Sagittaria Sagittaria polysaccharide protects against retinal vascular damage in diabetic mice by suppressing TLR4 signaling pathway and microglial activation

Sagittaria Sagittaria polysaccharide protects against retinal vascular damage in diabetic mice by suppressing TLR4 signaling pathway and microglial activation | 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 Sagittaria Sagittaria polysaccharide protects against retinal vascular damage in diabetic mice by suppressing TLR4 signaling pathway and microglial activation Yang Jiang, Manyu Zhou, Jing Zhang, Yao Liang, Jiazhen Ding, Wenyong Liao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7204796/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Molecular Neurobiology → Version 1 posted 9 You are reading this latest preprint version Abstract Sagittaria sagittifolia polysaccharide (SSP) exhibits anti-inflammatory, antioxidant, lipid-regulating, and hypoglycemic properties, demonstrating therapeutic potential against diabetic retinopathy (DR). This study aimed to investigate the intervention efficacy of SSP in DR mice and its regulatory effects on retinal microglia activation. The type 2 diabetic mouse model was established by high-fat diet feeding combined with streptozotocin (STZ) intravenous injection. At week 9, retinal vascular pathology was assessed via fundus photography and fluorescein angiography. Serum lipid metabolism TG, CHO, LDL and HDL were quantified using an automated biochemical analyzer. Retinal histopathology and thickness were evaluated through HE staining combined with evans blue staining. Microglial activation adjacent to retinal vasculature was visualized by immunofluorescence, while retinal apoptosis was examined using immunohistochemistry and TUNEL staining. Co-localization of TLR4 and Iba was analyzed by immunofluorescence. Protein expression levels of TLR4, Myd88, P-p65, and total p65 in retinal tissues were determined by Western blot. SSP treatment significantly attenuated DR progression, as evidenced by preserved retinal vascular integrity, restored retinal thickness, reduced vascular leakage, lowered fasting blood glucose, and regulated lipid metabolism (reduced TG/TC/LDL-C, increased HDL-C). Furthermore, SSP suppressed pathological recruitment of microglia to retinal vasculature and inhibited their pro-inflammatory morphological transition. Mechanistically, SSP downregulated TLR4/Iba co-expression and inhibited downstream Myd88/NF-κβ signaling pathway. The study results demonstrated that SSP can delay the progression of retinopathy in type 2 diabetic mice. This mechanism seems to be associated with SSP's blood glucose-lowering and lipid-regulating effects, along with its inhibition of microglia-mediated inflammatory responses. diabetic retinopathy Sagittaria Sagittaria polysaccharide microglia TLR4 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Diabetic retinopathy (DR) is one of the most common ocular complications in patients with diabetes, affecting approximately one-third of diabetic patients to varying degrees of severity[ 1 ]. In its early stage, known as non-proliferative diabetic retinopathy (NPDR), the condition typically presents with no obvious symptoms. however, NPDR is likely to progress to proliferative diabetic retinopathy (PDR), a sight-threatening stage that can culminate in severe vision loss and irreversible blindness. Currently, DR has become one of the leading causes of irreversible vision loss among adults worldwide [ 2 ], imposing substantial health and economic burdens on society. The pathological mechanisms of diabetic retinopathy (DR) are highly complex, with inflammatory responses serving as a critical factor influencing disease outcomes. During the non-proliferative diabetic retinopathy (NPDR) stage, substantial evidence indicates that the destruction of the blood-retinal barrier (BRB), degradation of tight junction proteins, and increased vascular permeability in the retina are closely associated with the accumulation of inflammatory factors (IL-1β, IL-6, TNF-α, etc.) [ 3 – 5 ]. These inflammatory mediators promote apoptosis of vascular endothelial cells and pericytes, leukocyte aggregation and adhesion within vessels, and microthrombus formation, ultimately resulting in local retinal ischemia and hypoxia [ 4 , 6 ]. Furthermore, in the proliferative diabetic retinopathy (PDR) stage, inflammatory responses not only synergize with VEGF to drive pathological neovascularization [ 7 ] but also perpetuate chronic inflammation, activate the complement system, and enhance leukocyte adhesion [ 8 ]. These processes culminate in vision-threatening pathological changes such as neovascularization rupture, vitreous hemorrhage, and retinal detachment [ 9 ]. Therefore, controlling the progression of inflammatory responses at early stages is crucial for delaying DR advancement. Microglia, the primary resident immune cells in the retina, play a central role in retinal inflammatory responses. Under physiological conditions, microglia remain in a resting state characterized by a branched morphology, continuously surveilling changes in the microenvironment. Upon injury or pathological stimulation, microglia become activated, manifesting morphological changes, proliferation, migration, and the release of cytokines and chemokines to mediate inflammation [ 10 ]. In retinal diseases, microglial activation often coincides with BRB disruption. Studies demonstrate that under pathological conditions such as retinal ischemia or hypoxia, activated microglia migrate to injury sites and release pro-inflammatory factors (e.g., TNF-α, IL-1β), which directly increase vascular endothelial permeability, exacerbating BRB disruption [ 11 ]. In an in vitro hyperglycemia-induced microglial model, elevated expression of TNF-α and IL-1β was observed in the supernatant [ 12 , 13 ]. To visually assess the impact of such pro-inflammatory microglial supernatants on BRB permeability, Wang et al. co-cultured pericytes and endothelial cells in a Transwell system to simulate BRB. Treatment with microglial supernatant induced significant apoptosis in both cell types and markedly increased BRB permeability [ 12 , 13 ]. Furthermore, NF-κβ, a key pro-inflammatory transcription factor in microglia, is regulated by the classical TLR4 signal. In the inflammatory mechanism of DR, the TLR4/Myd88/NF-κβ signaling pathway has been identified as a critical target for drugs (e.g., asiatic acid, paeoniflorin) and mesenchymal stem cell therapies to suppress inflammatory polarization of retinal microglia [ 11 , 12 , 14 ]. Thus, targeting microglia-driven inflammatory responses and their upstream signaling events—TLR4/Myd88/NF-κβ signaling pathway—represents a promising therapeutic strategy for mitigating DR progression. Sagittaria sagittifolia polysaccharide (SSP), the primary active component of Sagittaria sagittifolia, has demonstrated significant hypoglycemic effect in diabetic mouse models as early as a decade ago [ 15 ]. By reducing blood glucose levels, SSP theoretically holds potential for intervening in diabetic retinopathy (DR). Furthermore, our research team has focused on exploring SSP's applications in ocular and hepatic diseases, revealing its marked anti-inflammatory, antioxidant, and lipid-regulating properties. For instance, SSP has been shown to reduce lens opacity in selenite-induced cataract rats through anti-inflammatory and antioxidant mechanisms [ 16 ], and to mitigate isoniazid-rifampicin combination-induced liver injury in mice via the Nrf2 antioxidant pathway [ 17 ]. However, no studies have specifically investigated SSP's role in DR intervention. Given SSP's dual hypoglycemic-hypolipidemic capacity and anti-inflammatory effects, we aim to explore SSP's potential in DR intervention and elucidate its anti-inflammatory mechanisms within the DR retina. Materials and methods Drug preparation The SSP was extracted from fresh Sagittaria sagittifolia L. [ 17 , 16 ], with Sagittaria sagittifolia L. being an accepted name in the plant list ( http://www.worldfloraonline.org ). SSP solutions were formulated daily in physiological saline at concentrations of 80 mg/mL, 40 mg/mL, and 20 mg/mL for experimental use. Calcium dobesilate (CD), purchased from Beijing Jingfeng Pharmaceutical Group Co., Ltd., was prepared as a 23 mg/mL solution. Gavage volume = body weight (g) × 0.1ml/10g. Grouping, modeling and treatments A total of 36 healthy male C57BL/6J mice (8 weeks old, 20-25g) were purchased from Sibeifu Biotechnology Co., Ltd. (Laboratory Animal Production License No.: SCXK(JING)2024-0001). The experimental protocol of this study has been reviewed and approved by the Animal Experiment Ethics Committee of Beijing University of Chinese Medicine (BUCM-2024102805-4090). After 3 days of acclimatization, the mice were randomly divided into a control group (n = 6) and a high-fat diet (HFD) group (n = 30) using a random number table. The control group was fed a standard diet, while the HFD group received a 45% high-fat diet (Sumeidisen Biomedical Co., Ltd., Art.No.: MD12032) for 2 weeks. All mice were then fasted for 12 hours (overnight fasting with water ad libitum) and subjected to intraperitoneal injection of 50 mg/kg streptozotocin (STZ, Sigma, dissolved in 0.1 M sodium citrate buffer) daily for 5 consecutive days to induce type 2 diabetic models. 