Multimodal Assessment of Gentiopicroside's Protective Effect in CDCA-induced HepG2 Cell Injury

Summary

This study demonstrated that gentiopicroside can treat cholestatic liver injury by controlling the production and metabolism of bile acids and lipids and preventing oxidative stress and inflammatory responses.

Abstract

The primary active component of Swertia chirayita, gentiopicroside (GPS), has been shown to shield the liver in cholestasis instances. In order to elucidate the protective mechanism of GPS on cholestatic liver injury, HepG2 cells were treated with chenodeoxycholic acid (CDCA) to simulate the cholestasis environment in vitro, and the CCK-8 method was used to determine the protective effect of GPS on HepG2 cells. Using a biological kit, the contents of intracellular TBA, T-CHO, TG, ALP, ALT, AST, GSH, IL-1β, IL-6, TNF-α, SOD, and MDA were detected. Confocal laser microscopy was used to confirm the level of ROS. HepG2 cells' expressions of SHP-2, Tgr5, CYP7A1, and NTCP were assessed using immunofluorescence and Western blot analysis. Autodock software was then used to verify the molecular docking of its main active ingredients. The findings demonstrated that GPS significantly prevented the damage that CDCA caused to HepG2 cells. This protective effect may have been achieved by controlling the production and metabolism of bile acids and lipids within the cell and preventing oxidative stress and inflammatory responses.

Introduction

The body's digestive system's metabolic site is the liver. Bile secretion is a key mechanism in the hepatobiliary system's fat catabolism process. The primary constituents of bile, bile acids, are released by liver cells and are crucial for preserving the equilibrium of fat metabolism. For both healthy individuals and sick cases, the liver's physiological and pathological processes depend on the balance of free and conjugated bile acids in the liver. If this equilibrium is upset, bile acid circulation will not function normally, which can lead to intrahepatic cholestasis and subsequent liver damage¹. According to research already conducted, cholestatic liver injury (CLI) is a condition linked to metabolic disorders, intestinal microbiota imbalance, and liver cell necrosis². It is brought on by an obstruction of bile flow and the buildup of bile acids in the liver cells for CLI. Severe cases of CLI can progress to liver cirrhosis, liver fibrosis, and liver failure³. Currently, the mechanism underlying CLI is still somewhat unclear and complex, and the majority of patients receive low efficacy from medication therapy⁴ along with significant side effects⁵. As a result, one of the most pressing issues facing the medical community today is the search for effective CLI medications.

Current research has demonstrated the significance of a few nuclear receptors in the metabolism of bile acid⁶. Among these, the bile acid receptor is a crucial regulatory receptor of bile acid that may bind to BAs and activate protein tyrosine phosphatase (SHP)⁷. On the other hand, excessive accumulation of BAs in hepatocytes that results in cholestasis would negatively inhibit the SHP receptor and activate the CYP7A1 (cytochrome P450 7A1) receptor, which will lead to the production of bile acid⁸. Additionally, it blocked the G-protein-coupled bile acid receptor TGR5 from secreting BAs and stimulated the sodium taurocholate cotransporting polypeptide (NTCP) receptor to transport BAs. It causes bile acids to build up in liver cells, which damages the liver⁹. Consequently, additional research into this mechanism may shed light on more viable CLI treatment options.

With a long history of usage in medicine, Swertia chirayita is a Chinese traditional Tibetan medicine. It is regarded as the best Tida and is frequently used by the locals to treat gallbladder and liver conditions¹⁰. It is frequently used to treat gallstones, cholecystitis, cirrhosis, hepatitis, and other liver illnesses¹¹. Recent research has revealed that some iridoid chemical components of Swertia, as well as its active ingredients, have a good therapeutic effect on liver illnesses such as liver fibrosis and acute liver injury^12,13,14. The active ingredients of Swertia chirayita alleviate liver disease by lowering inflammation¹⁵ and its antioxidant properties¹⁶. Our preliminary research has demonstrated that the Tibetan medicine compound formula with Swertia chirayita as the main ingredient exhibits a favorable therapeutic effect on CLI, suggesting that Swertia chirayita holds potential as a treatment for CLI¹⁷. The primary active ingredient that is extracted and purified from Swertia chirayita is GPS (Gentiopicroside)¹⁸, which has a number of pharmacological actions, such as liver protection¹⁹, glucose metabolism regulation²⁰, gut microbiome improvement²¹, and anti-tumor properties²². Previous research has demonstrated that GPS ameliorates liver damage in cholestatic conditions and decreases intrahepatic cholestasis by upregulating the expression of bile acid transporters^23,24. Similar to this, research conducted both in vivo and in vitro has demonstrated that GPS can lessen liver damage by enhancing lipid metabolism, reducing inflammation, and reducing oxidative stress^25,26.

