Qiming granules regulate Müller cell pyroptosis and the P2X7R/NLRP3 immune inflammatory pathway in diabetic retinopathy
Qi Zhou, Min Tang, Yaping Wang, Hongbin Lv, Guiqi Yang, Fang Wang, Hejiang Ye
TL;DR
Qiming Granules reduce early diabetic retinopathy neurodegeneration by inhibiting Müller cell pyroptosis and immune inflammation via the P2X7R/NLRP3 pathway.
Contribution
This study identifies Qiming Granules as a multi-target therapy for diabetic retinopathy by regulating Müller cell pyroptosis and immune inflammation through the P2X7R/NLRP3 pathway.
Findings
Qiming Granules inhibited RGC apoptosis and retinal cell pyroptosis in a DR neurodegeneration model.
Astragaloside IV and puerarin, absorbed components of QMG, effectively reduced high glucose-induced Müller cell pyroptosis.
QMG suppressed the P2X7R/NLRP3 pathway, reducing immune inflammatory responses in Müller cells.
Abstract
Müller cell pyroptosis and immune inflammation-induced retinal ganglion cell (RGC) damage are the core pathological markers and potential therapeutic targets for neurodegeneration in early diabetic retinopathy (DR). Qiming Granules (QMG)—recommended by the traditional Chinese medicine guidelines for DR owing to their multi-target immunomodulatory, antioxidant, and microvascular protective effects. This study aimed to clarify the protective effect of QMG on early DR neurodegeneration, reveal their neuroprotective mechanism by regulating the P2X7R/NLRP3 pathway to inhibit Müller cell pyroptosis and immune inflammation, identify the chemical components of QMG and its absorbed components in rat plasma, study the effects of major absorbed components on inhibiting Müller cells pyroptosis and immune inflammatory response, and investigate the potential pharmacodynamic substances of QMG. An in…
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TopicsInflammasome and immune disorders · Retinal Diseases and Treatments · Ocular Diseases and Behçet’s Syndrome
Background
Diabetic retinopathy (DR)—a chronic, progressing neurovascular disorder associated with diabetes—is the main cause of visual impairment in in people of working age groups [1]. DR pathogenesis is complex, with inflammation potentially playing a pivotal role. Although hormone-based therapies have shown certain improvements in visual and anatomical outcomes in advanced-stage DR, they cannot reverse permanent vision damage [2]. Therefore, identifying early biomarkers and developing preventive strategies are paramount. Neuronal degeneration, characterized by neuroglial dysfunction and increased apoptosis of retinal ganglion cells (RGCs), precedes microvascular lesions in DR, suggesting a neurodegenerative disease state. Targeting early neurodegenerative changes remains a critical scientific challenge.
Inflammation mediated by Müller cell pyroptosis is a key factor in RGC damage and neurodegenerative pathology. Closely linked to innate immunity, inflammation is the primary response to retinal environmental changes [3]. Müller cells span the entire retinal layer and are critical for protecting neurons and maintaining retinal homeostasis. Hyperglycemia activates Müller cells to secrete inflammatory mediators that amplify innate immune responses and recruit other immune cells to eliminate stressors. However, persistent Müller cell activation creates a self-sustaining immune loop, perpetuating chronic inflammation that ultimately damages RGCs [4]. The purinergic ligand-gated ion channel 7 receptor (P2X7R)/NOD-like receptor family pyrin domain containing 3 (NLRP3) pathway may be a central mediator of Müller cell pyroptosis and immune-inflammatory imbalances. Under high adenosine triphosphate (ATP) stimulation, P2X7R cation channels open, inducing K^+^ efflux and Na^+^/Ca^2+^ influx. Potassium efflux facilitates pannexin-1 pore formation on cell membranes, allowing NLRP3 ligands to enter cells and initiate NLRP3 inflammasome assembly [5]. Apoptosis-associated speck-like protein containing CARD (ASC), pro-caspase-1, and NLRP3 comprise the NLRP3 inflammasome [6]. Hyperglycemia promotes interactions between these components, forming cytoplasmic complexes that cleave pro-caspase-1 into active caspase-1. Gasdermin D (GSDMD) is cleaved by activated caspase-1, which triggers N-terminal oligomerization to create membrane holes and cause pyroptosis. Pro-interleukin (IL)-1β and pro-IL-18 are likewise converted into mature cytokines by caspase-1 [7]. Excessive inflammatory cytokine production triggers an inflammatory cascade, damaging retinal tissue. Studies have demonstrated increased caspase-1 activity and IL-1β production in Müller cells exposed to hyperglycemia, while caspase-1/IL-1β inhibition reduces pyroptosis [8]. P2X7R and NLRP3 suppression protects retinal tissue in diabetic mice, although current research remains limited to microvascular pathology without addressing Müller cell-mediated RGC injury mechanisms [9].
Qiming Granules (QMG) is a traditional Chinese herbal preparation composed of Huangqi (Astragalus mongholicus Bunge), Gegen (Pueraria montana var. lobata), Dihuang (Rehmannia glutinosa Libosch.), Gouqizi (Lycium barbarum L.), Juemingzi (Senna obtusifolia L.), Chongweizi (Leonurus japonicus Houtt.), Puhuang (Typha angustifolia L.), Shuizhi (Whitmania pigra Whitman). It was included in the Pharmacopoeia of the People’s Republic of China (ChP) (2020) and the plant name has been checked with http://mpns.kew.org in August 3, 2025. Among these ingredients, Astragalus mongholicus Bunge and Pueraria montana var. lobata act as monarch drugs, with astragaloside IV and puerarin as their primary active constituents. QMG is recommended in traditional Chinese medicine guidelines for DR treatment [10]. Clinical trials have demonstrated its efficacy in reducing retinal microaneurysms, hemorrhages, and exudates, while delaying vision loss [10]. Pharmacological studies have revealed that Astragaloside IV alleviates endothelial dysfunction caused due to high hyperglycemia by inhibiting P2X7R-dependent p38 MAPK pathway and that puerarin inhibits oxidative stress and NLRP3 inflammasome activation to suppress RGC apoptosis [11, 12]. Although QMG has shown therapeutic effects in early DR, their potential to modulate Müller cell pyroptosis, immune–inflammatory responses, and RGC degeneration remains unexplored.
