The current study was granted by &#34;Study on the key issues of curative effect of Koumiss on regional diseases of Mongolian medicine&#34; in 2018 Supported Project of the science and technology program of the Department of Science and Technology of Inner Mongolia Autonomous Region.

NOTE: See the Table of Materials for details related to all materials, instruments, and reagents used in this protocol.
1. Cell culture
<ol>
	<li>Culture HepG2 cells in culture flasks containing Dulbecco&#39;s Modified Eagle Medium (DMEM) (containing 10% fetal bovine serum [FBS], 100 units/mL penicillin, and 100 &#181;g/mL streptomycin). Maintain the culture flasks at 37 &#176;C in a 5% CO2 incubator.</li>
</ol>
2. Effect of oleic acid on cell viability as measured by cell counting kit-8
<ol>
	<li>Dissolve a specific volume of OA in dimethyl sulfoxide (DMSO) to achieve a concentration of 200 mM. Store the solution at -20 &#176;C for future use.</li>
	<li>Seed HepG2 cells in a 96-well plate at a cell density of 6 &#215; 103 cells per well. Add 100 &#181;L of DMEM to each well. Incubate and culture the cells at 37 &#176;C in a 5% CO2 incubator for 24 h. Divide the HepG2 cells into two groups:
	<ol>
		<li>Control group: add cell culture medium.</li>
		<li>OA group: add OA to the cell culture medium to achieve final concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM.</li>
	</ol>
	</li>
	<li>After 24 h of initial incubation, discard the supernatant from each well and add OA according to the specified grouping, adding 100 &#181;L per well. For the control group, add 100 &#181;L of cell culture medium to each well. Continue to incubate the cells for another 24 h. 
	NOTE: Ensure each group has six replicate wells. To prevent evaporation, add 100 &#181;L of phosphate-buffered saline (PBS) to the outer ring of wells in the 96-well plate.</li>
	<li>After 24 h of incubation, add 10 &#181;L of cell counting kit-8 (CCK-8) to each well, mix gently, and incubate for 2 h in the dark. Remove the 96-well plate from the incubator, place it in the microplate reader, and measure the absorbance value at 450 nm (A450). The GI50 values were counted according to the OD.</li>
</ol>
3. Oil Red O staining to observe intracellular lipid droplet formation
<ol>
	<li>Seed HepG2 cells in 6-well cell culture plates at a density of 5 &#215; 105 cells per well and culture the plates in a constant-temperature incubator for 24 h. Refer to Figure 1 for a visual representation of the described steps.</li>
	<li>After 24 h of cell culture, add 2 mL of cell culture medium containing OA to each well, achieving final concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM. After an additional 24 h, remove the cell culture medium from each well and wash twice with PBS. Add 1 mL of ORO fixative to each well and incubate for 30 min.</li>
	<li>Prepare the ORO staining solution by mixing staining solution A with staining solution B at a ratio of 3:2. Allow the mixture to stand at room temperature for 10 min, then filter it once through a 0.45 &#181;m filter. Store the filtered solution in a centrifuge tube protected from light until use.</li>
	<li>Discard the fixative and wash twice with distilled water. Add 1 mL of 60% isopropanol to each well and incubate for 30 s. Discard 60% of the isopropanol solution and add 1 mL of freshly prepared ORO stain solution to each well before incubating for 20 min. Discard the ORO staining solution, add 1 mL of 60% isopropanol to each well, and incubate for 30 s. Wash 5x with water to remove excess dye.</li>
	<li>Cover the cells with distilled water and observe under a microscope. Once the images are collected, discard the liquid in the plate and allow it to dry. Then, add 2 mL of isopropanol to each well and shake the plate on an orbital shaker for 10 min. Transfer the liquid to a new 96-well plate, with 16 wells in each group, adding 100 &#181;L per well. Calculate the lipid content by measuring the optical density (OD) of each well using a microplate reader at 510 nm (A510).</li>
</ol>
4. Effects of different concentrations of oleic acid on total triglyceride in HepG2 cell supernatant
<ol>
	<li>Equilibrate the kit at room temperature for 20 min and prepare the required plates for the experiment.</li>
	<li>Collect the cell supernatant and centrifuge at 1,570 &#215; g for 10 min. Set standard wells and testing sample wells. Add 50 &#181;L of standard ([S0 &#8594; S5] concentration followed by: 0, 0.5, 1, 2, 4, 8 mmol/L) to standard wells. In addition to the blank and standard wells, add 10 &#181;L of different samples to the sample wells, followed by adding 40 &#181;L of sample diluent to each well. Add 100 &#181;L of detection antibody-horseradish peroxidase to each well, seal with a plate membrane, and incubate at 37 &#176;C for 1 h in a constant temperature oven.</li>
	<li>Discard the supernatant, blot dry on dust-free paper, and wash each well with 1x washing solution. Leave to stand at room temperature for 1 min. Repeat the washing process 5x.</li>
	<li>Add 50 &#181;L of substrate A and 50 &#181;L of substrate B to each well. Mix gently and incubate for 15 min at 37 &#176;C. Add 50 &#181;L of termination solution to each well and measure the OD value of each well at 450 nm (A450) within 15 min.</li>
	<li>Plot the concentration of the standard along the x-axis and the corresponding absorbance (OD) value along the y-axis to perform linear regression and derive the curve equation to calculate the concentration value of each sample.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Determine significant differences in quantitative data.</li>
	<li>Calculate the mean &#177; standard deviation (SD) and graphically represent the data. Consider P &#60; 0.05 to be statistically significant.</li>
</ol>

