Aucubin mitigates the elevation of microglial aerobic glycolysis and inflammation in diabetic neuropathic pain via aldose reductase

Abstract

Background: Treatment of diabetic neuropathy is often limited by side effects. Aucubin, an iridoid glycoside derived from natural plants, exhibits notable anti-inflammatory and antioxidant properties.

Aim: To investigate the effects of aucubin on diabetic neuropathic pain (DNP) and glycolysis and inflammation in microglia.

Methods: Streptozotocin (STZ) was used to establish a DNP animal model. Blood glucose levels and body weight of mice were measured following STZ administration. Paw withdrawal threshold was calculated for mechanical allodynia. Paw withdrawal latency was recorded for thermal hyperalgesia. The open field test and elevated plus maze was used to assess locomotor activity and anxiety-like behavior. Western blotting was utilized for analysis of protein expression. Immunofluorescence staining was measured for morphometric analysis of microglia. Glycolysis and ATP synthesis in BV-2 cell lines were detected by metabolic extracellular flux analysis. The SwissTargetPrediction and STRING databases were used for comprehensive screening to identify potential target proteins for aucubin. The molecular docking between the possible target proteins and aucubin was investigated using Auto Dock Tool. The BV-2 cell line was transfected with lentiviral AKR1B1-shRNA to further ascertain the function of AKR1B1 in the impact of aucubin on aerobic glycolysis and inflammation during high glucose stimulation.

Results: Aucubin significantly improved pain and anxiety-like behavior in STZ-induced diabetic mice and restored microglial aerobic glycolysis and inflammation. Several public databases and molecular docking studies suggested that AKR1B1, MMP2 and MMP9 are involved in the effect of aucubin on DNP. Aucubin failed to restore aerobic glycolysis and inflammation in the context of AKR1B1 deficiency.

Conclusion: Aucubin has potential as a therapeutic agent for alleviating DNP by inhibiting expression of AKR1B1.

Keywords: Aldose reductase; Aucubin; Diabetic neuropathic pain; Glycolysis; Microglia.

Figure 1

Schematic diagram of the experimental protocol. PWT: Paw withdrawal threshold; STZ: Streptozotocin; CAB: Citric acid buffer; i.p.: Intraperitoneal injection; OFT: Open field test.

Figure 2

Aucubin alleviate streptozotocin-induced diabetic neuropathic pain and anxiety-like behavior. A: Blood glucose level of the diabetic neuropathic pain diabetic neuropathic pain (DNP): Mice after aucubin treatment; B: Body weight of the DNP mice after aucubin treatment; C: Mechanical paw withdrawal threshold in mice; D: Thermal paw withdrawal latency in mice; E: Total distance traveled in the open field test (OFT); F: Distance traveled by mice in the central area in the OFT after streptozotocin streptozotocin (STZ): Injection; G: Total distance traveled in the elevated plus maze (EPM); H: Number of entries of mice into the open arms in the EPM after STZ injection. Data are presented as means ± SEM (n = 10). ^aP < 0.05, ^bP < 0.01 control (CON) + vehicle (Veh) vs STZ + Veh; ^cP < 0.05, dP < 0.01 STZ + Veh vs STZ + aucubin. CON: Control; STZ: Streptozotocin; Veh: Vehicle; Auc: Aucubin; PWT: Paw withdrawal threshold; OFT: Open field test; EPM: Elevated plus maze.

Figure 3

Aucubin releases streptozotocin-induced inflammatory responses. A: Representative bands of tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6; B; Quantitative analysis of the relative expression of TNF-α, IL-1β, and IL-6 (n = 3); C: Representative bands of nuclear factor-κB (NF-κB) in cytoplasmic protein and nuclear protein; D: Quantitative analysis of the relative expression of NF-kappa B in cytoplasmic protein and nuclear protein (n = 3); E: Representative images of immunofluorescence of iba1; F-H: Sholl analysis of microglia of interscetion numbers (F); area of soma (G); and max branch length (H). Data are expressed as mean ± SEM. ^aP < 0.01 streptozotocin (STZ) + vehicle (Veh) vs STZ + aucubin; ^bP < 0.01 control + Veh vs STZ + Veh. CON: Control; STZ: Streptozotocin; Veh: Vehicle; Auc: Aucubin; TNF-α: Tumour necrosis factor-α; IL: Interleukin.

Figure 4

Aucubin reduced aerobic glycolysis and inflammation in the BV-2 cell. A: the mRNA levels of pro-inflammatory cytokine; B-E: The experimental program of the extracellular acidification rate (B and C) and oxygen consumption rate (D and E) of BV-2 measured by Seahorse XFe96 Extracellular Flux Analyzer. Data are expressed as mean ± SEM. n = 6. ^aP < 0.01 high glucose vs high glucose + auccubin (Auc); ^bP < 0.01 normal glucose vs high glucose; ^cP < 0.05 high glucose vs high glucose + Auc; ^dP < 0.05 normal glucose vs high glucose. ECAR: Extracellular acidification rate; 2-DG: 2-deoxy-d-glucose; OCR: Oxygen consumption rate; FCCP: Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Auc: Aucubin; TNF-α: Tumour necrosis factor-α; IL: Interleukin.

Figure 5

The potential target proteins of aucubin on the diabetic neuropathic pain. A: Venn diagram showing the potential target genes for aucubin in treating diabetic neuropathic pain; B: Kyoto Encyclopedia of Genes and Genomes pathway analysis of the potential target genes; C-E: Molecular docking between the target 3 proteins (MMP2, MMP9, and AKR1B1) and aucubin (Auc) conducted using Auto Dock Tool 1.5.7; F: mRNA levels of MMP9, MMP2, and AKR1B1 in mice after streptozotocin (STZ) and Auc treatment. (n = 6); G: Representative bands of Western blot for MMP2 and AKR1B1 in mice after STZ and Auc treatment; H: Quantitative analysis of the relative expression of MMP2 and AKR1B1 (n = 3). Data are expressed as mean ± SEM. ^aP < 0.05, ^bP < 0.01 CON + vehicle (Veh) vs STZ + Veh; ^cP < 0.05, ^dP < 0.01 STZ +Veh vs STZ + Auc. CON: Control; STZ: Streptozotocin; Auc: Aucubin; Veh: Vehicle.

Figure 6

Aucubin fails to restore aerobic glycolysis and inflammation in the context of AKR1B1 deficiency. A: The expression level of AKR1B1 after transfecting AKR1B1 shRNA lentivirus. The quantitative analysis of the relative expression of AKR1B1 below. Data are expressed as mean ± SEM (n = 4); B: The mRNA levels of pro-inflammatory cytokine; C-F: The experimental program of extracellular acidification rate (C and D) and the oxygen consumption rate (E and F) of BV-2 measured by Seahorse XFe96 Extracellular Flux Analyzer. Data are expressed as mean ± SEM (n = 6). ^aP < 0.01, ^bP < 0.05, high glucose vs high glucose + aucubin. NS: No significance; Auc: Aucubin; 2-DG: 2-deoxy-d-glucose; ECAR: Extracellular acidification rate; OCR: Oxygen consumption rate.