"","orcid":"","institution":"Shanghai General Hospital, Shanghai First People’s Hospital), Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chaoyang","middleName":"","lastName":"Zhang","suffix":""},{"id":302715034,"identity":"e7fcaae9-2d4f-4cc7-a8a3-4a05318cc746","order_by":7,"name":"Jingfa Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3PsWoCQRCA4VmETTPx2hGCeYWBFDnJgUVeZETRxoClVchxsBa5kDZdXsFHWFm46tJbeo8gabZIkesN7tml2L+ej5kBiMX+YToprPvxhOPPJj+Iz8KkT9XEXpfpDcO04Kach8kQlncW9TpjWJjBQbsOh0HNlpDwHnZmLegg2bzKedIzK8sp4SjPzV7SBVD9tQ1scVsr7RZwqiX4AExPISJsrW5JpcxKdK8LmfHupSVcKwOiHzsQqsSpknDwoQqalHMM/nL7Xrhv8M/jhK6ao/fZMNm8nScn4WXjsVgsFvuzX7uDR/PdmDtUAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai General Hospital, Shanghai First People’s Hospital), Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jingfa","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-05-11 14:05:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4405680/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4405680/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57427576,"identity":"e823faa4-c645-4cf9-bef7-697e1177e560","added_by":"auto","created_at":"2024-05-30 14:34:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253160,"visible":true,"origin":"","legend":"\u003cp\u003eThe alteration in the size of HEs among patients with DME following conbercept treatment. The area of the HEs exhibited minimal change after the 1st ICI, but experienced a significant decrease subsequent to the 2nd and 3rd ICIs. (A) Representative images of a 73-year-old female patient's HEs area at baseline and after each ICI. (B) Area of HEs at baseline and after each ICI in all 21 eyes. (C) Statistical analysis and normalization of the HEs area among above 4 groups (n = 21).Data are mean ± SEM; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ICI:\u003cstrong\u003e \u003c/strong\u003eintravitreal conbercept injection.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/f6231d17bf0d8bc161bb7c5d.jpg"},{"id":57427573,"identity":"f94963ce-439b-42cd-9367-25bc8469c929","added_by":"auto","created_at":"2024-05-30 14:34:25","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":715637,"visible":true,"origin":"","legend":"\u003cp\u003eThe rMC-l cells exhibited phagocytic activity towards Ox-LDL under high glucose conditions. (A) After co-incubation of Ox-LDL with cells for 24 hours, the immunofluorescence technique was employed to visualize the colocalization of GFAP (green) and Ox-LDL (red). The area enclosed by the dashed white line is magnified. (B) Dil-Ox-LDL was incubated with rMC-l cells for 4 hours and observed by immunofluorescence microscope. \u003cem\u003eScale bar\u003c/em\u003e: 20 μm. The measurements were performed at least three times.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/1d6e2ecc18b01c01902b8e6a.jpeg"},{"id":57427572,"identity":"d7bbb96b-c8e8-4072-a3d1-f2d9020d579c","added_by":"auto","created_at":"2024-05-30 14:34:25","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52731,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of conbercept treatment on the viability of rMC-l cells under high glucose conditions. The rMC-l cells were incubated with conbercept (0-500 μg/mL) for 24 hours, and the cell viability was assessed using the CCK-8 assay (n = 3). The measurements were performed at least three times. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, compared with control.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/3cefd1bd40c281bf13f2136f.jpeg"},{"id":57427577,"identity":"1ea7281d-da7f-4fd8-a1bb-f7c6e432c381","added_by":"auto","created_at":"2024-05-30 14:34:26","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":552502,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of conbercept on the phagocytic capacity in rMC-l cells under high glucose conditions. (A) Visualization of the uptake and distribution of Dil-Ox-LDL (red) in rMC-l cells incubated with or without conbercept for 4 hours, showing its presence surrounding the nucleus (blue). The area enclosed by the dashed white line is magnified. (B) Statistical analysis was conducted on the intensity of Dil-Ox-LDL in (A). (C) The alterations in phagocytosis among the control, Ox-LDL, and Ox-LDL + conbercept groups, following 24 hours of incubation, were assessed through Western blot analysis, as well as (D) the corresponding quantitative analysis(n = 3). \u003cem\u003eScale bar\u003c/em\u003e:20 μm. The measurements were performed at least three times. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/d6ed6ac4daa625c1250238f6.jpeg"},{"id":57427574,"identity":"87a35628-7c59-4f23-82c8-f8dfc4364af6","added_by":"auto","created_at":"2024-05-30 14:34:25","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":612252,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of Dil-Ox-LDL uptake by rMC-l cells with conbercept treatment under high glucose conditions using flow cytometry. (A) The cells were grouped and subjected to treatment with vehicle control, Ox-LDL alone, or Ox-LDL in combination with conbercept. Following a 4-hour incubation, flow cytometry analysis was conducted (n=3). (B) Uptake of Dil-Ox-LDL was quantified.. The measurements were performed at least three times. