To Explore The Protective Effect And Mechanism Of Luofengning No.0 On HCAEC From The NLRP3 Inflammasome

BackgroundIn-stent restenosis(ISR)and stent thrombosis(ST)are two difficult problems in percutaneous coronary intervention field.Both of them have complicated mechanisms involving with the mechanical injury caused by the stent on blood vessels,abnormal vasomotor function,endothelial injury,and local inflammation caused by metal foreign bodies and surface coated polymers,etc.The formation of neointimia with pathological proliferation of human coronary smooth muscle cells(HCASMC)as the core caused by various factors is the key mechanism of ISR and the delay of re-endothelialization caused by antiproliferative drugs is an important cause of ST.Therefore,on the one hand,in order to reduce the influence of metal stent to blood vessels,the product of the concept of "leave nothing left "—drug-coated balloon(DCB)favored by interventional physicians gradually.But due to the lack of large-scale randomized controlled trial,except ISR lesions,evidence-based medical evidence of DCB treatment for other types is still insufficient.On the other hand,the drugs on the surface of scaffolds and balloons in clinical application are single anti-cell proliferation agents,which have no specific selectivity to the inhibition of cell activity.In order to reduce the damage of agents to human coronary endothelial cells(HCAEC)and develop the" endothelial-friendly"scaffolds and balloons,the development of new coated agents has also become research hotspots.From now on,the development of single-component drugs is currently at a bottleneck period.Rational compatibility of multiple drugs in traditional Chinese medicine prescriptions has the advantage of "enhancing effect and reducing toxicity",which it could provide ideas for the research and development of new coating agents.Based on theory of "endogenous collateral wind syndrome",Pro.Wang Xian made a prescription—Luofengning 0,which includes composed the effective mono-components of Taxus chinensis and leech,namely paclitaxel and bivarudin(recombinant hirudin),for the development of agents coating of DES and DCB.Previous studies have shown that it can reduce HCASMC proliferation level by inhibiting inflammatory response.The injury and death of HCAEC caused by inflammation are important factors in the delay of re-endothelialization.Therefore,we took HCAEC as the research platform to explore the effect and possible mechanism of Luofengning 0 on NLRP3 inflammasome during HCAEC injuryAimsOn one hand,this study conducted a meta-analysis of existing literature to explore the efficacy and safety of DCB in de novo small coronary artery(SCV)lesions;On the other hand,this study screened the optimal concentration ratio of paclitaxel and bivarudin in Luofengning 0,and make an intervention in Ang?-induced HCAEC injury model.To observe the effect of Luofengning 0 on NLRP3 inflammasome during HCAEC injury and investigated the protective effect of Luofengning 0 on HCAEC from the angle of inflammation and pyroptosis.It will provide theoretical basis for the development of a new generation of endothelium friendly DCBMethodPart 1.Comparison of efficacy and safety between DCB and DES in the treatment of de novo SCV lesionsIn this part,a total of 9 medical databases were searched to screen out randomized controlled trials conforming to the inclusion and exclusion criteria for meta-analysis.Part 2.The protective effect and mechanism of Luofengning 0 on Ang?-induced HCAEC damageExperiment 1.Selecting the optimal concentration ratio between paclitaxel and bivarudin in Luofengning 0The 4th?6th generation HCASMC and HCAEC were selected as research platforms By setting up a series of different concentrations ratio of paclitaxel and bivarudin and performing intervention on culture of both types of cells.MTT assay was used to detect the proliferative activity of the cells after intervention.We selected a concentration ratio that can significantly inhibit HCASMC activity but has no influence on HCAEC(90%of cell relative activity is taken as the judgment threshold).Experiment 2.Ang?-induced HCAEC damage model.The 4th?6th generation HCAEC were selected as research platforms.By setting up a series of different concentrations of Ang? and performing intervention on culture of HCAEC.We selected an Ang? concentration that could significantly inhibit HCAEC activity(with 70%relative cell activity as the judgment threshold).Experiment 3.The protective effect of Luofengning 0 on Ang?-induced HCAEC damageThe Ang?-induced HCAEC injury model was interfered with above Luofengning 0 drug ratio concentration.LDH release assay was used to detect the cell activity of each group.And HO-PI immunofluorescence staining was used to detect the cell death.Experiment 4.Effect of Luofengning 0 on NLRP3 inflammatome pathway in Ang??induced HCAEC injury modelThe Ang?