72 hours post-injection, fasting blood glucose (FBG) was measured via tail vein sampling. Mice with FBG levels > 11.1 mmol/L were confirmed as successful type 2 diabetic models [ 18 ]. Following successful modeling, the HFD group was further randomized into five subgroups: Model group (untreated diabetic mice), Positive group (CD, 0.23 g/kg), SSP-L (low-dose SSP, 200 mg/kg), SSP-M (medium-dose SSP, 400 mg/kg), SSP-H (high-dose SSP, 800 mg/kg). The Control and model groups received an equivalent volume of physiological saline. Treatments continued for 9 weeks, after which retinal pathology was assessed. Throughout the experiment, diabetic mice remained on the HFD, and the normal group maintained a standard diet. The study protocol and grouping are depicted in Fig. 1 . Fundus photography and fluorescein angiography. At 9 weeks post-successful induction of diabetes in murine models, mice from all groups were anesthetized using isoflurane (4% for induction, 2% for maintenance). Compound Tropicamide Eye Drops (Shenyang Xingqi Ophthalmology Co., Ltd.) were administered to dilate the pupils, followed by topical anesthesia with Oxybuprocaine Hydrochloride (Santen Pharmaceutical Co., Ltd.). Carbomer gel was applied to the ocular surface, and mice were positioned on the animal platform. Fundus photography of both eyes was performed using the Micron-III Small Animal Retinal Imaging System (Optoprobe, Model: OPTO-RIS). For retinal fluorescein angiography, 10% sodium fluorescein (0.04 mL/mouse) was injected intraperitoneally. Serum biochemical analysis 9 weeks after the establishment of the type 2 diabetic model, the levels of triglycerides (TG), total cholesterol (T-CHO), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in mouse serum were measured using a fully automated serum biochemical analyzer. Evans blue staining Three mice from each group were selected for retinal Evans Blue (EB) staining. A 3% EB solution was prepared. After tail vein injection of EB for 30 min, the mice were anesthetized and euthanized. The right eye was enucleated and fixed in 4% paraformaldehyde for 30 min. The fixed eyeball was immersed in PBS for 10 min. The retina was dissected on a glass slide, and the cup-shaped ocular wall was divided into 3–4 evenly spaced sections centered on the optic nerve head (ONH) to prepare retinal flat mounts. The left eye was processed for paraffin embedding. HE staining and retinal thickness measurement The left eyes of mice from each group (including Evans Blue-stained eyeballs, totaling 6 eyeballs) were fixed in 4% paraformaldehyde for 48 hours. Subsequently, the tissues were trimmed and rinsed overnight to remove residual paraformaldehyde. The samples underwent dehydration and clearing through a graded series of ethanol (60% for 2 h, 80% for 2 h, 90% for 4 h, 95% I for 2 h, 95% II for 2 h, 100% I for 2 h, 100% II for 2 h) followed by xylene (xylene I for 1 h, xylene II for 2 h). Tissue infiltration was performed with paraffin (three times, 30 min each) before embedding. Sections were prepared using a microtome and dried for subsequent use. For staining, the sections were deparaffinized and rehydrated sequentially in xylene I (20 min), xylene II (20 min), 100% ethanol I (5 min), 100% ethanol II (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 60% ethanol (5 min), and finally deionized water (3 min). Hematoxylin staining was performed for 5 min, followed by immediate differentiation in 1% acid alcohol for 3 sec and thorough rinsing under running tap water for 10 min. Counterstaining was carried out with eosin for 3 min, followed by rapid dehydration in 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II (30 sec each). Finally, the sections were cleared and mounted for microscopic observation. Retinal thickness was measured at six locations (0.2 mm, 0.6 mm, 1 mm, 1.4 mm, 1.8 mm, and 2.2 mm from the ONH), with full-thickness retinal thickness recorded at each point. TUNEL staining Paraffin sections undergo dewaxing and hydration. A hydrophobic barrier pen was used to outline the tissue sections. Staining was performed according to the manufacturer’s instructions of the TUNEL Apoptosis Detection Kit (LabLead, China). The reagents (Proteinase K, 3% H 2 O 2 , and fluorescein-labeled TUNEL reaction mixture reagents) were prepared in sufficient quantities. Then, these slices underwent permeabilization (Proteinase K, 1:50, 30 min), blocking (3% H 2 O 2 , 20 min), and TUNEL reaction (fluorescein-labeled TUNEL reaction mixture reagents, 60 minutes), sealed with an anti-fluorescence quencher containing DIPA. Apoptotic cells in the retina were visualized and analyzed under a fluorescence microscope. Immunohistochemistry Deparaffinized and rehydrated sections were prepared using standard protocols, and tissue boundaries were outlined with a hydrophobic barrier pen. After hydration, sections were rinsed three times with deionized water (1 min each). Antigen retrieval was performed using preheated EDTA buffer (pH 9.0, 100°C) twice (5 min each). Endogenous peroxidase activity was blocked with 2% H 2 O 2 for 10 min, followed by three PBS washes (5 min each). Non-specific binding sites were blocked with 10% BSA. Sections were incubated overnight at 4°C with primary antibodies: Iba (1:100, CST, USA), Caspase-3 (1:50, CST, USA). After incubation, sections were washed three times with PBS (1 min each) and incubated with HRP-conjugated secondary antibody (goat anti- rabbit IgG, 1:1000, Proteintech, China) for 1 h. Further, stain the sections using DAB and hematoxylin for 5 min, followed by gradient dehydration, clearing, and mounting. Brown precipitate indicating target protein expression was visualized under a light microscope. Immunofluorescence Immunofluorescence staining was performed on both paraffin sections and evans blue-stained retinal flat mounts. For paraffin sections, the enhanced immunofluorescence Kit (Product: AFIHC033, Hunan Aifang Biotechnology Co., Ltd., China) was used. The processing steps for paraffin sections included deparaffinization, rehydration, antigen retrieval, peroxidase blocking, and 10% BSA blocking, consistent with the immunohistochemistry protocol. Sections were incubated with Iba antibody (1:100, CST, USA) at 37 ℃ for 2 h, followed by three PBS washes (5 min each). A Polymer-HRP-conjugated secondary antibody was then applied for 30 min, followed by PBS washes (3 × 5 min). Signal amplification was achieved using TSA fluorescent dye (TYR-520Plus). To remove non-covalently bound primary-secondary-HRP complexes, sections underwent heat-induced antigen retrieval in EDTA buffer (pH 9.0, 100°C, 30 min). Repeat the steps of peroxidase blockade, BSA blocking, primary antibody incubation (TLR4, 1:200, Proteintech, China), Polymer HRP secondary antibody incubation, TSA fluorescent dye (TYR-570Plus fluorescent dye) incubation for the second round of labeling. Sections were cover slipped with an anti-fluorescence quencher containing DAPI and analyzed under a fluorescence microscope to assess co-localization of Iba and TLR4. Retinal flat mounts were prepared as described in the part 2.5 and subjected to immunofluorescence staining. Briefly, the retinas were incubated in 0.3% Triton X-100 at 4°C for 1 h. After blotting excess Triton X-100 solution with filter paper, 10% BSA blocking buffer was applied and incubated at 37 ℃ for 2 h. The blocking buffer was removed, and the primary antibody diluent (Iba, 1:100, CST, USA) was added, followed by incubation at 37 ℃ for 3 h. Unbound primary antibody was blotted with filter paper, and the samples were washed three times with PBS. A FITC-conjugated goat anti-rabbit secondary antibody (1:200, Abcam, UK) was applied and incubated at 37 ℃ for 1 h. After washing with PBS, the retinas were mounted with an anti-fluorescence quencher containing DAPI. Perivascular expression of Iba was visualized using a fluorescence microscope. Western blot Total proteins were isolated from retina using RIPA buffer (containing 1% PMSF and protease inhibitors). Equal amounts of protein samples were subjected to electrophoretic separation using SDS-PAGE, and the resulting products were then transferred onto polyvinylidene difluoride (PVDF) membranes from Millipore. The membrane was blocked with 5% skim milk/TBST for 1 h at room temperature on a shaker, followed by three TBST washes (10 min each). After washing, the membranes were incubated overnight at 4 ◦C with primary antibodies, including anti-TLR4 (1:1000, CST, USA), anti-Myd88 (1:1000, Abcam, UK), anti-p65/p-p65 (1:1000, CST, USA), and anti-β-actin (1:5000, Proteintech, China). After TBST washes (3 × 10 min), the membrane was incubated with HRP-conjugated secondary antibody (goat anti-rabbit IgG, 1:5000, Proteintech, China) for 1 h at room temperature and washed again. Protein bands were visualized using an Enhanced Chemiluminescence (ECL) Detection Kit (Lablead, China) and imaged with a chemiluminescence system. Statistical analysis Data were analyzed using SPSS 20.0 (IBM Corp., Armonk, NY), and histograms were plotted with GraphPad Prism 5 (GraphPad, San Diego, CA). Continuous variables are expressed as mean ± standard deviation (mean ± SD). If data met normality (Shapiro-Wilk test) and homogeneity of variance (Levene’s test), one-way ANOVA was applied. For datasets with violated variance assumptions, the non-parametric rank-sum test (Mann-Whitney U test) was used. P-values < 0.05 were considered statistically significant. Result SSP protects the retinal vasculature in diabetic mice The success of the diabetic retinopathy (DR) model was assessed using fundus photography and fundus fluorescein angiography (FFA). In fundus images (Fig. 2 A), retinal vessels in the control group radiated outward from the optic disc with clear and smooth vessel walls. Arteries appeared light red, while veins were dark red. In contrast, the model group exhibited grayish-white exudates around retinal veins. FFA revealed fluorescent leakage in regions corresponding to the grayish-white exudates, indicating disruption of retinal vascular integrity and pathological changes such as extravasation of plasma components (Fig. 2 A). In fundus images, all groups except the control group showed varying degrees of grayish-white exudates. Compared to the model group, the extent of exudates was reduced in both the CD and SSP groups ( Fig. 2 B). CD, a clinically used vasoprotective agent for DR treatment [ 19 ], served as a positive drug. Our results suggest that SSP exhibits similar vasoprotective effects to CD, though its underlying mechanisms require further investigation. SSP reduces FBG, serum TG, T-CHO, LDL levels, and increases HDL levels in diabetic mice. To explore whether SSP delays DR progression via glycemic control, FBG levels were monitored throughout the experiment (Fig. 2 C). All mice exhibited normal FBG levels prior to STZ induction (Fig. 2 D). At 72 hours post-STZ injection, diabetic models were confirmed successful when FBG > 11.1 mmol/L (Fig. 2 E). SSP with high dose (SSP-H) effectively reduced FBG in diabetic mice (Fig. 2 F), with statistical significance observed only at week 8 (P < 0.05). Hyperlipidemia, a common comorbidity in type 2 diabetes, was assessed in our experiment. Compared to the control group, all other groups showed significant increases in serum total cholesterol (TC), cholesterol (T-CHO), and low-density lipoprotein (LDL), alongside reduced high-density lipoprotein (HDL) levels. SSP administration notably regulated lipid metabolism (reduced TG/TC/LDL-C, increased HDL-C) (Figs. 2 G–J). SSP reduces blood leakage in the retina of diabetic mice Following tail vein injection of EB solution, eyeballs from each group were paraffin-embedded, sectioned, and subjected to HE staining. Under light microscopy, while retinal micromorphology in diabetic mice (Model group) showed no significant structural changes compared to controls, vascular walls appeared thinner, with red exudates observed around the vessels (Fig. 3 , Model group; red triangles: vessels, yellow triangles: exudates). To confirm whether these exudates originated from intravascular components, fluorescence microscopy (532nm) revealed red fluorescence around the vessels (principle: evans blue binds rapidly to plasma albumin and appears red under 532nm excitation light stimulation). Notably, both the CD group and SSP-H group effectively increased vascular wall thickness. Fluorescence microscopy further demonstrated a significant reduction in perivascular exudates in these groups (Fig. 3 , CH/SSP-H groups). These findings indicate that SSP protects vascular integrity