Inhibition Of Vascular Endothelial Cell Scorching By Rosmarinic Acid Through The NRF2/NLRP3 Signaling Pathway

Objective: Cardiovascular disease(CVD)has become the number one killer of human health today,making its prevention and treatment an urgent priority.Atherosclerosis(AS)is the pathological basis of CVD and endothelial damage is the initial factor,so preventing and treating atherosclerosis due to endothelial damage is essential for cardiovascular disease.In recent years,with the development of scientific research,more and more studies have confirmed that the development of AS is inextricably linked to cellular scorching.Pyroptosis is thought to be a newly discovered form of programmed cell death distinct from necrosis and apoptosis,characterised by swollen cells with large bubbles protruding from the plasma membrane and cell lysis.This death pathway is mediated by the pore formation of gasdermin D(GSDMD),which is activated by human Caspase-1/Caspase-4/Caspase-5(or mouse Caspase-11)and subsequently releases cell contents and proinflammatory cytokines(cytokines).Therefore,the active search for new mechanisms and possible drug targets to reduce or inhibit endothelial cell death and the endothelial inflammatory response is a priority in the management of AS.Rosmarinic acid(RA)is a polyphenolic compound with the chemical name a-ocaffeoyl-3,4-dihydroxyphenyl lactic acid.RA has been observed to have many interesting biological activities such as anti-viral,anti-rheumatic,anti-infective,anti-bacterial,antioxidant and anti-cancer cell effects,but its molecular mechanisms have not yet been elucidated.Recent studies have found that rosmarinic acid inhibits ox-LDL(Oxidized modified LDL)-induced inflammatory vesicle activation under high glucose conditions through downregulation of the p38-FOXO1-TXNIP pathway,thereby attenuating the development of pyrogenic-induced atherosclerosis.Therefore,this experiment focused on the mechanism by which rosmarinic acid ameliorates lipopolysaccharide(LPS)-induced scorching of human umbilical vein endothelial cells(HUVECS)through the NRF2-NLRP3(nuclear transcription factor E2-related factor 2-NOD-like receptor protein 3)axis,thereby reducing atherosclerosis.Methods: Part 1: The expression of Caspase-1,GSDMD-N and NLRP3 was measured by Western Blot to determine the optimal concentration of LPS and to establish the optimal cell scorch model.Part 2: In this scorch death model,RA was added at different concentration gradients and its effect on vascular endothelial cell viability was also observed by the cck8 method and the expression of NLRP3 scorch death vesicles in HUVECS cells was measured by Western Blot to determine the optimal concentration of RA.Part 3: Based on the second part,the m RNA expression and protein expression of NRF2 in HUVECS cells were observed,and the role of NRF2 in the scorch death process was tentatively determined.Part 4: On the basis of the third part,NRF2 was transfected and its effect on the viability of vascular endothelial cells was observed by the cck8 method.Its effect on the expression of Caspase-1,GSDMD-N and NLRP3 proteins,which are indicators of scorch death,was observed by Western Blot.The m RNA expression of Caspase-1,GSDMD-N and NLRP3 was observed by PCR.Fluorescent probe for the detection of reactive oxygen species(ROS)in HUVECS cells.Cellular immunofluorescence staining for NLRP3 expression in HUVECS.Results: Protein expression of Caspase-1,GSDMD-N and NLRP3 were increased after LPS action on HUVECS.After LPS-induced scorch death occurred and RA was added,a decrease in both protein expression and m RNA expression of NRF2 was observed compared to the previous levels.After transfection with NRF2,there was a significant decrease in the viability of vascular endothelial cells,and the protein expression and m RNA expression of Caspase-1,GSDMD-N and NLRP3 were significantly increased,and the trend of scorching death was more obvious.Increased ROS levels in HUVECS cells were detected by fluorescent probes and increased fluorescent expression of NLRP3 in HUVECS was detected by cellular immunofluorescent staining.Conclusion: Lipopolysaccharide induces the expression of Caspase-1,GSDMD-N and NLRP3 in vascular endothelial cells and induces endothelial cell scorching.Rosmarinic acid has a protective effect against cellular scorching by inhibiting the protein expression of NLRP3 scorching vesicles in vascular endothelial cells,thereby increasing the viability of vascular endothelial cells in a concentration-dependent manner.Low expression of NRF2 leads to increased ROS content,which promotes NLRP3 expression.In combination with the above findings,rosmarinic acid may inhibit vascular endothelial cell pyroptosis via the NRF2-NLRP3 pathway.