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/5ed6045630fc2ac5fe7727f0.jpeg"},{"id":57427578,"identity":"44b5d2ed-9011-4a89-a255-97c187003753","added_by":"auto","created_at":"2024-05-30 14:34:26","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":782930,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of conbercept on the expression of scavenger receptors and cholesterol efflux transporters in rMC-l cells induced by Ox-LDL for 24 hours under high glucose conditions. The expression of RAGE, SR-AII, CD36, LOX-1, TLR-4 and ABCG-1 of rMC-l cells among different groups were determined by qRT-PCR (n = 3). The measurements were performed at least three times. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/aa0733b9ae9e298d8a68aaa0.jpeg"},{"id":57427575,"identity":"8678fd91-77ac-4ee2-a8a9-4effec17819a","added_by":"auto","created_at":"2024-05-30 14:34:26","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":224516,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation of the gene expression levels of CD36, SR-AII, RAGE, and CD36, LOX-1, TLR-4 in rMC-l cells were analyzed. The correlation analysis based on gene expression data obtained from qRT-PCR. The correlation graphs were generated using GraphPad Prism 8 software, with the correlation coefficient 'r' indicating the strength and direction of the linear relationship between the variables in the sample set.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/585279c36ce282482384c92c.jpeg"},{"id":57427579,"identity":"669c64aa-24c6-4427-8a01-bac4b9b8225f","added_by":"auto","created_at":"2024-05-30 14:34:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1248295,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of enhanced phygocytosis of HEs by Müller glia treated with conbercept. The illustration was generated using BioRender.com to visualize the cellular interactions and process flow.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/f7d1a682e6451aeb9665a578.png"},{"id":67903401,"identity":"f657efc1-03fd-4ccc-91b2-71e206d52864","added_by":"auto","created_at":"2024-10-31 02:38:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4643244,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4405680/v1/5863b45d-aee9-49c9-9915-3a45ad9e2bfb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Conbercept enhances the phagocytic activity of retinal Müller glia towards hard exudates in diabetic retinopathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHard exudates (HEs) are lipid-rich deposits often observed in various retinal vascular diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], including diabetic retinopathy (DR) and diabetic macular edema (DME), retinal vein occlusion (RVO), and etc. The standard treatment for above conditions is intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents. Recent clinical studies have suggested that anti-VEGF treatment not only reduces macular edema but also diminishes HEs [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], leading to speculation that these drugs might promote HEs absorption by modulation of retinal phagocytic cells.\u003c/p\u003e \u003cp\u003eConbercept, a recombinant fusion protein with potent anti-VEGF activity, has shown efficacy in reducing retinal vascular leakage and improving visual outcomes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While its primary mechanism of action is inhibiting VEGF-mediated vascular permeability and neovascularization, recent studies have indicated that conbercept might also exert differential effects on retinal cells, such as enhancing the autophagy of retinal RF/6A cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRetinal M\u0026uuml;ller cells, the predominant glial cells in the retina, play a crucial role in maintaining retinal homeostasis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Their cell bodies are primarily located in the Outer Plexiform Layer (OPL) and Inner Nuclear Layer (INL), which correspond to the sites where HEs typically accumulate as observed on OCT. As macrophage-like cells within the central nervous system, one of their physiological functions is phagocytosis, which is essential for preventing the accumulation of HEs. The phagocytic function of retinal microglia has been extensively documented [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and there is growing interest among researchers regarding the phagocytic capacity of M\u0026uuml;ller glia in the retinal metabolism. Previous studies have shown that M\u0026uuml;ller glia can eliminate degenerated photoreceptors before microglia intervention, a process modulated by phosphatidylserine and Rac1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, in the absence of microglia, M\u0026uuml;ller glia can engulf other apoptotic cells in the retina, underscoring their significant phagocytic role [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, under diabetic conditions, this phagocytic function of M\u0026uuml;ller glia might be compromised, thereby contributing to the accumulation of HEs. Currently, there are no reports regarding the effect of conbercept on the phagocytosis of M\u0026uuml;ller glia as well as the related mechanism(s), and there is also a lack of \u003cem\u003ein vitro\u003c/em\u003e studies to demonstrate its phagocytotic effect treated with or without conbercept.\u003c/p\u003e \u003cp\u003eIn this study, clinical samples from diabetic patients were utilized to investigate the impact of conbercept on retinal hard exudates. Additionally, we explored the phagocytosis of Ox-LDL by M\u0026uuml;ller glia \u003cem\u003ein vitro\u003c/em\u003e treated with or without conbercept treatment under diabetic condition. The scavenger receptors and transporters involved in lipid metabolism were also explored.