and mitigates Pathological leakage of blood vessels. SSP increases retinal thickness in diabetic mice Retinal thinning is an early hallmark of diabetic retinopathy (DR), preceding microangiopathy [ 20 ]. In this study, retinal morphology was assessed using HE staining, and the retinal thickness was measured at six locations relative to the optic nerve head (ONH): 0.2 mm, 0.6 mm, 1 mm, 1.4 mm, 1.8 mm, and 2.2 mm (Fig. 4 A). Compared to the control group, diabetic mice exhibited significant retinal thinning (Fig. 4 B). The CD and SSP effectively increased retinal thickness of diabetic mice (Fig. 4 C), with statistical significance observed at 2.2 mm (Fig. 4 D) from the ONH (P < 0.05). SSP suppresses retinal apoptotic cells in diabetic mice Apoptosis is a primary mechanism of retinal cell death in early-stage of DR and a key contributor to retinal thinning [ 20 , 21 ]. To evaluate apoptosis in the retina of diabetic mice, we assessed caspase-3 + cells and TUNEL-positive cells in the retina. Caspase-3 expression was quantified in retinal regions approximately 200 µm from the ONH. The results demonstrated that the number of Caspase-3 + cells was significantly increased in the model group in the inner nuclear layer (INL) of the retina, while SSP markedly reduced the quantity of these cells (Fig. 5 A). TUNEL-positive cells were significantly increased in the model group, while SSP administration effectively suppressed apoptosis of retinal cells (Fig. 5 B). We used different methods to display the apoptotic cells of each group of mice, and found that SSP could stably inhibit the number of apoptotic cells in the diabetic mice retina. SSP reduces perivascular microglial accumulation in diabetic mice Retinal flat mounts were prepared from mice injected with EB solution. Microglia adjacent to blood vessels were visualized via immunofluorescence. EB dye emitted red fluorescence under fluorescence microscopy to demarcate blood vessels (white dashed box), while Iba (a microglial marker) was labeled with green fluorescence. In diabetic mice (Fig. 6 , Model group), a significant increase in perivascular microglial density was observed. SSP-H treatment effectively reduced microglial clustering around retinal vessels (Fig. 6 , SSP-H group). These findings suggest that retinal vessels in diabetic mice are surrounded by increased microglia, though their detrimental effects on vascular integrity and the BRB require further validation. SSP modulates microglial morphology in diabetic mice Microglial chemotaxis toward blood vessels is accompanied by functional and morphological changes [ 10 ]. To investigate this, we analyzed microglial morphology on retinal cross-sections using immunohistochemistry. In model group ( Fig. 7A ), microglia exhibited shortened processes, enlarged soma, and reduced total branch length per field ( Fig. 7C , quantified as cumulative process length), along with decreased branch points ( Fig. 7D ). Compared to the model group, SSP-H treatment ( Fig. 7B/C/D ) significantly increased microglial process length ( P < 0.01) and branch points ( P 0.05). These results suggest that SSP promotes microglial process elongation and arborization, highlighting a morphological shift toward a surveillant and non-inflammatory state. SSP inhibits TLR4 expression on the retinal microglia and the Myd88/NF-κβ signaling pathway in diabetic mice Activated microglia undergo morphological changes (enlarged soma, shortened processes, and reduced branch points) and promote retinal inflammatory responses in diabetic retinopathy (DR) [ 10 , 22 ]. In diabetic retinas, damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) activate microglia via pattern recognition receptors (PRRs). Toll-like receptor 4 (TLR4), a key PRR for DAMPs/PAMPs, was investigated to determine its role in microglial activation. Immunofluorescence revealed significantly increased TLR4 expression in retinal microglia of diabetic mice (white boxes, Model, Fig. 8 A). The SSP treatment decreased co-localization of Iba and TLR4 (white boxes, SSP-H, Fig. 8 A), suggesting SSP may suppresses microglial activation by downregulating TLR4. What’s more, western blot analysis (Fig. 8 B–E) demonstrated that SSP significantly reduced protein levels of TLR4, Myd88 and the radio of p-p65 to p65. These findings indicate that SSP may modulate microglial activity by suppressing the TLR4/Myd88/NF-κβ signaling pathway, thereby attenuating neuroinflammation in the retina of diabetes mice. Discussion Our findings demonstrate that SSP delays DR progression in mice by protecting retinal vasculature, increasing retinal thickness, suppressing apoptosis, and preventing vascular leakage. To elucidate SSP’s anti-DR mechanisms, we first evaluated its effects on primary drivers of DR—hyperglycemia and dyslipidemia. Chronic hyperglycemia induces systemic microvascular complications, including diabetic foot, nephropathy, and retinopathy, which progress to microcirculatory dysfunction, tissue ischemia, and irreversible damage [ 23 – 25 ]. Glycemic control mitigates vascular pathology and associated tissue injury. In our study, SSP-treated mice maintained lower FBG levels than the model group, though statistical significance was limited by sample size at most time points. Hyperlipidemia further exacerbates DR. Preclinical and clinical studies consistently link elevated TG, LDL, CHO, and reduced HDL to DR pathogenesis [ 26 , 27 ], with the TG/HDL ratio serving as a key predictor of retinopathy in type 2 diabetes [ 28 ]. Our prior work showed SSP significantly reduces serum TG and CHO levels in a non-alcoholic fatty liver disease (NAFLD) rat model [ 29 ]. Similarly, SSP corrected dyslipidemia in diabetic mice in our experiments. These results suggested SSP’s dual modulation of glucose and lipid metabolism contributes to DR amelioration. Our results demonstrate that SSP exerts significant protective effects on retinal vasculature in diabetic mice, including maintaining vascular wall integrity and preventing plasma extravasation. However, given the persistent disparity in serum FBG levels between the SSP-treated and control groups, we propose that SSP’s protective mechanisms extend beyond glycemic and lipid regulation. Inflammation, particularly microglial-mediated neuroinflammation, plays a pivotal role in DR pathogenesis. We observed microglial accumulation around retinal vessels in diabetic mice, consistent with findings by Mills et al., who reported that perivascular microglial adhesion and aggregation exacerbate BRB disruption and vascular hyperpermeability [ 30 ]. Notably, SSP treatment markedly reduced perivascular microglial accumulation, suggesting that microglia may serve as a critical cellular target for SSP’s vaso-protective effects. Microglia, the resident immune cells of the retina, play a central role in retinal inflammatory responses. Upon sensing injury signals, microglia transition from a resting "ramified" morphology (characterized by branched processes) to an activated "amoeboid" state, which is associated with phagocytic activity and pro-inflammatory functions [ 10 ]. Immunohistochemical analysis revealed that retinal microglia in diabetic mice exhibited enlarged soma diameter, shortened processes, and reduced process arborization—morphological changes linked to their pro-inflammatory phenotype. SSP treatment effectively inhibited this shift toward an inflammatory morphology. The TLR4/Myd88/NF-κβ signaling pathway is a key upstream regulator of microglial activation and subsequent inflammatory damage [ 31 ]. In diabetes, damage-associated molecular patterns (DAMPs, e.g., advanced glycation end products [AGEs] induced by hyperglycemia) and pathogen-associated molecular patterns (PAMPs, e.g., gut-derived LPS) activate TLR4/MyD88/NF-κβ signaling pathway in microglia [ 32 , 33 ]. To investigate TLR4 activation in DR retinal microglia, immunofluorescence demonstrated increased co-localization of Iba (microglial marker) and TLR4, which was significantly attenuated by SSP. Consistent with this, downstream proteins (Myd88 and NF-κβ) were also downregulated in SSP-treated mice. These findings suggest that SSP may suppress microglial activation in DR by inhibiting the TLR4/MyD88/NF-κβ signaling pathway (Fig. 9 ). However, several issues require further exploration in upcoming research. First, further investigation is warranted to elucidate SSP’s effects on inflammatory responses in activated microglia, including the expression of critical downstream cytokines. Second, the role of microglia in BRB disruption requires deeper exploration. While our study observed microglial clustering near retinal vessels, the direct impact of microglia on BRB integrity was not addressed. Future studies could employ ex vivo BRB models to dissect microglial contributions to barrier dysfunction. Finally, microglial polarization—specifically the balance between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes—should be characterized in diabetic retinas. Unraveling the polarization states and associated molecular mechanisms will clarify SSP’s anti-inflammatory actions in DR. In conclusion, our findings demonstrate that SSP ameliorates diabetic retinopathy (DR) progression in mice, exerting protective effects including preservation of retinal vasculature, attenuation of retinal thinning, suppression of intraretinal apoptosis, and prevention of vascular leakage. These benefits are likely mediated by SSP’s glucose- and lipid-lowering properties, coupled with its inhibition of microglia-driven inflammatory responses. Declarations Ethics approval The experimental protocol for this study was reviewed and approved by the Ethics Committee for Animal Experimentation of Beijing University of Chinese Medicine (approval no. BUCM-2024102805-4090). Conflict of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was supported by the National Natural Science Foundation of China [grant number 82174474] Author Contribution YJ, MZ were responsible for conducting the experiments, analyzing data, generating figures, and writing the manuscript. JZ was responsible for designing the methodology. YL (Yao Liang), JD and WL were responsible for data collection and conducting experiments. 