In this study, we aimed to investigate the protective effect of QMG against early DR-associated neurodegeneration and explore its role in inhibiting pyroptosis and immune inflammation in retinal Müller cells. In addition, we aimed to elucidate the mechanisms by which QMG regulates the P2X7R/NLRP3 pathway to suppress Müller cell pyroptosis and neuroinflammation. Additionally, we aimed to identify the chemical constituents of QMG and their blood-entry components. The inhibitory effects of these major blood-entry components on high glucose-induced pyroptosis and immune inflammation in Müller cells were examined, thereby providing insights into the potential pharmacodynamic material basis of QMG.
Methods
Chemicals and reagents
Streptozotocin (STZ) was purchased from Sigma-Aldrich (St. Louis, MO, USA); brilliant blue G (BBG) from Technology Co., Ltd. (Beijing, China); a Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit from Hoffmann-La Roche (Basel, Switzerland); enzyme-linked immunosorbent assay (ELISA) kits for IL-1β and IL-18 from Elabscience (Wuhan, China); fetal bovine serum (FBS) and HG Dulbecco’s modified Eagle’s medium (DMEM) from Gibco (USA); a cell counting kit-8 (CCK-8) from Lanjieke Technology Co., LTD (Beijing, China); a lactate dehydrogenase (LDH) kit from Jianglai Biotechnology Co., LTD (Shanghai, China); antibodies against polyclonal GSDMD, GSDMD-N, Caspase 1, CASPASE 1 P10, and NLRP3 from Invitrogen (Carlsbad, CA, USA); antibodies against ASC, IL-18, and IL-1β from Proteintech (Wuhan, China); antibodies against P2X7R from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); and BzATP triethylammonium salt and A438079 hydrochloride were purchased from MedChemExpress (USA); Astragaloside IV from Wansheng Pharmaceutical Co., Ltd (Zhejiang, China) and Puerarin from Must Bio-Technology Co., Ltd. (Chengdu, China).
QMG preparation and HPLC analysis
QMG was obtained from Zhejiang Wansheng Pharmaceutical Co. LTD (Batch Number: Z20090036). The quality of QMG was evaluated by high-performance liquid chromatography (HPLC) systems. QMG (0.6 g) with 50 mL of methanol was extracted for 45 min in an ultrasonic water bath. The extract weight loss was compensated for with methanol prior to filtration. The resulting filtrate served as the test solution. Astragaloside IV (6.80 mg) and Puerarin (4.54 mg) reference standards were precisely weighed and dissolved in methanol to prepare reference solutions with concentrations of 0.68 mg/mL and 0.454 mg/mL, respectively. The components were analyzed using a PDA detector with a Diamonsil Plus C18 column (4.6 × 250 mm2, 5 µm). The mobile phase consisted of acetonitrile and 0.1% phosphoric acid aqueous solution with 0–10 min and 20–25% gradient elution. The detection wavelength, column temperature, and flow rate were maintained at 250 nm, 30 °C, and 1.0 mL/min, respectively. Both the reference standard and test solutions were precisely injected (10 µL each) into the HPLC system for analysis.
Animals and experimental design
All animal experimental procedures were approved by the Medical Ethics Committee of Chengdu University of Traditional Chinese Medicine (Grant Number: 2021–70). Male Sprague–Dawley rats (6–8 weeks old, 140–160 g) were provided by Chengdu Dashuo Experimental Animal Co. Ltd. (Chengdu, China). The rats were maintained in a specific-pathogen-free room under a standard 12/12 h light/dark cycle and 55–60% humidity at 22–24 °C with ad libitum feeding.
After a 1-week adaptation period, the rats were randomly divided into blank control and vehicle groups and fed normal chow and high-fat diets, respectively. After four weeks of feeding, all animals were fasted for 12 h. The vehicle group received an intraperitoneal injection of 1% STZ solution (35 mg/kg body weight), whereas the control group received an equivolume of sterile citrate buffer. Seventy-two hours after STZ injection, rats with fasting blood glucose levels > 16.7 mmol/L were considered successfully established type 2 diabetes mellitus (T2DM) models. After 4 weeks of feeding, hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM) were conducted to confirm diabetic retinal neurodegeneration model establishment.
Animal experiments were conducted in two stages. First, model rats were randomly divided into four groups (n = 10 per group): model group and three QMG groups (0.61 g/kg, 1.22 g/kg, and 2.44 g/kg QMG by oral gavage). Second, 52 rats were divided into four groups (n = 14 per group): normal control group; model group; QMG group, high-dose QM granules by oral gavage; and BBG group, P2X7R antagonist BBG (50 mg/kg/d) intraperitoneal injection. The control and model groups were administered equal volumes of saline by oral gavage. All groups were treated consecutively once daily for 12 weeks. Rats’ eyeballs were collected for subsequent experiments.
Cell cultures and treatments
Retinal Müller cells (rMC-1) were purchased from Beina Chuanglian Biotechnology Research Institute (Beijing, China) and cultured in DMEM containing 10% FBS at 37 °C in 5% CO_2_ humidified air. The cells were randomly divided into the control, model, QMG, BzATP, A438079, BzATP + QMG, and A438079 + QMG groups.
