[Study on potential hepatotoxicity of main monomers of Polygonum multiflorum based on liver micro-tissue].

In this study, we aimed to establish a rat liver micro-tissue evaluation system to evaluate the hepatotoxicity of the main monomers in Polygonum multiflorum. Rat primary hepatocytes were isolated and purified by two-step in situ perfusion method to prepare hepatic parenchymal cells. The ultra-low adsorption plate and the inverted model were used to establish an in vitro hepatotoxicity evaluation system. After the system was established, the main monomer components(monanthone with emodin type, rhein, emodin, emodin-8-O-ß-D-glucopyranoside, physcion) of P. multiflorum were selected for in vitro hepatotoxicity evaluation. This study showed that the primary cells of the liver can form liver micro-tissues in the low adsorption plate method and the mold perfusion method, with good liver structure and function, which can be used to evaluate the hepatotoxicity of the drug to be tested after long-term administration. The five monomers to be tested in P. multiflorum can significantly affect the proliferation of primary liver micro-tissues in rats in a dose-and time-dependent manner. The hepatotoxic effects were as follows: monanthone with emodin type > rhein > emodin > emodin-8-O-ß-D-glucopyranoside > physcion. The results suggested that the emodin-type monoterpene and rhein might be the potential hepatotoxic components, while the metabolites of emodin-8-O-ß-D-glucoside and emodin methyl ether showed more toxic risks. The rat primary hepatocyte micro-tissue model system established in this experiment could be used to achieve long-term drug administration in vitro, which was consistent with the clinical features of liver injury caused by long-term use of P. multiflorum. The experimental results provided important information and reference on the clinical application and toxic component of P. multiflorum.

Identification of zinc-finger BED domain-containing 3 (Zbed3) as a novel Axin-interacting protein that activates Wnt/beta-catenin signaling.

Axin, a key modulator of the Wnt/beta-catenin pathway, acts as a scaffold protein in phosphorylating and degrading cytoplasmic beta-catenin. Canonical Wnt proteins appear to stabilize beta-catenin by inducing the interaction of LRP5/6 with Axin. This interaction requires the phosphorylation of the Ser or Thr residues in the PPPP(S/T)PX(T/S) motifs at the intracellular domain of LRP5/6. In this work, we identified a novel Axin-interacting protein, zinc-finger BED domain-containing 3 (Zbed3), by yeast two-hybrid screening. The interaction was confirmed in co-immunoprecipitation experiment in mammalian cells and in vitro pulldown assays. Moreover, we found Zbed3 also contains a PPPPSPT motif, which is crucial to its binding to Axin. The Ser and Thr residues in the motif appear to be also phosphorylated by glycogen synthase kinase 3beta (GSK3beta) and the CKI family kinases, as GSK3beta and CKIepsilon could enhance the interaction of Zbed3 with Axin. Mutation of the Ser (SA) or Thr (TA) residue to Ala in the motif markedly impaired its ability to interact with Axin. Expressing Zbed3, but not these mutants, led to inhibition of GSK3beta-mediated beta-catenin phosphorylation, cytoplasmic beta-catenin accumulation, and activation of lymphoid enhancer binding factor-1-dependent reporter gene transcription. Furthermore, knockdown of Zbed3 with RNA interference attenuated Wnt-induced beta-catenin accumulation, lymphoid enhancer binding factor-1-dependent luciferase reporter activity, and the Wnt target gene expression. These results together indicate that Zbed3 is a novel Axin-binding protein that is involved in Wnt/beta-catenin signaling modulation.