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Ann Med 54 (1):1089-1111. doi:10.1080/07853890.2022.2064541 Santos FM, Ciordia S, Mesquita J, Cruz C, Sousa J, Passarinha LA, Tomaz CT, Paradela A (2023) Proteomics profiling of vitreous humor reveals complement and coagulation components, adhesion factors, and neurodegeneration markers as discriminatory biomarkers of vitreoretinal eye diseases. Front Immunol 14:1107295. doi:10.3389/fimmu.2023.1107295 Zou G, Que L, Liu Y, Lu Q (2024) Interplay of endothelial-mesenchymal transition, inflammation, and autophagy in proliferative diabetic retinopathy pathogenesis. Heliyon 10 (3):e25166. doi:10.1016/j.heliyon.2024.e25166 Wolf SA, Boddeke HW, Kettenmann H (2017) Microglia in Physiology and Disease. Annu Rev Physiol 79:619-643. doi:10.1146/annurev-physiol-022516-034406 Fang M, Wan W, Li Q, Wan W, Long Y, Liu H, Yang X (2021) Asiatic acid attenuates diabetic retinopathy through TLR4/MyD88/NF-kappaB p65 mediated modulation of microglia polarization. Life Sci 277:119567. doi:10.1016/j.lfs.2021.119567 Tong J, Yao G, Chen Y, Xie H, Zheng X, Sun L, Huang Z, Xie Z (2024) Mesenchymal Stem Cells Regulate Microglial Polarization via Inhibition of the HMGB1/TLR4 Signaling Pathway in Diabetic Retinopathy. Inflammation 47 (5):1728-1743. doi:10.1007/s10753-024-02005-6 Wang X, Xu C, Bian C, Ge P, Lei J, Wang J, Xiao T, Fan Y, Gu Q, Li HY, Xu J, Hu Z, Xie P (2024) M2 microglia-derived exosomes promote vascular remodeling in diabetic retinopathy. J Nanobiotechnology 22 (1):56. doi:10.1186/s12951-024-02330-w Zhu SH, Liu BQ, Hao MJ, Fan YX, Qian C, Teng P, Zhou XW, Hu L, Liu WT, Yuan ZL, Li QP (2017) Paeoniflorin Suppressed High Glucose-Induced Retinal Microglia MMP-9 Expression and Inflammatory Response via Inhibition of TLR4/NF-kappaB Pathway Through Upregulation of SOCS3 in Diabetic Retinopathy. Inflammation 40 (5):1475-1486. doi:10.1007/s10753-017-0571-z Luo YH, Zhang CX, Xu CP (2012) Research on Hypoglycemic Effect of the Sagittaria Sagittifolin 521 Polysaccharide. JOURNAL OF DALI UNIVERSITY 11: 4-5+9, 2012. ( Chinese ) Zhou MY, Liu BQ, Gao X, Zhang SJ, Jiang Y, Yang T, Sun JB, Zhang X, Liao Y (2025) Sagittaria sagittifolia polysaccharide extract regulates Nrf2 to improve endoplasmic reticulum stress-mediated apoptosis in rat cataracts and HLEB3 cells. Int J Biol Macromol 300:140270. doi:10.1016/j.ijbiomac.2025.140270 Wang J, Luo W, Li B, Lv J, Ke X, Ge D, Dong R, Wang C, Han Y, Zhang C, Yu H, Liao Y (2018) Sagittaria sagittifolia polysaccharide protects against isoniazid- and rifampicin-induced hepatic injury via activation of nuclear factor E2-related factor 2 signaling in mice. J Ethnopharmacol 227:237-245. doi:10.1016/j.jep.2018.09.002 Reed MJ, Meszaros K, Entes LJ, Claypool MD, Pinkett JG, Gadbois TM, Reaven GM (2000) A new rat model of type 2 diabetes: the fat-fed, streptozotocin-treated rat. Metabolism 49 (11):1390-1394. doi:10.1053/meta.2000.17721 Zhang X, Liu W, Wu S, Jin J, Li W, Wang N (2015) Calcium dobesilate for diabetic retinopathy: a systematic review and meta-analysis. Sci China Life Sci 58 (1):101-107. doi:10.1007/s11427-014-4792-1 Vujosevic S, Muraca A, Alkabes M, Villani E, Cavarzeran F, Rossetti L, De Cilla S (2019) Early Microvascular and Neural Changes in Patients with Type 1 and Type 2 Diabetes Mellitus without Clinical Signs of Diabetic Retinopathy. Retina 39 (3):435-445. doi:10.1097/IAE.0000000000001990 van Dijk HW, Verbraak FD, Kok PH, Garvin MK, Sonka M, Lee K, Devries JH, Michels RP, van Velthoven ME, Schlingemann RO, Abramoff MD (2010) Decreased retinal ganglion cell layer thickness in patients with type 1 diabetes. Invest Ophthalmol Vis Sci 51 (7):3660-3665. doi:10.1167/iovs.09-5041 Pfeifer CW, Walsh JT, Santeford A, Lin JB, Beatty WL, Terao R, Liu YA, Hase K, Ruzycki PA, Apte RS (2023) Dysregulated CD200-CD200R signaling in early diabetes modulates microglia-mediated retinopathy. Proc Natl Acad Sci U S A 120 (45):e2308214120. doi:10.1073/pnas.2308214120 Mohsin F, Javaid S, Tariq M, Mustafa M (2024) Molecular immunological mechanisms of impaired wound healing in diabetic foot ulcers (DFU), current therapeutic strategies and future directions. Int Immunopharmacol 139:112713. doi:10.1016/j.intimp.2024.112713 Hu Q, Jiang L, Yan Q, Zeng J, Ma X, Zhao Y (2023) A natural products solution to diabetic nephropathy therapy. Pharmacol Ther 241:108314. doi:10.1016/j.pharmthera.2022.108314 Jonas JB (2024) Diabetic retinopathy. Asia Pac J Ophthalmol (Phila) 13 (3):100077. doi:10.1016/j.apjo.2024.100077 Xu W, Xu X, Zhang M, Sun C (2024) Association between HDL cholesterol with diabetic retinopathy in diabetic patients: a cross-sectional retrospective study. BMC Endocr Disord 24 (1):65. doi:10.1186/s12902-024-01599-0 Chen S, Zhang M, Yang P, Guo J, Liu L, Yang Z, Nan K (2024) Genetic Association between Lipid-Regulating Drug Targets and Diabetic Retinopathy: A Drug Target Mendelian Randomization Study. J Lipids 2024:5324127. doi:10.1155/2024/5324127 Nakashima R, Ikeda S, Shinohara K, Matsumoto S, Yoshida D, Ono Y, Nakashima H, Miyamoto R, Matsushima S, Kishimoto J, Itoh H, Komuro I, Tsutsui H, Abe K (2025) Triglyceride/high density lipoprotein cholesterol index and future cardiovascular events in diabetic patients without known cardiovascular disease. Sci Rep 15 (1):9217. doi:10.1038/s41598-025-92933-6 Deng X, Ke X, Tang Y, Luo W, Dong R, Ge D, Han L, Yang Y, Liu H, Reyila T, Liao Y (2020) Sagittaria sagittifolia polysaccharide interferes with arachidonic acid metabolism in non-alcoholic fatty liver disease mice via Nrf2/HO-1 signaling pathway. Biomed Pharmacother 132:110806. doi:10.1016/j.biopha.2020.110806 Mills SA, Jobling AI, Dixon MA, Bui BV, Vessey KA, Phipps JA, Greferath U, Venables G, Wong VHY, Wong CHY, He Z, Hui F, Young JC, Tonc J, Ivanova E, Sagdullaev BT, Fletcher EL (2021) Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy. Proc Natl Acad Sci U S A 118 (51). doi:10.1073/pnas.2112561118 Zusso M, Lunardi V, Franceschini D, Pagetta A, Lo R, Stifani S, Frigo AC, Giusti P, Moro S (2019) Ciprofloxacin and levofloxacin attenuate microglia inflammatory response via TLR4/NF-kB pathway. J Neuroinflammation 16 (1):148. doi:10.1186/s12974-019-1538-9 Nielsen TB, Pantapalangkoor P, Yan J, Luna BM, Dekitani K, Bruhn K, Tan B, Junus J, Bonomo RA, Schmidt AM, Everson M, Duncanson F, Doherty TM, Lin L, Spellberg B (2017) Diabetes Exacerbates Infection via Hyperinflammation by Signaling through TLR4 and RAGE. mBio 8 (4). doi:10.1128/mBio.00818-17 Sangaran PG, Ibrahim ZA, Chik Z, Mohamed Z, Ahmadiani A (2021) LPS Preconditioning Attenuates Apoptosis Mechanism by Inhibiting NF-kappaB and Caspase-3 Activity: TLR4 Pre-activation in the Signaling Pathway of LPS-Induced Neuroprotection. Mol Neurobiol 58 (5):2407-2422. doi:10.1007/s12035-020-02227-3 Additional Declarations No competing interests reported. Supplementary Files wb.docx Cite Share Download PDF Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 21 Aug, 2025 Reviews received at journal 20 Aug, 2025 Reviews received at journal 20 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers invited by journal 06 Aug, 2025 Editor assigned by journal 29 Jul, 2025 Submission checks completed at journal 29 Jul, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7204796","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498191088,"identity":"995d652c-7ae0-42ac-bc22-4a01b99c4a64","order_by":0,"name":"Yang Jiang","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Jiang","suffix":""},{"id":498191089,"identity":"9fefcf3b-6ddd-46cb-b540-449ce8ff8bda","order_by":1,"name":"Manyu Zhou","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Manyu","middleName":"","lastName":"Zhou","suffix":""},{"id":498191090,"identity":"8a16005f-cba3-4f18-b356-fcb3b97e60d3","order_by":2,"name":"Jing Zhang","email":"","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":498191091,"identity":"567627a4-c2c0-4702-8c4f-b393c8b653d0","order_by":3,"name":"Yao Liang","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Liang","suffix":""},{"id":498191092,"identity":"56f68dba-2732-42d9-a7d2-51733209bb58","order_by":4,"name":"Jiazhen Ding","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiazhen","middleName":"","lastName":"Ding","suffix":""},{"id":498191093,"identity":"a47ba6e5-2ce6-467a-9f5e-415afb95447e","order_by":5,"name":"Wenyong Liao","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenyong","middleName":"","lastName":"Liao","suffix":""},{"id":498191094,"identity":"dba4d7bd-5c7a-4fa6-9b12-832ee656f1a9","order_by":6,"name":"Yan Liao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACPmYInQDEjA8SKmoIa2FD0sJs8ODMMSK0MCC0sEk+bGEmQgs788PHPDV38gyOn06rSGxgY+Bv704g4DA2Y2OeY8+KDc7kbruRuEOGQeLM2Q0EtDCYSfOwHU7ccIMXqOUMG4OBRC4hLezfpHn+QbQUJLYxE6OFx0yatw2ihYFYLcWGc/sOF0ueyd0skXDmGA9Bv/DzH9/44M23w3l8x89u/PijokaOv70XvxYQYOJB4vDgVIYMGH8QpWwUjIJRMApGLAAAI7VHK94UxF4AAAAASUVORK5CYII=","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2025-07-24 10:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7204796/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7204796/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-025-05388-1","type":"published","date":"2025-11-25T15:58:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88919787,"identity":"30e2df53-e326-4099-91e1-bbadc96e55f3","added_by":"auto","created_at":"2025-08-12 17:03:50","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1666086,"visible":true,"origin":"","legend":"\u003cp\u003eStudy protocol and grouping\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/3b0e5026e705b085ba28ff1a.jpeg"},{"id":88920476,"identity":"917ffe08-ae03-42c4-9f5f-d831860657aa","added_by":"auto","created_at":"2025-08-12 17:11:51","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1896156,"visible":true,"origin":"","legend":"\u003cp\u003eFundus photography, fluorescein angiography (FFA), fasting blood glucose (FBG), and lipid in mice (n = 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes:\u003c/strong\u003e A:\u0026nbsp;Fundus photography and FFA images of the control and model groups. Red boxes indicate perivascular gray-white plaques or hypofluorescent shadows; B:\u0026nbsp;Fundus photography of each group. Red boxes indicate perivascular gray-white plaques; C:\u0026nbsp;Line graph of weekly fasting blood glucose (FBG) levels post-STZ injection; D:\u0026nbsp;FBG levels of each group of mice before STZ injection; E:\u0026nbsp;FBG levels at 72 hours post-STZ injection; F:\u0026nbsp;FBG levels at week 8; G:\u0026nbsp;High-density lipoprotein (HDL); H:\u0026nbsp;Low-density lipoprotein (LDL); I:\u0026nbsp;Triglycerides (TG); J:\u0026nbsp;Total cholesterol (T-CHO). \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 vs. the Control group; \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003eP \u0026lt; 0.05 vs. the Model group; \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003eP \u0026lt; 0.01 vs. the Model group.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/9658fae3cad4497579b2de8a.jpeg"},{"id":88919786,"identity":"8ed50f1c-f177-4296-8614-de35b539795d","added_by":"auto","created_at":"2025-08-12 17:03:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":553499,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining and EB staining (n-3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes: \u003c/strong\u003eThe red triangle points to the blood vessel and the yellow triangle points to the exudates.