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 common therapy are studied. The findings of the presence study will improve advantages in the future and 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 lead lowering effect of Crohn's. 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 region 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 GI`50 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 1 millimolar with statistical significance observed at 0.5 and 1 millimolar. To begin, seed overnight grown culture of HepG2 in DMEM in a 6-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, HepG2 cells treated with oleic acid showed increased red staining intensity indicating higher lipid droplet formation.

The lipid droplets and lipids in HepG2 cells increased with rising oleic acid concentrations. To begin, equilibrate the total triglyceride kit at room temperature for 20 minutes. Collect the supernatant of overnight grown HepG2 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 Horseradish 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 HepG2 cells with varying concentrations of oleic acid resulted in a significant increase in the total triglycerides content of the cell supernatant.