<em>In Vitro</em> Modeling of Fat Deposition in Metabolic Dysfunction-associated Steatotic Liver Disease

Qinglan Bao; Xiaoxiao Zhang; Yingsong Chen; Tegexibaiyin Wang; Bateer Siqin

doi:10.3791/66810

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Biology

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

Published: July 19, 2024

doi:

10.3791/66810

Qinglan Bao, Xiaoxiao Zhang, Yingsong Chen*^1,3,4, Tegexibaiyin Wang*⁵, Bateer Siqin*⁶

¹Inner Mongolia Medical University, ²Xilin Gol League Mongolian Medical Hospital, ³National and local Joint Mongolian Medicine R & D Engineering Center, ⁴Inner Mongolia Minzu University, ⁵Affiliated Hospital of Inner Mongolia Minzu University, ⁶Traditional Chinese & Mongolian Medical Research Institute of Inner Mongolia

Summary

This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

Abstract

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading to significant health challenges. Previous reports have studied the establishment of animal and cellular models for MASLD, highlighting differences between them. In this study, a cellular model was created by inducing fat accumulation in MASLD. HepG2 cells were stimulated with the unsaturated fatty acid oleic acid at various concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) to emulate MASLD. The model's efficacy was assessed using cell counting kit-8 assays, Oil Red O staining, and lipid content analysis. This study aimed to create a simple-to-operate cellular model for MASLD cells. Results from the cell counting kit-8 assays showed that the survival of HepG2 cells was dependent on the concentration of oleic acid, with a GI₅₀ of 1.875 mM. Cell viability in the 0.5 mM and 1 mM groups were significantly lower than those in the control group (P < 0.05). Furthermore, Oil Red O staining and lipid content analysis examined fat deposition at varying oleic acid concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) on HepG2 cells. The lipid content of the 0.25 mM, 0.5 mM, and 1 mM groups was significantly higher than that of the control group (P < 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P < 0.05).

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a range of conditions, including simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma¹^,²^,³^,⁴^,⁵^,⁶, all attributed to factors other than alcohol consuption⁷. MASLD is the most prevalent liver disease caused by metabolic liver injury, affecting nearly one-quarter of the global population⁸^,⁹^,¹⁰^,¹¹^,¹². While the precise pathogenesis of MASLD has not yet been elucidated, various theories attempt to explain its development. One prevailing notion suggests a departure from the classic "two-hit" theory towards a "multiple-hit" model¹. Central to these hypotheses is the role of insulin resistance, which is believed to be pivotal in MASLD pathogenesis¹³. Research indicates that insulin resistance in hepatocytes leads to increased levels of free fatty acids, subsequently forming triglycerides stored within the liver¹⁴^,¹⁵.

Researchers have used both in vivo and in vitro models to simulate fat deposition in MASLD; yet fully replicating its pathomechanism remains challenging. Despite this limitation, these models have been instrumental in studying potential therapeutic targets for MASLD. However, the development of a stable model of MASLD is crucial. While animal models are effective, they are time-consuming and expensive, thus highlighting the growing interest in in vitro cellular models. These models often use single or multiple free fatty acids such as oleic acid (OA) and palmitic acid to recreate diet-induced MASLD. Among these, the human hepatoblastoma cell line HepG2 is often used to establish in vitro cellular models of MASLD.

OA induction stimulates HepG2 cells to replicate fatty deposition akin to MASLD, a method with a well-established history. The aim of this study was to demonstrate the viability, Oil Red O (ORO) staining, lipid content, and triglyceride (TG) level of HepG2 cells treated with 0.25 mM OA. The objective of this experiment was to provide further evidence for the development of MAFLD modeling studies.

Protocol

NOTE: See the Table of Materials for details related to all materials, instruments, and reagents used in this protocol. 1. Cell culture Culture HepG2 cells in culture flasks containing Dulbecco's Modified Eagle Medium (DMEM) (containing 10% fetal bovine serum [FBS], 100 units/mL penicillin, and 100 µg/mL streptomycin). Maintain the culture flasks at 37 °C in a 5% CO2 incubator. 2. Ef…

Representative Results

Effect of oleic acid on cell viability HepG2 cells were exposed to varying concentrations of OA (0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM), resulting in a decrease in cell survival rates at 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM compared to 0 mM. Statistical significance was observed at 0.5 mM (P < 0.05) and 1 mM (P < 0.05) when compared to 0 mM. The results of OA's impact on cell viability, as assessed by the CCK-8 kit, are shown in Fig…

Discussion

MASLD is a clinicopathological syndrome characterized by excessive intracellular fat deposition in hepatocytes due to factors beyond alcohol and other established liver-damaging agents¹⁸. MASLD is intricately linked to acquired metabolic stress liver injury, notably associated with insulin resistance and genetic susceptibility. To effectively study and screen drugs for MASLD, it is crucial to select an appropriate experimental model. Establishing a cell model is particularly vital in MASLD researc…

Disclosures

The authors have nothing to disclose.

Acknowledgements

The current study was granted by "Study on the key issues of curative effect of Koumiss on regional diseases of Mongolian medicine" in 2018 Supported Project of the science and technology program of the Department of Science and Technology of Inner Mongolia Autonomous Region.

Materials

0.22 µm filter	Millex
0.25% Trypsin-EDTA (1x) Trypsin-EDTA	Gibco	25200-056
0.45 µm filter	Millex
2 mL Crygenic Vials	CORNING	430659
25 cm² Cell Culture Flask	CORNING	430639
6-well cell culture plate	CORNING	3516
96-well cell culture plate	CORNING	3599
Blood Count Plate	Shanghai Jing Jing Biochemical Reagent & Instrument Co.	02270113
Cell Counting Kit-8 assays	Beijing Solarbio Science & Technology Co.,Ltd.	CA1210-1000T
CO₂ incubator	NUAIRE	NU-5710E
DMSO Dimethyl sulfoxide	Beijing Solarbio Science & Technology Co.,Ltd.	D8371
Dulbecco's Modified Eagle Medium	Gibco	8122691
Enzyme Labeling Equipment	Tecan	Spark
Fetal Bovine Serum, Qualified	Gibco	10099141
HepG2 cells line	Beijing North China Chuanglian Biotechnology Research Institute (BNCC)	221031
Human Triglyceride (TG) ELISA instruction	Nanjing Jiacheng Bioengineering Institute	20170301
Inverted Microscope for Cell Culture	Leica	DMi1
Isopropanol	Tianjin Zhiyuan Chemical Reagent Co.	2021030141
Oil Red Stain Kit, For Cultured Cells	Beijing Solarbio Science & Technology Co.,Ltd.	G1262
Oleic acid	Sangon Biotech (Shanghai) Co., Ltd.	A502071
Penicillin Streptomycin	Gibco	15140122
SPSS 24.0		Statistics software

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Bao, Q., Zhang, X., Chen, Y., Wang, T., Siqin, B. In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-associated Steatotic Liver Disease. J. Vis. Exp. (209), e66810, doi:10.3791/66810 (2024).