Cordycepin Attenuates NLRP3/Caspase-1/GSDMD-Mediated LPS-induced Macrophage Pyroptosis

Zige Liu Li Lv Jiao Wei Yuli Xie Mujia Jili Yian Huang RIRONG YANG Yu Luo ^*

• Guangxi Medical University, Nanning, Guangxi Zhuang Region, China

Pyroptosis, a form of programmed cell death driven by the NLRP3 inflammasome, is a key contributor to inflammation in various diseases. This study aimed to investigate the anti-inflammatory mechanisms of cordycepin, focusing on its role in macrophage pyroptosis. Molecular docking analysis was performed to evaluate the binding affinity of cordycepin to key pyroptosis-related proteins, including NLRP3, Caspase-1, and GSDMD. RAW264.7 cells were pre-treated with cordycepin to assess its effects on pyroptosis. Key measurements included reactive oxygen species (ROS) levels, xanthine oxidase (XO) activity, and the expression of NLRP3, Caspase-1, and GSDMD. Additionally, lactate dehydrogenase (LDH) release, interleukin (IL)-1β and IL-18 levels in the culture supernatant, and macrophage cell death rates were evaluated using Hoechst 33342/PI dual staining. The results demonstrated that cordycepin exhibits strong binding affinity for NLRP3, Caspase-1, and GSDMD. Cordycepin pre-treatment significantly reduced ROS levels and XO activity, inhibited the expression of NLRP3, cleaved-Caspase-1, and cleaved-GSDMD, and decreased pyroptosis-associated inflammatory cytokines IL-1β and IL-18, along with Caspase-1 activity. Furthermore, cordycepin reduced the macrophage pyroptosis rate. In conclusion, cordycepin inhibits macrophage pyroptosis by reducing XO activity, suppressing ROS production, and regulating the expression of key molecules in the NLRP3/Caspase-1/GSDMD pathway. These findings provide a strong experimental basis for the potential development of cordycepin as a novel anti-inflammatory agent.