(W1130-03-18) Study on the Anti-Lipid Accumulation Effects of Compounds Derived from Herbs Based on High-Throughput Intracellular Lipid Droplet Phenotypic Analysis

Purpose: This study aimed to establish a high-content screening platform to systematically evaluate the anti-steatotic effects of bioactive components from Ling-Gui-Zhu-Gan Decoction (LGZGD) and Danshen (DS) against non-alcoholic fatty liver disease (NAFLD) and to elucidate their underlying molecular mechanisms through phenotypic analysis and gene expression profiling.

Methods: A free fatty acid-induced steatosis model was established in AML12 hepatocytes to simulate NAFLD pathology. High-content imaging and analysis system was employed as the primary technological platform to quantitatively assess multiple cellular parameters simultaneously. Eight phytochemical compounds derived from LGZGD and DS were screened using BODIPY staining with comprehensive morphometric analysis of lipid droplet characteristics, including area, count, and size. Transcriptional analysis via quantitative PCR was subsequently performed to investigate the molecular targets of the identified active compound.

Results: High-content phenotypic screening revealed that luteolin exhibited the most potent anti-steatotic activity among all tested LGZGD and DS components, significantly reducing lipid droplet accumulation in hepatocytes. The high-resolution multiparametric analysis demonstrated that luteolin treatment decreased lipid droplet area, count, and size in a concentration-dependent manner. Further mechanistic investigations using the same platform identified that luteolin significantly downregulated the transcription of key lipid droplet regulators, including Rpn1 (droplet number regulator), Taf2 (droplet area modulator), and Nadk.

Conclusion: This study demonstrates that luteolin, a bioactive component of DS, effectively attenuates hepatic steatosis by suppressing the transcription of Rpn1, Taf2, and Nadk. Our findings validate high-content phenomic screening as a valuable approach for investigating the multi-component, multi-pathway mechanisms of traditional Chinese medicine formulations, establishing an integrated methodological framework that bridges chemical biology with molecular cell biology for complex herbal medicine research.


Figure 1. Effects of 8 compounds derived from LGZGD and DS on lipid droplet accumulation in an in vitro model of NAFLD.
A. Representative fluorescent images of lipid droplets accumulation (BODIPY staining in green) in AML12 cells under different treatments. (scale bars =80 μm). Control: without 360 μM FFA treatment; Model: with 360 μM FFA treatment; Quercetin: with 360 μM FFA and 10 μM quercetin treatment; Isorhamnetin: with 360 μM FFA and 10 μM isorhamnetin treatment; Kaempferol: with 360 μM FFA and 10 μM kaempferol treatment; Formononetin: with 360 μM FFA and 10 μM formononetin treatment; Naringenin: with 360 μM FFA and 10 μM naringenin treatment; Epicatechin: with 360 μM FFA and 10 μM epicatechin treatment; Ursolic acid: with 360 μM FFA and 10 μM ursolic acid treatment; Luteolin: with 360 μM FFA and 10M luteolin treatment. B. Statistics of lipid droplet in AML12 cells under different treatments. The data are expressed as the mean ± SEM. n=3, one-way ANOVA, ####P <0.0001 versus control, **P <0.01 versus model, ****P <0.0001 versus model. Mean granule area (MGA) was calculated by total granule area/cell number in each view, and granules per cell (GPC) was calculated by number of granules/cell number.


Figure 2. Effects of luteolin on lipid droplet accumulation in an in vitro model of NAFLD.
A. Representative fluorescent images of lipid droplets accumulation (BODIPY staining in green) in AML12 cells under different concentration luteolin treatments. (scale bars =100 μm). Control: without 360 μM FFA treatment; Model: with 360 μM FFA treatment; 0.625 μM LUT: with 360 μM FFA and 0.625 μM luteolin treatment; 1.25 μM LUT: with 360 μM FFA and 1.25 μM luteolin treatment; 2.5 μM LUT: with 360 μM FFA and 2.5 μM luteolin treatment. B. Statistics of lipid droplet in AML12 cells under different concentration luteolin treatments. The data are expressed as the mean ± SEM. n=3, one-way ANOVA, ####P <0.0001 versus control, **P <0.01 versus model, ****P <0.0001 versus model. Mean granule area (MGA) was calculated by total granule area/cell number in each view, and granules per cell (GPC) was calculated by number of granules/cell number, the MGA/GPC to assess the mean particle size of lipid droplets. C. Expression levels of lipid droplet-related genes with luteolin treatment. *P < 0.05.