This method demonstrates the use of AML-12 cells which will allow for model construction and investigating the protective effects of platycodin D on NAFLD. Over reliance on the use of animal models increases in the burden of therapeutic drug development. Using cell models in the early stage of NAFLD drug development is more practical and cost effective.
This TCM model may help improve the understanding of the biological function of platycodin D and help study the use of other TCM for the treatment of NAFLD. Begin by seeding one milliliter of AML-12 cells per well in 12-well plates. Then divide the 12-well plate into four different treatment groups and label them as control, PD treated, PA treated, and PA plus PD treated group.
Add one milliliter of the normal cell culture media per well to the control and the PD treated group. Add one milliliter of the normal cell culture media supplemented with 300 micromolar palmitic acid to the PA treated and PA plus PD treated group. After 24 hours of incubation, remove the culture medium of the cells and then wash the cells with one milliliter of serum-free media two times.
Next, add one milliliter of normal cell culture media supplemented with a vehicle to the control group and one milliliter of normal cell culture media supplemented with one micromolar platycodin D to the PD treated group. Add one milliliter of normal cell culture media supplemented with 300 micromolar palmitic acid to the PA treated group and one milliliter of normal cell culture media supplemented with 300 micromolar palmitic acid and one micromolar platycodin D to the PA plus PD treated group. After treating the cells for 24 hours, wash the cells with one milliliter of 1X PBS per well three times.
Then stain the cells with 100 microliters of 10 micromolar DCFH-DA per well. Incubate the cells in the dark for 30 minutes. After incubation, wash the cells with 1X PBS three times and add one milliliter of 1X PBS to each well.
Place the 12-well plate on the microscope stage and use a 20X objective to observe the morphology of the cells at 200X magnification. To measure the mitochondrial membrane potential, wash the treated cells prepared earlier with one milliliter of 1X PBS per well three times. Then stain the cells with 100 microliters of five micrograms per milliliter JC-1 working solution and incubate the cells for 30 minutes at 37 degrees Celsius in the dark.
Then wash the cells with 1X PBS three times and add one milliliter of 1X PBS to each well. Place the 12-well plate on the microscope stage and use a 20X objective to observe the morphology of the cells at 200X magnification. Lyse the treated cells and collect the cell lysate into 1.5 milliliter microcentrifuge tubes.
Centrifuge the tubes at 12, 000 G for 20 minutes at four degrees Celsius. Then add 5X SDS-PAGE sample loading buffer to the cell lysate supernatant at a volume ratio of one to four. Next, place the prepared 12%SDS-PAGE gel with 12 wells into the electrophoresis tank and add 1X SDS up sample buffer diluted with ultrapure water to the recommended height limit.
After SDS-PAGE electrophoresis, carry out the electro transfer of the proteins to a 0.45 micron PVDF membrane. Incubate the membrane in five milliliters of the primary antibodies diluted in blocking buffer. Use antibodies specific for LC-III, p62, and beta-actin.
Incubate overnight at four degrees Celsius. Then incubate the PVDF membrane with rabbit anti-mouse LGG HRP secondary antibody diluted one to 10, 000 in blocking buffer. Incubate the membrane at room temperature protected from light for two hours.
After incubation, place the PVDF membrane on a plastic wrap. Add an appropriate amount of ECL working solution and incubate for two minutes. Then remove the ECL working solution and expose the PVDF membrane in the imaging system for image development.
DCFH-DA staining showed that platycodin D could significantly reduce the level of intracellular ROS in the ALM-12 cells, indicating that this treatment can reduce cellular oxidative stress. Moreover, the green fluorescence representing JC-1 monomers or depolarized mitochondria was higher in the palmitic acid treated cells than in the untreated cells, whereas the red fluorescence representing the JC-1 dimers or polarized mitochondria was lower in the palmitic acid treated cells than the untreated cells, indicating palmitic acid-induced MMP depolarization. In addition, compared with the model group, the ratio of JC-1 monomers to dimers decreased in the platycodin D treatment group, indicating that it could ameliorate the palmitic acid-induced MMP.
Protein expression levels of autophagy-related proteins LC-III and p62 were investigated by western blotting. The ratio of LC-32 to LC-31 significantly decreased and the protein expression level of p62 increased after being treated with palmitic acid. On the other hand, platycodin D significantly reduced the protein expression level of p62 and increased the ratio of LC-32 to LC-31, indicating restoration of the autophagic flux induced by palmitic acid.
If the fluorescence value of the cells in the negative control group is relatively high, the working concentrations of the fluorescent probes can be adjusted as needed.
