Cigarette Smoke Extract of Heated Tobacco Product Induces Necroptosis <i>via</i> Receptor-Interacting Protein 1-Mediated Phosphorylation of Mixed-Lineage Kinase Domain-Like Protein in Vascular Smooth Muscle Cells

Heated tobacco products (HTPs) are alternatives to traditional combustible cigarettes and are designed to reduce the generation of substances harmful to health. Recently, we reported that nicotine- and tar-free cigarette smoke extract (CSE) prepared from HTP aerosols (HTP-CSE) induces ferroptosis and non-ferroptotic cell death in rat vascular smooth muscle A7r5 cells. However, the non-ferroptotic mechanism underlying HTP-CSE-induced cell death remains unclear. Here, we demonstrate that HTP-CSE causes necroptosis, a type of programmed cell death mediated by the receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL) signaling pathway. Treatment of A7r5 cells with HTP-CSE at concentrations ranging from 10 to 80% for 4 h decreased mitochondrial metabolic activity and increased lactate dehydrogenase leakage in a concentration-dependent manner. The cytotoxic effects at concentrations of 10 and 20% were partially inhibited by Necrostatin-1s, an RIPK1 inhibitor. Treatment with 20% HTP-CSE for 3 h facilitated MLKL phosphorylation, and the phosphorylation level persisted for up to 6 h after HTP-CSE treatment. The facilitation of MLKL phosphorylation in response to 3 h of exposure to 20% HTP-CSE was almost completely depressed by Necrostatin-1s. These results suggest that HTP-CSE activates the RIPK1/MLKL signaling pathway, resulting in necroptosis associated with mitochondrial dysfunction and cell membrane damage.