Tubuloside B ameliorates blood-brain barrier disruption and cognitive deficits after cerebral ischemia by inhibiting TRIC protein ubiquitination and degradation methods
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BCCAO model and drug administration
The CCH model was induced via permanent bilateral common carotid artery occlusion under isoflurane anesthesia. Sham-operated rats underwent identical procedures without ligation. Drug treatments began 24 h post-surgery and continued for 8 weeks.
Blood-brain barrier permeability
BBB integrity was evaluated using Evans blue (EB; 2%, 4 mL/kg) extravasation. EB was quantified fluorometrically (620/680 nm) in formamide-extracted brain tissues.
Methods
Specific pathogen-free male Sprague-Dawley rats (8-10 weeks, 250-300 g; Shanghai SLAC Laboratory Animal Co., Ltd.) were housed under standard conditions. All experimental protocols were approved by the Experimental Animal Ethics Committee of Xuzhou Medical University (XYDW-2023-001). Rats were randomly divided into four groups ( n = 10/group) using a computer-generated randomization sequence: Sham (sham surgery + saline), CCH (BCCAO + saline), CCH + Tub-B-Low (BCCAO + 5 mg/kg Tub-B), and CCH + Tub-B-High (BCCAO + 20 mg/kg Tub-B). The doses of Tub-B (5 mg/kg and 20 mg/kg) were selected based on prior pharmacokinetic studies and preliminary dose-response experiments showing significant neuroprotective effects without toxicity. Tub-B and TCGs (purity > 98%...
Cell culture and drug treatment
PC12 cells were maintained in DMEM with 10% FBS and 1% penicillin-streptomycin. Aβ₂₅-₃₅ oligomers were prepared by incubation at 37 °C for 7 days. Cells were pretreated with Tub-B (1, 5, 10 µM) for 2 h before Aβ₂₅-₃₅ (20 µM, 24 h) exposure. Cell viability was measured using CCK-8 assay.
Tub-B improves cognitive function and BBB integrity in CCH rats
In the Morris water maze test, CCH rats exhibited significantly prolonged escape latency and reduced time in the target quadrant compared to sham controls ( P < 0.05; Fig. ). Treatment with high-dose Tub-B (20 mg/kg) significantly improved spatial memory, increasing platform crossings and target quadrant occupancy ( P < 0.05). Evans blue extravasation assays confirmed that Tub-B dose-dependently reduced BBB leakage, with the high-dose group showing near-normal permeability (0.038 ± 0.007 µg/g vs. CCH: 0.15 ± 0.02 µg/g, P < 0.001; Fig. ).
Tub-B inhibits the expression of pro-inflammatory cytokines in the hippocampal tissue of CCH rats
ELISA showed a significant elevation of TNF-α and IL-1β levels in the CCH group compared to the Sham group (all *** P < 0.001). Treatment with Tub-B particularly at the high dose, markedly suppressed this increase in pro-inflammatory cytokines (TNF-α: * P < 0.05 for CCH + Tub-B-Low vs. CCH, *** P < 0.001 for CCH + Tub-B-High vs. CCH; IL-1β: * P < 0.05 for CCH + Tub-B-Low vs. CCH, *** P < 0.001 for CCH + Tub-B-High vs. CCH). Conversely, Tub-B administration enhanced the production of the anti-inflammatory cytokines IL-10 and TGF-β (*** P < 0.001 for CCH + Tub-B-High vs. CCH). Furthermore, the levels of IL-10 and TGF-β in the CCH + Tub-B-High group were significantly higher than those in the CCH +...
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The CCH model was induced via permanent bilateral common carotid artery occlusion under isoflurane anesthesia. Sham-operated rats underwent identical procedures without ligation. Drug treatments began 24 h post-surgery and continued for 8 weeks.
BBB integrity was evaluated using Evans blue (EB; 2%, 4 mL/kg) extravasation. EB was quantified fluorometrically (620/680 nm) in formamide-extracted brain tissues.
Specific pathogen-free male Sprague-Dawley rats (8-10 weeks, 250-300 g; Shanghai SLAC Laboratory Animal Co., Ltd.) were housed under standard conditions. All experimental protocols were approved by the Experimental Animal Ethics Committee of Xuzhou Medical University (XYDW-2023-001). Rats were randomly divided into four groups ( n = 10/group) using a computer-generated randomization sequence: Sham (sham surgery + saline), CCH (BCCAO + saline), CCH + Tub-B-Low (BCCAO + 5 mg/kg Tub-B), and CCH + Tub-B-High (BCCAO + 20 mg/kg Tub-B). The doses of Tub-B (5 mg/kg and 20 mg/kg) were selected based on prior pharmacokinetic studies and preliminary dose-response experiments showing significant neuroprotective effects without toxicity. Tub-B and TCGs (purity > 98% and > 50%, respectively; Xi'an Leiberei Biotechnology) were administered by gavage starting 24 h after surgery for 8 weeks.
PC12 cells were maintained in DMEM with 10% FBS and 1% penicillin-streptomycin. Aβ₂₅-₃₅ oligomers were prepared by incubation at 37 °C for 7 days. Cells were pretreated with Tub-B (1, 5, 10 µM) for 2 h before Aβ₂₅-₃₅ (20 µM, 24 h) exposure. Cell viability was measured using CCK-8 assay.
In the Morris water maze test, CCH rats exhibited significantly prolonged escape latency and reduced time in the target quadrant compared to sham controls ( P < 0.05; Fig. ). Treatment with high-dose Tub-B (20 mg/kg) significantly improved spatial memory, increasing platform crossings and target quadrant occupancy ( P < 0.05). Evans blue extravasation assays confirmed that Tub-B dose-dependently reduced BBB leakage, with the high-dose group showing near-normal permeability (0.038 ± 0.007 µg/g vs. CCH: 0.15 ± 0.02 µg/g, P < 0.001; Fig. ).
ELISA showed a significant elevation of TNF-α and IL-1β levels in the CCH group compared to the Sham group (all *** P < 0.001). Treatment with Tub-B particularly at the high dose, markedly suppressed this increase in pro-inflammatory cytokines (TNF-α: * P < 0.05 for CCH + Tub-B-Low vs. CCH, *** P < 0.001 for CCH + Tub-B-High vs. CCH; IL-1β: * P < 0.05 for CCH + Tub-B-Low vs. CCH, *** P < 0.001 for CCH + Tub-B-High vs. CCH). Conversely, Tub-B administration enhanced the production of the anti-inflammatory cytokines IL-10 and TGF-β (*** P < 0.001 for CCH + Tub-B-High vs. CCH). Furthermore, the levels of IL-10 and TGF-β in the CCH + Tub-B-High group were significantly higher than those in the CCH + Tub-B-Low group (all * P < 0.05). These results collectively demonstrate that Tub-B effectively attenuates neuroinflammation in the hippocampus following CCH ( Fig. ).
Machine-readable layer
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