Tubuloside B ameliorates blood-brain barrier disruption and cognitive deficits after cerebral ischemia by inhibiting TRIC protein ubiquitination and degradation methods
Aim. Evidence-backed execution summary for Tubuloside B ameliorates blood-brain barrier disruption and cognitive deficits after cerebral ischemia by inhibiting TRIC protein ubiquitination and degradation methods from Tubuloside B ameliorates blood-brain barrier disruption and cognitive deficits after cerebral ischemia by inhibiting TRIC protein ubiquitination and degradation.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Immunohistochemistry
reagent used in the protocol.
- Use
- Brain Sect. (4 µm) were deparaffinized, subjected to antigen retrieval, and incubated with anti-TRIC antibody (1:200). HRP-conjugated secondary antibody and DAB were used for detection. Images were acquired with a light microscope.
BCCAO model and drug administration
reagent used in the protocol.
- Use
- 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.
Western blot and co-immunoprecipitation
reagent used in the protocol.
- Use
- Brain tissues or cells were lysed in RIPA buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against TRIC (1:1000), ubiquitin (1:1000), ZO-1 (1:1000), claudin-5 (1:1000), occludin (1:1000), and β-actin (1:5000). For Co-IP, lysates were immunoprecipitated with...
Enzyme-linked immunosorbent assay
reagent used in the protocol.
- Use
- Following the completion of the Morris water maze test, the rats were deeply anesthetized by an intraperitoneal injection of 2% sodium pentobarbital. The brains were then rapidly removed after decapitation. Bilateral hippocampal tissues were dissected immediately on an ice-cold plate. The tissues were weighed and ho...
Cell culture and drug treatment
reagent used in the protocol.
- Use
- 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 µ...
Tub-B preserves TRIC expression and suppresses its ubiquitination
reagent used in the protocol.
- Use
- Immunofluorescence and Western blot analyses revealed that TRIC expression was significantly downregulated in the cortex of CCH rats and in OGD-treated rBMECs ( P < 0.05; Figs. and A). Tub-B treatment effectively restored TRIC protein levels. Co-immunoprecipitation demonstrated increased TRIC ubiq...
Immunohistochemistry
Brain Sect. (4 µm) were deparaffinized, subjected to antigen retrieval, and incubated with anti-TRIC antibody (1:200). HRP-conjugated secondary antibody and DAB were used for detection. Images were acquired with a light microscope.
- Use
- Brain Sect. (4 µm) were deparaffinized, subjected to antigen retrieval, and incubated with anti-TRIC antibody (1:200). HRP-conjugated secondary antibody and DAB were used for detection. Images were acquired with a light microscope.
Morris water maze
Spatial learning and memory were assessed over 5 training days with a hidden platform. A probe test was conducted on day 6. Escape latency, path length, and time in the target quadrant were recorded. Behavioral tests and data analysis were performed by investigators blinded to the group assignments.
- Use
- Spatial learning and memory were assessed over 5 training days with a hidden platform. A probe test was conducted on day 6. Escape latency, path length, and time in the target quadrant were recorded. Behavioral tests and data analysis were performed by investigators blinded to the group assignments.
Methods
The main plasma metabolites of total glycosides of Cistanche (TCGs) were identified by UPLC-QQQ-MS. A bilateral common carotid artery occlusion (BCCAO) rat model was used to induce chronic cerebral hypoperfusion (CCH). Cognitive function was assessed using the Morris water maze, and BBB permeability was evaluated by...
- Use
- The main plasma metabolites of total glycosides of Cistanche (TCGs) were identified by UPLC-QQQ-MS. A bilateral common carotid artery occlusion (BCCAO) rat model was used to induce chronic cerebral hypoperfusion (CCH). Cognitive function was assessed using the Morris water maze, and BBB permeability was evaluated by...
Enzyme-linked immunosorbent assay
Following the completion of the Morris water maze test, the rats were deeply anesthetized by an intraperitoneal injection of 2% sodium pentobarbital. The brains were then rapidly removed after decapitation. Bilateral hippocampal tissues were dissected immediately on an ice-cold plate. The tissues were weighed and ho...
- Use
- Following the completion of the Morris water maze test, the rats were deeply anesthetized by an intraperitoneal injection of 2% sodium pentobarbital. The brains were then rapidly removed after decapitation. Bilateral hippocampal tissues were dissected immediately on an ice-cold plate. The tissues were weighed and ho...
In vitro BBB model and OGD
Primary rat brain microvascular endothelial cells (rBMECs) were cultured, and barrier integrity was monitored by transendothelial electrical resistance (TEER). For OGD, cells were placed in glucose-free DMEM in a hypoxic chamber (1% O₂, 5% CO₂, 94% N₂) for 4 h, then reoxygenated with or withou...
