Rhein suppresses African swine fever virus replication in vitro via activating the caspase-dependent mitochondrial apoptosis pathway

African swine fever (ASF) is a virulent infectious diseases of pigs caused by the African swine fever virus (ASFV) that can spread widely and cause high fatality rates. Currently, there is no effective way to treat the disease, and there is no effective vaccine to prevent it. Rhein, an anthraquinone compound extracted from many traditional Chinese medicines, exhibits anti-inflammatory, anti-tumor, and anti-viral activities. However, the anti-viral effects of rhein on ASFV remain unclear. Therefore, this study aimed to investigate the anti-ASFV activity of rhein in porcine alveolar macrophages (PAMs) and the underlying mechanisms. In this study, we confirmed that rhein inhibits ASFV replication significantly in a dose-dependent manner in vitro. Moreover, rhein could alter the susceptibility of PAMs to ASFV and promoted the production of superoxide in the mitochondria, which induced the loss of mitochondrial membrane potential, leading to the activation of caspase-9, caspase-3, and apoptosis. Mito-TEMPO, a mitochondria-targeted antioxidant, blocked rhein-induced mitochondrial superoxide generation and loss of mitochondrial membrane potential, prevented caspase-9 and caspase-3 activation, alleviated apoptosis, and suppressed the anti-ASFV activity of rhein. Altogether, our results suggested that rhein could play an anti-ASFV role by inducing apoptosis through the activation of the caspase-dependent mitochondrial apoptotic pathway and may provide a novel compound for developing anti-ASFV drugs.