Lin Qiu-Ru, Li Chen-Guang, Zha Qing-Bing, Xu Li-Hui, Pan Hao, Zhao Gao-Xiang, Ouyang Dong-Yun, He Xian-Hui
Department of Immunobiology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Department of Fetal Medicine, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China.
Toxicol Appl Pharmacol. 2016 Feb 1;292:56-64. doi: 10.1016/j.taap.2015.12.027. Epub 2016 Jan 4.
Gossypol, a polyphenolic compound isolated from cottonseeds, has been reported to possess many pharmacological activities, but whether it can influence inflammasome activation remains unclear. In this study, we found that in mouse macrophages, gossypol induced cell death characterized by rapid membrane rupture and robust release of HMGB1 and pro-caspase-11 comparable to ATP treatment, suggesting an induction of pyroptotic cell death. Unlike ATP, gossypol induced much low levels of mature interleukin-1β (IL-1β) secretion from mouse peritoneal macrophages primed with LPS, although it caused pro-IL-1β release similar to that of ATP. Consistent with this, activated caspase-1 responsible for pro-IL-1β maturation was undetectable in gossypol-treated peritoneal macrophages. Besides, RAW 264.7 cells lacking ASC expression and caspase-1 activation also underwent pyroptotic cell death upon gossypol treatment. In further support of pyroptosis induction, both pan-caspase inhibitor and caspase-1 subfamily inhibitor, but not caspase-3 inhibitor, could sharply suppress gossypol-induced cell death. Other canonical pyroptotic inhibitors, including potassium chloride and N-acetyl-l-cysteine, could suppress ATP-induced pyroptosis but failed to inhibit or even enhanced gossypol-induced cell death, whereas nonspecific pore-formation inhibitor glycine could attenuate this process, suggesting involvement of a non-canonical pathway. Of note, gossypol treatment eliminated thioglycollate-induced macrophages in the peritoneal cavity with recruitment of other leukocytes. Moreover, gossypol administration markedly decreased the survival of mice in a bacterial sepsis model. Collectively, these results suggested that gossypol induced pyroptosis in mouse macrophages via a non-canonical inflammasome pathway, which raises a concern for its in vivo cytotoxicity to macrophages.
棉酚是一种从棉籽中分离出的多酚类化合物，据报道具有多种药理活性，但它是否能影响炎性小体激活仍不清楚。在本研究中，我们发现，在小鼠巨噬细胞中，棉酚诱导的细胞死亡具有快速膜破裂以及HMGB1和前半胱天冬酶-11大量释放的特征，这与ATP处理相当，提示诱导了焦亡性细胞死亡。与ATP不同，棉酚诱导经脂多糖预处理的小鼠腹腔巨噬细胞分泌的成熟白细胞介素-1β（IL-1β）水平低得多，尽管它引起的前IL-1β释放与ATP相似。与此一致，在棉酚处理的腹腔巨噬细胞中未检测到负责前IL-1β成熟的活化半胱天冬酶-1。此外，缺乏凋亡相关斑点样蛋白（ASC）表达且无半胱天冬酶-1激活的RAW 264.7细胞在棉酚处理后也发生焦亡性细胞死亡。为进一步支持焦亡诱导，泛半胱天冬酶抑制剂和半胱天冬酶-1亚家族抑制剂而非半胱天冬酶-3抑制剂可显著抑制棉酚诱导的细胞死亡。其他典型的焦亡抑制剂，包括氯化钾和N-乙酰-L-半胱氨酸，可抑制ATP诱导的焦亡，但未能抑制甚至增强棉酚诱导的细胞死亡，而非特异性成孔抑制剂甘氨酸可减弱这一过程，提示涉及非经典途径。值得注意的是，棉酚处理消除了腹腔中巯基乙酸盐诱导的巨噬细胞，并募集了其他白细胞。此外，在细菌败血症模型中，棉酚给药显著降低了小鼠的存活率。总体而言，这些结果表明棉酚通过非经典炎性小体途径在小鼠巨噬细胞中诱导焦亡，这引发了对其在体内对巨噬细胞细胞毒性的担忧。