- Use
- Primary rat brain microvascular endothelial cells (rBMECs) were cultured, and barrier integrity was monitored by transendothelial electrical resistance (TEER). For OGD, cells were placed in glucose-free DMEM in a hypoxic chamber (1% O₂, 5% CO₂, 94% N₂) for 4 h, then reoxygenated with or withou...
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 tar...
- Use
- 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 tar...
Tub-B improves cognitive function and BBB integrity in CCH rats
Fig. 5 Tub-B improves spatial learning and memory in CCH rats. A - C Escape latency during acquisition phase in Morris water maze. D Time spent in target quadrant during probe trial. E Number of platform crossings. F Swimming Trajectories. Data are mean ± SD ( n = 10). Two-way rep...
- Use
- Fig. 5 Tub-B improves spatial learning and memory in CCH rats. A - C Escape latency during acquisition phase in Morris water maze. D Time spent in target quadrant during probe trial. E Number of platform crossings. F Swimming Trajectories. Data are mean ± SD ( n = 10). Two-way rep...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data are presented as mean ± SD from at least three independent experiments (biological replicates, n = 3). Analyses were performed using GraphPad Prism 9.0. One-way or two-way ANOVA followed by Tukey's or Bonferroni's post hoc test was used. Statistical significance is indi...
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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 +...
Measurement outputs
What raw and processed outputs should exist?
Fig. 7 Tub-B suppressed pro-inflammatory cytokines (PICs) production. A - D The concentrations of TNF-α, IL-1β, IL-10, and TGF-β were determined using ELISA kits....
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Spatial learning and memory were assessed over 5 training days with a hidden platform. A probe test was conducted on day 6. Escape latency, path length, and time in the target q...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Brain tissues or cells were lysed in RIPA buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against TRIC (1:1000), ubiquitin...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The main plasma metabolites of total glycosides of Cistanche (TCGs) were identified by UPLC-QQQ-MS. A bilateral common carotid artery occlusion (BCCAO) rat model was used to ind...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Specific pathogen-free male Sprague-Dawley rats (8-10 weeks, 250-300 g; Shanghai SLAC Laboratory Animal Co., Ltd.) were housed under standard conditions.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Fig. 7 Tub-B suppressed pro-inflammatory cytokines (PICs) production. A - D The concentrations of TNF-α, IL-1β, IL-10, and TGF-β were determined using ELISA kits....; Spatial learning and memory were assessed over 5 training days with a hidden platform. A probe test was conducted on day 6. Escape latency, path length, and time in the target q...; Brain tissues or cells were lysed in RIPA buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against TRIC (1:1000), ubiquitin...; The main plasma metabolites of total glycosides of Cistanche (TCGs) were identified by UPLC-QQQ-MS. A bilateral common carotid artery occlusion (BCCAO) rat model was used to ind....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
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 ex...; Data are presented as mean ± SD from at least three independent experiments (biological replicates, n = 3). Analyses were performed using GraphPad...; Network pharmacology analysis identified five overlapping targets between Tub-B and chronic cerebral hypoperfusion (CCH), including KDR (VEGFR2) and MARVELD2 (TRIC) (Supplementa...; Fig. 5 Tub-B improves spatial learning and memory in CCH rats. A - C Escape latency during acquisition phase in Morris water maze. D Time spent in target quadrant during p...
from paperReporting output
Report representative outputs alongside summary comparisons for Fig. 7 Tub-B suppressed pro-inflammatory cytokines (PICs) production. A - D The concentrations of TNF-α, IL-1β, IL-10, and TGF-β were determined using ELISA kits...., Spatial learning and memory were assessed over 5 training days with a hidden platform. A probe test was conducted on day 6. Escape latency, path length, and time in the target q..., Brain tissues or cells were lysed in RIPA buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against TRIC (1:1000), ubiquitin..., The main plasma metabolites of total glycosides of Cistanche (TCGs) were identified by UPLC-QQQ-MS. A bilateral common carotid artery occlusion (BCCAO) rat model was used to ind....
inferred from protocolStructured statistical 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 ex...; Data are presented as mean ± SD from at least three independent experiments (biological replicates, n = 3). Analyses were performed using GraphPad...; Network pharmacology analysis identified five overlapping targets between Tub-B and chronic cerebral hypoperfusion (CCH), including KDR (VEGFR2) and MARVELD2 (TRIC) (Supplementa...; Fig. 5 Tub-B improves spatial learning and memory in CCH rats. A - C Escape latency during acquisition phase in Morris water maze. D Time spent in target quadrant during p...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
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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