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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model.

Abstract

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a traditional ethnomedicine for the treatment of various diseases and is a typical functional food that can be incorporated into the everyday diet. Studies have suggested that platycodin D (PD), one of the main active ingredients in Platycodon grandiflorum, has high bioavailability and significantly mitigates the progress of NAFLD, but the underlying mechanism of this is still unclear. This study aims to investigate the therapeutic effect of PD against NAFLD in vitro. AML-12 cells were pretreated with 300 µM palmitic acid (PA) for 24 h to model NAFLD in vitro. Then, the cells were either treated with PD or received no PD treatment for 24 h. The levels of reactive oxygen species (ROS) were analyzed using 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining, and the mitochondrial membrane potential was determined by the JC-1 staining method. Moreover, the protein expression levels of LC3-II/LC3-I and p62/SQSTM1 in the cell lysates were analyzed by western blotting. PD was found to significantly decrease the ROS and mitochondrial membrane potential levels in the PA-treated group compared to the control group. Meanwhile, PD increased the LC3-II/LC3-I levels and decreased the p62/SQSTM1 levels in the PA-treated group compared to the control group. The results indicated that PD ameliorated NAFLD in vitro by reducing oxidative stress and stimulating autophagy. This in vitro model is a useful tool for studying the role of PD in NAFLD.

Introduction

Platycodon grandiflorus (PG), which is the dried root of Platycodon grandiflorus (Jacq.) A.DC., is used in traditional Chinese medicine (TCM). It is mainly produced in the northeast, north, east, central, and southwest regions of China1. The PG components include triterpenoid saponins, polysaccharides, flavonoids, polyphenols, polyethylene glycols, volatile oils, and minerals2. PG has a long history of being used as a food and a herbal medicine in Asia. Traditionally, this herb was used to make medicine against lung diseases. Modern pharmacology also provides evidence of the efficacy of PG for treating other diseases. Studies have shown that PG has a therapeutic effect on a variety of drug-induced liver injury models. The dietary supplementation of PG or platycodin extracts can ameliorate high-fat diet-induced obesity and its related metabolic diseases3,4,5. Polysaccharides from PG can be used for the treatment of acute liver injury caused by LPS/D-GalN in mice6. Moreover, saponins from the roots of PG ameliorate high-fat diet-induced non-alcoholic steatohepatitis (NASH)7. Furthermore, platycodin D (PD), one of the most important therapeutic components of PG, can enhance low-density lipoprotein receptor expression and low-density lipoprotein uptake in human hepatocellular carcinoma (HepG2) cells8. Furthermore, PD can also induce apoptosis and inhibit adhesion, migration, and invasion in HepG2 cells9,10. Thus, in this study, mouse hepatoma AML-12 cells are used for in vitro model construction and to further study the pharmacological effects and underlying mechanisms of PD in this model.

The term non-alcoholic fatty liver disease (NAFLD) refers to a group of liver diseases that includes simple steatosis, NASH, cirrhosis, and hepatocellular carcinoma11. Although the pathogenesis of NAFLD is incompletely understood, from the classic "two-hit" theory to the current "multiple-hit" theory, insulin resistance is considered to be central in the pathogenesis of NAFLD12,13,14. Studies have demonstrated that insulin resistance in hepatocytes could lead to increased free fatty acids, which form triglycerides that are deposited in the liver and cause the liver to become fatty15,16. The accumulation of fat can lead to lipotoxicity, oxidative stress-induced mitochondrial dysfunction, endoplasmic reticulum stress, and inflammatory cytokine release, resulting in the pathogenesis and progression of NAFLD17,18. In addition, autophagy also plays a role in the pathogenesis of NAFLD, as it is involved in regulating cellular insulin sensitivity, cellular lipid metabolism, hepatocyte injury, and innate immunity19,20,21.

A variety of animal models and cellular models have been established to provide a basis for exploring the pathogenesis and potential therapeutic targets of NAFLD22,23. However, single animal models cannot fully mimic all the pathological processes of NAFLD24. Individual differences between animals lead to different pathological features. Using liver cell lines or primary hepatocytes in in vitro studies of NAFLD ensures maximum consistency in the experimental conditions. Hepatic lipid metabolism dysregulation can lead to higher levels of hepatocyte lipid droplet accumulation in NAFLD25. Free fatty acids such as oleic acid and palm oil have been used in the in vitro model to mimic NAFLD caused by a high-fat diet26,27. The human hepatoblastoma cell line HepG2 is often used in the construction of NAFLD models in vitro, but, as a tumor cell line, the metabolism of HepG2 cells is significantly different from that of liver cells under normal physiological conditions28. Therefore, using primary hepatocytes or mouse primary hepatocytes to construct the in vitro NAFLD model for drug screening is more advantageous than using tumor cell lines. Comparing the synergistic examination of drug effects and therapeutic targets in both animal models and in vitro hepatocyte models, it seems that using mouse hepatocytes to construct the in vitro NAFLD model has better application potential.