HepG2 cells are frequently utilized in in vitro studies of liver disease because they exhibit hepatocyte metabolic enzyme activity²⁷. In the meantime, hepatocytes can secrete more bile when exposed to one of the four forms of free bile acids, which include CDCA²⁸. As a result, CDCA can be employed to create a cell model of cholestatic liver injury and mimic the cholestatic environment in vitro. Given that CDCA-induced hepatocyte injury is critically mediated by oxidative stress and inflammatory pathways and considering the documented anti-oxidative and anti-inflammatory effects of GPS in other hepatic contexts²⁶, we hypothesize that GPS may confer protection against cholestatic liver injury by alleviating CDCA-induced cytotoxicity in HepG2 cells. This work investigated the protective effect of GPS on CLI and its molecular mechanism using a model of CDCA-induced damage to HepG2 cells.

Protocol

Cell culture and treatment
HepG2 cells and MEM media were purchased from a commercial vendor. HepG2 cells were grown in MEM media supplemented with 10% FBS, 1% 100 U/mL penicillin, and 100 U/mL streptomycin, at 37 °C and 5% CO₂ atmosphere. After cell adhesion, the culture media were changed. The cells were cultivated at a 1:3 ratio after 2-3 days, and they were utilized in the experiment once the cell culture achieved 80% confluency.

Cell viability assay
On day 1, cells were seeded at 2 x 10⁴ HepG2 cells (2 x 10⁵ cells/mL, 100 µL per well) in a 96-well plate in six replicates²⁹. On day 2, post 24 h of inoculation, the cells were treated with CDCA at different concentrations of CDCA (0, 20, 40, 60, 80, 100 µM) for 12 h. Cell viability was detected by the CCK-8 method. After 24 h of initial cell seeding, the cells were treated with CDCA at different concentrations of GPS (0, 25, 50, 100, 200, 400 and 800 µM) for 24 h. On day 3, cell viability was detected by the CCK-8 method. On day 4, 2 x 10⁴ HepG2 cells (2 x 10⁵ cells/mL, 100 µL per well) were seeded in a 96-well plate in six replicates for 24 h. On day 5, cells are separated into six groups: Control, Model, Ursodeoxycholic acid (UDCA), and GPS: 25 µM, 50 µM, and 100 µM. Excluding the control group, 60 µM CDCA solution was added to all groups for 12 h. Then UDCA solution (60 µM) and GPS solution (25 µM, 50 µM, and 100 µM) were added for 24 h, to UDCA and the 3 GPS groups, respectively. On day 6, cell viability was detected by the CCK-8 method.

Cytokine assay
According to the reported literature, TNF-α, IL-6, and IL-1β kits were used to detect the levels of inflammatory factors in HepG2 cells after different treatments³⁰. After 24 h of CDCA or CDCA+GPS intervention, discard the culture medium and rinse it with PBS. Centrifuge the cell suspension at 69 x g for 10 min, discard the supernatant and take the cell precipitate. Add PBS solution and homogenize under ice water bath conditions to prepare cell homogenates. After a 10 min centrifugation at 10,005 x g and 4 °C, the supernatant was removed, and the BCA technique was used to measure the total protein in the cell contents. According to the manufacturer's instructions, the levels of TNF-α, IL-6, and IL-1β in the cell supernatant were determined by ELISA. The absorbance was measured at 450 nm using a multifunctional microplate reader.

