This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model.
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.
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.
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
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.
3. Measurement of the change in mitochondrial membrane potential
NOTE: The changes in mitochondrial membrane potential are monitored by the JC-1 staining assay.
4. Measurement of the protein expression levels of LC3-II/LC3-I and p62/SQSTM1
5. Statistical analysis
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 intracellular ROS in the cells incubated with 300 µM of PA (P < 0.01), indicating that PD can reduce cellular oxidative stress.
Mitochondrial membrane potential of the cells
The mitochondrion is the central organelle governed by the intrinsic apoptosis pathway48,49,50. Therefore, the mitochondrial membrane potential (MMP) of AML-12 cells that had been incubated with PD for 24 h was observed under a fluorescence microscope after JC-1 staining. As shown in Figure 2, the green fluorescence (JC-1 monomers, treated as depolarized mitochondria39,40,41) in the PA-treated cells was higher than in the untreated cells. On the other hand, the red fluorescence (JC-1 dimers, treated as polarized mitochondria39,40,41) in the PA-treated cells was lower than in the untreated cells, indicating PA-induced MMP depolarization in the cells. In addition, compared with the model group, the ratio of JC-1 monomers:dimers decreased in the PD treatment group (P < 0.01), indicating that PD could ameliorate PA-induced MMP depolarization.
The protein expression levels of LC3-II/LC3-I and p62
This study also investigated the potential role of PD on the autophagy pathway by investigating the protein expression levels of autophagy-related proteins LC3 and p62 in the cells by western blotting. As shown in Figure 3, the ratio of LC3-II:LC3-I significantly decreased (P < 0.005), and the protein expression level of p62 increased after being treated with PA for 48 h (P < 0.01). On the other hand, PD could significantly reduce the protein expression level of p62 (P < 0.01) and increase the ratio of LC3-II:LC3-I (P < 0.05), indicating that PD may restore the autophagic flux inhibited by PA.
Figure 1: Effects of PD on intracellular ROS in the cells as assessed by DCFH-DA staining. (A) ROS generation was detected by fluorescence microscopy, 200x. Scale bar: 20 µm. (B) Relative fold changes in the ROS levels in different groups. Each column represents mean ± SD (n = 3). **P < 0.01. Please click here to view a larger version of this figure.
Figure 2: Effects of PD on MMP depolarization in the cells as assessed by JC-1 staining. (A) MMP depolarization was detected by fluorescence microscopy, 200x. Scale bar: 20 µm. (B) Relative changes in the JC-1 monomers:dimers ratio in the different groups. Each column represents mean ± SD (n = 3). **P < 0.01. Please click here to view a larger version of this figure.
Figure 3: Effects of PD on the protein expression levels of LC3-II/LC3-I and p62 in the cells as assessed by western blotting. (A) The protein levels of LC3, p62, and β-actin were detected by western blotting. (B) Relative changes in the LC3-II:LC3-I ratio in the different groups. (C) Relative fold changes in the expression of p62 in the different groups. Each column represents mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. Please click here to view a larger version of this figure.
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 method described here involves an in vitro model of NAFLD established by PA-stimulated AML-12 cells. We investigated the protective effects of PD against NAFLD in this model, and we found that PD attenuated the PA-induced increase in ROS. Besides, PD also ameliorated PA-induced MMP depolarization. Furthermore, PD could significantly reduce the protein expression level of p62 and increase the ratio of LC3-II:LC3-I. These experimental results could provide a basis for the application of PD as an active pharmaceutical ingredient for NAFLD treatment.
As a major intracellular degradation pathway, autophagy realizes the physiological process of recycling and reusing nutrient raw materials by degrading excess components in cells19,54. Autophagy is also a key repair mechanism for the body to maintain "yin-yang balance", and it is the microscopic embodiment of "qi transformation" in TCM55,56,57. In the present protocol, in addition to detecting the changes in ROS production and MMP depolarization, the potential role of PD on the autophagy pathway was investigated by detecting the protein expression levels of autophagy-related proteins LC-3 and p62 by western blotting. After the treatment of PA-induced cells with PD, the ratio of LC3-II:LC3-I decreased, and the accumulation of p62 increased, indicating that the level of autophagy decreased. The experimental results are consistent with other literature reports20,34,58,59. This in vitro cell model may help improve the understanding of the biological function of PD and help study the use of other active TCM substances for the treatment of NAFLD.
There are some critical steps in the experimental process. To determine whether the in vitro NAFLD model is successfully established, the lipid droplet deposits can be compared in the normal control and PA-treated group cells by Oil Red O staining, as described previously60,61. After DCFH-DA staining or JC-1 staining, the remaining probes that do not enter the cells must be washed off; otherwise, this will cause a high background when taking the fluorescence images. In addition, the time (except for incubation time) between the loading and measurement of the fluorescent probes (DCFH-DA or JC-1) must be shortened to reduce the possibility of experimental errors. Moreover, the working concentrations of the fluorescent probes (DCFH-DA or JC-1) can be adjusted as needed if the fluorescence value of the cells in the negative control group is relatively high. Alternatively, the incubation time of the JC-1 working solution and the cells can be appropriately adjusted within a timeframe of 15-60 min according to the actual experimental conditions.
There are also some limitations in this in vitro cell model. Cells such as hepatic parenchymal cells, hepatic stellate cells, Kupffer cells, and vascular endothelial cells are present in liver tissues62. Therefore, experiments only on hepatic parenchymal cells may not be adequate to explain the effects of drugs. Other cell models also need to be used in anti-NAFLD drug discovery. In addition, three-dimensional (3D) cell culture systems have been used to construct liver models and test hepatotoxicity63,64. Future studies will focus on developing in vitro models using co-culture and 3D cell culture techniques for potential anti-NAFLD drug screening.
As a multi-system disease, many factors play a role in the development and progression of NAFLD, meaning a single animal model or cell model cannot fully mimic all the pathological processes of this disease65. Over-reliance on the use of animal models increases the burden of therapeutic drug development. Using in vitro cell models in the early stages of anti-NAFLD drug development is more practical and cost-effective. In this study, a normal mouse hepatocyte cell line AML-12 was used to construct the in vitro model of NAFLD, and the therapeutic effect of PD was validated in this model. Notably, the results of this study provide a basis for further research on the anti-NAFLD effects of PD in mice hepatocyte and primary hepatocyte models.
In conclusion, an in vitro model to study the protective effects of PD against NAFLD is presented here. This also could be a useful in vitro model for investigating the efficacy of other active substances of TCM for the treatment of NAFLD.
The authors have nothing to disclose.
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).
5% BSA Blocking Buffer | Solarbio, Beijing, China | SW3015 | |
AML12 (alpha mouse liver 12) cell line | Procell Life Science&Technology Co., Ltd, China | AML12 | |
Beyo ECL Plus | Beyotime, Shanghai, China | P0018S | |
Bio-safety cabinet | Esco Micro Pte Ltd, Singapore | AC2-5S1 A2 | |
cellSens | Olympus, Tokyo, Japan | 1.8 | |
Culture CO2 Incubator | Esco Micro Pte Ltd, Singapore | CCL-170B-8 | |
Dexamethasone | Beyotime, Shanghai, China | ST125 | |
Dimethyl sulfoxide | Solarbio, Beijing, China | D8371 | |
DMEM/F12 | Hyclone, Logan, UT, USA | SH30023.01 | |
Foetal Bovine Serum | Hyclone, Tauranga, New Zealand | SH30406.05 | |
Graphpad software | GraphPad Software Inc., San Diego, CA, USA | 8.0 | |
HRP Goat Anti-Mouse IgG (H+L) | ABclonal, Wuhan, China | AS003 | |
Hydrophobic PVDF Transfer Membrane | Merck, Darmstadt, Germany | IPFL00010 | |
Insulin, Transferrin, Selenium Solution, 100× | Beyotime, Shanghai, China | C0341 | |
MAP LC3β Antibody | Santa Cruz Biotechnology (Shanghai) Co., Ltd | SC-376404 | |
Mitochondrial Membrane Potential Assay Kit with JC-1 | Solarbio, Beijing, China | M8650 | |
Olympus Inverted Microscope IX53 | Olympus, Tokyo, Japan | IX53 | |
Palmitic Acid | Sigma, Germany | P0500 | |
Penicillin-Streptomycin Solution (100x) | Hyclone, Logan, UT, USA | SV30010 | |
Phenylmethanesulfonyl fluoride | Beyotime, Shanghai, China | ST506 | |
Phosphate Buffered Solution | Hyclone, Logan, UT, USA | BL302A | |
Platycodin D | Chengdu Must Bio-Technology Co., Ltd, China | CSA: 58479-68-8 | |
Protease inhibitor cocktail for general use, 100x | Beyotime, Shanghai, China | P1005 | |
Protein Marker | Solarbio, Beijing, China | PR1910 | |
Reactive Oxygen Species Assay Kit | Solarbio, Beijing, China | CA1410 | |
RIPA Lysis Buffer | Beyotime, Shanghai, China | P0013E | |
SDS-PAGE Gel Quick Preparation Kit | Beyotime, Shanghai, China | P0012AC | |
SDS-PAGE Sample Loading Buffer, 5x | Beyotime, Shanghai, China | P0015 | |
Sigma Centrifuge | Sigma, Germany | 3K15 | |
SQSTM1/p62 Antibody | Santa Cruz Biotechnology (Shanghai) Co., Ltd | SC-28359 | |
Tecan Infinite 200 PRO | Tecan Austria GmbH, Austria | 1510002987 | |
WB Transfer Buffer,10x | Solarbio, Beijing, China | D1060 | |
β-Actin Mouse mAb | ABclonal, Wuhan, China | AC004 |