A simple, reliable and easily generalizable cell-based assay for screening potential drugs that inhibit lipid accumulation.

Abstract

Ectopic lipid deposition in the hepatocyte plays an important role in the development of nonalcoholic fatty liver disease (NAFLD), which has become one of the most common causes of chronic liver disease worldwide yet no approved drugs are currently available. In this study, a cell-based method was developed to screen potential drugs with low toxicity that inhibit lipid accumulation. In the same 96-well plate, cytotoxicity was measured using CCK8 assay, followed by lipid content detection using BODIPY 493/503 via fluorometry assay, a lipid droplet-specific fluorescent dye commonly used in microscopy and flow cytometry, but not previously reported in fluorometry. Lipid content was normalized to DAPI staining to control for cell number. The results of this assay were highly consistent with the fluorescence microscopy, with significantly lower intra-group variability in detecting lipid accumulation induced by free fatty acids in Huh7 cells. Validation was conducted using 10 well documented steatotic compounds and 5 negative controls, all of which were correctly identified by the assay. In addition, the inhibitory effect of ML261, a well-known inhibitor of hepatic lipid droplets formation, was also confirmed by the assay both in AML12 cells and Hepa1-6 cells. To our knowledge, this study is the first to quantify lipid droplets using BODIPY 493/503 by fluorometry assay, and to demonstrate that CCK8 does not interfere with subsequent BODIPY 493/503 staining, both of which will reduce the cost and increase the efficiency. In conclusion, the method is simple, reliable, efficient and does not rely on expensive instruments, making it an easily generalizable approach to identify potential drug candidates for NAFLD treatment.