Detection of TBA, TG, and T-CHO
The TBA, TG, and T-CHO tests were utilized in accordance with the kit's instructions to identify HepG2 cells' bile acid and lipid metabolism³¹. TBA, TG, and T-CHO were purchased commercially. After the cell precipitates were broken up ultrasonically in an ice water bath, the cell homogenate was prepared by adding lysate RIPA for 30 min.Absorbance was measured at 450 nm using a multifunctional microplate reader.Following the addition of BCA working solutions to each well and the homogenization of the cells, the absorbance was measured at 562 nm using an enzymoleter, and the incubation period was 30 min The absorbance of the standard product was used to build the standard curve, and the following formula was used to get the absorbance value:
TBA content (µmol/gprot) = (A_sample-A_blank)/(A_standard-A_blank)×C_calibration/Cpr
T-CHO, TG content (mmol/gprot) = (A_sample-A_blank)/(A_standard-A_blank)×C_standard/Cpr

Evaluation of liver function
Liver function was assessed using ALT, AST, and ALP. ALT, AST, and ALP were purchased commercially. Following two rounds of PBS buffer cleaning on the collected cells, the cells were centrifuged for 10 min at 69 x g, the supernatant was removed, the cells were precipitated, PBS buffer was added, and the homogenate was created by ultrasonic crushing in an ice water bath. The test and control holes were then set. The operation table was followed while adding the reagent, shaking and mixing the hole plate gently, and letting it sit at room temperature for 15 min. Each hole's OD value was determined using an enzymoleter with a 505 nm wavelength. The absolute OD value was then entered into the standard curve and additional quantitative analysis was carried out using the formula.

Determination of SOD, MDA, GSH activities
SOD activity, MDA content, and GSH content were assessed in accordance with the manufacturer's instructions, respectively. SOD, MDA, and GSH were purchased commercially. To put it briefly, a six-well plate was used to inoculate the cells. Following treatment, the cells underwent pretreatment (collection and breaking) in accordance with the kit's experimental instructions. The appropriate working solutions were then poured to the cell detection plate after blank holes, standard holes, and measuring holes had been set up in accordance with the operation steps of the instructions. Subsequently, the cells are cultured at the temperature and duration mentioned in the kit instructions. A BCA detection kit was then used to quantify the protein content. Ultimately, the absorbance was determined at the appropriate wavelength, and a more thorough quantitative analysis of the oxidative stress index was conducted.

Detection of changes in ROS in HepG2 cells using DCFH-DA probe
Routine culture for cell counts was carried out.HepG2 Cells with a density of 1 x 10⁵ cells/mL were inoculated into glass-bottom dishes at 1.5 mL each, and cultured according to the above method. After culture, the medium was discarded and the cells were washed with PBS. The DCFH-DA was diluted to a concentration of 1:1000, added at 1.5 mL diluent per well, and incubated for 20 min. Following incubation, the cells were rinsed 3x with MEM basic culture solution. Then, 100 µL of antifade mounting medium with DAPI was added to each dish. A fluorescence confocal microscope was used to view and take pictures at 488 nm for excitation and 525 nm for emission³².

Immunofluorescence imaging
The cells were washed 3x with PBS buffer following incubation. After being fixed for 30 min with 4% paraformaldehyde, the cells were cleaned using PBS buffer on a shaking table. The diluent was added, then refrigerated at 4 °C away from light for overnight. After that, the cells were coated with fluorescent diluent and incubated away from light for 1 h at 37 °C. Following incubation, cells were washed three times with PBS buffer. After fixation with 4% paraformaldehyde for 30 min, cells were rinsed with PBS buffer on a rotary shaker. Subsequently, 150 µL of primary antibody diluent was added and incubated overnight at 4 °C under light-protected conditions. The primary antibody diluent was recovered, followed by the addition of an adequate volume of PBS buffer and shaking for 5 min; this washing procedure was repeated three times. Each dish then received 150 µL of fluorescent secondary antibody diluent and was incubated at 37 °C for 1 h in a light-protected incubator. An appropriate volume of PBS buffer was added to each dish and washed on a rotary shaker for 8 min, repeated for three cycles. Finally, cells were covered with sufficient DAPI staining solution for 10 min, mounted with antifade mounting medium, and imaged using a super-resolution imaging system³³.

