Extracts from <i>Tenebrio molitor</i> Larvae and <i>Protaetia brevitarsis</i> Larvae Reduced Cadmium-induced Ferroptosis and Cytotoxicity in Hep3B Cells

Background Cadmium is a hepatotoxic heavy metal. Cadmium is known to induce ceramide generation and ferroptosis. Edible insects have recently been touted as a potential functional food. Various studies are being conducted in this regard. Purpose This study was designed to investigate whether two types of insects, Tenebrio molitor larvae (TM) and Protaetia brevitarsis larvae (PB), can inhibit cadmium-induced hepatotoxicity. Materials and Methods TM and PB powder were extracted using 70% ethanol and diluted with phosphate-buffered saline. TM and PB extracts were cotreated with cadmium in Hep3B cells. To investigate the mechanism, desipramine (an acid sphingomyelinase (ASMase) inhibitor), ferrostatin-1 (a ferroptosis inhibitor), and GW4869 (a neutral sphingomyelinase (NSMase) inhibitor) were used with cadmium. Anti-ferroptosis mechanisms of the TM and PB extracts were demonstrated using Western blot, enzyme-linked immunosorbent assay, MTT cell viability assay, real-time polymerase chain reaction analysis, and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assay. Results Cadmium increased ceramide generation and ferroptosis, concomitant with increased ASMase and NSMase expressions. To understand which mechanism is the upstream pathway, ferrostatin-1, desipramine, and GW4869 were treated with cadmium. Cadmium-induced hepatotoxicity was partially inhibited by ferrostatin-1, which did not affect ASMase expression and ceramide levels. Desipramine, but not GW4869, reduced cadmium-induced cell death and ferroptosis by reducing ceramide levels. Therefore, ceramide generation by ASMase can be an upstream pathway of ferroptosis in cadmium-induced cell death. Interestingly, TM and PB extracts reduced cadmium-induced ceramide increase and ASMase expression, leading to reduced ferroptosis and cell death. TM and PB extracts also had antioxidant properties due to their ability to scavenge reactive oxygen species. Conclusion TM and PB have an anti-ferroptosis mechanism against cadmium, which is due to inhibition of ceramide generation by reducing ASMase expression. Therefore, TM and PB can inhibit cadmium-induced hepatotoxicity as functional foods.