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eTwenty-one eyes in 17 patients with DR or DME were diagnosed by comprehensive ophthalmologic examinations in the Department of Ophthalmology, Yijishan Hospital affiliated to Wannan Medical College, Wuhu, China, between January 2022 and January 2024. This study was approved by the Clinical Research Ethical Committee of Wannan Medical College Yijishan Hospital and adhered to the principles of the Declaration of Helsinki. All individual participants provided written informed consent.\u003c/p\u003e \u003cp\u003eAll patients underwent routine ophthalmic examinations before and after treatment, including best corrected visual acuity, intraocular pressure, slit lamp microscope and fundus examination. Patients were reexamined 1 week after each ICI.\u003c/p\u003e \u003cp\u003eInclusion criteria: (1) Diagnosis of diabetic retinopathy (with or without diabetic macular edema); (2) Presence of hyperreflective foci in the macular center with a size greater than 30 \u0026micro;m, exhibiting artifacts visible on fundus photography; (3) Diagnosis of type 2 diabetes by an endocrinologist.\u003c/p\u003e \u003cp\u003eExclusion criteria: (1) History of previous anti-VEGF treatment or other ocular surgeries; (2) Retinal laser therapy within the past six months; (3) Presence of any other eye diseases that may cause hard exudates in the macula; (4) Coexistence of eye conditions such as glaucoma, retinal detachment, and retinal vein occlusion that may lead to retinal pathology apart from diabetic retinopathy; (5) Inability to obtain clear images due to media opacities such as cataracts; (6) Unstable systemic condition or incomplete clinical data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIntravitreal injection of conbercept\u003c/h2\u003e \u003cp\u003eAn experienced ophthalmologist performed all intravitreal injections aseptically at the temporal limbus through the eyeball\u0026rsquo;s pars plana. All patients received intravitreal injections of conbercept (10 mg/0.2 ml) using a 30-gauge needle. The interval between each injection was more than 1 month. A 1-week variation was allowed for every injection interval.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOCT evaluation\u003c/h2\u003e \u003cp\u003eRetinal imaging was conducted using a Spectralis OCT system (Heidelberg Engineering GmbH, Germany) across a 2 mm\u0026times;2 mm area centered on the fovea. HEs are defined as OCT-detected lesions with a size larger than 30 \u0026micro;m and the presence of artifacts, resembling the retinal pigment epithelium-Bruch's complex. These HEs are predominantly located in the inner retinal layers, spanning from the nerve fiber layer to the outer nuclear layer. The measurement of HEs was performed using ImageJ software (version 1.46r, Rawak Software Inc., Stuttgart, Germany). OCT scans were imported into ImageJ, converted to 8-bit, and then sharpened to enhance clarity. Subsequently, relevant areas were selected for measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eThe Dil-Ox-LDL was purchased from Invitrogen (L34358; Carlsbad, CA, USA). The Ox-LDL and primary antibodies against anti-Ox-LDL were purchased from Yubo Biomed Technologies (Shanghai, China). The conbercept was purchased from Chengdu Kanghong Pharmaceutical Group Co., Ltd. (Sichuan, China). The DMEM High Glucose Medium (SH30021.01B) was purchased from HyClone (Logan, UT, USA). CY3 goat anti-rabbit IgG (GB21303), FITC goat anti-mouse IgG (GB22301) and β-actin were purchased from Servicebio (Wuhan, China). The primers were purchased from Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRat M\u0026uuml;ller cell (rMC-l) culture\u003c/h2\u003e \u003cp\u003eTransformed rat retinal M\u0026uuml;ller cell line (rMC-l) was kindly supplied by Sarthy (Northwestern University, Chicago, IL, USA). The cells were cultured in high glucose (4.5 g/L) DMEM containing 10% fetal bovine serum (10099158; Gibco, Shanghai, China) and 1% penicillin/streptomycin (C0222; Beyotime Biotechnology) at 37\u0026deg;C with 5% CO₂ in a humidified incubator. Upon reaching\u0026thinsp;~\u0026thinsp;80% confluence in a 10-cm dish, the cells were divided into three groups: vehicle control, Ox-LDL (10 \u0026micro;g/mL)-treated group, and Ox-LDL (10 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;conbercept (100 \u0026micro;g/mL)-treated group. Two types of Ox-LDL were used in different experiments: 10 \u0026micro;g/mL Dil-Ox-LDL (ThermoFisher, USA) for immunofluorescence, flow cytometry and qRT-PCR, and 10 \u0026micro;g/mL Ox-LDL (Yubo, Shanghai, China) for Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8; C0037; Beyotime Biotechnology). Cells were seeded in 96-well plates and treated with varying concentrations of conbercept (ranging from 10 to 500 \u0026micro;g/mL) for 24 hours. Post-treatment, 10 \u0026micro;L of CCK-8 solution was added to each well and incubated in the dark for 1 hour. Subsequently, the optical density was quantified using a microplate reader by measuring absorbance at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eImmunofluorescence of Ox-LDL in rMC-l cells\u003c/b\u003e\u003c/h2\u003e \u003cp\u003erMC-l cells were incubated with Ox-LDL, either with or without conbercept, for 24 hours. The cells were then fixed in cold methanol for 30 minutes and blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour. Subsequently, the cells were first incubated with anti-Ox-LDL (1:100) and glial fibrillary acidic protein (GFAP) (1:500) overnight at 4℃. And then, after three 5-minute washes in PBS, incubated with the corresponding secondary antibody (1:1,000, anti-rabbit CY3; 1:1,000, anti-mouse FITC) for 2 hours at room temperature. Dil-Ox-LDL was used in accordance with the manufacture\u0026rsquo;s protocol. Then, the cells were further incubated with 4'6-diamidino-2-phenylindole (DAPI,100 ng/mL) for 5 minutes, followed by three washes (5 minutes each wash). Finally, the slides were visualized with a confocal microscope (LSM 800; Zeiss Microsystems, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and Western blot\u003c/h2\u003e \u003cp\u003eThe rMC-l cells were incubated for 24 hours with Ox-LDL, in conditions with and without conbercept supplementation. Cells were lysed in protein extraction radioimmunoprecipitation assay (RIPA; P0013B; Beyotime Biotechnology) buffer on ice. After 15-second ultrasonic treatment, the samples were placed on ice for 30 minutes before centrifugation. Protein concentrations were determined with bicinchoninic acid (BCA) Protein Assay Kit (A5586; Thermo Scientific, Shanghai, China). Equal amounts of protein were resolved on 12% SDS-polyacrylamide gels and subsequently transferred electrophoretically onto nitrocellulose membranes. The membranes were blocked in 5% PBS buffered nonfat milk at room temperature for 30 minutes, and then separately incubated with anti-Ox-LDL (1:1,000) or β-actin (1:2,000) overnight at 4℃. After being washed three times with 0.1% TBS-buffered Tween-20 (TBST), the membranes were incubated with the corresponding secondary antibodies (1:2,000, anti-rabbit) at room temperature for 2 hours, followed by washes with TBST (three times). Detection of the bands was done using Chemi Doc Touch System (Bio-Rad). Immunoblots were visualized by enhanced chemiluminescence (ECL) and analyzed using ImageJ software version 1.37 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"http://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Samples were normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eDil-Ox-LDL, either with or without conbercept, was added to the medium and incubated for 4 hours. After three 5-minute washes in PBS, adherent rMC-l cells were collected, then digested with trypsin. The fluorescence of cells was analyzed with a flow cytometer (PE-A; Beckman CytoFLEX). Data were analyzed with FlowJo software (Tree Star Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time Polymerase Chain Reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThe rMC-l cells were treated with Ox-LDL and Ox-LDL\u0026thinsp;+\u0026thinsp;conbercept for 24 hours. The mRNA expression levels were analyzed by qRT-PCR (CXF96, Bio-Rad) using SYBR Green (208054, QIAGEN, Germany) based gene expression. In brief, adherent rMC-l cells were lysed and the total RNA was extracted for cDNA synthesis (K16225, Thermo, USA). Levels of mRNA expressions were normalized by the intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data were expressed as a relative to the controls. The information for primers was provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of patients with DR or DME and HEs area comparisons after anti-VEGF treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaseline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1st ICI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2nd ICI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3rd ICI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEyes (No.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients (No.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (male/female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean ages (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHEs area (mm\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eICI:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eintravitreal conbercept injection\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe information of rat primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNucleotide Sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eLOX-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CCACAAGACTGGATCTGGCAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGATAGGCAATTCTCCCGACT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCD36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGCTGCACCACATATCTACACA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGAATGGATCTTTGTAACCCCAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eTNF-α\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATGGGCTGTACCTTATCTACTCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GAGGCTGACTTTCTCCTGGTA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eABCG1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TCGAATCTCGTGCCGTACCTG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCTGTTCTGATCACCGTACTCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eSRA2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CTTGGCACTGCTTCACGAAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TTCTTGTACCAGCAGTGCCAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eTLR4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATCCCTGCATAGAGGTACTTCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATCCAGCCACTGAAGTTGTGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GACATGCCGCCTGGAGAAAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGCCCAGGATGCCCTTTAGT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and analyzed using pairwise t-tests or one-way ANOVA. A \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. For data requiring statistical analysis, the experiments were repeated at least 3 times.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePatient characteristics and the impact of Anti-VEGF therapy on reduction of HEs\u003c/h2\u003e \u003cp\u003eTwenty-one eyes from 17 patients with DR or DME were retrospectively reviewed in this study and baseline characteristics are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The participants included 10 females and 7 males, and the average age was 56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2 years old. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the OCT image of a 72-year-old female patient with DME exhibited a progressive reduction in macular edema size following an increasing number of conbercept injections. Notably, there was a concurrent decrease in HEs within the inner nuclear layer (INL), corresponding to the escalating frequency of conbercept injections. This observation suggests potential activation of M\u0026uuml;ller cells' phagocytic function, considering their anatomical localization within the INL. Statistically, there was no significant change in the area of HEs between baseline and the first follow-up, with measurements ranging from 1.39 mm\u0026sup2; \u0026plusmn; 1.41 mm\u0026sup2; to 1.38 mm\u0026sup2; \u0026plusmn; 1.3 mm\u0026sup2; (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.938). However, a significant reduction was observed at the second and third follow-ups, with the HEs area measured at 0.77 mm\u0026sup2; \u0026plusmn; 0.9 mm\u0026sup2; (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021) and 0.45 mm\u0026sup2; \u0026plusmn; 0.66 mm\u0026sup2; (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003erMC-l cells demonstrated phagocytosis of Ox-LDL\u003c/h2\u003e \u003cp\u003eUnder high glucose conditions, confocal microscopy analysis revealed that Ox-LDL engulfment occurred in the cytoplasm of GFAP-labeled rMC-l cells, with close proximity to the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, 2.5D reconstruction of Dil-Ox-LDL signals demonstrated their localization near the nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), confirming the occurrence of phagocytosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConbercept decreased rMC-l cells viability in a dose-dependent manner\u003c/h2\u003e \u003cp\u003eThe viability of rMC-l cells under high glucose conditions exhibited a dose-dependent response upon treatment with different concentrations of conbercept. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the cell viability of rMC-l cells was observed to be 94.40% (10 \u0026micro;g/mL, n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), 88.49% (50 \u0026micro;g/mL, n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 92.23% (100 \u0026micro;g/mL, n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 80.42% (300 \u0026micro;g/mL, n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and 70.76% (500 \u0026micro;g/mL, n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of the control, respectively, when treated with increasing concentrations of conbercept. Considering that conbercept exhibits a comparable and relatively modest (less than 12%) decline in rMC-l cell viability within the concentration range of 10\u0026ndash;100 \u0026micro;g/mL, with a notable increase in inhibition to 20% at 300 \u0026micro;g/mL, it can be inferred that the concentration range of 0-100 \u0026micro;g/mL of conbercept is relatively safe for rMC-l cells. Consequently, a treatment concentration of 100 ug/mL was chosen for subsequent cellular experiments.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eConbercept significantly enhanced the phagocytosis of Ox-LDL by rMC-l cells\u003c/h2\u003e \u003cp\u003eTo further verify the increased uptake of Ox-LDL by rMC-l cells under high glucose conditions and explore the impact of conbercept, we employed immunofluorescence and western blot techniques to visualize and quantify the internalized amounts of Dil-Ox-LDL and Ox-LDL within the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The analysis showed that conbercept treatment led to a 23% increase (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in intracellular Dil-Ox-LDL levels by immunofluorescence, as well as a 30% increase (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in Ox-LDL levels in rMC-l cells by western blot. Additionally, flow cytometry analysis confirmed a significant 21% enhancement (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in Dil-Ox-LDL uptake with conbercept treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results indicated that conbercept could effectively enhance the phagocytotic activity of rMC-l cells towards Ox-LDL under diabetic conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eConbercept upregulated the expression of scavenger receptors and cholesterol efflux transporters in Ox-LDL-induced rMC-l cells\u003c/h2\u003e \u003cp\u003eqRT-PCR analysis revealed that treatment with Ox-LDL upregulated the expressions of several receptors and transporters involved in lipid metabolism, including RAGE (receptor for advanced glycation end products), LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), TLR-4 (toll-like receptor 4), CD36 (cluster of differentiation 36), SR-AⅡ (scavenger receptor class A, type II), and ABCG-1 (ATP binding cassette subfamily G member 1). Notably, the addition of conbercept to Ox-LDL-treated cells further amplified the expressions of these genes, except for ABCG-1 which exhibited a decrease. This