Oleic Acid-Induced HepG2 Cells for Metabolic Disease Analysis 

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The authors declare no conflicts of interest.

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...

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 &#34;two-hit&#34; theory towards a &#34;multiple-hit&#34; 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.

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	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
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			<td>0.25% Trypsin-EDTA (1x) Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
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			<td>0.45 &#181;m filter</td>
			<td>Millex</td>
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			<td>2 mL Crygenic Vials</td>
			<td>CORNING</td>
			<td>430659</td>
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			<td>25 cm2 Cell Culture Flask</td>
			<td>CORNING</td>
			<td>430639</td>
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			<td>6-well cell culture plate</td>
			<td>CORNING</td>
			<td>3516</td>
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			<td>96-well cell culture plate</td>
			<td>CORNING</td>
			<td>3599</td>
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			<td>Blood Count Plate</td>
			<td>Shanghai Jing Jing Biochemical Reagent &#38; Instrument Co.</td>
			<td>02270113</td>
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			<td>Cell Counting Kit-8 assays</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>CA1210-1000T</td>
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			<td>CO2 incubator</td>
			<td>NUAIRE</td>
			<td>NU-5710E</td>
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			<td>&#160;DMSO Dimethyl sulfoxide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>D8371</td>
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			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>8122691</td>
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			<td>Enzyme Labeling Equipment</td>
			<td>Tecan</td>
			<td>Spark</td>
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			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10099141</td>
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			<td>HepG2 cells line</td>
			<td>Beijing North China Chuanglian Biotechnology Research Institute (BNCC)</td>
			<td>221031</td>
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		<tr>
			<td>Human Triglyceride (TG) ELISA instruction</td>
			<td>Nanjing Jiacheng Bioengineering Institute</td>
			<td>20170301</td>
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		<tr>
			<td>Inverted Microscope for Cell Culture</td>
			<td>Leica</td>
			<td>DMi1&#160;</td>
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		<tr>
			<td>Isopropanol</td>
			<td>Tianjin Zhiyuan Chemical Reagent Co.</td>
			<td>2021030141</td>
			<td></td>
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		<tr>
			<td>Oil Red Stain Kit, For Cultured Cells</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>G1262</td>
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		<tr>
			<td>Oleic acid&#160;</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A502071</td>
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		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
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		<tr>
			<td>SPSS 24.0</td>
			<td></td>
			<td>Statistics software</td>
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	</tbody>
</table>

in vitro modeling of fat deposition in metabolic dysfunction-associated steatotic liver disease

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&#39;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 &#60; 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 &#60; 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P &#60; 0.05).

Author Spotlight: Establishing MASLD Cell Models for Investigating Disease Mechanisms and the Lipid-Lowering Effects of Koumiss

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

Our research direction is the study of traditional Mongolian medicine by establishing a metabolic dysfunction-associated steatotic liver disease cell model. The role and the mechanism of koumiss therapy are studied. The findings of the presence study will prove advantageous in the future, enabling the establishment of metabolic dysfunction-associated steatotic liver disease cell models and the investigation of the disease underlying mechanisms.A future study will be conducted based on the metabolic-dysfunction associated steatotic liver disease cell model to investigate the mechanism of action of the lipid-lowering effect of koumiss.

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 &#60; 0.05) and 1 mM (P &#60; 0.05) when compared to 0 mM. The results of OA&#39;s impact on cell viability, as assessed by the CCK-8 kit, are shown in Fig...