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/cc4f4081bc5783b9860dfbab.jpeg"},{"id":88920472,"identity":"d24e41bf-f4f5-46ee-b49e-a4f4056f6689","added_by":"auto","created_at":"2025-08-12 17:11:50","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3041614,"visible":true,"origin":"","legend":"\u003cp\u003eRetinal thickness of each group (n = 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes: \u003c/strong\u003eA:\u0026nbsp;Schematic diagram of retinal thickness measurement; B:\u0026nbsp;Schematic diagram of retinal thickness at 2.2 mm from the ONH; C:\u0026nbsp;Retinal thickness curve. D:\u0026nbsp;Quantification of retinal thickness at 2.2 mm from the ONH. \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs. the Control group; \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the Model group; \u003csup\u003e\u003cstrong\u003e** \u003c/strong\u003e\u003c/sup\u003eor \u003csup\u003e\u003cstrong\u003e\u0026amp;\u0026amp;\u003c/strong\u003e\u003c/sup\u003e P \u0026lt; 0.01 vs. the Model group.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/07fd843ad04d2e853baa03df.jpeg"},{"id":88919793,"identity":"a5db76f2-3397-404b-a8ce-815a96fb4454","added_by":"auto","created_at":"2025-08-12 17:03:51","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3669354,"visible":true,"origin":"","legend":"\u003cp\u003eThe apoptotic cells of mice in each group (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e: A: Schematic diagram of Caspase-3\u003csup\u003e+\u003c/sup\u003ecells in the retina; B: Schematic diagram of TUNEL\u003csup\u003e+\u003c/sup\u003ecells in the retina.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/b0929a8dd0a23c489e5ad414.jpeg"},{"id":88920475,"identity":"468b4eee-25f1-43eb-85a6-8ccfe2268076","added_by":"auto","created_at":"2025-08-12 17:11:51","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":737407,"visible":true,"origin":"","legend":"\u003cp\u003ePerivascular microglia (n=3).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/bdeeca8b44d3857df74f575e.jpeg"},{"id":88920473,"identity":"42f16c9c-076a-41d0-93c4-4e0fa91165dc","added_by":"auto","created_at":"2025-08-12 17:11:51","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1306030,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of retinal microglia of each group (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes:\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e: Representative microglial morphology images from each group; \u003cstrong\u003eB\u003c/strong\u003e: Quantitative analysis of maximum soma diameter; \u003cstrong\u003eC\u003c/strong\u003e: Quantitative analysis of process length; \u003cstrong\u003eD\u003c/strong\u003e: Quantitative analysis of branch points.\u003csup\u003e ##\u003c/sup\u003eP \u0026lt; 0.01 vs. the Control group; \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e P \u0026lt; 0.01 vs. the Model group.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/254d89fe7536fecbd1197652.jpeg"},{"id":88920918,"identity":"f5d302cb-66fd-434f-ab61-008ca6d11f9c","added_by":"auto","created_at":"2025-08-12 17:19:51","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1123322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe TLR4/Myd88/NF-κβ signaling pathway in the retina of each group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes:\u003c/strong\u003e A: Co-localization of Iba and TLR4 (n=6); B: Representative western blot images of TLR4, Myd88, p-P65, and P65 (n=3); C: Quantitative image of TLR4 (n=3); D: Quantitative image of Myd88 (n=3); E: Quantitative image of p-P65 to P65 ratio (n=3). \u003csup\u003e\u003cstrong\u003e## \u003c/strong\u003e\u003c/sup\u003eP \u0026lt; 0.01 vs. the Control group; \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e P \u0026lt; 0.05 vs. the Model group; \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e P \u0026lt; 0.01 vs. the Model group.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/91c0e7028154002a317a6ada.jpeg"},{"id":88920477,"identity":"a1b396fa-19d5-4533-b2b4-3493f49c1bd2","added_by":"auto","created_at":"2025-08-12 17:11:51","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4580934,"visible":true,"origin":"","legend":"\u003cp\u003e(1)\u003cstrong\u003e \u003c/strong\u003eIn DR retinas, DAMPs and PAMPs bind to TLR4 on microglial cell surfaces, mediating inflammatory responses through the TLR4/Myd88/NF-κβ signaling pathway, thereby causing BRB destruction; (2)\u003cstrong\u003e \u003c/strong\u003eActivated microglia migrate to the BRB injury site; (3)\u003cstrong\u003e \u003c/strong\u003eSSP suppresses microglia-mediated inflammation.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/830c788460631b8f9574406f.jpeg"},{"id":97178946,"identity":"5f776035-01ce-4ece-ab1c-4a30f3b695b1","added_by":"auto","created_at":"2025-12-01 16:14:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19490416,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/4739c53f-5022-4d03-a5a8-a2aa545c0f24.pdf"},{"id":88920917,"identity":"8bdb288d-3ffb-4b94-a92e-56aad0986c17","added_by":"auto","created_at":"2025-08-12 17:19:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1703687,"visible":true,"origin":"","legend":"","description":"","filename":"wb.docx","url":"https://assets-eu.researchsquare.com/files/rs-7204796/v1/8e1616bf68767dbe3d18562e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sagittaria Sagittaria polysaccharide protects against retinal vascular damage in diabetic mice by suppressing TLR4 signaling pathway and microglial activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic retinopathy (DR) is one of the most common ocular complications in patients with diabetes, affecting approximately one-third of diabetic patients to varying degrees of severity[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In its early stage, known as non-proliferative diabetic retinopathy (NPDR), the condition typically presents with no obvious symptoms. however, NPDR is likely to progress to proliferative diabetic retinopathy (PDR), a sight-threatening stage that can culminate in severe vision loss and irreversible blindness. Currently, DR has become one of the leading causes of irreversible vision loss among adults worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], imposing substantial health and economic burdens on society.\u003c/p\u003e\u003cp\u003eThe pathological mechanisms of diabetic retinopathy (DR) are highly complex, with inflammatory responses serving as a critical factor influencing disease outcomes. During the non-proliferative diabetic retinopathy (NPDR) stage, substantial evidence indicates that the destruction of the blood-retinal barrier (BRB), degradation of tight junction proteins, and increased vascular permeability in the retina are closely associated with the accumulation of inflammatory factors (IL-1β, IL-6, TNF-α, etc.) [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These inflammatory mediators promote apoptosis of vascular endothelial cells and pericytes, leukocyte aggregation and adhesion within vessels, and microthrombus formation, ultimately resulting in local retinal ischemia and hypoxia [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, in the proliferative diabetic retinopathy (PDR) stage, inflammatory responses not only synergize with VEGF to drive pathological neovascularization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] but also perpetuate chronic inflammation, activate the complement system, and enhance leukocyte adhesion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These processes culminate in vision-threatening pathological changes such as neovascularization rupture, vitreous hemorrhage, and retinal detachment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, controlling the progression of inflammatory responses at early stages is crucial for delaying DR advancement.\u003c/p\u003e\u003cp\u003eMicroglia, the primary resident immune cells in the retina, play a central role in retinal inflammatory responses. Under physiological conditions, microglia remain in a resting state characterized by a branched morphology, continuously surveilling changes in the microenvironment. Upon injury or pathological stimulation, microglia become activated, manifesting morphological changes, proliferation, migration, and the release of cytokines and chemokines to mediate inflammation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In retinal diseases, microglial activation often coincides with BRB disruption. Studies demonstrate that under pathological conditions such as retinal ischemia or hypoxia, activated microglia migrate to injury sites and release pro-inflammatory factors (e.g., TNF-α, IL-1β), which directly increase vascular endothelial permeability, exacerbating BRB disruption [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In an in vitro hyperglycemia-induced microglial model, elevated expression of TNF-α and IL-1β was observed in the supernatant [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To visually assess the impact of such pro-inflammatory microglial supernatants on BRB permeability, Wang et al. co-cultured pericytes and endothelial cells in a Transwell system to simulate BRB. Treatment with microglial supernatant induced significant apoptosis in both cell types and markedly increased BRB permeability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, NF-κβ, a key pro-inflammatory transcription factor in microglia, is regulated by the classical TLR4 signal. In the inflammatory mechanism of DR, the TLR4/Myd88/NF-κβ signaling pathway has been identified as a critical target for drugs (e.g., asiatic acid, paeoniflorin) and mesenchymal stem cell therapies to suppress inflammatory polarization of retinal microglia [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, targeting microglia-driven inflammatory responses and their upstream signaling events\u0026mdash;TLR4/Myd88/NF-κβ signaling pathway\u0026mdash;represents a promising therapeutic strategy for mitigating DR progression.\u003c/p\u003e\u003cp\u003eSagittaria sagittifolia polysaccharide (SSP), the primary active component of Sagittaria sagittifolia, has demonstrated significant hypoglycemic effect in diabetic mouse models as early as a decade ago [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By reducing blood glucose levels, SSP theoretically holds potential for intervening in diabetic retinopathy (DR). Furthermore, our research team has focused on exploring SSP's applications in ocular and hepatic diseases, revealing its marked anti-inflammatory, antioxidant, and lipid-regulating properties. For instance, SSP has been shown to reduce lens opacity in selenite-induced cataract rats through anti-inflammatory and antioxidant mechanisms [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and to mitigate isoniazid-rifampicin combination-induced liver injury in mice via the Nrf2 antioxidant pathway [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, no studies have specifically investigated SSP's role in DR intervention. Given SSP's dual hypoglycemic-hypolipidemic capacity and anti-inflammatory effects, we aim to explore SSP's potential in DR intervention and elucidate its anti-inflammatory mechanisms within the DR retina.