suggests a differential regulatory effect of conbercept on genes associated with lipid metabolism and phagocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additionally, our study results indicate that there is a correlation between the expression levels of RAGE and SR-AⅡ, CD36, as well as between TLR-4 and LOX-1, CD36 genes in rMC-l cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur results revealed a novel mechanism of action of conbercept in the high glucose environment, offering innovative insights for the resolution of HEs in DR or DME. Clinical studies have demonstrated that retinal photocoagulation therapy, intravitreal injection of steroid hormones or anti-VEGF therapy can effectively alleviate HEs, and the mechanism involves the regulation of microvascular leakage, lipid accumulation and inflammatory response [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The results of our study are consistent with previous findings, demonstrating a significant reduction in the area of HEs within the macular region following ICI. This reduction was particularly prominent at the INL, which corresponds to the location of M\u0026uuml;ller soma, indicating the activation of phagocytic activity of M\u0026uuml;ller by conbercept (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Subsequent \u003cem\u003ein vitro\u003c/em\u003e investigations confirmed the phagocytic effect of M\u0026uuml;ller glia on Ox-LDL, which can be enhanced by conbercept (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), thereby providing a more comprehensive understanding of the mechanism underlying reduction in HEs following anti-VEGF treatment and offering significant clinical implications.\u003c/p\u003e \u003cp\u003eHEs formation results from increased vascular permeability due to lesions in the microvessels in the retina. Normally, endothelial cells of retinal blood vessels form tight junctions that prevent plasma components from entering retinal tissue [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. But in DR or DME, impaired vascular barrier function leads to leakage of lipids and proteins from the blood into the parenchymal tissue of the retina. These leakages usually accumulate between the OPL and the INL of the retina, because the anatomy and vascular supply of this region make it a reservoir for leakages. Specifically, the OPL is the synaptic region of neurons with a rich microvascular network, while the INL contains the nucleus of retinal cells, and the structural characteristics of these layers lead to the easy deposition of exudates to form HEs here.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated the phagocytic functions of both M\u0026uuml;ller glia and microglia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. M\u0026uuml;ller cells are more likely to be directly involved in HEs clearance due to their anatomic location. The cell body of M\u0026uuml;ller glia is situated in the INL, with its fibers extending from the inner limiting membrane to the outer nuclear layer. This unique cellular morphology not only provides structural support for maintaining retinal homeostasis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], but also facilitates direct contact between M\u0026uuml;ller glia and the region where HEs typically accumulate, specifically between the OPL and INL. Therefore, it is reasonable to speculate that M\u0026uuml;ller glia, owing to their strategic location and functional role, are likely to be the primary mediators of exudative material clearance in the retina when HEs accumulate. Our study further substantiated the phagocytic function of M\u0026uuml;ller glia, particularly for lipid products (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a finding that may have positive implications for improving retinal lipid metabolism and alleviating hard exudate accumulation, among others.\u003c/p\u003e \u003cp\u003eThe upregulation of scavenger receptors such as RAGE, SR-AⅡ, and CD36 in M\u0026uuml;ller glia in response to conbercept treatment implies a direct influence on the cells' lipid handling capabilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), crucial for mediating the clearance of Ox-LDL, a key factor in the formation of HEs. Indeed, hyperglycemia has been reported to induce the upregulation of RAGE, leading to RAGE signaling and subsequent pro-inflammatory responses by retinal M\u0026uuml;ller glia, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e upon exposure to high glucose [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, advanced glycation end-products (AGEs) stimulate the uptake of Ox-LDL into macrophages through the Cdk5-CD36 pathway via RAGE-mediated oxidative stress [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Findings suggest that SR-AII could directly prevent the activation of the RAGE-MAPK-NF-κB signaling pathway and diminish the secretion of proinflammatory cytokines in microglia [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our study revealed a positive correlation between the gene expression levels of RAGE with CD36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting that conbercept potentially enhanced the uptake of Ox-LDL into rMC-l cells by promoting the CD36 pathways, which are induced by the AGEs-RAGE axis. The increased expression of SR-AⅡ by conbercept can reduce the effects of inflammation activation caused by RAGE. This upregulation is pivotal, as scavenger receptors, particularly CD36 and SR-AⅡ, are known to be the principal receptors for binding and uptake of Ox-LDL in macrophages, exhibiting specific recognition and binding capabilities for Ox-LDL that facilitate lipid engulfment, intracellular degradation, and metabolism [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This process typically involves endosome formation and cytoskeletal modifications, where SR-AⅡ functions to dampen lipid-related inflammatory responses, and CD36 actively participates in intracellular lipid transport and metabolism [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. By enhancing the phagocytotic capacity of M\u0026uuml;ller glia through these pathways, conbercept may help mitigate the buildup of lipid-rich deposits, a hallmark of diabetic retinal diseases like DR, and contribute to the reduction of lipid deposition and plaque formation.