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 Table of Materials) at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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 &#181;M of PA to the PA-treated and PA + PD-treated group.</li>
	<li>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.</li>
	<li>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 &#181;M PD to the PD-treated group; add normal cell culture media (1 mL/well) supplemented with 300 &#181;M PA to the PA-treated group; add normal cell culture media (1 mL/well) supplemented with 300 &#181;M PA and 1 &#181;M PD to the PA + PD-treated group.</li>
	<li>After incubation for an additional 24 h, investigate the protective effects of PD on cells using 2&#8242;,7&#8242;-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining (step 2), JC-1 staining (step 3), and western blotting (step 4). 
	&#8203;NOTE: All the incubation operations are done at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
</ol>
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.
<ol>
	<li>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 &#181;L of 10 &#181;M 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).</li>
	<li>Wash the cells with 1x PBS (1 mL/well) three times. Add 1 mL of 1x PBS to each well.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: The technical details of the fluorescence microscope and Image J software used for the imaging work have been described previously37,38.</li>
</ol>
3. Measurement of the change in mitochondrial membrane potential
NOTE: The changes in mitochondrial membrane potential are monitored by the JC-1 staining assay.
<ol>
	<li>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 &#181;L of 5 &#181;g/mL JC-1 working solution (see Table of Materials) for 30 min at 37 &#176;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).</li>
	<li>Wash the cells with 1x PBS (1 mL/well) three times. Add 1 mL of 1x PBS to each well.</li>
	<li>Place the 12-well plate on the microscope stage, and then use a 20x objective to observe the morphology of the cells (magnification: 200x).</li>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: The technical details of the fluorescence microscope and Image J software used for the imaging work have been described previously37,38.</li>
</ol>
4. Measurement of the protein expression levels of LC3-II/LC3-I and p62/SQSTM1
<ol>
	<li>After treatment (step 1.8), wash the cells three times with 1x PBS (1 mL/well) that has been precooled at 4 &#176;C.</li>
	<li>Add RIPA lysis buffer (100 &#181;L/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.</li>
	<li>Collect the cell lysate into 1.5 mL microcentrifuge tubes, and centrifuge at 12,000 x g for 20 min at 4 &#176;C. Determine the protein concentration of the supernatants by the BCA method following the standard procedures42.</li>
	<li>Add SDS-PAGE sample loading buffer (5x, see Table of Materials) to the cell lysate supernatants (volume ratio = 1:4).</li>
	<li>Mix by vortexing (at high speed for ~15 s), and heat the mixed samples at 100 &#176;C for 5 min to denature the protein.</li>
	<li>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&#39;s instructions.</li>
	<li>Add the protein marker (5 &#181;L/well) and samples (20 &#181;g/well) into different wells of the SDS-PAGE gel.</li>
	<li>Set the stable voltage mode at 100 V, and perform the electrophoresis for 80 min.</li>
	<li>After the SDS-PAGE electrophoresis, carry out the electrotransfer of the proteins to a polyvinylidene fluoride (PVDF) membrane (0.45 &#181;M, see Table of Materials) following previously published reports43,44.</li>
	<li>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.</li>
	<li>Block the PVDF membrane with 5 mL of bovine serum albumin (BSA, 5%) in a shaker at room temperature for 1 h.</li>
	<li>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 &#946;-actin (mouse mAb, 1:2,000) (see Table of Materials) overnight at 4 &#176;C.</li>
	<li>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.</li>
	<li>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 &#181;L/membrane) (see Table of Materials), and incubate for 2 min.</li>
	<li>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. 
	&#8203;NOTE: The technical details of the western blotting and Image J software used for the imaging work have been described previously45,46.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Present the data from the experiments as the mean &#177; standard deviation (SD).</li>
	<li>Perform the analysis of significance with the statistical software tool described previously47.</li>
	<li>Calculate the statistical differences between the two groups using a t-test. A P-value below 0.05 is considered statistically significant: *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.005.</li>
</ol>

The authors declare no conflicts of interest.

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 &#34;yin-yang balance&#34;, and it is the microscopic embodiment of &#34;qi transformation&#34; 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.

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 intracellular ROS in the cells incubated with 300 &#181;M of PA (P &#60; 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 &#60; 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 &#60; 0.005), and the protein expression level of p62 increased after being treated with PA for 48 h (P &#60; 0.01). On the other hand, PD could significantly reduce the protein expression level of p62 (P &#60; 0.01) and increase the ratio of LC3-II:LC3-I (P &#60; 0.05), indicating that PD may restore the autophagic flux inhibited by PA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64816/64816fig01.jpg" /> 
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 &#181;m. (B) Relative fold changes in the ROS levels in different groups. Each column represents mean &#177; SD (n = 3). **P &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64816/64816fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64816/64816fig02.jpg" /> 
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 &#181;m. (B) Relative changes in the JC-1 monomers:dimers ratio in the different groups. Each column represents mean &#177; SD (n = 3). **P &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64816/64816fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64816/64816fig03.jpg" /> 
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 &#946;-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 &#177; SD (n = 3). *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64816/64816fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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JoVE Journal

Medicine

1.8K Views.  Chongqing Academy of Chinese Materia Medica. This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model.

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

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

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

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Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

In Vitro Model of Coronary Angiogenesis

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology