ABSTRACT
Isobavachalcone (IBC) is a flavonoid component of the traditional Chinese medicine Psoraleae Fructus, with a range of pharmacological properties. However, IBC causes some hepatotoxicity, and the mechanism of toxicity is unclear. The purpose of this paper was to investigate the possible mechanism of toxicity of IBC on HepG2 cells and zebrafish embryos. The results showed that exposure to IBC increased zebrafish embryo mortality and decreased hatchability. Meanwhile, IBC induced liver injury and increased expression of ALT and AST activity. Further studies showed that IBC caused the increase of ROS and MDA the decrease of CAT, GSH, and GSH-Px; the increase of Fe2+ content; and the changes of ferroptosis related genes (acsl4, gpx4, and xct) and iron storage related genes (tf, fth, and fpn) in zebrafish embryos. Through in vitro verification, it was found that IBC also caused oxidative stress and increased Fe2+ content in HepG2 cells. IBC caused depolarization of mitochondrial membrane potential (MMP) and reduction of mitochondrial ATP, as well as altered expression of ACSl4, SLC7A11, GPX4, and FTH1 proteins. Treatment of HepG2 cells with ferrostatin-1 could reverse the effect of IBC. Targeting the System Xc--GSH-GPX4 pathway of ferroptosis and preventing oxidative stress damage might offer a theoretical foundation for practical therapy and prevention of IBC-induced hepatotoxicity.
ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus is a well-known Traditional Chinese Medicine which has long been used to warm and tonify the kidney and treat diseases such as osteoporosis and diarrhea. However, it may cause multiorgan injury, which limited its use. AIM OF THE STUDY: The aim of this study was to identify the components of ethanol extract of salt-processed Psoraleae Fructus (EEPF) and systematically investigate its acute oral toxicity and the mechanism underlying its acute hepatotoxicity. MATERIALS AND METHODS: In this study, the UHPLC-HRMS analysis was carried out for components identification. Followed by acute oral toxicity test in Kunming mice, which received oral gavage of EEPF from 3.85 to 78.00 g/kg. Body weight, organ indexes, biochemical analysis, morphology, histopathology, oxidative stress state, TUNEL, mRNA and protein expression of NLRP3/ASC/Caspase-1/GSDMD signaling pathway were evaluated to study the EEPF-induced acute hepatotoxicity and its underlying mechanisms. RESULTS: The results showed that 107 compounds such as psoralen and isopsoralen were identified in EEPF. And the acute oral toxicity test demonstrated the LD50 of EEPF was 15.95 g/kg in Kunming mice. The survival mice displayed non-significant difference in body weight compared with Control at the end of the observation period. And the organ indexes of heart, liver, spleen, lung, and kidney showed no significant difference. However, the morphological and histopathological changes of these organs in high-dose-groups mice indicated that the liver and kidney might be the main target toxic organs of EEPF, which showed hepatocyte degeneration with lipid droplets and protein cast in kidney. It could be confirmed by the significant increases of liver and kidney function parameters such as AST, ALT, LDH, BUN, and Crea. In addition, the oxidative stress markers, MDA in the liver and kidney was significantly increased while SOD, CAT, GSH-Px (only liver), and GSH were significantly decreased. Furthermore, EEPF increased the TUNEL-positive cells and the mRNA and protein expression of NLRP3, Caspase-1, ASC and GSDMD in liver with increased protein expression of IL-1ß and IL-18. Notably, cell viability test showed that the specific inhibitor of Caspase-1 could reverse the Hep-G2 cell death induced by EEPF. CONCLUSION: To summarize, this study analyzed the 107 compounds of EEPF. The acute oral toxicity test demonstrated the LD50 value of EEPF was 15.95 g/kg in Kunming mice and the liver and kidney might be the main target toxic organs of EEPF. It caused liver injury through oxidative stress and pyroptotic damage via NLRP3/ASC/Caspase-1/GSDMD signaling pathway.
