The Role Of ROS/NLRP3/caspase-1 Pathway In Pyroptosis And Apoptosis Induced By NiSO₄ In H9c2

Objective:To treat H9c2 cells with nickel sulfate（NiSO₄）,observe the pyroptosis caused by NiSO₄,and observe the role of ROS/NLRP3/caspase-1 signaling pathway in the process of pyroptosis and its relationship with apoptosis.Methods:（1）Selected H9c2 cells with good growth status from 7 to 20generations were treated with varying concentrations of NiSO₄（0,62.5,250 and1200μmol/L）for 24 hours;H9c2 cells were pretreated with the reactive oxygen species（ROS）inhibitor N-acetylcysteine（1000μmol/L NAC）for 1 hour and the caspase-1 inhibitor Z-YVAD-fluoromethyl ketone（20μmol/L Z-YVAD-FMK,YVAD）for 4 hours,followed by treatment of H9c2 cells in combination with 1000μmol/L NiSO₄ for 24 hours.（2）The CCK-8 kit was employed to detect changes in the activity of H9c2 cells under NiSO₄ induction.（3）Biochemical assay kits were used to detect intracellular reactive oxygen species（ROS）level and lactate dehydrogenase（LDH）level of the cell supernatant.（4）Reverse transcription-quantitative polymerase chain reaction（RT-q PCR）was performed to detect the m RNA levels of pyroptosis（ASC,NLRP3,GSDMD,caspase-1,IL-18 and IL-1β）and apoptosis（P53,Cyt C,caspase-9,Bcl-2,Bak-1,Bax and caspase-3）in H9c2 cells.（5）Western blot was being used to detect the expression levels of pyroptosis-related proteins（NLRP3,ASC,caspase-1,GSDMD,GSDMD-N,IL-18 and IL-1β）and apoptosis-related proteins（P53,Bcl-2,Bax,caspase-3,caspase-9 and cleaved-caspase-3）.（6）Annexin V-FITC/PI assay detected the changes of total apoptosis rate in H9c2cells.Results:（1）The CCK-8 assay of NiSO₄-induced activity changes in H9c2 cells showed that:after 24 hours of exposure to H9c2 cells with different doses of NiSO₄（0,37.5,75,150,300,600 and 1200μmol/L）,the survival rate of H9c2 cells in the NiSO₄（75～1200μmol/L）group showed a significant decrease,and their IC₅₀ for 24 hours of exposure was 857.7μmol/L（P<0.05）.（2）According to the toxicity test results and IC₅₀,62.5,250 and 1000μmol/L of NiSO₄ were finally selected to infect H9c2 cells for 24 hours to observe the effect of NiSO₄ on the pyroptosis of H9c2 cells.（1）The biochemical experiment results showed that compared with the control group,the ROS fluorescence intensity of the NiSO₄ treatment group was enhanced,with statistically significant differences in fluorescence intensity between the 250 and 1000μmol/L NiSO₄ groups（P<0.05）;Compared with the control group,NiSO₄ treatment significantly increased the release of LDH in the cell supernatant in a dose dependent manner（P<0.05）.（2）RT-q PCR results confirmed that the m RNA levels of ASC,NLRP3,GSDMD,caspase-1,IL-18and IL-1βin the NiSO₄ treatment were higher than the control group（P<0.05）.（3）Western blot revealed that the protein expressions of NLRP3,ASC,caspase-1,GSDMD,GSDMD-N,IL-18 and IL-1βsignificantly elevated with the increase of NiSO₄ dos（P<0.05）.（3）In order to further investigate the important role of ROS/NLRP3/caspase-1signaling pathway in NiSO₄-induced H9c2 cells pyroptosis,this study selected the ROS inhibitor NAC to treat H9c2 cells for the experiment.（1）The biochemical experiment results showed that the intracellular fluorescence intensity of ROS and the LDH content were significantly lower in the NAC+NiSO₄ group compared with the1000μmol/LNiSO₄ group（P<0.05）.（2）RT-q PCR results showed that the expression levels of ASC,NLRP3,GSDMD,caspase-1,IL-18 and IL-1βm RNA were significantly down-regulated in H9c2 cells in the NAC+NiSO₄-treated group compared with the NiSO₄ group（P<0.05）.（3）Western blot results showed that the protein levels of NLRP3,ASC,caspase-1,GSDMD,GSDMD-N,IL-18 and IL-1βwere significantly decreased in the NAC+NiSO₄-treated group compared with the NiSO₄ group（P<0.05）,indicating that NAC could alleviate NiSO₄-induced pyroptosis in H9c2 cells.（4）In order to further explore the relationship between pyroptosis and apoptosis in H9c2 cells injured by NiSO₄,this study selected caspase-1 inhibitor YVAD to treat H9c2 cells for experiments.（1）The biochemical experiment results showed that compared with the 1000μmol/L NiSO₄ group,the intracellular LDH content in the YVAD+NiSO₄ group significantly decreased（P<0.05）;（2）RT-q PCR results showed that the expression levels of GSDMD,caspase-1,IL-18 and IL-1βm RNA were significantly down-regulated in the YVAD+NiSO₄-treated group compared with the NiSO₄ group in H9c2 cells（P<0.05）;（3）Western blot results showed that the protein levels of GSDMD,GSDMD-N,caspase-1,IL-18 and IL-1βwere significantly decreased in H9c2 cells in the YVAD+NiSO₄-treated group compared to the NiSO₄group（P<0.05）.This indicates that YVAD significantly inhibited the expression of caspase-1 and slowed down the occurrence of NiSO₄-induced pyroptosis of H9c2cells.（4）Flow results revealed that the total apoptosis rate of cells in the 1000μmol/L NiSO₄ treatment group was significantly higher than that in the control group（P<0.05）;Compared with NiSO₄group,the apoptosis rate of YVAD+NiSO₄ group was significantly lower（P<0.05）.（5）RT-q PCR results revealed that the m RNA levels of P53,Cyt C,caspase-9,Bak-1,Bax and caspase-3 were significantly down-regulated in the YVAD+NiSO₄ group compared with the NiSO₄ group,while only Bcl-2 m RNA expression levels were up-regulated in the YVAD+NiSO₄ group（P<0.05）.（6）Western blot results showed that the intracellular protein expression of P53,Bax,caspase-3,caspase-9 and cleaved-caspase-3 was significantly inhibited in the YVAD+NiSO₄ group compared with the NiSO₄ group,while only Bcl-2 protein expression was significantly increased in the YVAD+NiSO₄ group（P<0.05）.This indicates that YVAD significantly slowed down the occurrence of NiSO₄-induced apoptosis in H9c2 cells.Conclusion:NiSO₄exposure could induce H9c2 cells pyroptosis through ROS/NLRP3/caspase-1 signaling pathway,and inhibiting pyroptosis of caspase-1dependence might weaken NiSO₄-induced apoptosis.