Inhibition of calpain reduces oxidative stress and attenuates pyroptosis and ferroptosis in Clostridium perfringens Beta-1 toxin-induced macrophages

Clostridium perfringens Beta-1 toxin (CPB1) is a lethal toxin, which can lead to necrotic enteritis, but the pathological mechanism has not been elucidated. We investigated whether Reactive Oxygen Species (ROS) participated in CPB1-induced Pyroptosis and Ferroptosis, and investigated the effects of calpain on CPB1-induced oxidative stress and inflammation. Scavenging ROS by N-Acetyl-L cysteine (NAC) led to the reduction of ROS, inhibited the death of macrophages, cytoplasmic swelling and membrane rupture, the expression of pyroptosis-related proteins and proinflammatory factor, while increased the expression of anti-inflammatory factors in cells treated with rCPB1. Adenosine triphosphate (ATP) synthase, H⁺ transporting, mitochondrial F1 complex, alpha subunit 1 (ATP5A1) was identified specifically interact with rCPB1. Silencing ATP5A1 inhibited accumulation of ATP and ROS, leaded to less cytoplasmic swelling and membrane rupture, attenuated Pyroptosis and inflammation in rCPB1-treated cells. We also found that rCPB1 induces Ferroptosis in macrophages, and the level of Ferroptosis was similar with H₂O₂. Of note, H₂O₂ is a major ROS source, indicated that ROS production may play a major role in the regulation of Ferroptosis in macrophages treated with rCPB1. This finding was further corroborated in rCPB1- induced human acute monocytic leukemia cells, which were treated with NAC. In addition, the inhibition of Ferroptosis using liproxstatin-1 inhibited the shriveled mitochondrial morphology, increased the expression of Glutathione Peroxidase 4, nicotinamide adenine dinucleotide (phosphate) hydrogen: quinone oxidoreductase 1 and cysteine/glutamic acid reverse transport solute carrier family 7 members 11, decreased the expression of heme oxygenase 1, nuclear receptor coactivator 4 and Transferrin Receptor proteins, reduced malondialdehyde and lipid peroxidation levels, and increased intracellular L-glutathione levels in cells treated with rCPB1. Furthermore, calpain inhibitor PD151746 was used to investigate how Pyroptosis and Ferroptosis were involved simultaneously in rCPB1-treated macrophages. We showed that PD151746 inhibited ATP and ROS production, reversed the representative Pyroptosis/Ferroptosis indicators and subsequently reduced inflammation. The above findings indicate that rCPB1 might lead to macrophage Pyroptosis and Ferroptosis through the large and sustained increase in intracellular calpain and oxidative stress, further lead to inflammation.

Calpain; Clostridium perfringens Beta-1 toxin; Ferroptosis; Pyroptosis; ROS.