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eDrug preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe SSP was extracted from fresh \u003cem\u003eSagittaria sagittifolia\u003c/em\u003e L. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], with \u003cem\u003eSagittaria sagittifolia\u003c/em\u003e L. being an accepted name in the plant list (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.worldfloraonline.org\u003c/span\u003e\u003cspan address=\"http://www.worldfloraonline.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). SSP solutions were formulated daily in physiological saline at concentrations of 80 mg/mL, 40 mg/mL, and 20 mg/mL for experimental use. Calcium dobesilate (CD), purchased from Beijing Jingfeng Pharmaceutical Group Co., Ltd., was prepared as a 23 mg/mL solution. Gavage volume = body weight (g) × 0.1ml/10g.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrouping, modeling and treatments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 36 healthy male C57BL/6J mice (8 weeks old, 20-25g) were purchased from Sibeifu Biotechnology Co., Ltd. (Laboratory Animal Production License No.: SCXK(JING)2024-0001). The experimental protocol of this study has been reviewed and approved by the Animal Experiment Ethics Committee of Beijing University of Chinese Medicine (BUCM-2024102805-4090). After 3 days of acclimatization, the mice were randomly divided into a control group (n = 6) and a high-fat diet (HFD) group (n = 30) using a random number table. The control group was fed a standard diet, while the HFD group received a 45% high-fat diet (Sumeidisen Biomedical Co., Ltd., Art.No.: MD12032) for 2 weeks. All mice were then fasted for 12 hours (overnight fasting with water ad libitum) and subjected to intraperitoneal injection of 50 mg/kg streptozotocin (STZ, Sigma, dissolved in 0.1 M sodium citrate buffer) daily for 5 consecutive days to induce type 2 diabetic models. 72 hours post-injection, fasting blood glucose (FBG) was measured via tail vein sampling. Mice with FBG levels \u0026gt; 11.1 mmol/L were confirmed as successful type 2 diabetic models [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Following successful modeling, the HFD group was further randomized into five subgroups: Model group (untreated diabetic mice), Positive group (CD, 0.23 g/kg), SSP-L (low-dose SSP, 200 mg/kg), SSP-M (medium-dose SSP, 400 mg/kg), SSP-H (high-dose SSP, 800 mg/kg). The Control and model groups received an equivalent volume of physiological saline. Treatments continued for 9 weeks, after which retinal pathology was assessed. Throughout the experiment, diabetic mice remained on the HFD, and the normal group maintained a standard diet. The study protocol and grouping are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFundus photography and fluorescein angiography.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 9 weeks post-successful induction of diabetes in murine models, mice from all groups were anesthetized using isoflurane (4% for induction, 2% for maintenance). Compound Tropicamide Eye Drops (Shenyang Xingqi Ophthalmology Co., Ltd.) were administered to dilate the pupils, followed by topical anesthesia with Oxybuprocaine Hydrochloride (Santen Pharmaceutical Co., Ltd.). Carbomer gel was applied to the ocular surface, and mice were positioned on the animal platform. Fundus photography of both eyes was performed using the Micron-III Small Animal Retinal Imaging System (Optoprobe, Model: OPTO-RIS). For retinal fluorescein angiography, 10% sodium fluorescein (0.04 mL/mouse) was injected intraperitoneally.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSerum biochemical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e9 weeks after the establishment of the type 2 diabetic model, the levels of triglycerides (TG), total cholesterol (T-CHO), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in mouse serum were measured using a fully automated serum biochemical analyzer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvans blue staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThree mice from each group were selected for retinal Evans Blue (EB) staining. A 3% EB solution was prepared. After tail vein injection of EB for 30 min, the mice were anesthetized and euthanized. The right eye was enucleated and fixed in 4% paraformaldehyde for 30 min. The fixed eyeball was immersed in PBS for 10 min. The retina was dissected on a glass slide, and the cup-shaped ocular wall was divided into 3–4 evenly spaced sections centered on the optic nerve head (ONH) to prepare retinal flat mounts. The left eye was processed for paraffin embedding.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHE staining and retinal thickness measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe left eyes of mice from each group (including Evans Blue-stained eyeballs, totaling 6 eyeballs) were fixed in 4% paraformaldehyde for 48 hours. Subsequently, the tissues were trimmed and rinsed overnight to remove residual paraformaldehyde. The samples underwent dehydration and clearing through a graded series of ethanol (60% for 2 h, 80% for 2 h, 90% for 4 h, 95% I for 2 h, 95% II for 2 h, 100% I for 2 h, 100% II for 2 h) followed by xylene (xylene I for 1 h, xylene II for 2 h). Tissue infiltration was performed with paraffin (three times, 30 min each) before embedding. Sections were prepared using a microtome and dried for subsequent use.\u003c/p\u003e\u003cp\u003eFor staining, the sections were deparaffinized and rehydrated sequentially in xylene I (20 min), xylene II (20 min), 100% ethanol I (5 min), 100% ethanol II (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 60% ethanol (5 min), and finally deionized water (3 min). Hematoxylin staining was performed for 5 min, followed by immediate differentiation in 1% acid alcohol for 3 sec and thorough rinsing under running tap water for 10 min. Counterstaining was carried out with eosin for 3 min, followed by rapid dehydration in 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II (30 sec each). Finally, the sections were cleared and mounted for microscopic observation.\u003c/p\u003e\u003cp\u003eRetinal thickness was measured at six locations (0.2 mm, 0.6 mm, 1 mm, 1.4 mm, 1.8 mm, and 2.2 mm from the ONH), with full-thickness retinal thickness recorded at each point.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTUNEL staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eParaffin sections undergo dewaxing and hydration. A hydrophobic barrier pen was used to outline the tissue sections. Staining was performed according to the manufacturer’s instructions of the TUNEL Apoptosis Detection Kit (LabLead, China). The reagents (Proteinase K, 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and fluorescein-labeled TUNEL reaction mixture reagents) were prepared in sufficient quantities. Then, these slices underwent permeabilization (Proteinase K, 1:50, 30 min), blocking (3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 20 min), and TUNEL reaction (fluorescein-labeled TUNEL reaction mixture reagents, 60 minutes), sealed with an anti-fluorescence quencher containing DIPA. Apoptotic cells in the retina were visualized and analyzed under a fluorescence microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDeparaffinized and rehydrated sections were prepared using standard protocols, and tissue boundaries were outlined with a hydrophobic barrier pen. After hydration, sections were rinsed three times with deionized water (1 min each). Antigen retrieval was performed using preheated EDTA buffer (pH 9.0, 100°C) twice (5 min each). Endogenous peroxidase activity was blocked with 2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 min, followed by three PBS washes (5 min each). Non-specific binding sites were blocked with 10% BSA. Sections were incubated overnight at 4°C with primary antibodies: Iba (1:100, CST, USA), Caspase-3 (1:50, CST, USA). After incubation, sections were washed three times with PBS (1 min each) and incubated with HRP-conjugated secondary antibody (goat anti- rabbit IgG, 1:1000, Proteintech, China) for 1 h. Further, stain the sections using DAB and hematoxylin for 5 min, followed by gradient dehydration, clearing, and mounting. Brown precipitate indicating target protein expression was visualized under a light microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmunofluorescence staining was performed on both paraffin sections and evans blue-stained retinal flat mounts. For paraffin sections, the enhanced immunofluorescence Kit (Product: AFIHC033, Hunan Aifang Biotechnology Co., Ltd., China) was used. The processing steps for paraffin sections included deparaffinization, rehydration, antigen retrieval, peroxidase blocking, and 10% BSA blocking, consistent with the immunohistochemistry protocol. Sections were incubated with Iba antibody (1:100, CST, USA) at 37 ℃ for 2 h, followed by three PBS washes (5 min each). A Polymer-HRP-conjugated secondary antibody was then applied for 30 min, followed by PBS washes (3 × 5 min). Signal amplification was achieved using TSA fluorescent dye (TYR-520Plus). To remove non-covalently bound primary-secondary-HRP complexes, sections underwent heat-induced antigen retrieval in EDTA buffer (pH 9.0, 100°C, 30 min). Repeat the steps of peroxidase blockade, BSA blocking, primary antibody incubation (TLR4, 1:200, Proteintech, China), Polymer HRP secondary antibody incubation, TSA fluorescent dye (TYR-570Plus fluorescent dye) incubation for the second round of labeling. Sections were cover slipped with an anti-fluorescence quencher containing DAPI and analyzed under a fluorescence microscope to assess co-localization of Iba and TLR4.\u003c/p\u003e\u003cp\u003eRetinal flat mounts were prepared as described in the part 2.5 and subjected to immunofluorescence staining. Briefly, the retinas were incubated in 0.3% Triton X-100 at 4°C for 1 h. After blotting excess Triton X-100 solution with filter paper, 10% BSA blocking buffer was applied and incubated at 37 ℃ for 2 h. The blocking buffer was removed, and the primary antibody diluent (Iba, 1:100, CST, USA) was added, followed by incubation at 37 ℃ for 3 h. Unbound primary antibody was blotted with filter paper, and the samples were washed three times with PBS. A FITC-conjugated goat anti-rabbit secondary antibody (1:200, Abcam, UK) was applied and incubated at 37 ℃ for 1 h. After washing with PBS, the retinas were mounted with an anti-fluorescence quencher containing DAPI. Perivascular expression of Iba was visualized using a fluorescence microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal proteins were isolated from retina using RIPA buffer (containing 1% PMSF and protease inhibitors). Equal amounts of protein samples were subjected to electrophoretic separation using SDS-PAGE, and the resulting products were then transferred onto polyvinylidene difluoride (PVDF) membranes from Millipore. The membrane was blocked with 5% skim milk/TBST for 1 h at room temperature on a shaker, followed by three TBST washes (10 min each). After washing, the membranes were incubated overnight at 4 ◦C with primary antibodies, including anti-TLR4 (1:1000, CST, USA), anti-Myd88 (1:1000, Abcam, UK), anti-p65/p-p65 (1:1000, CST, USA), and anti-β-actin (1:5000, Proteintech, China). After TBST washes (3 × 10 min), the membrane was incubated with HRP-conjugated secondary antibody (goat anti-rabbit IgG, 1:5000, Proteintech, China) for 1 h at room temperature and washed again. Protein bands were visualized using an Enhanced Chemiluminescence (ECL) Detection Kit (Lablead, China) and imaged with a chemiluminescence system.