Histopathology
The left eyeballs were immersed in FAS eye fixative solution for 24 h. After dehydration using an ethanol gradient, eyeballs were embedded in paraffin and cut into 4-µm slices. HE staining was employed to outline the retina’s general architecture. The retinal tissue was measured and analyzed. The first measurement point was set at approximately 500 μm from the center of the optic nerve. Subsequently, the second, third, fourth, and fifth measurement points were determined at intervals of approximately 500 μm each. A total of five measurement points were identified on one side, resulting in ten measurement points bilaterally. At each point, the thickness of the ganglion cell layer was measured, while the number of ganglion cells were counted.
TUNEL assay
TUNEL staining was applied to observe RGC apoptosis. Frozen sections were embedded in 4% paraformaldehyde and washed with TBST. Sections were incubated with anti-NeuN overnight at 4 °C. The next day, after several washes with phosphate-buffered saline (PBS), sections were incubated with Alexa Fluor^®^594 donkey anti-rabbit lgG (H + L) and further washed in TBST. A fluorescent microscope (Leica, Japan) was used to photograph the sections after the nuclei were stained with DAPI. Six sections from each group were randomly selected to count the number of TUNEL-positive NeuN-labeled RGCs in the retina.
TEM
Retinal eyecups were prefixed with 2.5% glutaraldehyde and then fixed with 1% osmium tetroxide. After dehydration, embedding, and slicing, 60–90-nm sections were stained with uranyl acetate and lead citrate. Ultrastructural changes in the RGCs, capillary lumen, and Müller cells were observed using a JEOL TEM (Tokyo, Japan). Under electron microscopy, the nuclear morphology of ganglion cells and mitochondrial swelling in Müller cells were observed. In each group, five random photographic fields were selected for analysis at identical magnification, with three ganglion cells and ten mitochondria analyzed per field. Nuclear morphology was scored as follows: 0-chromatin evenly distributed, nuclear membrane smooth and continuous, nucleolus intact; 1-chromatin mildly aggregated, nuclear membrane smooth, nucleolus normal or mildly enlarged; 2-chromatin noticeably aggregated, nuclear membrane invaginated, indented, or irregular, nucleolus enlarged; 3-chromatin aggregated, nuclear membrane invaginated and fragmented, nucleolus absent. Mitochondrial swelling was graded using the Flameng scoring criteria: 0-normal appearance; 1-structure largely intact, matrix density reduced; 2-mitochondria swollen, cristae separated; 3-cristae fractured, mitochondria severely swollen; 4-inner and outer membranes ruptured, mitochondria severely swollen and vacuolated.
ELISA
IL-1β and IL-18 levels in the retinal and treated cells were quantified using ELISA kits according to the manufacturer’s instructions. Color change was determined at 450 nm. A standard curve was plotted according to the standard wells’ absorbance values and the standard’s concentration. Each sample’s concentration was calculated according to its optical density value.
Cell viability assay
The effects of glucose and QMG on rMC-1 viability were evaluated using a CCK-8 assay. Specific testing steps were performed according to the manufacturer’s instructions. Cells were seeded in 96-well plates at a density of 5 × 10^3^ cells/well and cultured in complete medium supplemented with glucose (0, 5, 10, 25, 50, 75, 100, and 150 mM) for 48 h. Mannitol was added to the 5.5 mM group for osmotic pressure adjustment to correspond with that of G10, G25, G50, G75, G100, and G150, thus forming M10, M25, M50, M75, M100, and M150 (mannitol concentrations 4.5, 19.5, 44.5, 71.5, 94.5, and 134.5 mmol/L, respectively) [13]. The cells were treated with different QMG concentrations (0, 10, 25, 50, 100, 150, 300, 600 μg/mL). Absorbance was measured at 450 nm using a microplate spectrophotometer. Each experiment was repeated six times.
To determine the optimal intervention concentrations of astragaloside IV and puerarin, the primary systemic components of QMG, Müller cells were treated with each compound at concentrations of 0, 6.25, 12.5, 25, 50, 75, 100, 150, and 200 μmol/L for 12 h, followed by cell viability assessment using the CCK-8 assay.
Lactate dehydrogenase determination
Live cells contain cytoplasmic lactate dehydrogenase (LDH). Normally, the cell membrane prevents LDH translocation; however, LDH is released from cells when the cell membrane is compromised. Microglial activity was evaluated to verify the cytomembrane integrity. Supernatants of cultured Müller cells were collected and measured using an LDH assay kit according to the manufacturer’s protocol. An absorbance plate reader quantified LDH release from Müller cells.
Western blot analysis
Retina or Müller cells were lysed using radioimmunoprecipitation assay lysis buffer. Lysates were clarified by centrifugation at 13,500 rpm for 15 min at 4 °C. The total protein concentration was determined using the BCA protein assay kit; the proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies against GSDMD, GSDMD-N, caspase-1, cleaved caspase-1, IL-18, IL-1β, P2X7R, NLRP3, and ASC overnight at 4 °C. Protein band intensities were quantified by densitometric analysis using ImageJ software and expressed as relative values normalized to β-actin.
Immunofluorescence
Müller cells were fixed in 4% paraformaldehyde for 15 min, washed twice with PBS, and incubated with 0.25% Triton-X-100 for 5 min. Subsequently, 10% goat serum solution was applied for blocking for 30 min, followed by overnight incubation with primary antibodies at 4 °C. The next day, after three washes with PBS with Tween 20, fluorescently labeled goat anti-rabbit/mouse secondary antibodies were added and incubated for 1 h. After washing with PBS with Tween 20, the samples were observed and imaged using a fluorescence microscope, and fluorescence intensity values were recorded using a software interface.