-induced HCAEC injury model was interfered with above Luofengning 0 drug ratio concentration.ELISA,RT-qPCR and Western Blot were used to detect the influence of major information transfer sites,gene and protein expression levels of downstream inflammatory products in NLRP3 inflammatome pathway.ResultPart 1.Comparison of efficacy and safety between DCB and DES in the treatment of de novo SCV lesionsMeta-analysis of the literature indicated that DCB approach and DES approach had similar efficacy and safety in the treatment of de novo SCV lesions,and the incidence of major cardiovascular events(MACEs)was similar between them.In addition,DCB approach was associated with better angiographic outcomes such as loss of vessels(LLL)and minimum vessel diameter(MLD).Therefore,DCB approach may be a new option to treat de novo SCV lesionsPart 2.The protective effect and mechanism of Luofengning 0 on Ang?-induced HCAEC damageExperiment 1.Selecting the optimal concentration ratio between paclitaxel and bivarudin in Luofengning 0The concentration of paclitaxel in Luofengning 0 was fixed at 1?mol/L.With the increase of the concentration of bivarudin,the inhibition degree of HCASMC and HCAEC increased significantly compared with the control group(p<0.05),the inhibition was concentration dependent and the inhibition degree of HCAEC was significantly lower than that of HCASMC.When the two drugs were combined with paclitaxel 1?mol//L+bivarudin 0.625mg/ml.the relative activity of HCAEC and HCASMC was approximately 92%and 63%respectivelyExperiment 2.Ang?-induced HCAEC damage model.With the increase of Ang? concentration,the inhibition of HCAEC activity was aggravated,which was significantly different from that of the control group(P<0.05).The inhibition was concentrate-dependent.When Ang? concentration was 10-5mol/L,the relative activity of HCAEC was about 71.9%.Experiment 3.The protective effect of Luofengning 0 on Ang?-induced HCAEC damage1)The LDH release test results showed that compared with the control group,the LDH release level in the model group was significantly increased(p<0.05).The LDH release level in the paclitaxel group and Luofengning 0 group was significantly lower than that in the model group,and in Luofengning 0 group was more significantly decreased(p<0.05).2)HO-PI immunofluorescence staining results showed that compared with control group,model group show strong red fluorescence and strong blue fluorescence.The red and blue fluorescence of paclitaxel group and Luofengning group were significantly reduced compared with the model group,and the survival number of Luofengning 0 group was better than that of Paclitaxel group.Experiment 4.Effect of Luofengning 0 on NLRP3 inflammatome pathway in Ang?-induced HCAEC injury model.1)The ELISA test results showed that compared with the control group,the levels of IL-1? and IL-18 in the model group were significantly increased(p<0.05),the LDH release levels in the paclitaxel group and Luofengning group 0 were significantly lower than those in the model group,and in Luofengning 0 group were more significant(p<0.05).2)The RT-qPCR test results showed that compared with the control group,the levels of NF-?B,NLRP3,ASC,Caspase-1,GSDMD,IL-1 and IL-18 were significantly expressed more in the model group(p<0.05),the expression levels in the paclitaxel group and Luofengning 0 group were significantly lower than those in the model group,and in Luofengning 0 group were more significant(p<0.05).3)Western Blot results showed that the levels of NF-B,NLRP3,ASC,Caspase-1 and GSDMD in the model group were significantly higher than those in the control group(p<0.05).LDH release levels in paclitaxel group and Luofengning group 0 were significantly lower than those in the model group.The protein level of paclitaxel group and Luofengning 0 group was significantly lower than that of the model group,and the decrease of Luofengning 0 group was more significant(p<0.05).Conclusion1.DCB approach and DES approach have similar efficacy and safety in the treatment of de novo SCV lesions,and DCB angiographic follow-up results are better.Therefore,DCB alone may be a better choice for the treatment of primary SCV lesions.2.When the combination of paclitaxel 1?mol/L with pivarudin 0.0625mg/ml was used as the final concentration ratio of Luofengning 0,the activity of HCAEC was not affected significantly and the proliferation of HCASMC was significantly inhibited.3.Through inhibiting the NLRP3 inflammasome pathway,Luofengning 0 could alleviate the inflammatory damage and pyroptosis induced by Ang II and protect HCAEC.And the results were better than that of applying paclitaxel alone,which provided a theoretical basis for the research and development of the new generation DCB.