A novel cholesterol-reducing mechanism of polygonati rhizoma: Dual action via Bacteroides-mediated cholesterol sulfonation and feedback inhibition of ACAT2 by sulfated metabolite.

Polygonati Rhizoma (PR) has the function of "invigorating spleen and tonifying kidney", and is historically applied as a homology of medicine and food to prevent and treat dyslipidemia in China. However, there is limited experimental evidence to support this application, and the underlying mechanism has not been fully deciphered. To analyze the composition and illuminate the cholesterol-lowering potential and molecular mechanism of PR's aqueous extract (PRE) in high-fat emulsion (HFE)-induced hypercholesterolemia mouse model. Ion chromatograph was employed to determine the monosaccharide composition of PRE. HFE-induced Kunming mouse model was constructed to evaluate the anti-hypercholesterolemia effect of PRE. Metagenomic sequences and liquid chromatography-mass spectrometry (LC-MS) analysis were performed to elucidate the mechanism through which PR regulated cholesterol metabolism. Antibiotic cocktail (ABX) intervention and fecal microbiota transplantation (FMT) were used to validate whether PRE regulated cholesterol metabolism through the intestinal microbiota. The cholesterol-reducing effect of cholesterol sulfate (CS) was explored in poloxamer 407 (P407)-induced mouse model of dyslipidemia. Molecular docking and molecular dynamics (MD) simulation were also employed to elucidate the underlying mechanisms. Furthermore, a combination of qRT-PCR, Western blot, and surface plasmon resonance (SPR) were employed to delineate its mechanism. Our study indicated that the polysaccharides of PRE were mainly composed of fructose (92.33 %) and glucose (5.25 %). PRE treatment effectively blocked body weight gain, significantly decreased serum and hepatic levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDL-C) level. Additionally, PRE ameliorated hepatic lipid accumulation in mice with HFE-elicited hypercholesterolemia. Notably, metagenomic sequencing and LC-MS analysis indicated that PRE markedly increased the abundance of intestinal genera Bacteroides and significantly elevated the fecal CS concentration in HFE mice. Genome-based functional analysis further indicated that cofactors of sulfonation (ATP sulfurylase CysD and CysN, BT0414-BT0415) were significantly upregulated after treatment with PRE. The cholesterol-lowering effect of PRE was largely contingent upon microbial conversion of cholesterol-to-CS mediated by Bacteroides, as validated by antibiotics-induced intestinal microbiota depletion in pseudo-germ-free model and restoration of gut microbiota through FMT. In vitro study also showed that PRE promoted the growth of Bacteroides thetaiotaomicron. Furthermore, CS markedly alleviated serum, hepatic, bile, and fecal levels of TG, TC, LDL-C, HDL-C, and TBA, indicative of appreciable lipid-lowering effect. MD simulation and SPR results indicated that CS directly bound to ACAT2. Consistent with this interaction, CS greatly downregulated the mRNA and protein expression of ACAT2 in small intestinal tissue. These findings for the first time suggested that PR acted as a prebiotic agent to ameliorate hypercholesterolemia, at least in part, via dual mechanism involving modulation of Bacteroides-mediated sulfonation metabolic pathway and feedback inhibition of ACAT2 by CS, highlighting its therapeutic potential for cholesterol-related disorders. This work might also offer novel mechanistic insight and further buttressed the ethnopharmacological application of PR in the therapy of hypercholesterolemia.