UPLC-Q-Orbitrap HRMS analysis
Eight rats were randomly divided into two groups: a blank control group and a QMG group. The QMG group received oral administration of QMG suspension at a dose of 2.44 g/kg (1 mL/100 g body weight), while the blank control group received an equal volume of distilled water. Administration was performed twice daily for three consecutive days. One hour after the last administration, rats were anesthetized with 1% sodium pentobarbital (50 mg/kg). Blood was collected from the abdominal aorta into heparinized vacuum tubes and centrifuged at 3000 rpm for 10 min at 4 °C to obtain plasma. Plasma from rats within the same group was pooled to minimize individual variations and stored at − 80 °C until analysis. For sample pretreatment, 400 μL of plasma was mixed with 1200 μL methanol, vortexed for ≥ 30 s, and incubated at 4 °C for ≥ 20 min to precipitate proteins. After centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatant was collected, dried under nitrogen, reconstituted in 1000 μL methanol, and filtered through a 0.22 μm membrane before UPLC-MS analysis.
Test solutions of QMG, as well as reference standard solutions of astragaloside IV and puerarin, were prepared. Chromatographic separation was performed using a gradient elution with mobile phase A (acetonitrile) and B (0.1% formic acid in water) as follows: 10–15% A (0–3 min), 15–20% A (3–5 min), 20–26% A (5–8 min), 26–35% A (8–15 min), 35–70% A (15–40 min), and 70–95% A (40–55 min). The column temperature was 30 °C, flow rate 0.4 mL/min, and detection wavelengths were set at 210, 250, 284, and 330 nm. The injection volume was 5 μL. Mass spectrometry was conducted using ESI ionization with positive/negative switching mode. Key parameters: capillary voltage ± 3.5/–3.0 kV, capillary temperature 320 °C, heater temperature 300 °C, sheath gas 35 arb, and auxiliary gas 10 arb. Full scan resolution was 35,000 FWHM (m/z 100–1500), and dd-MS2 resolution was 17,500 FWHM with stepped NCE (20, 40, 60 eV).
Statistical analysis
Regarding the CCK-8 assay, experiments were performed with eight replicates per group and repeated in three independent experiments. Other in vitro experiments were performed in triplicate and repeated three independent times. Quantitative data are presented as mean ± standard error of mean. All analyses were performed using GraphPad Prism 8.0. One-way analysis of variance with post hoc Tukey's test was used to determine between-group differences, followed by the Bonferroni post hoc test. P < 0.05 was considered statistically significant.
Results
Quality evaluation of QMG by HPLC
According to the Chinese Pharmacopoeia 2020 edition, the contents of Astragaloside IV and puerarin in each bag of QMG (4.5 g) must exceed 1.1 mg and 32.0 mg, respectively. Our results showed that Astragaloside IV in the QMG was detected at 11.470 min during injection, with a peak area of 1389, whereas the reference standard for Astragaloside IV exhibited a peak area of 255,859. Using the single-point external standard method, the calculated Astragaloside IV content in QG was 1.3875 mg/bag. Similarly, puerarin in QMG was detected approximately 3.604 min after injection, displaying a peak area of 2,966,983, whereas the reference standard for puerarin showed a peak area of 5,849,100. The puerarin content was 57.57 mg/bag (Table 1). Both results complied with the Chinese Pharmacopoeia, 2020 edition, specifications. Table 1. The contents of astragaloside IV and puerarin in Qiming Granules identified by high-performance liquid chromatographyNameTime(min)Peak aeraContent(mg/4.5 g)Standard(mg/4.5 g)Astragaloside IV11.4713891.39 > 1.10Puerarin3.602,966,98357.57 > 32.00
QMG reduces neurodegeneration and inflammation in DR rats
The protective effects of QMG against diabetic retinal neurodegeneration were evaluated. Using HE staining, thinner ganglion cell layer, reduced number of retinal RGCs and vacuolar degeneration were observed in the model group, along with loosely arranged inner and outer nuclear layers. However, after QMG administration, the thickness of the retinal ganglion cell layer in the model rats increased; and the retinal RGC number increased, with more substantial effects observed in the high-dose group (Fig. 1A, B).Fig. 1. Qiming Granules alleviate retinal neurodegeneration and inflammation in DR rats. A HE-stained retinal sections, magnification 40 × , scale bar = 20 µm. Vacuolar degeneration of RGCs were marked with black arrows, and disorganized arrangement in the inner nuclear layer (INL) and outer nuclear layer (ONL) were marked with the yellow and red arrows, respectively. B GCL thickness and RGCs counts in each group. C TEM of RGCs, capillaries, and Müller cells in INL. Red arrows: chromatin condensation in RGCs. Yellow arrows: mitochondrial vacuolization in Müller cells, scale bars, 2 μm (lower magnification), 500 nm (higher magnification). D RGCs Nucleus morphology score and mitochondria score. E Immunofluorescent staining of NeuN with TUNEL, magnification 40× , scale bar = 20 µm. F Apoptotic RGCs counts in each group. G The expression levels of IL-1β and IL-18 in the retina of DR rats. (mean ± SEM, ****P < 0.0001 compared to control group; ^#^P < 0.05, ^##^P < 0.01, ^####^P < 0.0001 compared to model group, ns P > 0.05)
TEM revealed that RGCs in the model group exhibited apoptotic features, such as nuclear shrinkage, chromatin condensation, and cytoplasmic fragmentation, whereas Müller cells showed mitochondrial swelling and vacuolar changes in the cytoplasm. In the high- and medium-dose QMG groups, RGCs displayed intact, rounded nuclei with uniformly distributed chromatin, and vacuolar-like changes in Müller cell mitochondria in the inner nuclear layer were alleviated. No group exhibited abnormal manifestations, such as basement membrane thickening or endothelial cell/pericyte abnormalities in the capillaries (Fig. 1C, D). These results demonstrate that high- and medium-dose QMG can significantly improve retinal RGC and Müller cell ultrastructure in rat models.
