Fig 1: Schematic diagram of the therapeutic mechanism of combined use of bicyclol and berberine against NAFLD. Bicyclol enhanced lipolysis through p62-Nrf2-CES2 signaling axis and improved ß-oxidation through p62-Nrf2-PPARa signaling axis, while berberine suppressed the de novo lipogenesis by inhibiting protein expressions of ACC and FAS, along with the regulation of lipid metabolisms via the gut microbiota, and thus co-facilitating to ameliorate the occurrence and progress of NAFLD. The green arrow shows a stimulatory effect, the red flathead shows an inhibitory effect.
Fig 2: Bicyclol with or without berberine enhances the lipolysis and ß-oxidation through p62-Nrf2-CES2/PPARa axis. Male C57BL/6J mice were freely fed with WD or WD plus bicyclol and/or berberine for 16 weeks (A,C), or induced with WD/CCl4 for 4 weeks and then treated with drugs for 8 weeks (B,D). The protein levels of PPARa, CES2, Nrf2, p62, CD36, FABP1, ATGL, HSL, ACSL1, and ACSL5 were detected with Western blot and quantified using a Gel-Pro analyzer. Results were presented as mean ± SD, and a representative band was shown. n = 5–9 for each group, *p < 0.05 and **p < 0.01, the model group vs. control group; #p < 0.05 and ##p < 0.01 vs. the model group. PPARa, proliferator-activated receptor a; CES2, carboxylesterase 2; Nrf2, NF-E2-related factor 2; p62, ubiquitin-binding protein p62; CD36, cluster determinant 36; FABP1, fatty acid binding protein 1; ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase; ACSL, acyl-CoA synthetase long chain family member.
Fig 3: Combined use of bicyclol and berberine decreases lipid accumulation via regulating lipid metabolism-related gene expression in FFA-induced HepG2 cells. HepG2 cells were induced by 0.1 mM FFA and simultaneously treated for 24 h with 2 µM berberine, 2 µM bicyclol, or their combinations. The cells were assayed with an MTT method to show the cytotoxicity (A), or carried out Nile Red staining to show LDs (B) and the level of LDs in cells were quantified using Image-Pro Plus 6.0 (C). Total proteins were extracted and detected with Western blot (D). n = 3, **p < 0.01, the model group vs. control group; #p < 0.05 and ##p < 0.01 vs. the model group or the monotherapy group. FFA, free fatty acid; LDs, lipid droplets; PPARa, proliferator-activated receptor a; CES2, carboxylesterase 2; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase.
Fig 4: Carboxylesterase 2 (CES2/Ces2) antioxidant response to ROS in the presence of nanoparticles.a Computational simulations of molecular docking of normal CES2/Ces2h or mutated CES2p.G193A/Ces2hp.G148A with cholesterol ester and ·OH. Blue stick: oxyanion hole; Green sticks: spatial configuration of ligands with·OH in the oxyan hole; Pink sticks: spatial configuration of ligands without ·OH in the oxyan hole; Black dashes: hydrogen-bond interactions. b In vitro catalytic activity and corresponding ·OH concentration changes of normal CES2/Ces2h or mutated CES2p.G193A/Ces2hp.G148A enzymes on catalyzing PNPB hydrolysis under different conditions. c Intracellular lipid fraction analysis of normal Ces2h or mutated Ces2hp.G148A hepatocyte cell lines upon treatments with TiO2, NaYF4, and Au nanoparticles, respectively. d Incorporation of [1-13C]oleate into cellular lipids. e–g Chase experiments evaluating the turnover of lipid species, including triglycerides (TG), cholesterol esters (CE), and phospholipids (PL). Statistics for the chase period were analyzed as a percentage of the pulse. The group setting was the same in (c–g) as indicated in (d). Different letters indicate the significant difference (P < 0.05) analyzed by one-way ANOVA. Data in (b–g) are presented as mean values ± SEM. n = 6. Source data are provided as a Source Data file.
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