Andrographolide-induced PANoptosis underlies its multiple organ toxicity in mice.

Andrographolide (Andro), the major bioactive component of Andrographis paniculata, exhibits potent anti-inflammatory properties but has raised safety concerns due to reported organ toxicity. This study aimed to investigate the mechanisms underlying Andro's in vitro and in vivo toxicity. In mice, single dose (≤100 mg/kg) Andro administration showed no acute toxicity, with no overt histopathological organ injury. But repeated administration of the same dose of Andro triggered damage in lung, liver, uterus, and kidney, characterized by pulmonary alveolar disruption, renal tubular edema, and elevated serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT). Concurrent with systemic injury, PANoptosis was induced by Andro in these organs, as evidenced by the activation of caspase-1/-8/-3 (apoptosis), gasdermin D/E (GSDMD/E, pyroptosis), and MLKL (necroptosis), indicating the correlation between PANoptosis and organ toxicity. In vitro, Andro caused lytic cell death in macrophages and other cells in a time- and dose-dependent manner. During this process, Andro induced rapid activation of caspase-8, followed by caspase-1/-3 and GSDME cleavage and phosphorylation of MLKL (p-MLKL), indicative of the activation of the PANoptosis signaling pathway. Consistent with this, Andro induced lytic cell death was markedly attenuated by caspase-1 inhibitor VX-765, pan-caspase inhibitors (IDN-6556, Z-VAD-FMK) and GSDMD/E inhibitor (disulfiram). In addition, RIPK1 inhibition (by Nec-1) partially reduced cell death, confirming RIPK1-dependent necroptosis as a minor contributor. In conclusion, our data establish PANoptosis as an important mechanism of Andro-induced organ injury, providing a mechanistic framework for Andro's dichotomous bioactivity, informing evidence-based dosing strategies to maximize therapeutic efficacy while mitigating toxicity risks in clinical practice.