TUNEL staining combined with NeuN immunofluorescence revealed that RGC apoptosis in the model group was significantly increased compared with that in the control group. Treatment with high-, medium-, and low-dose QMG significantly reduced RGC apoptosis in the model rats, with the high-dose group showing the most pronounced effect (Fig. 1E, F).
ELISA results demonstrated that retinal inflammatory cytokine levels (IL-1β and IL-18) in model rats were significantly elevated compared with those in the control group. QMG administration at high, medium, and low doses markedly suppressed the expression of these inflammatory factors, with the high-dose group showing the most prominent inhibitory effect (Fig. 1G). These findings further verified the therapeutic potential of QMG in attenuating inflammatory responses during early-stage DR.
QMG inhibits HG-induced Müller cells pyroptosis and immune-inflammatory responses
QMG inhibited RGC apoptosis and inflammatory responses in a DR neurodegeneration model; however, its specific targets and molecular mechanisms of action remain unclear. Müller cells are the primary source of inflammatory factors under HG stimulation; furthermore, sustained Müller cell pyroptosis exacerbates inflammatory storms and RGC damage [4]. Therefore, we hypothesized that QMG could suppress inflammation by inhibiting Müller cell pyroptosis.
The CCK-8 assay showed that when the glucose concentration reached 75 mmol/L, cell viability decreased, whereas no significant change was observed at the same mannitol concentration. Thus, 75 mM glucose was selected for subsequent experiments (Fig. 2A). After QMG treatment at varied concentrations, Müller cell viability remained stable within the range of 10—100 μg/mL. However, at 150 μg/mL concentration, cell viability significantly decreased. Therefore, 100 μg/mL was chosen as the optimal intervention concentration for follow-up experiments (Fig. 2B).Fig. 2QMG inhibited high-glucose-induced rMC-1 pyroptosis and an immune–inflammatory response. A rMC-1 activity following incubation with glucose or mannitol for 48 h by CCK-8. B rMC-1 activity following incubation with Qiming Granules at different concentrations by CCK-8. C Western blot analysis and quantitative results of relative expressions of pyroptosis related proteins. mean ± SEM, n = 3, ^++^P < 0.01, ^+++^P < 0.001, compared to LPS + ATP group. D The expression of pyroptosis-related proteins detected by western blot analysis. E The expression of GSDMD-N and cleaved caspase-1 detected by immunofluorescent staining. F LDH activity detected by reagents to verify membrane integrity. G IL-1β and IL-18 levels detected by ELISA. (mean ± SEM, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control group; ^#^P < 0.05, ^##^P < 0.01, ^####^P < 0.0001 compared to model group)
In a previous study, lipopolysaccharide (LPS) and ATP were combined to induce pyroptosis [14]. The HG + LPS + ATP group served as the control. The expression levels of GSDMD-N/GSDMD, cleaved caspase-1/caspase-1, IL-1β, and IL-18 were significantly elevated in the HG + LPS + ATP group (Fig. 2C), suggesting that HG conditions promote pyroptosis pathway activation. Thus, the HG + LPS + ATP model was confirmed to be an ideal HG-induced Müller cell pyroptosis model.
Further investigations examined the inhibitory effects of QMG on HG-induced Müller cell pyroptosis. Western blotting demonstrated that QMG reduced the expression levels of key pyroptosis-related proteins, including GSDMD-N/GSDMD, cleaved caspase-1/caspase-1, IL-1β, and IL-18, in the model group (Fig. 2D). Immunofluorescence assays revealed that the fluorescence intensities of GSDMD-N and cleaved caspase-1 were significantly lower in the QMG group than in the model group (Fig. 2E). LDH and ELISA assays indicated that LDH, IL-1β, and IL-18 levels were significantly elevated in the model group, whereas QMG reduced cell membrane damage and inflammatory factor release (Fig. 2F, G). These findings collectively demonstrate that QMG inhibits HG-induced Müller cell pyroptosis and immune–inflammatory responses.
