The Effect And Mechanism Of Nitrosamines-induced Activation Of NLRP3 Inflammasome

Background:Esophageal cancer is an environment-related tumor,which seriously threatens human life and health.Epidemiological and toxicological data showed that nitrosamines were a kind of potential environmental carcinogens for esophageal cancer,but so far the carcinogenic mechanism of nitrosamines-induced esophageal cancer is still unclear.The tumor-related inflammatory microenvironment promotes tumor progression by promoting tumor cell proliferation,migration and assisting immune escape.Inflammasomes are considered to be precursor events of inflammatory response and play an important role in a variety of inflammation-related diseases,including tumors.Studies have confirmed that NLRP3 inflammasome are highly expressed in esophageal cancer tissues.Up-regulating the expression of NLRP3 can play a role in promoting cancer by promoting the proliferation and migration of esophageal cancer cells.However,there is no report about the role and mechanism of inflammasomes in the occurrence and development of esophageal cancer caused by nitrosamines exposure.Objective:1.Explore whether exposure to nitrosamines can lead to the activation of NLRP3inflammasome;2.To explore whether nitrosamines exposure mediates the activation of NLRP3 inflammasome through mitochondrial reactive oxygen species in esophageal epithelial cells;3.Explore the mechanism of nitrosamines-induced Het-1A pyrolysis of esophageal epithelial cells.Methods:1.Nitrosamines-induced the activation of NLRP3 inflammasomeHuman-derived immortalized esophageal epithelial cells are used as target cells.Setting up 4,20,100,500,2500 ng/m L nitrosamines poisoning groups respectively and a control group at the same time.The cytotoxicity of Het-1A cells exposed to different concentrations of nitrosamines was evaluated by the release of CCK8 and LDH.Western blot was used to detect NLRP3 inflammasome related proteins expression levels including NLRP3,caspase-1,pro-caspase-1,Pro-IL-1β and IL-1β.2.The role of mt ROS in the activation of NLRP3 inflammasome induced by nitrosamines in Het-1A cellsThe DCFH-DA and Mito SOX probes were used to detect the levels of ROS and mt ROS respectively,and the JC-1 probe was used to detect the mitochondrial membrane potential.The role of mt ROS in the activation of NLRP3 inflammasome was verified by adding ROS scavenger NAC and mt ROS scavenger Mito-TEMPO.3.NLRP3 inflammasome activation leads to caspase-1-dependent pyrolysisThe pyrolysis of Het-1A cells was observed by PI staining,LDH release,and GSDMD protein expression.At the same time,the caspase-1 inhibitor Z-YVAD-FMK and NLRP3 low-expressing cell lines were added to investigate the NLRP3/caspase-1/GSDMD pathway of pyrolysis.Results:1.Nitrosamines-induced activation of NLRP3 inflammasome1.1 Toxic effects of nitrosamines on Het-1A cells(1)Compared with the control group,after 48 hours of exposure,the Het-1A cell viability of 20,100,500,and 2500 ng/m L nitrosamines groups decreased to 84.31%,79.01%,75.41%,and 69.02%,respectively.The cell activity of the 4ng/m L exposure group did not change significantly compared with the control group(P > 0.05).(2)Compared with the control group,after 48 hours of exposure,the amount of cellular LDH released gradually increased with the increase of nitrosamines concentration.There were statistical differences between the 20,100,500 and 2500ng/m L nitrosamines groups and the control group(P < 0.05).Compared with the control group,the amount of LDH released in the 4ng/m L treatment group did not change significantly(P > 0.05).This is consistent with the result of decreased cell viability.1.2 Nitrosamines-induced activation of NLRP3 inflammasome in Het-1A cellsThe protein expression of NLRP3,caspase-1,pro-caspase-1,pro-IL-1β and IL-1β increased with the increase of nitrosamines exposure(P < 0.05),which indicates that nitrosamines induce NLRP3 inflammasome activation.2.mtROS mediates the activation of NLRP3 inflammasome2.1 Nitrosamines cause oxidative damage to Het-1A cells(1)Compared with the control group,the relative expression level of ROS increased with the increase of nitrosamines exposure concentration(P < 0.05).It shows that nitrosamines can induce the generation of ROS in Het-1A cells.