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Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response
Published on: September 15, 2017
Berberine alleviates NLRP3 inflammasome induced endothelial junction dysfunction through Ca2+ signalling in
Linfeng Dai1, Li Zhu1, Shiyu Ma2
1School of Pharmaceutical, Guangzhou University of Chinese Medicine, No. 232 Waihuan Dong Rd., Guangzhou University Town, Panyu District, Guangzhou, 510000, China.
Insights
Berberine (BBR) protects against inflammatory vascular injury by targeting the NLRP3 inflammasome and calcium signaling. BBR restores endothelial junction proteins and inhibits inflammasome activation, offering therapeutic potential for cardiovascular disease.
Area of Science:
- Cardiovascular Research
- Inflammation Biology
- Molecular Pharmacology
Background:
- Berberine (BBR) shows promise for cardiovascular disease due to its anti-inflammatory properties.
- Endothelial NLRP3 inflammasome activation drives inflammatory vascular injury, a precursor to cardiovascular disease.
- Endothelial calcium signaling is critical for NLRP3 inflammasome activation and endothelial dysfunction.
Purpose of the Study:
- To investigate whether Berberine (BBR) modulates the endothelial NLRP3 inflammasome pathway.
- To determine BBR's effect on endothelial junction integrity in inflammatory vascular injury.
Main Methods:
- Assessed therapeutic effects of BBR in LPS-induced inflammatory vascular injury in mice and mouse microvascular endothelial cells (MECs).
- Evaluated endothelial junction proteins (VE-cadherin, ZO-1), permeability (FITC-dextran, TEER), and NLRP3 inflammasome activation (Western blot, immunofluorescence).
- Utilized molecular docking and calcium imaging to explore BBR's interaction with the P2X7 receptor (P2X7R).
Main Results:
- BBR treatment restored VE-cadherin and ZO-1 expression and inhibited NLRP3 inflammasome activation in endothelial cells.
- BBR suppressed TXNIP binding to NLRP3, and extracellular calcium depletion mimicked BBR's protective effects.
- Molecular docking and calcium imaging indicated BBR binds to P2X7R, inhibiting ATP-induced calcium influx.
Conclusions:
- Berberine (BBR) demonstrates therapeutic potential for inflammatory vascular injury.
- BBR acts by targeting endothelial calcium signaling and the NLRP3 inflammasome pathway.
- The P2X7R receptor is a potential molecular target for BBR's beneficial effects.
Background:
Berberine has received rising attention for its application in cardiovascular disease because of its relationship with inflammation. The endothelial NLRP3 inflammasome triggers inflammatory vascular injury which would lead to cardiovascular disease. Endothelial calcium signalling plays a crucial role in both the activation of NLRP3 inflammasome and endothelial cells dysfunction. However, the efficacy of BBR on the endothelial NLRP3 inflammasome in inflammatory vascular injury remains unknown.
Purpose:
In this study, we focused on the NLRP3 pathway to determine whether BBR regulates endothelial junction function in inflammatory vascular injury.
Methods:
The integrity of the junction proteins VE-cadherin (VEC) and zonula occludens-1 (ZO-1) detected by immunofluorescence and immunoblotting was used to determine the therapeutic effect of BBR (50, 100, or 200 mg/kg/day) in LPS (100 μg/kg/day)-induced inflammatory vascular injury in mice and mouse microvascular endothelial cells (MECs) treated with LPS (1 μLPS ) and ATP (5 mM). Endothelial permeability was assessed by FITC-labelled dextran and trans-endothelial electrical resistance (TEER) in vitro. The assembly and activation of NLRP3 inflammasomes were detected by western blotting and immunofluorescence. Pharmacophore-based virtual molecular docking studies and calcium imaging analyses were used to determine the interaction of BBR with the ATP-gated Ca2+ channel P2X7R (purinergic P2X receptor 7) in the context of inflammatory vascular injury.
Results:
BBR recovered the expression of ZO-1 and VEC and inhibited endothelial NLRP3 inflammasome activation in coronary microvascular endothelium and in MECs. These results suggested a crucial role of the NLRP3 inflammasome in BBR-regulated endothelial integrity. Further analysis demonstrated that BBR treatment suppressed the binding of TXNIP (thioredoxin interacting protein) with NLRP3. Intriguingly, eliminating extracellular Ca2+ showed a similar effect as BBR. Virtual docking analysis indicated that R574 of P2X7R is a potential target for BBR binding. Ca2+ imaging showed that BBR inhibited the Ca2+ influx in response to ATP, supporting the potential interaction of BBR with P2X7R.
Conclusions:
These findings suggest that BBR exhibits potential and specific therapeutic value by targeting calcium signals and the endothelial NLRP3 inflammasome in inflammatory vascular injury.
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