QMG alleviates Müller cell pyroptosis by modulating the P2X7R/NLRP3 signaling pathway
We previously demonstrated that QMG suppresses Müller cell pyroptosis; consequently, we hypothesized that QMG could modulate Müller cell pyroptosis via the P2X7R/NLRP3 pathway. As shown in Fig. 3A, B, QMG downregulated P2X7R, NLRP3, and ASC expression in the model group. The expression of pyroptosis-related proteins increased after the addition of the P2X7R agonist, BzATP. However, QMG inhibited pyroptotic pathway activation induced by BzATP (Fig. 3 C, D). Furthermore, the combined use of QMG and the P2X7R inhibitor, A-438079, enhanced the inhibitory effect of A-438079 on cellular pyroptosis (Fig. 3E, F). Meanwhile, LDH and ELISA assays demonstrated that QMG could reduce BzATP-induced membrane damage in the model group and decrease IL-1β and IL-18 release. Additionally, QMG enhanced the protective effects of A-438079 against membrane injury in the model group (Fig. 3 G, H). Collectively, these results indicated that QMG alleviated Müller cell pyroptosis and immune–inflammatory responses via the P2X7R/NLRP3 pathway.Fig. 3. Qiming Granules alleviate Müller cells pyroptosis by modulating the P2X7R/NLRP3 signaling pathway. A ASC, P2X7R, and NLRP3 protein expression levels detected by WB analysis. B The expressions of P2X7R and NLRP3 were detected by immunofluorescent staining. C After adding BzATP, the expression levels of pyroptosis-related proteins were detected by western blotting. D After adding BzATP, cleaved caspase-1 and GSDMD-N were detected by immunofluorescent staining. E After adding A-438079, the expression levels of pyroptosis-related proteins were detected by western blotting. F After adding A-438079, cleaved caspase-1 and GSDMD-N were detected by immunofluorescent staining. G LDH activity detected by reagents to verify membrane integrity after adding BzATP and A-438079. H IL-1β and IL-18 levels detected by ELISA after adding BzATP and A-438079. (mean ± SEM, **P < 0.0001 compared to control group; ^#^P < 0.05, ^##^P < 0.01, ^###^P < 0.001, ^####^P < 0.0001 compared to model group, ^++^P < 0.01, ^++++^P < 0.0001 compared to BzATP group, ^△^P < 0.05, ^△△^P < 0.01, ^△△△^ P < 0.001, ^△△△△^P < 0.0001 compared to A-438079 group)
QMG inhibit pyroptosis via the P2X7R/NLRP3 signaling pathway in DR rats
In vitro experiments confirmed that QMG inhibited Müller cell pyroptosis and inflammatory factor release via P2X7R/NLRP3 pathway regulation. This experiment was based on an in vivo early DR neurodegeneration model using a high dose of QMG, with the P2X7R inhibitor BBG as the control group. Accordingly, the protein expression levels of pyroptosis-related proteins—cleaved caspase-1/caspase-1, GSDMD-N/GSDMD, IL-1β, and IL-18—were significantly elevated in the model group. In contrast, both the high-dose QMG and BBG groups exhibited significantly reduced expression levels of these pyroptosis-related proteins compared with those in the model group (Fig. 4 A). ELISA assays of retinal IL-1β and IL-18 levels were consistent with the Western blot results (Fig. 4 B), suggesting that QMG inhibits pyroptosis pathway activation in the retinas of DR rats.Fig. 4. Qiming Granules inhibit pyroptosis via the P2X7R/NLRP3 signaling pathway in DR rats. A The expression levels of pyroptosis-related proteins in the retina of diabetic rats were detected by western blot analysis. B ELISA was performed to measure the expression levels of IL-1β and IL-18 in the retina of DR rats. C ASC, P2X7R, and NLRP3 protein levels in the retina of diabetic rats were detected by western blot analysis. (mean ± SEM, **P < 0.0001 compared to control group; ^##^P < 0.01, ^###^P < 0.001, ^####^P < 0.0001 compared to model group, ^△^P < 0.05, ^△△^P < 0.01, ^△△△^ P < 0.001, compared to QMG H-dose group, ns P > 0.05)
Further examination of the P2X7R/NLRP3 pathway revealed that compared with the control group, the model group showed significantly increased retinal protein expression levels of P2X7R, NLRP3, and ASC. Both the high-dose QMG and BBG groups demonstrated significantly reduced expression levels of P2X7R, NLRP3, and ASC, with the BBG group exhibiting a more pronounced reduction (Fig. 4 C). Therefore, QMG alleviated pyroptosis and immune–inflammatory responses in model rats’ retinas by inhibiting the P2X7R/NLRP3 signaling axis.
Analysis of main blood components of QMG
Total ion chromatograms of QMG, blank plasma, and drug-containing plasma were obtained in positive and negative ion detection modes (Fig. 5). A total of 70 compounds were identified in QMG, including 2 amino acids, 3 phenylpropanoids, 2 anthraquinones, 1 anthraquinone glycoside, 1 phenol, 22 flavonoids, 15 flavonoid glycosides, 1 aldehyde, 3 alkaloids, 1 sugar, 3 terpenoids, 6 coumarins, 1 isoflavone, 2 isoflavone glycosides, 5 organic acids, and 2 saponins (supplementary material). After subtracting blank plasma, 9 compounds were identified in the drug-containing plasma: 1 phenol, 2 flavonoids, 2 flavonoid glycosides, 1 alkaloid, 2 terpenoids, and 1 organic acid. Puerarin, astragaloside IV, betaine, vitexin, aconitic acid, formononetin, and daidzein were prototype components, while wilforlide A and gingerol were metabolites (supplementary material). Among them, astragaloside IV and puerarin originate from the monarch medicines Astragalus membranaceus and Pueraria lobata, respectively.Fig. 5. Total ion chromatogram in negative ion mode. A Qiming Granules. B Blank plasma. C Medicated plasma. D Chromatogram of mixed reference substance. 2.62 min: Pueraria, 15.81 min: Astragaloside IV
Effects of astragaloside IV and puerarin on inhibiting high glucose-induced müller cell pyroptosis
When the concentration of astragaloside IV reached 75 μmol/L or higher, and puerarin reached 50 μmol/L or higher, Müller cell viability significantly decreased and progressively declined with increasing drug concentrations (Fig. 6 A). Therefore, 50 μmol/L astragaloside IV and 25 μmol/L puerarin were selected as the ideal intervention concentrations for subsequent experiments.Fig. 6. Astragaloside IV and puerarin alleviated müller cell pyroptosis. A rMC-1 activity following incubation with astragaloside IV and pueraria at different concentrations. B LDH activity after adding astragaloside IV and pueraria. C IL-1β and IL-18 levels detected by ELISA after adding astragaloside IV and puerarin. D The expression of pyroptosis-related proteins detected by western blot analysis. E Cleaved caspase-1 and GSDMD-N were detected by immunofluorescent staining. (mean ± SEM, *P < 0.05, ***P < 0.001, **P < 0.0001 compared to control group; ^#^P < 0.05, ^##^P < 0.01, ^###^P < 0.001, ^####^P < 0.0001 compared to model group, ^△^P < 0.05, ^△△^P < 0.01, ^△△△^ P < 0.001compared to puerarin group, ns P > 0.05)
LDH and ELISA results indicated that both astragaloside IV and puerarin reduced LDH release and levels of IL-1β and IL-18 in the supernatant of the model group (Fig. 6 B, C). Astragaloside IV exhibited superior effects in inhibiting cell membrane damage and inflammatory cytokine release than puerarin. Western blot analysis revealed that the pyroptosis pathway protein levels were significantly reduced in the astragaloside IV group compared with the model group. The puerarin group decreased cleaved caspase-1/caspase-1, GSDMD-N/GSDMD, and IL-1β protein levels, but no significant difference was observed in the IL-18 levels between the two groups (Fig. 6 D). Immunofluorescence results demonstrated that the average fluorescence intensities of GSDMD-N and cleaved caspase-1 were significantly lower in both the intervention groups compared with the model group, with a more pronounced effect in the astragaloside IV group (Fig. 6 E). These findings suggest that both astragaloside IV and puerarin effectively attenuate high glucose-induced Müller cell membrane damage, inflammatory cytokine release, and expression of pyroptosis-related factors. Astragaloside IV exhibits stronger anti-pyroptotic effects than puerarin. Both compounds are active ingredients of QMG in exerting therapeutic effects.