Free fatty acids entering the liver are oxidized to produce energy or stored as triglycerides. Significantly, free fatty acids have a certain lipotoxicity and may induce cellular dysfunction and apoptosis12. Palmitic acid (PA) is the most abundant saturated fatty acid in human plasma29. When cells in non-adipose tissue are exposed to high concentrations of PA for a long time, this stimulates the production of reactive oxygen species (ROS) and causes oxidative stress, lipid accumulation, and even apoptosis30. Therefore, many researchers use PA as an inducer to stimulate liver cells to produce ROS and, thus, construct the in vitro fatty liver disease model and evaluate the protective effects of certain active substances on cells31,32,33,34. This study introduces a protocol for investigating the protective effects of PD on a cell model of NAFLD induced by PA.

Protocol

AML-12 cells (a normal mouse hepatocyte cell line) are used for the cell-based studies. The cells are obtained from a commercial source (see Table of Materials).

1. Pretreatment of the AML-12 cells to model NAFLD in vitro

  1. Maintain the cells in normal cell culture media (DMEM plus Ham's F12 [1:1] containing 0.005 mg/mL insulin, 5 ng/mL selenium, 0.005 mg/mL transferrin, 40 ng/mL dexamethasone, and 10% fetal bovine serum [FBS], see Table of Materials) at 37 °C in a humidified atmosphere with 5% CO2.
  2. Seed the cells in 12-well plates (1 mL/well) with a density of 2.8 x 105 cells/well.
    NOTE: All the cell digestion and medium exchange operations are performed in a biosafety cabinet to avoid contaminating the cells.
  3. Remove the culture medium of the cells after the overnight incubation (~10 h), and then wash the cells two times with 1 mL of serum-free media (per well).
  4. Divide the 12-well cell plate into four different treatment groups from left to right, including (1) the control group, (2) the PD-treated group, (3) the PA-treated group, and (4) the PA + PD-treated group.
    NOTE: Three replicates of each experimental treatment group are arranged from top to bottom on the same 12-well cell plate.
  5. Add the normal cell culture media (1 mL/well) to the control group and the PD-treated group, and add the normal cell culture media (1 mL/well) supplemented with 300 µM of PA to the PA-treated and PA + PD-treated group.
  6. Remove the culture medium of the cells after 24 h of incubation, and then wash the cells with 1 mL of serum-free media (per well) two times.
  7. Add normal cell culture media (1 mL/well) supplemented with a vehicle (dimethyl sulfoxide, DMSO, 0.1% v/v) to the control group; add normal cell culture media (1 mL/well) supplemented with 1 µM PD to the PD-treated group; add normal cell culture media (1 mL/well) supplemented with 300 µM PA to the PA-treated group; add normal cell culture media (1 mL/well) supplemented with 300 µM PA and 1 µM PD to the PA + PD-treated group.
  8. After incubation for an additional 24 h, investigate the protective effects of PD on cells using 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining (step 2), JC-1 staining (step 3), and western blotting (step 4).
    ​NOTE: All the incubation operations are done at 37 °C in a humidified atmosphere with 5% CO2.

2. Measurement of the change in ROS production

NOTE: The intracellular ROS levels in the cells are assessed based on the DCFH-DA staining assay.

  1. At the end of the treatment period (step 1.8), wash the cells with 1 mL of phosphate-buffered saline per well (1x PBS, pH 7.4) three times, and then stain the cells with 100 µL of 10 µM DCFH-DA per well (see Table of Materials). Incubate the cells in the dark for 30 min.
    NOTE: If no obvious green fluorescence is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately increased (30-60 min). If overexposure of the green fluorescence value is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately reduced (15-30 min).
  2. Wash the cells with 1x PBS (1 mL/well) three times. Add 1 mL of 1x PBS to each well.
  3. Place the 12-well plate on the microscope stage (see Table of Materials), and then use a 20x objective to observe the morphology of the cells (magnification: 200x).
  4. Capture three representative fluorescence images (magnification: 200x) for each well on the fluorescence microscope using the green fluorescent channel with an excitation wavelength of 480 nm and an emission wavelength of 530 nm.
    NOTE: The green fluorescent channel with an excitation wavelength of 480 nm and an emission wavelength of 530 nm is recommended to detect the DCFH-DA. Additionally, DCFH-DA can be detected with the parameter settings for GFP and FITC in the fluorescence microscope35,36.
  5. Finally, process the images with image-acquisition software (see Table of Materials), and then use ImageJ software to calculate the mean fluorescence intensity of each group or the ratios of the different groups.
    ​NOTE: The technical details of the fluorescence microscope and Image J software used for the imaging work have been described previously37,38.

