Bergenin mitigates neuroinflammatory damage induced by high glucose: insights from Zebrafish, murine microbial cell line, and rat models

Abstract

Background: The escalating global burden of diabetes and its associated cognitive impairment underscores the urgency for effective interventions. Bergenin shows promise in regulating glucose metabolism, mitigating inflammation, and improving cognitive function. Zebrafish models offer a unique platform for assessing drug efficacy and exploring pharmacological mechanisms, complemented by subsequent investigations in cell and rat models.

Methods: The experimental subjects included zebrafish larvae (CZ98:Tg (mpeg1:EGFP) ^ihb20Tg/+ ), adult zebrafish (immersed in 2% glucose), BV2 cell line (50 mM glucose + 10 μm Aβ_1-42), and a streptozotocin (STZ) bilateral intracerebroventricular injection rat model. Bergenin's effects on the toxicity, behavior, and cognitive function of zebrafish larvae and adults were evaluated. The Morris water maze assessed cognitive function in rats. Neuronal histopathological changes were evaluated using HE and Nissl staining. qPCR and Western blot detected the expression of glycolysis enzymes, inflammatory factors, and Bergenin's regulation of PPAR/NF-κB pathway in these three models.

Results: 1) In zebrafish larvae, Bergenin interventions significantly reduced glucose levels and increased survival rates while decreasing teratogenicity rates. Microglial cell fluorescence in the brain notably decreased, and altered swimming behavior tended to normalize. 2) In adult zebrafish, Bergenin administration reduced BMI and blood glucose levels, altered swimming behavior to slower speeds and more regular trajectories, enhanced recognition ability, decreased brain glucose and lactate levels, weakened glycolytic enzyme activities, improved pathological changes in the telencephalon and gills, reduced expression of pro-inflammatory cytokines, decreased ins expression and increased expression of irs1, irs2a, and irs2b, suggesting a reduction in insulin resistance. It also altered the expression of pparg and rela. 3) In BV2 cell line, Bergenin significantly reduced the protein expression of glycolytic enzymes (GLUT1, HK2, PKFKB3, and PKM2), lowered IL-1β, IL-6, and TNF-α mRNA expression, elevated PPAR-γ protein expression, and decreased P-NF-κB-p65 protein expression. 4) In the rat model, Bergenin improves learning and memory abilities in STZ-induced rats, mitigates neuronal damage in the hippocampal region, and reduces the expression of inflammatory factors IL-1β, IL-6, and TNF-α. Bergenin decreases brain glucose and lactate levels, as well as glycolytic enzyme activity. Furthermore, Bergenin increases PPARγ expression and decreases p-NF-κB p65/NF-κB p65 expression in the hippocampus.

Conclusion: Bergenin intervenes through the PPAR-γ/NF-κB pathway, redirecting glucose metabolism, alleviating inflammation, and preventing high glucose-induced neuronal damage.

Keywords: Zebrafish; bergenin; diabetes-associated cognitive impairment (DACI); glycolysis; neuroinflammation.

FIGURE 1

Effects of Bergenin on High-Glucose-Induced Zebrafish Larvae. (A) Glucose Content in Zebrafish Larvae; Hatchability, Survival Rate, Teratogenicity Rate of Zebrafish Larvae; (B) Teratogenicity in zebrafish larvae; The arrow points to teratogenic effects, such as organ edema or tail malformation; (C) Fluorescence Expression in the Brains of Zebrafish Larvae and Relative Fluorescence Intensity; (D) Behavioral Trajectory Plot of Zebrafish Larvae and Statistical Analysis of Zebrafish Larval Behavior Data: Total Swimming Distance; Low-Speed Distance (<2 mm/s); Medium-Speed Distance (2–10 mm/s); High-Speed Distance (>10 mm/s). “ns” indicates no statistical significance; ^## P < 0.01 indicates significance compared to the control group; *P < 0.05, **P< 0.01 indicates significance compared to the model group. (One-way ANOVA and Pearson Chi-square test, Mean ± SD, N = 30).

FIGURE 2

Effects of Bergenin on High-Glucose-Induced Zebrafish Adults. (A) Imaging of Adult Zebrafish After Modeling and Drug Administration; (B) Body Mass Index (BMI) of Zebrafish Adults; (C) Blood Glucose of Zebrafish Adults; (D) Zebrafish Behavioral Trajectories; (E) Statistical Analysis of Zebrafish Behavior Data: Low-Speed Distance (<2 cm/s); Medium-Speed Distance (2–5 cm/s); High-Speed Distance (>5 cm/s); Total Swimming Distance; Swimming Speed; (F) Actual Scene of T-Maze; (G) Zebrafish Behavioral Trajectories in the T-maze. (H) Statistical Analysis of Zebrafish T-Maze Data: Total Swimming Distance; Mean Swimming Speed; Swimming Distance in EC area; Swimming Time in EC area. ^# P < 0.05, ^## P < 0.01, ^#### P < 0.001 indicates significance compared to the control group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicates significance compared to the model group. (One-way ANOVA, Mean ± SD, N = 20).