Discussion
DR is a common, severe ocular complication of diabetes mellitus. The increasing prevalence of DR has not only impaired patients' visual function and quality of life but has also imposed a significant medical and economic burden on society [1]. QMG have demonstrated remarkable efficacy in treating DR [10]. To investigate the mechanism by which QMG slow DR progression, we established DR neurodegeneration and HG-induced Müller cell pyroptosis models. Our results indicate that QMG inhibit Müller cell pyroptosis and immune–inflammatory responses via P2X7R/NLRP3 pathway regulation, thereby reducing RGC apoptosis and neurodegeneration in early-stage DR.
Neurodegenerative changes preceding microvascular lesions have been observed in donors with diabetes and in DR animal models [15, 16]. Neurodegeneration in DR is primarily characterized by neuronal apoptosis and glial dysfunction. Studies have reported that STZ-induced diabetic models exhibit apoptosis of RGCs and photoreceptor cells as early as four weeks after modeling [17]. Retinal neurodegeneration exacerbates microvascular damage and disrupts the neurovascular unit, promoting DR progression. Recent research has explored neurotrophic factors, the glutamate antagonists memantine, and brimonidine eye drops for the treatment of early DR neurodegeneration [18–20]. QMG are a clinically empirical, herbal formula for DR treatment and have shown to effectively improve visual function in rats with DR, reduce the thickening of retinal capillary basement membranes, inhibit polyol pathway activation and free radical production, alleviate retinal oxidative damage, and prevent osmotic damage to retinal tissue cells [21]. Additionally, QMG reduced blood–retinal barrier leakage in DM rats and prevented early DR lesions [22]. Clinical studies have demonstrated that QMG reduces retinal microaneurysms, hemorrhages, and exudates in patients with DR; delay DR-induced blindness; and lower the risk of vision loss, providing strong evidence-based support for early DR treatment with traditional Chinese medicine [23]. Modern pharmacological studies have indicated that astragaloside IV can ameliorate diabetic vascular inflammatory lesions by suppressing NLRP3 and IL-1β levels [24], and exert neuroprotective effects by inhibiting RGC apoptosis in db/db mice [25]. In the present study, puerarin inhibited oxidative stress and NLRP3 inflammasome activation, thereby reducing RGC apoptosis [11]. In preliminary experiments, we simulated T2DM using a 4-week high-fat, high-sugar diet combined with an intraperitoneal injection of STZ (35 mg/kg). Accordingly, RGC apoptosis occurred 4 weeks after DM modeling, consistent with early DR neurodegenerative features. These findings align with domestic and international literature and provide a foundation for subsequent pharmacodynamic evaluations and mechanistic studies [17, 26]. In the current study—focused on "treatment"—formal experiments involved administering different QMG doses via oral gavage 4 weeks after DM modeling to evaluate their therapeutic effects on early DR neurodegeneration. Pharmacodynamic studies demonstrated that QMG improves retinal histomorphology in model rats, reduce RGC apoptosis, and decrease inflammatory cytokine expression, including IL-1β and IL-18, in the retina. The neuroprotective effects of QMG on DR neurodegeneration may be achieved by suppressing inflammatory responses.
