Name: investigating the protective effects of platycodin d on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model
Uploaded: 2022-12-02T15:00:00
Description: Watch this Scientific Journal Video about Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model at JoVE.com

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).

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
<ol>
	<li>Maintain the cells in normal cell culture media (DMEM plus Ham&#39;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 <str...

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...

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 &#34;two-hit&#34; theory to the current &#34;multiple-hit&#34; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5% BSA Blocking Buffer</td>
			<td>Solarbio, Beijing, China</td>
			<td>SW3015</td>
			<td></td>
		</tr>
		<tr>
			<td>AML12 (alpha mouse liver 12) cell line</td>
			<td>Procell Life Science&#38;Technology Co., Ltd, China</td>
			<td>AML12</td>
			<td></td>
		</tr>
		<tr>
			<td>Beyo ECL&#160;Plus</td>
			<td>Beyotime, Shanghai, China</td>
			<td>P0018S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-safety cabinet</td>
			<td>Esco Micro Pte Ltd, Singapore</td>
			<td>AC2-5S1 A2&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cellSens</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>1.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture CO2 Incubator</td>
			<td>Esco Micro Pte Ltd, Singapore</td>
			<td>CCL-170B-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Beyotime, Shanghai, China</td>
			<td>ST125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Solarbio, Beijing, China</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Hyclone,&#160;Logan,&#160;UT,&#160;USA</td>
			<td>SH30023.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Hyclone, Tauranga, New Zealand</td>
			<td>SH30406.05</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad software</td>
			<td>GraphPad Software Inc., San Diego, CA, USA</td>
			<td>8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat Anti-Mouse IgG (H+L)</td>
			<td>ABclonal, Wuhan, China</td>
			<td>AS003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic PVDF Transfer Membrane</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium Solution, 100&#215;</td>
			<td>Beyotime, Shanghai, China</td>
			<td>C0341</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP LC3&#946; Antibody</td>
			<td>Santa Cruz Biotechnology (Shanghai) Co., Ltd</td>
			<td>SC-376404</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial Membrane Potential Assay Kit with JC-1</td>
			<td>Solarbio, Beijing, China</td>
			<td>M8650</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Inverted Microscope IX53</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>IX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Palmitic Acid</td>
			<td>Sigma, Germany</td>
			<td>P0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution (100x)</td>
			<td>Hyclone,&#160;Logan,&#160;UT,&#160;USA</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl&#160;fluoride</td>
			<td>Beyotime, Shanghai, China</td>
			<td>ST506</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution</td>
			<td>Hyclone,&#160;Logan,&#160;UT,&#160;USA</td>
			<td>BL302A</td>
			<td></td>
		</tr>
		<tr>
			<td>Platycodin D</td>
			<td>Chengdu Must Bio-Technology Co., Ltd, China</td>
			<td>CSA: 58479-68-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail for general use, 100x</td>
			<td>Beyotime, Shanghai, China</td>
			<td>P1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Marker</td>
			<td>Solarbio, Beijing, China</td>
			<td>PR1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive Oxygen Species Assay Kit</td>
			<td>Solarbio, Beijing, China</td>
			<td>CA1410</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA&#160;Lysis&#160;Buffer</td>
			<td>Beyotime, Shanghai, China</td>
			<td>P0013E</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE Gel Quick Preparation Kit</td>
			<td>Beyotime, Shanghai, China</td>
			<td>P0012AC</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE Sample Loading Buffer, 5x</td>
			<td>Beyotime, Shanghai, China</td>
			<td>P0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma Centrifuge</td>
			<td>Sigma, Germany</td>
			<td>3K15</td>
			<td></td>
		</tr>
		<tr>
			<td>SQSTM1/p62 Antibody</td>
			<td>Santa Cruz Biotechnology (Shanghai) Co., Ltd</td>
			<td>SC-28359</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Infinite 200 PRO&#160;&#160;</td>
			<td>Tecan Austria GmbH, Austria</td>
			<td>1510002987</td>
			<td></td>
		</tr>
		<tr>
			<td>WB Transfer Buffer,10x</td>
			<td>Solarbio, Beijing, China</td>
			<td>D1060</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Actin Mouse mAb</td>
			<td>ABclonal, Wuhan, China</td>
			<td>AC004</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating 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 &#181;M 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&#8242;,7&#8242;-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.

Intracellular ROS in the cells 
AML-12 cells were induced with 300 &#181;M 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...

Watch this Scientific Journal Video about Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model at JoVE.com

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

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

Investigating 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 ...

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.

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