Activation Of NLRP3 Inflammasome By NDV In ECA109 Tumor Cells

NLRP3 inflammasome is involved in the innate immune response of the body and serves as a key component in regulating the development of diseases.Its activation is not only influenced by pathogens such as bacteria and viruses,but also by certain danger signals or metabolic products.Upon activation,NLRP3 inflammasome can promote the release of inflammatory factors from cells into the extracellular space,causing a series of reactions.In this study,we mainly investigated whether the NLRP3 inflammasome is activated after Newcastle disease virus（NDV）acts on esophageal cancer ECA109 cells,how K⁺changes in cells after its activation,the impact of the ATP/P2X7 receptor（P2X7R）axis on the mechanism of NLRP3inflammasome activation.Regarding the activation of NLRP3 inflammasome after treatment with NDV F3 in ECA109 tumor cells,we examined the expression levels of NLRP3 inflammasome-related components.We performed Western Blot analysis to detect the expression of NLRP3 and IL-1βat different time points after treatment with NDV F3.The results showed that the protein levels of NLRP3 and IL-1βwere upregulated in a time-dependent manner compared to the control group（P<0.05）.We further measured the secretion of IL-1βin the cell culture supernatant by ELISA and found that the secretion of IL-1βincreased with the treatment time of NDV F3 compared to the control group（P<0.05）.ASC is an important intermediate component for the assembly and activation of NLRP3 inflammasome.We examined the formation of ASC specks in NDV F3-treated cells using fluorescence immunostaining and found that the formation of ASC aggregates increased compared to the control group（P<0.05）.Our findings suggest that NDV F3 treatment can activate NLRP3 inflammasome in ECA109 cells.We also investigated the mechanism of NLRP3 inflammasome activation and focused on the role of ion changes,specifically potassium（K⁺）.We used a fluorescence probe technique to detect changes in intracellular K⁺concentration and found that the intracellular K⁺concentration decreased with the treatment time of NDV F3 compared to the control group（P<0.05）.This suggests that changes in intracellular K⁺occur during NDV F3 treatment,we further studied the underlying mechanism of the decrease in intracellular K⁺concentration.The presence of Na-K-ATPase in cells is crucial for regulating the balance of intracellular Na⁺and K⁺.We measured the activity of Na-K-ATPase using the molybdate method and found that the activity of Na-K-ATPase decreased with the treatment time of NDV F3 compared to the control group（P<0.05）.Due to the existence of P2X7 receptors on the cell membrane surface,ATP can bind to these receptors and promote the opening of K⁺channels.We wanted to investigate whether K⁺efflux in NDV F3-treated ECA109 tumor cells is related to this mechanism.We used an ATPase,ATP,and a P2X7receptor inhibitor to intervene in the ATP/P2X7R signaling pathway to study whether the ATP/P2X7R signaling pathway affects the activation of the NLRP3 inflammasome through changes in intracellular K⁺concentration.We measured the changes in K⁺concentration,IL-1βsecretion in the cell supernatant,the expression of NLRP3 and IL-1βproteins,as well as the formation of ASC specks in ECA109 cells treated with NDV F3 under the influence of the three interventions.We used the P2X7 receptor inhibitor A-740003 solution to intervene in NDV F3-infected ECA109 cells and measured intracellular K⁺concentration using a fluorescent probe.The results showed that compared with the control group,intracellular K⁺concentration increased significantly after P2X7R was inhibited by A-740003 solution（P<0.05）,indicating that K⁺efflux was blocked.We also found that K⁺efflux was most significantly inhibited at a concentration of 10μmol·L^-1 of the inhibitor（P<0.05）.ELISA results showed that compared with the control group,IL-1βsecretion decreased significantly after P2X7R was inhibited by A-740003 solution（P<0.05）,and the inhibition was greatest at a concentration of 10μmol·L^-1 of the inhibitor（P<0.05）.Western blot analysis showed that compared with the control group,the expression of NLRP3 and IL-1βproteins was downregulated after P2X7R was inhibited by A-740003 solution（P<0.05）,the greatest inhibition was observed at a concentration of 10μmol·L^-1 of the inhibitor.The results of the ATPase and ATP interventions showed that compared with the control group,K⁺efflux was inhibited by the ATPase and promoted by ATP（P<0.05）.Western blot analysis showed that compared with the control group,the expression of NLRP3 and IL-1βproteins was decreased after NDV F3-infected ECA109cells were treated with the ATPase（P<0.05）,the expression of these proteins was upregulated after ATP treatment.The greatest upregulation was observed at a concentration of 3 mmol·L^-1 of ATP（P<0.05）.ELISA results showed that ATP promoted IL-1βsecretion（P<0.05）.We also used immunofluorescence to detect the expression of ASC protein in the cells and found that compared with the control group,the expression of ASC protein was downregulated after P2X7R was inhibited（P<0.05）and upregulated after ATP treatment（P<0.05）.In summary,after infecting ECA109 cells,NDV F3 upregulates the protein expression of NLRP3,ASC,and IL-1β,increases the secretion of IL-1βin the cell supernatant,indicating the activation of the NLRP3inflammasome.The study found that the activation of the NLRP3inflammasome is associated with a significant decrease in intracellular K⁺,suggesting a correlation with K⁺efflux.The intracellular low potassium environment is related to the activity of Na-K-ATPase and the ATP/P2X7R axis.By intervening in the ATP/P2X7R axis,we found that it can affect the expression of NLRP3,ASC,and IL-1βproteins,the secretion of IL-1βin the cell supernatant by affecting changes in intracellular K⁺concentration.Therefore,our study confirmed that NDV F3 can activate the NLRP3inflammasome in ECA109 cells,its mechanism may be related to the ATP/P2X7R axis affecting intracellular K⁺concentration.