Study On The Molecular Mechanism Of Extracellular ATP(eATP) Promoting Inflammation Through Activating The Notch1 Signaling Pathway

Adenosine triphosphate(ATP)is a major pro-inflammatory molecule that regulates a variety of cellular activities.Under conditions such as cell necrosis,tissue stress or injury,cell rupture releases high intracellular ATP concentration into the extracellular environment,which increases the concentration of ATP in the extracellular environment to a higher level in a short time,activates the NLRP3 inflammasome and has adverse effects on surrounding cells.As one of the basic inflammatory pathways,Notch1 signaling pathway is involved in the immune response of macrophages,regulates the inflammatory response of macrophages and the phenotype of macrophages,and is closely related to the NLRP3 inflammasome and NF-κB signaling pathway.In this study,the molecular mechanism of extracellular ATP(eATP)activation of Notch1 signaling pathway to induce inflammation was studied in detail,providing scientific basis for the development and utilization of drugs using Notch1 pathway as anti-inflammatory targets in the future.In order to investigate the effect of EATP-induced macrophage inflammation,the group without eATP cells was selected as the control group,and the group with different time of EATP-induced cells addition(5 min,10 min,15 min,30 min,45 min,60min)was selected as the treatment group.The levels of macrophage inflammatory cytokines(IL-1β,TNF-α,IL-6),m RNA transcription and NLRP3 inflammatory body protein expression were measured.The results show that: Compared with the control group,the content of IL-1β was extremely significant at 15 min induction of macrophages(P<0.001),and the contents of TNF-α and IL-6 were extremely significant at 10 min induction of macrophages(P<0.001).The m RNA transcription levels of IL-1β,TNF-α and IL-6 in macrophages were extremely significant at 45 min induction(P<0.005),and the expression level of NLRP3 inflammasome protein in macrophages was significantly increased at 10 min induction(P<0.001).In conclusion,eATP treatment of macrophages for a short period of time can significantly increase the secretion and m RNA expression of macrophage inflammatory cytokines IL-1β,TNF-α and IL-6,activate NLRP3 inflammasome,and induce acute cellular inflammation.In order to further explore the mechanism of eATP inducing macrophage inflammatory response,the expression of P2X7 receptor protein was detected.The results showed that there was no change in P2X7 expression in macrophages induced by ATP within 60 min,and the difference was not significant,indicating that ATP promotes acute inflammation in macrophages and there may be other potential inflammatory receptors.Based on this,we discussed the influence of eATP on Notch1 pathway,and the results showed that: ATP induced macrophages increased the expression of protein,Cleaved Notch1,HEes-1,and NF-κB P-P65 within 60 min,with significant differences(P<0.005),indicating that ATP induced activation of Notch1 signal and its downstream pathway within macrophages within a short time.In order to explore the molecular mechanism of ATP activation of Notch1 signal in macrophages,this study observed the binding situation of ATP and Notch NRR1 region and its influence on disulfide bond.The results showed that ATP and Notch NRR1 region were combined,and could affect disulfide bond with concentration hierarchy.After pretreatment of macrophages with DTNB as disulfide bond inhibitor,the activation of Notch1 signal was observed.The results showed that the expression of Cleaved Notch1 and HEES-1 protein was significantly decreased(P<0.005),and eATP may activate the Notch1 signaling pathway through disulfide bonding with the NRR1 region of Notch1 protein.Finally,the response of food functional components EGCG and alpha-twist(α-MG)to EATP-induced acute macrophage inflammation was investigated.The results showed that EGCG treatment could effectively reduce the secretion of TNF-α induced by ATP(P<0.01),and α-MG treatment could significantly reduce the expression of NLRP3protein(P<0.001).It was confirmed that EGCG and α-MG can significantly lower expression of Cleaved Notch1 and HES-1 protein(P<0.001),and inhibit ATP-induced activation of Notch1 signal in macrophages.Conclusions: In this study,for the first time,the interaction between eATP and Notch signal NRR1 region of macrophages activates Notch1 signaling pathway,thereby inducing the release of macrophage inflammatory cytokines,up-regulating NLRP3 inflammatorome,and inducing acute inflammation.