Berberine alleviates NLRP3 inflammasome induced endothelial junction dysfunction through Ca2+ signalling in inflammatory vascular injury

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 mu g/kg/day)-induced inflammatory vascular injury in mice and mouse microvascular endothelial cells (MECs) treated with LPS (1 mu LPS ) 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 immunofluo rescence. 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.

Automated summary

This study aimed to investigate the regulatory effect of berberine (BBR) on endothelial junction function in inflammatory vascular injury. The results showed that BBR restored the expression of junction proteins and inhibited the activation of NLRP3 inflammasomes in endothelial cells. Additionally, BBR exhibited a similar effect to eliminating extracellular calcium. These findings suggest that BBR has therapeutic potential in inflammatory vascular injury by targeting calcium signals and the NLRP3 inflammasome.