3. Measurement of the change in mitochondrial membrane potential

NOTE: The changes in mitochondrial membrane potential are monitored by the JC-1 staining assay.

  1. At the end of the treatment period (step 1.8), wash the cells with 1 mL of 1x PBS per well three times, and then stain the cells with 100 µL of 5 µg/mL JC-1 working solution (see Table of Materials) for 30 min at 37 °C in the dark.
    NOTE: If no obvious green fluorescence is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately increased (30-60 min). If overexposure of the green fluorescence value is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately reduced (15-30 min).
  2. Wash the cells with 1x PBS (1 mL/well) three times. Add 1 mL of 1x PBS to each well.
  3. Place the 12-well plate on the microscope stage, and then use a 20x objective to observe the morphology of the cells (magnification: 200x).
  4. Capture three representative fluorescence images (magnification: 200x) for each well on the fluorescence microscope using the green fluorescent channel with excitation and emission wavelengths of 485 nm and 535 nm, respectively, and the red fluorescent channel with excitation and emission wavelengths of 550 nm and 600 nm, respectively.
    NOTE: The green fluorescent channel with an excitation wavelength of 485 nm and an emission wavelength of 535 nm is used to detect the JC-1 monomer, which is treated as depolarized mitochondria39,40,41, and the red fluorescent channel with an excitation wavelength of 550 nm and an emission wavelength of 600 nm is used to detect the JC-1 dimer, which is treated as polarized mitochondria39,40,41.
  5. Finally, process the images with image-acquisition software, and then use Image J software to calculate the mean fluorescence intensity of each group or the ratios of the different groups.
    ​NOTE: The technical details of the fluorescence microscope and Image J software used for the imaging work have been described previously37,38.

4. Measurement of the protein expression levels of LC3-II/LC3-I and p62/SQSTM1

  1. After treatment (step 1.8), wash the cells three times with 1x PBS (1 mL/well) that has been precooled at 4 °C.
  2. Add RIPA lysis buffer (100 µL/well) supplemented with protease and a phosphatase inhibitor cocktail (1x) (see Table of Materials) to the 12-well plate, and lyse on ice for 5 min.
  3. Collect the cell lysate into 1.5 mL microcentrifuge tubes, and centrifuge at 12,000 x g for 20 min at 4 °C. Determine the protein concentration of the supernatants by the BCA method following the standard procedures42.
  4. Add SDS-PAGE sample loading buffer (5x, see Table of Materials) to the cell lysate supernatants (volume ratio = 1:4).
  5. Mix by vortexing (at high speed for ~15 s), and heat the mixed samples at 100 °C for 5 min to denature the protein.
  6. Put the 12-well-prepared 12% SDS-PAGE gel into the electrophoresis tank, and then add the SDS up-sample buffer (1x) diluted with ultra-pure water to the height limit position.
    NOTE: The SDS-PAGE gel is prepared using a commercial kit (see Table of Materials) according to the manufacturer's instructions.
  7. Add the protein marker (5 µL/well) and samples (20 µg/well) into different wells of the SDS-PAGE gel.
  8. Set the stable voltage mode at 100 V, and perform the electrophoresis for 80 min.
  9. After the SDS-PAGE electrophoresis, carry out the electrotransfer of the proteins to a polyvinylidene fluoride (PVDF) membrane (0.45 µM, see Table of Materials) following previously published reports43,44.
  10. After the electrotransfer of the proteins, wash the PVDF membrane with 10 mL of TBST (1x TBS, 0.1% Tween 20) two times (5 min/time) in a shaker at room temperature.
  11. Block the PVDF membrane with 5 mL of bovine serum albumin (BSA, 5%) in a shaker at room temperature for 1 h.
  12. Wash the PVDF membrane with 10 mL of TBST three times (10 min/time). Then, incubate the PVDF membrane in 5 mL of the specific primary antibodies diluted in blocking buffer for LC3 (mouse mAb, 1:2,000), p62/SQSTM1 (hereafter referred to as p62, mouse mAb, 1:2,000), and β-actin (mouse mAb, 1:2,000) (see Table of Materials) overnight at 4 °C.
  13. Wash the PVDF membrane with 10 mL of TBST three times (10 min/time) at room temperature. Then, incubate the PVDF membrane with rabbit anti-mouse IgG (HRP) secondary antibody diluted in blocking buffer (1:10,000) (see Table of Materials) at room temperature and sheltered from light for 2 h.
  14. Wash the PVDF membrane with 10 mL of TBST three times (10 min/time) at room temperature. Then, place the PVDF membrane on the plastic wrap, add an appropriate amount of ECL working solution (200 µL/membrane) (see Table of Materials), and incubate for 2 min.
  15. After incubation, remove the ECL working solution, and expose the PVDF membrane in the imaging system for image development. Finally, use the Image J software to analyze the gray values of each band.
    ​NOTE: The technical details of the western blotting and Image J software used for the imaging work have been described previously45,46.