Inflammatory responses are closely associated with innate immunity and represent the initial reaction to retinal environment changes [3]. Pattern recognition receptors (PRRs) recognize foreign pathogens and endogenous danger signals, triggering downstream immune–inflammatory responses to eliminate pathogenic microbial infections and repair damaged tissues. The NLRP3 inflammasome—composed of NLRP3, ASC, and pro-cysteinyl aspartate-specific proteinase (pro-caspase-1)—is a central intracellular PRR [5]. When the host is exposed to exogenous or endogenous stimuli, pro-caspase-1 and ASC are recruited and interact with NLRP3 to form a large cytoplasmic complex that undergoes autoactivation to hydrolyze pro-caspase-1 into active caspase-1. Activated caspase-1 cleaves pro-IL-1β and pro-IL-18, promoting IL-1β and IL-18 maturation and secretion, thereby regulating immune responses and inflammatory reactions to combat pathogen infections and stress-induced damage [27]. Additionally, activated caspase-1 cleaves GSDMD, triggering oligomerization of the GSDMD N-terminus and formation of cell membrane pores, leading to programmed cell death, known as pyroptosis. Müller cells span the entire retinal layer and play crucial roles in maintaining retinal homeostasis and protecting neurons. In DR, dying retinal Müller cells exhibit hypertrophy, a morphology similar to that observed during pyroptosis [28]. HG-induced pyroptosis in Müller cells results in a self-perpetuating immune response, sustaining a chronic inflammatory state that further damages RGCs [4]. Studies have shown increased caspase-1 activity and IL-1β production in Müller cells of HG-exposed rat retinas, whereas caspase-1/IL-1β inhibition can reduce Müller cell death [29]. In the present study, in vitro experiments confirmed that the model group exhibited pyroptotic characteristics in Müller cells, including cell membrane rupture, increased LDH release, and elevated levels of inflammatory factors, along with upregulated pyroptosis-related protein expression, demonstrating that an HG environment promotes Müller cell pyroptosis. QMG effectively alleviated HG-induced cell membrane damage and inflammatory factor release in Müller cells, suppressed the expression of pyroptosis pathway proteins, and mitigated pyroptosis. In vivo animal experiments further confirmed that QMG inhibited pyroptosis-related protein expression in the retinas of DR neurodegeneration models.
The P2X7 receptor (P2X7R) is a cation-gated channel expressed on the surface of Müller cells, with ATP as its ligand, enabling the passage of ions, such as K^+^, Na^+^, and Ca^2+^. Intracellular K^+^ efflux is a key signal for NLRP3 inflammasome activation, Müller cell pyroptosis, and inflammatory cytokine release [30]. Studies on DR have shown P2X7R/NLRP3 pathway upregulation in diabetic mice and HG-exposed retinal vascular endothelial cells. Inhibiting the activation of P2X7R and the NLRP3 inflammasome can reduce pyroptosis and inflammatory cytokine release, thereby protecting the inner blood–retinal barrier [31, 32]. The nucleoside reverse transcriptase inhibitor, lamivudine, can mitigate neuronal and vascular damage in diabetic mice by targeting P2X7R [33]. In experimental glaucoma models, Müller cell activation and ATP/P2X7R signaling contribute to RGC apoptosis [34]. Furthermore, the P2X7R/NLRP3 pathway’s role in chronic inflammatory neurodegenerative diseases, such as multiple sclerosis and Alzheimer's disease, has been confirmed [35]. Therefore, the P2X7R/NLRP3 pathway has emerged as a novel therapeutic target for neurodegenerative diseases. However, research on its mechanism of action in DR has been limited to microvascular pathologies [36]. The P2X7R/NLRP3 pathway, Müller cell pyroptosis, and immune–inflammatory responses may be critical factors in early DR-related neurodegeneration and RGC injury, thus representing potential therapeutic targets. Our in vivo DR model demonstrated that QMG effectively reduced P2X7R/NLRP3 pathway protein expression in the retinas of model rats. In vitro experiments employed the P2X7R-specific agonist, BzATP, and the P2X7R inhibitor, A-438079, to interfere with the P2X7R/NLRP3 pathway, followed by examination of the downstream pyroptosis pathways and inflammatory factors. These results confirmed that QMG alleviated HG-induced Müller cell pyroptosis and immune–inflammatory responses by modulating the P2X7R/NLRP3 pathway.
The efficacy of compound Chinese herbal medicines depends on their original components and metabolites reaching target organs via systemic circulation, and accumulating at effective therapeutic concentrations. QMG contains multiple components whose systemic absorption and pharmacological mechanisms remain unclear. Using UPLC-Q-Orbitrap HRMS, we identified 70 chemical compounds in QMG and 9 compounds in the bloodstream after administration. Astragaloside IV and puerarin, derived from the principal herbs Astragalus membranaceus and Pueraria lobata, were confirmed as the main prototype blood-borne compounds. Previous studies indicate that astragaloside IV reduces retinal ganglion cell apoptosis in db/db mice by inhibiting ERK1/2 phosphorylation and NF-κB expression [25], and mitigating high glucose-induced endothelial damage via antioxidative effects [37]. Puerarin injection has been shown to alleviate retinal hemorrhage, exudation, and edema in patients with DR, and delay DR progression by suppressing inflammation, oxidative stress, and apoptosis. Both compounds are known to inhibit pyroptosis and inflammatory responses; however, their effects in DR-related cell models remain unexplored. This study demonstrated that both astragaloside IV and puerarin significantly reduced LDH release and decreased of the IL-1β and IL-18 levels in high glucose-stimulated Müller cells. They suppressed membrane damage and key pyroptosis-related protein expression, with astragaloside IV exhibiting more potent effects. These findings confirm that astragaloside IV and puerarin are active ingredients of QG, attenuating Müller cell pyroptosis and inflammatory activation under high glucose conditions.
Conclusions
This study confirms that QMG can inhibit RGCs apoptosis and retinal inflammatory responses in a rat model of DR neurodegeneration. QMG exerts their effects by suppressing the P2X7R/NLRP3 pathway, thereby reducing HG-induced Müller cell pyroptosis and immunoinflammatory responses, ultimately alleviating RGC apoptosis and neurodegeneration (Fig. 7). A total of 70 compounds were identified in QMG, with 9 compounds detected in the bloodstream. Among these, astragaloside IV and puerarin were found to mitigate high glucose-induced Müller cell pyroptosis and immunoinflammation, with astragaloside IV exhibiting a more pronounced effect.Fig. 7. Diagram illustrating the mechanism by which Qiming Granules delay DR neuropathy through inhibiting Müller cells pyroptosis via the P2X7R/NLRP3 pathway
Supplementary Information
Supplementary Material 1.