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

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

in-vitro-modeling-fat-deposition-metabolic-dysfunction-associated

Research

JoVE Journal

Biology

617 Views.  Inner Mongolia Medical University. La nostra direzione di ricerca è lo studio della medicina tradizionale mongola stabilendo un modello cellulare di malattia epatica steatosica associata a disfunzione metabolica. Vengono studiati il ruolo e il meccanismo della terapia koumiss. I risultati dello studio di presenza si riveleranno vantaggiosi in futuro, consentendo la creazione di modelli cellulari di malattia steatosica epatica associata a disfunzione metabolica e lo studio dei meccanismi sotto...

Video: Autore in primo piano: Stabilire modelli cellulari MASLD per studiare i meccanismi della malattia e gli effetti ipolipemizzanti di Koumiss

Assessing the Effect of Oleic Acid on HepG2 Cell Viability Using Cell Counting Kit-8

To begin seed overnight-grown culture of HepG2 in 100 microliters of DMEM in each well of a 96-well plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day discard the supernatant and add 100 microliters of oleic acid per well according to the specified grouping.For the control group, add 100 microliters of DMEM to each well. Incubate the cells at 37 degrees Celsius for 24 hours. After incubation, add 10 microliters of cell counting reagent to each well, mix gently, and incubate for two hours in the dark.Place the plate in the microplate reader and measure the absorbance at 450 nanometers. Calculate the GI50 values of HepG2 cells according to the optical density. Compared to the control, oleic acid decreased HepG2 cell survival rates at concentrations of 0.125, 0.25, 0.5, and one millimolar, with statistical significance observed at 0.5 and one millimolar.

Oil Red O Staining for Visualizing Intracellular Lipid Droplet Accumulation in HepG2 Cells

To begin seed overnight grown culture of Hep G2 in DMEM in a six well plate. Culture the cells at 37 degrees Celsius for 24 hours. After incubation, add two milliliters of cell culture medium containing oleic acid to each well.Incubate the cells for 24 hours at 37 degrees Celsius. The next day, remove the culture medium and wash the cells twice with PBS. Add one milliliter of oil red O fixative to each well and incubate for 30 minutes.Discard the fixative and wash the cells twice with distilled water. Add one milliliter of 60%isopropanol to each well and incubate for 30 seconds. After discarding isopropanol, add one milliliter of freshly prepared oil red O stain solution and incubate for 20 minutes.Now, add one milliliter of 60%isopropanol and incubate for 30 seconds. Wash the cells five times with water to remove excess dye. After microscopic imaging, observe the cells covered with distilled water on a computer.After imaging, remove the water from the plate and allow it to dry. Add two milliliters of isopropanol to each well and shake the plate on an orbital shaker for 10 minutes. Transfer 100 microliters of cell suspension to a 96 well plate with 16 wells in each group.On a microplate reader at 510 nanometers, measure the optical density to calculate the lipid content. Compared to the control cells, Hep G2 cells treated with oleic acid showed increased red staining intensity, indicating higher lipid droplet formation. The lipid droplets and lipids in Hep G2 cells increased with rising oleic acid concentrations.

Effect of Oleic Acid Concentrations on Total Triglyceride Levels in HepG2 Cell Supernatant

To begin, equilibrate the total triglyceride kit at room temperature for 20 minutes. Collect the supernatant of overnight grown Hep G2 cells and centrifuge at 1, 570 G for 10 minutes. Set the standard and test sample wells.Add 50 microliters of the oleic acid to the standard labeled wells. In addition to the blank and standard wells, add 10 microliters of cell culture to the sample wells. Then add 40 microliters of sample diluent to each well.Now add 100 microliters of detection antibody, horse radish peroxidase to each well. Seal with a plate membrane and incubate at 37 degrees Celsius in a constant temperature oven. After one hour, discard the supernatant and blot dry on dust-free paper.Add washing solution to each well and incubate at room temperature for one minute. Now, add 50 microliters of substrate A and 50 microliters of substrate B to each well. Mix gently and incubate for 15 minutes at 37 degrees Celsius.Add 50 microliters of termination solution, and within 15 minutes, measure the optical density at 450 nanometers. Plot the concentration of the standard along the x axis and the corresponding absorbance value along the Y axis. Perform linear regression and derive the curve equation to calculate the concentration value of each sample.Compared to the control group, treatment of Hep G2 cells with varying concentrations of oleic acid resulted in a significant increase in the total triglycerides content of the cell supernatant.