Hesperidin alleviates ZBP1-drived PANoptosis induced by copper nanoparticles in immune organs of gallus.

With the application of copper nanoparticles (nano-Cu) in livestock and poultry feed addition, their biotoxicity has been gradually recognized. Therefore, it has become an urgent problem to find the effective natural antagonists to reduce the toxicity of copper nanoparticles. Here, we found that hesperidin could alleviate nano-Cu-induced pathological injury in the immune organs of chickens via the histopathological examination of the spleen, thymus, and bursa of Fabricius. Additionally, the results of western blot showed that nano-Cu exposure activated ZBP1-mediated PANoptosis in immune organs, with evidenced by the significant up-regulation of ZBP1 signal molecule and PANoptosis-related proteins (apoptosis: Caspase-7, Caspase-3, Caspase-8; pyroptosis: Caspase-1, GSDMD, GSDME; necroptosis: RIPK1 and MLKL). Besides that, immunohistochemistry and immunofluorescence also showed that the staining intensity of Caspase-9 and Caspase-8 proteins was observably elevated in nano-Cu group compared to control group, and the staining intensity of the hesperidin mixed nano-copper group was markedly lower than that of the nano-Cu group. Meanwhile, hesperidin effectively attenuated the ZBP1 expression and PANoptosis under nano-Cu exposure. These findings suggested that excessive nano-Cu could cause ZBP1-drived PANoptosis in immune organs, while hesperidin could alleviate toxic damage induced by nano-Cu exposure.