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using SPSS 20.0 (IBM Corp., Armonk, NY), and histograms were plotted with GraphPad Prism 5 (GraphPad, San Diego, CA). Continuous variables are expressed as mean ± standard deviation (mean ± SD). If data met normality (Shapiro-Wilk test) and homogeneity of variance (Levene’s test), one-way ANOVA was applied. For datasets with violated variance assumptions, the non-parametric rank-sum test (Mann-Whitney U test) was used. P-values \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cb\u003eSSP protects the retinal vasculature in diabetic mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe success of the diabetic retinopathy (DR) model was assessed using fundus photography and fundus fluorescein angiography (FFA). In fundus images (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), retinal vessels in the control group radiated outward from the optic disc with clear and smooth vessel walls. Arteries appeared light red, while veins were dark red. In contrast, the model group exhibited grayish-white exudates around retinal veins. FFA revealed fluorescent leakage in regions corresponding to the grayish-white exudates, indicating disruption of retinal vascular integrity and pathological changes such as extravasation of plasma components (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In fundus images, all groups except the control group showed varying degrees of grayish-white exudates. Compared to the model group, the extent of exudates was reduced in both the CD and SSP groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). CD, a clinically used vasoprotective agent for DR treatment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], served as a positive drug. Our results suggest that SSP exhibits similar vasoprotective effects to CD, though its underlying mechanisms require further investigation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSSP reduces FBG, serum TG, T-CHO, LDL levels, and increases HDL levels in diabetic mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore whether SSP delays DR progression via glycemic control, FBG levels were monitored throughout the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). All mice exhibited normal FBG levels prior to STZ induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). At 72 hours post-STZ injection, diabetic models were confirmed successful when FBG \u0026gt; 11.1 mmol/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). SSP with high dose (SSP-H) effectively reduced FBG in diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), with statistical significance observed only at week 8 (P \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003eHyperlipidemia, a common comorbidity in type 2 diabetes, was assessed in our experiment. Compared to the control group, all other groups showed significant increases in serum total cholesterol (TC), cholesterol (T-CHO), and low-density lipoprotein (LDL), alongside reduced high-density lipoprotein (HDL) levels. SSP administration notably regulated lipid metabolism (reduced TG/TC/LDL-C, increased HDL-C) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG–J).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSSP reduces blood leakage in the retina of diabetic mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing tail vein injection of EB solution, eyeballs from each group were paraffin-embedded, sectioned, and subjected to HE staining. Under light microscopy, while retinal micromorphology in diabetic mice (Model group) showed no significant structural changes compared to controls, vascular walls appeared thinner, with red exudates observed around the vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Model group; red triangles: vessels, yellow triangles: exudates). To confirm whether these exudates originated from intravascular components, fluorescence microscopy (532nm) revealed red fluorescence around the vessels (principle: evans blue binds rapidly to plasma albumin and appears red under 532nm excitation light stimulation). Notably, both the CD group and SSP-H group effectively increased vascular wall thickness. Fluorescence microscopy further demonstrated a significant reduction in perivascular exudates in these groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, CH/SSP-H groups). These findings indicate that SSP protects vascular integrity and mitigates Pathological leakage of blood vessels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSSP increases retinal thickness in diabetic mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRetinal thinning is an early hallmark of diabetic retinopathy (DR), preceding microangiopathy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, retinal morphology was assessed using HE staining, and the retinal thickness was measured at six locations relative to the optic nerve head (ONH): 0.2 mm, 0.6 mm, 1 mm, 1.4 mm, 1.8 mm, and 2.2 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared to the control group, diabetic mice exhibited significant retinal thinning (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The CD and SSP effectively increased retinal thickness of diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), with statistical significance observed at 2.2 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) from the ONH (P \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSSP suppresses retinal apoptotic cells in diabetic mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApoptosis is a primary mechanism of retinal cell death in early-stage of DR and a key contributor to retinal thinning [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To evaluate apoptosis in the retina of diabetic mice, we assessed caspase-3\u003csup\u003e+\u003c/sup\u003ecells and TUNEL-positive cells in the retina. Caspase-3 expression was quantified in retinal regions approximately 200 µm from the ONH. The results demonstrated that the number of Caspase-3\u003csup\u003e+\u003c/sup\u003e cells was significantly increased in the model group in the inner nuclear layer (INL) of the retina, while SSP markedly reduced the quantity of these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). TUNEL-positive cells were significantly increased in the model group, while SSP administration effectively suppressed apoptosis of retinal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eWe used different methods to display the apoptotic cells of each group of mice, and found that SSP could stably inhibit the number of apoptotic cells in the diabetic mice retina.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSSP reduces perivascular microglial accumulation in diabetic mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRetinal flat mounts were prepared from mice injected with EB solution. Microglia adjacent to blood vessels were visualized via immunofluorescence. EB dye emitted red fluorescence under fluorescence microscopy to demarcate blood vessels (white dashed box), while Iba (a microglial marker) was labeled with green fluorescence. In diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Model group), a significant increase in perivascular microglial density was observed. SSP-H treatment effectively reduced microglial clustering around retinal vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, SSP-H group). These findings suggest that retinal vessels in diabetic mice are surrounded by increased microglia, though their detrimental effects on vascular integrity and the BRB require further validation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSSP modulates microglial morphology in diabetic mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMicroglial chemotaxis toward blood vessels is accompanied by functional and morphological changes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To investigate this, we analyzed microglial morphology on retinal cross-sections using immunohistochemistry. In model group (\u003cb\u003eFig.\u0026nbsp;7A\u003c/b\u003e), microglia exhibited shortened processes, enlarged soma, and reduced total branch length per field (\u003cb\u003eFig.\u0026nbsp;7C\u003c/b\u003e, quantified as cumulative process length), along with decreased branch points (\u003cb\u003eFig.\u0026nbsp;7D\u003c/b\u003e). Compared to the model group, SSP-H treatment (\u003cb\u003eFig.\u0026nbsp;7B/C/D\u003c/b\u003e) significantly increased microglial process length (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) and branch points (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), although soma diameter remained unchanged (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). These results suggest that SSP promotes microglial process elongation and arborization, highlighting a morphological shift toward a surveillant and non-inflammatory state.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSSP inhibits TLR4 expression on the retinal microglia and the Myd88/NF-κβ signaling pathway in diabetic mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eActivated microglia undergo morphological changes (enlarged soma, shortened processes, and reduced branch points) and promote retinal inflammatory responses in diabetic retinopathy (DR) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In diabetic retinas, damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) activate microglia via pattern recognition receptors (PRRs). Toll-like receptor 4 (TLR4), a key PRR for DAMPs/PAMPs, was investigated to determine its role in microglial activation. Immunofluorescence revealed significantly increased TLR4 expression in retinal microglia of diabetic mice (white boxes, Model, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The SSP treatment decreased co-localization of Iba and TLR4 (white boxes, SSP-H, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), suggesting SSP may suppresses microglial activation by downregulating TLR4. What’s more, western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB–E) demonstrated that SSP significantly reduced protein levels of TLR4, Myd88 and the radio of p-p65 to p65. These findings indicate that SSP may modulate microglial activity by suppressing the TLR4/Myd88/NF-κβ signaling pathway, thereby attenuating neuroinflammation in the retina of diabetes mice.