Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western China, College of Life Science, Ningxia University, Yinchuan 750021, China.
Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western China, College of Life Science, Ningxia University, Yinchuan 750021, China.
Microbiol Res. 2024 Dec;289:127916. doi: 10.1016/j.micres.2024.127916. Epub 2024 Sep 27.
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 HO. Of note, HO 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.
产气荚膜梭菌β-1 毒素（CPB1）是一种致死性毒素，可导致坏死性肠炎，但病理机制尚不清楚。我们研究了活性氧（ROS）是否参与 CPB1 诱导的细胞焦亡和铁死亡，并研究了钙蛋白酶对 CPB1 诱导的氧化应激和炎症的影响。用 N-乙酰-L-半胱氨酸（NAC）清除 ROS 可减少 ROS，抑制 rCPB1 处理的巨噬细胞死亡、细胞质肿胀和膜破裂、细胞焦亡相关蛋白和促炎因子的表达，同时增加抗炎因子的表达。三磷酸腺苷（ATP）合酶、H 转运、线粒体 F1 复合物、α亚基 1（ATP5A1）被鉴定为与 rCPB1 特异性相互作用。沉默 ATP5A1 可抑制 ATP 和 ROS 的积累，导致细胞质肿胀和膜破裂减少，减弱 rCPB1 处理细胞的细胞焦亡和炎症。我们还发现 rCPB1 诱导巨噬细胞发生铁死亡，铁死亡水平与 HO 相似。值得注意的是，HO 是主要的 ROS 来源，表明 ROS 产生可能在 rCPB1 处理的巨噬细胞铁死亡调节中起主要作用。这一发现在 rCPB1 诱导的人急性单核细胞白血病细胞中得到了进一步证实，这些细胞用 NAC 处理。此外，使用 liproxstatin-1 抑制铁死亡会抑制皱缩的线粒体形态，增加谷胱甘肽过氧化物酶 4、烟酰胺腺嘌呤二核苷酸（磷酸）氢醌氧化还原酶 1 和半胱氨酸/谷氨酸反向转运溶质载体家族 7 成员 11 的表达，降低血红素加氧酶 1、核受体共激活因子 4 和转铁蛋白受体蛋白的表达，降低丙二醛和脂质过氧化水平，增加 rCPB1 处理细胞内 L-谷胱甘肽水平。此外，用钙蛋白酶抑制剂 PD151746 研究细胞焦亡和铁死亡如何同时参与 rCPB1 处理的巨噬细胞。我们发现 PD151746 抑制 ATP 和 ROS 的产生，逆转代表性的细胞焦亡/铁死亡指标，随后减轻炎症。上述发现表明，rCPB1 可能通过细胞内钙蛋白酶和氧化应激的大量和持续增加导致巨噬细胞细胞焦亡和铁死亡，进一步导致炎症。