(2)The relative expression level of mt ROS increased with the increase of nitrosamines exposure concentration(P < 0.05),which indicates that nitrosamines can promote the production of mt ROS in Het-1A cells,and it is concentration-dependent.(3)Compared with the control group,as the concentration of nitrosamines increased,the mitochondrial membrane potential decreased(P < 0.05),indicating that nitrosamines can cause mitochondrial damage in Het-1A cells.2.2 The mechanism of mt ROS-induced NLRP3 inflammasome activation(1)Compared with the nitrosamine treatment group,the intracellular ROS level of the NAC and nitrosamine co-treatment group and mito-TEMPO and nitrosamine co-treatment group was significantly reduced(P < 0.05),indicating that NAC/mito-TEMPO can be Effectively eliminate the production of ROS caused by nitrosamine poisoning.Compared with the nitrosamine treatment group,the red fluorescence intensity of the NAC and nitrosamine co-treatment group and mito-TEMPO and nitrosamine co-treatment group was significantly weaker,and the intracellular mt ROS level was significantly reduced(P < 0.05),indicating NAC/mito-TEMPO can effectively eliminate the production of mt ROS caused by nitrosamine poisoning.Compared with the nitrosamine treatment group,the green fluorescence intensity of the NAC and nitrosamine co-treatment group and mito-TEMPO and nitrosamine co-treatment group was significantly weakened,the red fluorescence intensity was significantly weakened,and the intracellular mitochondrial membrane potential level increased(P < 0.05),indicating that NAC/mito-TEMPO can effectively inhibit the oxidative damage caused by nitrosamine poisoning.(2)Compared with the nitrosamine treatment group,the protein expression level of NLRP3,caspase-1,pro-caspase-1,pro-IL-1β and IL-1β in the NAC and nitrosamine co-treatment group and mito-TEMPO and nitrosamine co-treatment group was significantly reduced(P < 0.05),indicating that reactive oxygen species,especially mitochondrial reactive oxygen species,mediate nitrosamine-induced NLRP3 inflammasome activation.3.NLRP3 inflammasome activation leads to caspase-1-dependent pyrolysis3.1 Nitrosamines induced pyrolysis of Het-1A cells(1)The results of PI staining showed that as the concentration of nitrosamines increased,the number of positive cells stained with PI gradually increased.(2)Western blot detection results showed that the protein expression level of GSDMD increased with the increase of nitrosamines exposure concentration,and the protein expression of GSDMD in Het-1A cells increased significantly(P < 0.05).Summarizing the results of LDH release in the first section of Chapter 1,indicates that nitrosamines can cause Het-1A cell pyrolysis.3.2 The relevant mechanism of nitrosamines-induced pyrolysis of Het-1A cells(1)Compared with the positive control group with nitrosamines alone,the LDH release in the Z-YVAD-FMK and nitrosamines co-treatment group was significantly reduced,the positive rate of PI staining in cells decreased,and the protein expression levels of GSDMD,NLRP3,caspase-1,pro-caspase-1,pro-IL-1β and IL-1β were also significantly reduced(P < 0.05).It shows that inhibiting the activation of caspase-1can affect the activation of NLRP3 inflammasomes,and then inhibit the pyrolysis of Het-1A cells induced by nitrosamines.(2)In cells with low NLRP3 expression,the release of LDH induced by nitrosamines was significantly reduced(P < 0.05),and the percentage of positive cells stained with PI decreased;The protein expression levels of GSDMD,caspase-1,pro-caspase-1,pro-IL-1β and IL-1β were also significantly reduced(P < 0.05).It shows that inhibiting the activation of NLRP3 inflammasome can inhibit nitrosamines-induced pyrolysis.Conclusion:1.Nitrosamines poisoning leads to the activation of NLRP3 inflammasome in esophageal epithelial Het-1A cells.2.Nitrosamines induce oxidative damage to Het-1A cells in the esophageal epithelium,and increase the production of ROS and mt ROS,which further mediate the activation of NLRP3 inflammasomes.3.Nitrosamines poisoning can cause pyrolysis of esophageal epithelial Het-1A cells.The NLRP3/caspase-1/GSDMD pathway is related to the occurrence of nitrosamines-induced pyrolysis.