Roles Of TRPM2in Caspase-1Dependent Pyroptosis

Objective:Pyroptosis-anew form of cell death is depend on activated cysteinyl aspartate-specific protease-1(caspase-1) and occupies a very important position in inflammatory immune diseases and ischemia-reperfusion injury. Transient receptor potential protein M2(TRPM2) is a selective cation channel involved in the regulation of many cellular functions. However the regulationary role of TRPM2in inflammatory immune diseases still remains unclear.Therefore, this study was designed to explore the role and its possible mechanism of TRPM2in activating caspase-1and caspase-1-dependent pyroptosis of mouse bone marrow-derived macrophages.Methods:We used the mouse bone marrow-derived macrophages from wide-type mice and TRPM2-/-mice as cell model. After lug/ml LPS incubation for4hours,5mM ATP was added, with or not with reactive oxygen species (ROS) inhibitor or caspase-1inhibitor, the cells were collected0.5hours later for western blotting of activated caspase-1. Assess the incidence of pyroptosis by4,6-diamidino-2-phenylindole (DAPI) and Ethidium Bromide (EtBr) or Ethidium Homodimer-2(EthD-2) staining. Cells harvested were cross-linked with disuccinimidyl suberate (DSS), then apoptosis-associated speck-like protein containing a CARD (ASC) oligomers were isolated by low-speed centrifugation, and detected by western blot. Results:Compared with wild-type mice, the activated caspase-1-P10levels of TRPM2-/-mice were significantly higher(64.39±9.74vs.22.27±2.19, P=0.002); percentage of EtBr positive cells was also significantly higher(84.06%±4.48%vs.38.17%±5.30%, P=0.000), and such a high percentage of EtBr positive cells can be blocked by specific caspase-1inhibitor Z-YVAD (6.88%±6.31%vs.2.71%±0.36%, P=0.235),and ROS inhibitors NAC and DPI (NAC:33.54%±11.52%vs.21.12%±12.62%, P=0.028; DPI:34.74%±11.55%vs.31.75%±14.65%, P=0.583). But no significant differences were found in percentage of EthD-2positive cells between the two groups (5.96%±1.99%vs.3.92%±3.24%, P=0.07). In addition, the value of ASC oligomerization strength in TRPM2-/-mice was also significantly higher.Conclusion:After stimulation of LPS and ATP, TRPM2-/-mice showed stronger activation of caspase-1, caspase-1-dependent pyroptosis incidence and the degree of oligomerization of ASC than in wild-type mice, and its possible mechanism may relate to the regulation of TRPM2in ROS generation. This study suggests that when cells are in a event of injury, such as sepsis, gene deletion of TRPM2can enhance the activation of caspase-1and caspase-1-dependent pyroptosis, and thus plays a role in immune adjustment.