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOur findings demonstrate that SSP delays DR progression in mice by protecting retinal vasculature, increasing retinal thickness, suppressing apoptosis, and preventing vascular leakage. To elucidate SSP\u0026rsquo;s anti-DR mechanisms, we first evaluated its effects on primary drivers of DR\u0026mdash;hyperglycemia and dyslipidemia. Chronic hyperglycemia induces systemic microvascular complications, including diabetic foot, nephropathy, and retinopathy, which progress to microcirculatory dysfunction, tissue ischemia, and irreversible damage [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Glycemic control mitigates vascular pathology and associated tissue injury. In our study, SSP-treated mice maintained lower FBG levels than the model group, though statistical significance was limited by sample size at most time points. Hyperlipidemia further exacerbates DR. Preclinical and clinical studies consistently link elevated TG, LDL, CHO, and reduced HDL to DR pathogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], with the TG/HDL ratio serving as a key predictor of retinopathy in type 2 diabetes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our prior work showed SSP significantly reduces serum TG and CHO levels in a non-alcoholic fatty liver disease (NAFLD) rat model [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, SSP corrected dyslipidemia in diabetic mice in our experiments. These results suggested SSP\u0026rsquo;s dual modulation of glucose and lipid metabolism contributes to DR amelioration.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOur results demonstrate that SSP exerts significant protective effects on retinal vasculature in diabetic mice, including maintaining vascular wall integrity and preventing plasma extravasation. However, given the persistent disparity in serum FBG levels between the SSP-treated and control groups, we propose that SSP\u0026rsquo;s protective mechanisms extend beyond glycemic and lipid regulation. Inflammation, particularly microglial-mediated neuroinflammation, plays a pivotal role in DR pathogenesis. We observed microglial accumulation around retinal vessels in diabetic mice, consistent with findings by Mills et al., who reported that perivascular microglial adhesion and aggregation exacerbate BRB disruption and vascular hyperpermeability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, SSP treatment markedly reduced perivascular microglial accumulation, suggesting that microglia may serve as a critical cellular target for SSP\u0026rsquo;s vaso-protective effects.\u003c/p\u003e\u003cp\u003eMicroglia, the resident immune cells of the retina, play a central role in retinal inflammatory responses. Upon sensing injury signals, microglia transition from a resting \"ramified\" morphology (characterized by branched processes) to an activated \"amoeboid\" state, which is associated with phagocytic activity and pro-inflammatory functions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Immunohistochemical analysis revealed that retinal microglia in diabetic mice exhibited enlarged soma diameter, shortened processes, and reduced process arborization\u0026mdash;morphological changes linked to their pro-inflammatory phenotype. SSP treatment effectively inhibited this shift toward an inflammatory morphology.\u003c/p\u003e\u003cp\u003eThe TLR4/Myd88/NF-κβ signaling pathway is a key upstream regulator of microglial activation and subsequent inflammatory damage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In diabetes, damage-associated molecular patterns (DAMPs, e.g., advanced glycation end products [AGEs] induced by hyperglycemia) and pathogen-associated molecular patterns (PAMPs, e.g., gut-derived LPS) activate TLR4/MyD88/NF-κβ signaling pathway in microglia [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To investigate TLR4 activation in DR retinal microglia, immunofluorescence demonstrated increased co-localization of Iba (microglial marker) and TLR4, which was significantly attenuated by SSP. Consistent with this, downstream proteins (Myd88 and NF-κβ) were also downregulated in SSP-treated mice. These findings suggest that SSP may suppress microglial activation in DR by inhibiting the TLR4/MyD88/NF-κβ signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHowever, several issues require further exploration in upcoming research. First, further investigation is warranted to elucidate SSP\u0026rsquo;s effects on inflammatory responses in activated microglia, including the expression of critical downstream cytokines. Second, the role of microglia in BRB disruption requires deeper exploration. While our study observed microglial clustering near retinal vessels, the direct impact of microglia on BRB integrity was not addressed. Future studies could employ ex vivo BRB models to dissect microglial contributions to barrier dysfunction. Finally, microglial polarization\u0026mdash;specifically the balance between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes\u0026mdash;should be characterized in diabetic retinas. Unraveling the polarization states and associated molecular mechanisms will clarify SSP\u0026rsquo;s anti-inflammatory actions in DR.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings demonstrate that SSP ameliorates diabetic retinopathy (DR) progression in mice, exerting protective effects including preservation of retinal vasculature, attenuation of retinal thinning, suppression of intraretinal apoptosis, and prevention of vascular leakage. These benefits are likely mediated by SSP\u0026rsquo;s glucose- and lipid-lowering properties, coupled with its inhibition of microglia-driven inflammatory responses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cp\u003e The experimental protocol for this study was reviewed and approved by the Ethics Committee for Animal Experimentation of Beijing University of Chinese Medicine (approval no. BUCM-2024102805-4090).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the National Natural Science Foundation of China [grant number 82174474]\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYJ, MZ were responsible for conducting the experiments, analyzing data, generating figures, and writing the manuscript. JZ was responsible for designing the methodology. YL (Yao Liang), JD and WL were responsible for data collection and conducting experiments. YL (Yan Liao) confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eThe data generated in the present study may be requested from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGurudas S, Vasconcelos JC, Prevost AT, Raman R, Rajalakshmi R, Ramasamy K, Mohan V, Rani PK, Das T, Conroy D, Tapp RJ, Sivaprasad S, Collaborators SM-IS (2024) National prevalence of vision impairment and blindness and associated risk factors in adults aged 40 years and older with known or undiagnosed diabetes: results from the SMART-India cross-sectional study. 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Mol Neurobiol 58 (5):2407-2422. doi:10.1007/s12035-020-02227-3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"diabetic retinopathy, Sagittaria Sagittaria polysaccharide, microglia, TLR4","lastPublishedDoi":"10.21203/rs.3.rs-7204796/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7204796/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSagittaria sagittifolia polysaccharide (SSP) exhibits anti-inflammatory, antioxidant, lipid-regulating, and hypoglycemic properties, demonstrating therapeutic potential against diabetic retinopathy (DR). This study aimed to investigate the intervention efficacy of SSP in DR mice and its regulatory effects on retinal microglia activation. The type 2 diabetic mouse model was established by high-fat diet feeding combined with streptozotocin (STZ) intravenous injection. At week 9, retinal vascular pathology was assessed via fundus photography and fluorescein angiography. Serum lipid metabolism TG, CHO, LDL and HDL were quantified using an automated biochemical analyzer. Retinal histopathology and thickness were evaluated through HE staining combined with evans blue staining. Microglial activation adjacent to retinal vasculature was visualized by immunofluorescence, while retinal apoptosis was examined using immunohistochemistry and TUNEL staining. Co-localization of TLR4 and Iba was analyzed by immunofluorescence. Protein expression levels of TLR4, Myd88, P-p65, and total p65 in retinal tissues were determined by Western blot. SSP treatment significantly attenuated DR progression, as evidenced by preserved retinal vascular integrity, restored retinal thickness, reduced vascular leakage, lowered fasting blood glucose, and regulated lipid metabolism (reduced TG/TC/LDL-C, increased HDL-C). Furthermore, SSP suppressed pathological recruitment of microglia to retinal vasculature and inhibited their pro-inflammatory morphological transition. Mechanistically, SSP downregulated TLR4/Iba co-expression and inhibited downstream Myd88/NF-κβ signaling pathway. The study results demonstrated that SSP can delay the progression of retinopathy in type 2 diabetic mice. This mechanism seems to be associated with SSP's blood glucose-lowering and lipid-regulating effects, along with its inhibition of microglia-mediated inflammatory responses.\u003c/p\u003e","manuscriptTitle":"Sagittaria Sagittaria polysaccharide protects against retinal vascular damage in diabetic mice by suppressing TLR4 signaling pathway and microglial activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 17:03:46","doi":"10.21203/rs.3.rs-7204796/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-21T10:06:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T18:26:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T04:38:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147656531592726285545140001304082701713","date":"2025-08-12T15:49:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249786026125176678918605493564643828184","date":"2025-08-07T03:24:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-07T02:55:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T10:11:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-29T10:07:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2025-07-24T10:51:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"299bfd5e-0a93-4218-8db6-0e6e1c1c301d","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:08:07+00:00","versionOfRecord":{"articleIdentity":"rs-7204796","link":"https://doi.org/10.1007/s12035-025-05388-1","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2025-11-25 15:58:25","publishedOnDateReadable":"November 25th, 2025"},"versionCreatedAt":"2025-08-12 17:03:46","video":"","vorDoi":"10.1007/s12035-025-05388-1","vorDoiUrl":"https://doi.org/10.1007/s12035-025-05388-1","workflowStages":[]},"version":"v1","identity":"rs-7204796","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7204796","identity":"rs-7204796","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}