Polygonum multiflorum Thunb. Induces hepatotoxicity in SD rats and hepatocyte spheroids by Disrupting the metabolism of bilirubin and bile acid

The liver damage associated with Polygonum multiflorum Thunb. (P. multiflorum) and its preparations have aroused widespread concern. Opinions on the toxicity mechanisms and targets of P. multiflorum vary, and the toxic components are even more controversial. However, based on the current research results, we believed that any single component in P. multiflorum could not directly lead to liver injury, but may be the synergistic effect of multiple components. In addition, the toxicity mechanism also involved multiple targets.This study aimed to elucidate the mechanism and target of the hepatotoxicity of P. multiflorum.In this study, the manifestations of liver injury triggered by P. multiflorum and the associated metabolic enzymes/transporters in the metabolic pathways of bilirubin and bile acid were investigated to elucidate the mechanism and target of the hepatotoxicity of P. multiflorum and related components. First, the hepatotoxicity and potential effect of P. multiflorum on both metabolic pathways were studied in rats administered P. multiflorum extracts (in 70% ethanol) for 42 days. Then, in vitro cultured hepatocyte spheroids were used to determine the hepatotoxicity of monomer components.This revealed that P. multiflorum could simultaneously block bilirubin(BIL) and bile acid(BA) metabolism pathways, subsequently leading to liver damage. The targets and modes of action include reducing the activity of UGT1A1, the only metabolic enzyme of BIL, downregulating BIL and BA uptake transporters NTCP, OATP1B1, OATP1B3, efflux transporters MRP2, and BSEP, and upregulating efflux transporter MRP3. Furthermore, our data indicated that 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucoside (TSG) and emodin-8-O-β-D-glucoside (EG) are the main toxic components in P. multiflorum. TSG accounts for 3.71% of the total content of P. multiflorum. In addition to markedly downregulating UGT1A1, TSG can upregulate OATP1B1/3 and promote the uptakes of bilirubin and bile acid, producing synergistic toxicity. EG accounts for 0.29% of the total content and demonstrates direct hepatotoxicity and extensive substrate overlap with bilirubin and bile acids. It can affect these two metabolic pathways simultaneously, promoting the accumulation of both bilirubin and bile acid for further toxic effects. Emodin is other major component, accounting for 0.01% of the total content, and its hepatotoxicity mechanisms include direct toxicity and inhibitory effects on bilirubin metabolizing enzymes. However, emodin is mainly distributed in the kidneys, so its hepatotoxicity risk is relatively low.The simultaneous blockade of bilirubin and bile acid metabolic pathways as the critical toxic mechanism of P. multiflorum-induced liver injury, and potential toxic components were TSG and EG.