\u003c/p\u003e \u003cp\u003eTLR-4 is mainly recognized for detecting bacterial lipopolysaccharides (LPS) and is vital in regulating metabolism, especially in lipid metabolism related to diseases like atherosclerosis and diabetes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Once activated, TLR-4 affects lipid uptake in different cells by altering the expression of genes related to lipid metabolism and transport. Additionally, TLR-4 activation can enhance the phagocytic activity of BV2 cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These receptors, central to the innate immune system, demonstrate intricate interactions, i.e., TLR-4 influences the expression or activity of other lipid receptors such as LOX-1 and CD36, thereby impacting cellular lipid uptake. Specifically, LOX-1 serves as a receptor for Ox-LDL, recognizing and binding it to facilitate cellular uptake, intracellular degradation, and metabolism, thereby reducing extracellular lipid accumulation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Concurrently, the synergistic effect between LOX-1 and TLR-4 mutually enhanced their activity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while TLR-4 further augmented the uptake of Ox-LDL by macrophages via CD36 upregulation and initiates inflammatory signaling pathways [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The observed modulation of LOX-1, CD36, and TLR-4 expression by conbercept suggests a multifaceted impact on the retinal immune response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our findings underscore a significant correlation between TLR-4 and the expression of LOX-1 and CD36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), underlining the complexity of conbercept's role in modulating lipid metabolism and the immune response in the context of DR and DME, potentially contributing to the reduction of chronic inflammation associated with these conditions.\u003c/p\u003e \u003cp\u003eOx-LDL typically enters lysosomes and is hydrolyzed into free cholesterol and fatty acids by neutral cholesteryl ester hydrolases (CEH), with subsequent metabolism occurring via ABCA1 and ABCG1 pathways [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The downregulation of ABCG1 expression may be associated with the cellular endeavor to maintain cholesterol homeostasis, potentially serving as a mechanism for attenuating cholesterol efflux in response to heightened phagocytosis of Ox-LDL. The observed downregulation of ABCG-1 suggests a strategic shift in the M\u0026uuml;ller glia' lipid metabolism towards enhanced phagocytic clearance of Ox-LDL, which could serve as a protective mechanism against oxidative damage and inflammation. This shift aligns with the broader role of M\u0026uuml;ller glia in retinal homeostasis and supports the hypothesis that conbercept's therapeutic benefits may include the modulation of lipid metabolism, enhancing cellular resilience against the diabetic milieu.\u003c/p\u003e \u003cp\u003eThe dual role of conbercept in both inhibiting pathological angiogenesis and modulating M\u0026uuml;ller glia function highlights its potential as a more comprehensive therapeutic agent for DR and DME. These findings suggest that the therapeutic effects of conbercept might not be limited to VEGF inhibition but could also include modulation of cellular metabolic and immune responses, which are crucial in the pathogenesis of diabetic retinal diseases. Considering the complex interplay of angiogenesis, inflammation, and lipid metabolism in the progression of DR and DME, our study underscores the need for a holistic approach in treatment strategies. Conbercept's ability to simultaneously influence various pathways offers a promising therapeutic avenue, potentially addressing multiple aspects of retinal pathology. Further investigation into this relationship can potentially lead to more effective and comprehensive treatment approaches for retinal vascular diseases associated with HEs.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, for the expression levels of scavenger receptors, only mRNA levels were detected, which need to be confirmed by their protein expressions. Furthermore, we did not investigate whether conbercept exerts its influence on these molecules through direct regulation or indirectly via antagonism of its own targets. Additionally, the primary mediator of M\u0026uuml;ller-cell phagocytosis among VEGFA, VEGFB, or PlGF targeted by conbercept was not explored in this study.