5. Statistical analysis

  1. Present the data from the experiments as the mean ± standard deviation (SD).
  2. Perform the analysis of significance with the statistical software tool described previously47.
  3. Calculate the statistical differences between the two groups using a t-test. A P-value below 0.05 is considered statistically significant: *P < 0.05, **P < 0.01, ***P < 0.005.

Results

Intracellular ROS in the cells
AML-12 cells were induced with 300 µM PA for 24 h, and a NAFLD cell model was established. Subsequently, the cells were treated with PD for 24 h. The cells were labeled with a DCFH-DA fluorescent probe, and ROS production was observed under a fluorescence microscope. The results of the DCFH-DA staining of intracellular ROS in the cells are shown in Figure 1. The results showed that PD could significantly reduce the level of intracell...

Discussion

Studies have highlighted the fact that NAFLD is a clinicopathological syndrome, ranging from fatty liver to NASH, that can progress to cirrhosis and liver cancer51. A high-fat diet and an inactive lifestyle are typical risk factors for NAFLD. Both non-drug therapies and drug therapies for NAFLD treatment have been researched51,52,53. However, the pathogenesis of NAFLD has not been fully elucidated. The me...

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This work is supported by grants from the Chongqing Science and Technology Commission (cstc2020jxjl-jbky10002, jbky20200026, cstc2021jscx-dxwtBX0013, and jbky20210029) and the China Postdoctoral Science Foundation (No. 2021MD703919).

Materials

NameCompanyCatalog NumberComments
5% BSA Blocking BufferSolarbio, Beijing, ChinaSW3015
AML12 (alpha mouse liver 12) cell lineProcell Life Science&Technology Co., Ltd, ChinaAML12
Beyo ECL PlusBeyotime, Shanghai, ChinaP0018S
Bio-safety cabinetEsco Micro Pte Ltd, SingaporeAC2-5S1 A2 
cellSensOlympus, Tokyo, Japan1.8
Culture CO2 IncubatorEsco Micro Pte Ltd, SingaporeCCL-170B-8
DexamethasoneBeyotime, Shanghai, ChinaST125
Dimethyl sulfoxideSolarbio, Beijing, ChinaD8371
DMEM/F12Hyclone, Logan, UT, USASH30023.01
Foetal Bovine SerumHyclone, Tauranga, New ZealandSH30406.05
Graphpad softwareGraphPad Software Inc., San Diego, CA, USA8.0
HRP Goat Anti-Mouse IgG (H+L)ABclonal, Wuhan, ChinaAS003
Hydrophobic PVDF Transfer MembraneMerck, Darmstadt, GermanyIPFL00010
Insulin, Transferrin, Selenium Solution, 100×Beyotime, Shanghai, ChinaC0341
MAP LC3β AntibodySanta Cruz Biotechnology (Shanghai) Co., LtdSC-376404
Mitochondrial Membrane Potential Assay Kit with JC-1Solarbio, Beijing, ChinaM8650
Olympus Inverted Microscope IX53Olympus, Tokyo, JapanIX53
Palmitic AcidSigma, GermanyP0500
Penicillin-Streptomycin Solution (100x)Hyclone, Logan, UT, USASV30010
Phenylmethanesulfonyl fluorideBeyotime, Shanghai, ChinaST506
Phosphate Buffered SolutionHyclone, Logan, UT, USABL302A
Platycodin DChengdu Must Bio-Technology Co., Ltd, ChinaCSA: 58479-68-8
Protease inhibitor cocktail for general use, 100xBeyotime, Shanghai, ChinaP1005
Protein MarkerSolarbio, Beijing, ChinaPR1910
Reactive Oxygen Species Assay KitSolarbio, Beijing, ChinaCA1410
RIPA Lysis BufferBeyotime, Shanghai, ChinaP0013E
SDS-PAGE Gel Quick Preparation KitBeyotime, Shanghai, ChinaP0012AC
SDS-PAGE Sample Loading Buffer, 5xBeyotime, Shanghai, ChinaP0015
Sigma CentrifugeSigma, Germany3K15
SQSTM1/p62 AntibodySanta Cruz Biotechnology (Shanghai) Co., LtdSC-28359
Tecan Infinite 200 PRO  Tecan Austria GmbH, Austria1510002987
WB Transfer Buffer,10xSolarbio, Beijing, ChinaD1060
β-Actin Mouse mAbABclonal, Wuhan, ChinaAC004

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