X-ray irradiation reduces ATP-dependent activation of NLRP3 inflammasome by inhibiting TWIK2 activity in macrophages

Background: The spleen, as the body's largest peripheral immune organ and a crucial source of circulating monocytes, plays a significant role in the acute inflammatory response of spleen-derived macrophages to diseases. Therefore, studying the impact and mechanism of X-ray irradiation on spleen-derived macrophages' inflammatory responses is of great importance.

Method: Extracted and identified mice splenic macrophages were divided into four groups: control group, LPS and ATP co-stimulated non-irradiated group, LPS and ATP co-stimulated group irradiated after 6h, and LPS and ATP co-stimulated group irradiated after 12h. In the LPS and ATP co-stimulated groups, LPS (1μg/ml) and ATP (5mmol/L) were added to establish an inflammatory model in mice splenic macrophages. The irradiated groups were exposed to a medical linear accelerator (Elekta Synergy), while the non-irradiated groups were placed under the light source for the same duration without irradiation. Protein extraction was performed in each group at 6h and 12h post-treatment for subsequent analysis using Western blot, ELISA, RT-qPCR and other relevant methods.

Results: (1) Compared with the non-irradiated group, the cell activity in the groups irradiated for 6h and 12h at 8Gy showed a significant increase (P<0.01). (2) In the LPS and ATP co-stimulated groups irradiated after 6h and 12h, the expression of NLRP3 mRNA and protein, IL-18 and IL-1β showed a notable decrease compared to the LPS and ATP co-stimulated non-irradiated group (P<0.05). Additionally, caspase-1 expression of caspase-1 mRNA and protein in the 12h post-irradiation group also decreased considerably when compared with the LPS and ATP co-stimulated non-irradiated group (P<0.05). In the groups irradiated after 6h and 12h, (3) there was a remarkable decrease in the expression of TWIK mRNA and TWIK2, (4) as well as Gq mRNA and protein, when compared to the LPS and ATP co-stimulated non-irradiated group (P<0.05). Particularly, the 12h post-irradiation group exhibited a notable reduction in PKC expression (P<0.05).

Conclusion: X-ray irradiation is capable of inhibiting the activation of ATP-dependent NLRP3 inflammasomes in splenic macrophages.