FIGURE 3

Effects of Bergenin on Inflammation and Insulin Resistance in High-Glucose-Induced Zebrafish Adults. (A) Pathological Changes in the Telencephalon Region of High-Glucose-Induced Zebrafish Adults: The arrow indicates activated and aggregated microglial cells (HE staining); (B) Pathological Changes in Gill Tissues: The arrow indicates inflammation response in the gill, characterized by capillary disorder, dilation, and congestion (HE staining); (C) The Effect of Bergenin on Inflammatory Factors and Insulin Resistance in High Glucose-Induced Zebrafish: Relative mRNA level of il1b, il6, tnfa, ins, irs1, irs2a, and irs2b. “ns” indicates no statistical significance; ^## P < 0.01, ^#### P < 0.0001 indicates significance compared to the control group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicates significance compared to the model group. (One-way ANOVA, Mean ± SD, N = 15).

FIGURE 4

Effects of Bergenin on Glycolysis and PPAR-γ/NF-κB pathway in High-Glucose-Induced Zebrafish Adults. (A–E): The Effect of Bergenin on Glucose, Lactic Acid, and Glycolytic Key Enzymes in High Glucose-Induced Zebrafish: (A) Glucose content; (B) Lactic acid content; (C) HK activity; (D) PFK activity; (E) PK activity; (F–K): The Effect of Bergenin on PPAR-γ/NF-κB pathway mRNA in High Glucose-Induced Zebrafish: (F–H) Relative mRNA levels of pparg in the brain, liver, and muscle of zebrafish; (I–K) Relative mRNA level of rela in the brain, liver and muscle of zebrafish. “ns” indicates no statistical significance; ^# P < 0.05, ^## P < 0.01 indicates significance compared to the control group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicates significance compared to the model group. (One-way ANOVA, Mean ± SD, N = 15).

FIGURE 5

Effects of Bergenin in High Glucose-Induced BV2 Cells. (A) The Effect of Bergenin on Inflammatory Factors in High Glucose-Induced BV2 Cells: Relative mRNA level of IL-1β, IL-6, TNF-α; B–F: The Effect of Bergenin on Glycolytic Key Enzymes in High Glucose-Induced BV2 Cells: The Western blot images of glycolysis-related enzymes (B). Data are represented as GLUT1/β-actin (C), HK2/β-actin (D), PFKFB3/β-actin (E) and PKM2/β-actin (F). (G–I): The Effect of Bergenin on the PPAR-γ/NF-κB Pathway in High Glucose-Induced BV2 Cells: The Western blot images of PPAR-γ/NF-κB Pathway (G). Data are represented as PPAR-γ/β-actin (H) and p-p65/p65 (I). “ns” indicates no statistical significance; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 indicates significance compared to the control group; *P < 0.05, **P < 0.01, ***P < 0.001 indicates significance compared to the model group. (One-way ANOVA, Mean ± SD, N = 3).

FIGURE 6

Effects of Bergenin in STZ-Induced Intracerebral Injection Rat Model. (A) The Morris Water Maze Trajectory Map evaluated the neurological functions, as well as the learning and memory abilities of rats. (B) Statistical data from the Morris Water Maze included the mean swimming speed, latency time to discover the platform, residence time in the quadrant, and frequency of crossing the target platform. (C–F): Effects of Bergenin on the Morphology of Hippocampal Neurons in the STZ-induced Intracerebral Injection Rat Model Detected by HE Staining (C, E) and Nissl Staining (D, F), The arrows respectively indicate pyknosis (C); (G) The Effect of Bergenin on Inflammatory Factors in STZ-Induced Intracerebral Injection Rat Model: Relative mRNA level of IL-1β, IL-6, TNF-α. ^# P < 0.05, ^## P < 0.01 indicates significance compared to the control group; *P< 0.05, **P< 0.01 indicates significance compared to the model group. (One-way ANOVA, Mean ± SD, N = 3).

FIGURE 7

Effects of Bergenin on Glycolysis and PPAR-γ/NF-κB Pathway in STZ-Induced Intracerebral Injection Rat Model. A–E: The Effect of Bergenin on Glucose, Lactic Acid, and Glycolytic Key Enzymes in STZ-Induced Intracerebral Injection Rat Model: (A) Glucose content; (B) Lactic acid content; (C) HK activity; (D) PFK activity; (E) PK activity; (F–H): The Effect of Bergenin on the PPAR-γ/NF-κB Pathway in STZ-Induced Intracerebral Injection Rat Model: The Western blot images of PPAR-γ/NF-κB Pathway (F). Data are represented as PPAR-γ/β-actin (G) and p-p65/p65 (H). ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 indicates significance compared to the control group; *P < 0.05, **P < 0.01,***P < 0.001 indicates significance compared to the model group. (One-way ANOVA, Mean ± SD, N = 3).

FIGURE 8

Bergenin regulates glycolysis process to inhibit microglial inflammatory activation. Image created by Figdraw (ID:OAAWI4291b).

FIGURE 9

The mechanism of action of bergenin in alleviating inflammation through the PPAR-γ/NF-κB pathway. Image created by Figdraw (ID: UOYRY932b3).