\u003c/p\u003e \u003cp\u003eIn conclusion, conbercept treatment enhanced the phagocytotic effect of retinal M\u0026uuml;ller glia under diabetic conditions, and this effect might be mediated by a series of key phagocytic receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This study broadens our understanding of conbercept's role in retinal health, particularly in the context of diabetes-induced retinal changes. The enhanced phagocytotic activity of M\u0026uuml;ller glia and the modulation of key receptors involved in lipid metabolism and innate immunity by conbercept could be critical in developing more effective treatment strategies for retinal diseases like DR and DME. Future research should aim to further elucidate the molecular mechanisms underpinning these effects and explore the long-term outcomes of such modulation on retinal health and disease progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China, No. 82171062 (to JFZ), No. 82301222 (to CYZ) and No. 32201244 (to XSL)\u003c/p\u003e\u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eThis study was approved by the Clinical Research Ethical Committee of Wannan Medical College Yijishan Hospital and adhered to the principles of the Declaration of Helsinki. All individual participants provided written informed consent.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.Z. wrote the manuscript. S.Q. contributed to the interpretation of these results for the manuscript.H.X. and Y.L. supervised the project.X.L. and Y.Z. provided essential theoretical insights and critically revised the manuscript for important intellectual content.C.Z. and J.Z. conducted a thorough review and final approval of the manuscript prior to submission.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYanko L, Ungar H, Michaelson IC. The exudative lesions in diabetic retinopathy with special regard to the hard exudate. Acta Ophthalmol (Copenh). 1974;52(1):150-160.\u003c/li\u003e\n\u003cli\u003eDomalpally A, Ip MS, Ehrlich JS. 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Med Res Rev. 2008;28(4):483-544. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Conbercept, Müller glia, oxidized low-density lipoprotein, phagocytosis, anti-VEGF","lastPublishedDoi":"10.21203/rs.3.rs-4405680/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4405680/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eHard exudates (HEs) are one of the main factors affecting vision in patients with diabetic retinopathy (DR) and diabetic macular edema (DME). Anti-vascular endothelial growth factor (anti-VEGF) is the main therapy for DME, but its effect on M\u0026uuml;ller glia phagocytosis remains unclear. The aim of this study was to investigate the effects of conbercept on phagocytosis of HEs by M\u0026uuml;ller glia in DR and elucidate the underlying mechanism(s).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTwenty-one eyes from 17 patients diagnosed with DR or DME underwent optical coherence tomography (OCT) imaging at baseline and one week after each consecutive intravitreal conbercept injection (ICI), administered three times with an interval of over one month between each injection, to assess changes in HEs. The rat M\u0026uuml;ller cell line (rMC-l) was cultured under high glucose conditions to mimic a diabetic environment. Cells were treated with oxidized low-density lipoprotein (Ox-LDL) alone or in combination with conbercept. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8). The phagocytosis of Ox-LDL by rMC-l cells with or without conbercept was examined via immunofluorescence, flow cytometry, and Western blot. Gene expressions of several scavenger receptors and transporters involved in lipid metabolism, including RAGE, LOX-1, TLR-4, CD36, SR-AII, and ABCG-1, were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe area of the HEs exhibited minimal changes following the initial ICI, whereas a significant decrease in area was observed after three consecutive injections. The viability of rMC-l cells was obviously reduced at higher concentrations of conbercept (\u0026gt;\u0026thinsp;100 \u0026micro;g/mL). Under high glucose conditions, rMC-l cells phagocytosed Ox-LDL, particularly locolized around the nucleus, and conbercept further enhanced this phagocytosis. Ox-LDL treatment increased the expression of the receptors and transporters involved in phagocytosis and lipid metabolism, while conbercept treatment further enhanced their expressions, except for ABCG-1 which was decreased.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study confirmed that Conbercept treatment can effectively reduce the area of HEs in DR and DME. This therapeutic effect may be attributed to the enhanced phagocytic capability of M\u0026uuml;ller glia towards HEs, which is mediated by the regulation of key lipid metabolism receptors and transport proteins. These findings provide novel mechanisms underlying the facilitation of HEs clearance in DR and DME by anti-VEGF therapy, thereby establishing a theoretical basis for future therapeutic strategies.\u003c/p\u003e","manuscriptTitle":"Conbercept enhances the phagocytic activity of retinal Müller glia towards hard exudates in diabetic retinopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-30 14:34:21","doi":"10.21203/rs.3.rs-4405680/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1a1c441-2a77-4052-8509-0a3c50792d36","owner":[],"postedDate":"May 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-31T02:38:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-30 14:34:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4405680","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4405680","identity":"rs-4405680","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}