Western blot analysis
After varying treatments, the whole protein of HepG2 cells was extracted using RIPA lysate, and the protein concentration was ascertained using the BCA technique. After isolating the target protein using 10% SDS-PAGE, it was transferred to a 0.45 µm PVDF membrane. After that, the membrane was sealed for 1.5 h at 25 °C using 5% BSA. Primary antibodies against SHP-2 (1:1000), CYP7A1 (1:1000), Tgr5 (1:1000), NTCP (1:1000), and β-actin (1:1000) were incubated overnight at 4 °C. Next, the membrane was incubated with goat anti-rabbit immunoglobulin G secondary antibody (1:10000) conjugated with horseradish peroxidase for 2 h at 25 °C. The peroxidase-coated bands were then seen using an ultrahigh sensitivity ECL chemiluminescent solution, and pictures were taken using an imaging system³⁴.

Molecular docking
As reported, the molecular docking process was carried out according to the following procedures³⁵. The drug small molecule.mol2 structure was downloaded from ChemicalBook platform, and the ligand protein .sdf structure was downloaded from Uniprot and RCSBPDB platforms. The ligand and water molecules were removed by Pymol software, and the ligand protein was imported into AutoDock software for hydrogenation and detection. The final output is in .pdbqt format. The docking box, RunAutoGrid, RunAutoDock and AutoDock Tools analysis were performed in AutoDock software, and the minimum binding energy of each group of docking results was saved. The conformation with the lowest free binding energy of each group was selected, and its ligand-receptor conformation was visualized by Pymol software.

Statistical analysis
The aforementioned data were statistically sorted using GraphPad Prism 9.2, and all experimental results were reported as Mean ± SD. In order to statistically compare the data between groups, One-way ANOVA was employed. The difference between the two groups was deemed significant whenp < 0.05.

Results

The concentration of CDCA was set to 0 µM, 20 µM, 40 µM, 60 µM, 80 µM, and 100 µM concentration gradient for experiments. When the concentration of CDCA was 60 µM, the cell activity was significantly different from that of the Control group, and finally 60 µM was used as the concentration of CDCA (Figure 1A). The results showed that after incubation for 24 h after drug administration, there was no significant difference in cell activity between each group and the Control group (

Discussion

CLI is a liver disease caused by the obstruction of bile formation, secretion, or excretion. It is clinically manifested as pruritus, osteoporosis, and even develops into liver fibrosis, cirrhosis, and other diseases, which seriously endanger life and health^36,37,38. UDCA is recognized as the most commonly used drug in the treatment of CLI³⁹. However, due to the large number of patients with poor response...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ALP	Nanjing Jiancheng Bioengineering Institute	A059-2-2
ALT	Nanjing Jiancheng Bioengineering Institute	C009-2-1
AST	Nanjing Jiancheng Bioengineering Institute	C010-2-1
BCA	Boster Biological Technology Co., Ltd	AR0146
CDCA	Chengdu Medesheng Technology Co., Ltd	474-25-9
cell counting kit-8	Boster Biological Technology Co., Ltd	AR1160-500
Cytochrome P450 7A1 Antibody	Abmart Shanghai Co.,Ltd.	TD2612S
DAPI	Beyotime Biotechnology	C1005
DMSO	Boster Biological Technology Co., Ltd	PYG0040
Goat Anti-Rabbit IgG (H&L)	Chengdu Zen-Bioscience Co., Ltd.	511202
Goat Anti-Rabbit IgG AF488	Abmart Shanghai Co.,Ltd.	M21012M
GPS	Chengdu Medesheng Technology Co., Ltd	RP210717
GSH	Nanjing Jiancheng Bioengineering Institute	A006-2-1
HepG2 cells	Procell Life Science & Technology Co., Ltd	CL-0103
HRP Goat Anti-Rabbit IgG	Wuhan ABclonal Biotechnology Co., Ltd.	AS014
IL-1β	Elabscience Biotechnology Co., Ltd	E-EL-H0149c
IL-6	Elabscience Biotechnology Co., Ltd	E-EL-H6156
MDA	Nanjing Jiancheng Bioengineering Institute	A003-1-2
MEM base medium	Procell Life Science & Technology Co., Ltd	PM150410
PBS	Boster Biological Technology Co., Ltd	PYG0021
Rapid Transfer Buffer (20×)	NCM Biotech	WB4600
RIPA	Boster Biological Technology Co., Ltd	BL651A
ROS	Beyotime Biotechnology	S0033S
SHP2 Rabbit pAb	Wuhan ABclonal Biotechnology Co., Ltd.	A12486
SLC10A1 Antibody	Abmart Shanghai Co.,Ltd.	PU276166S
SOD	Nanjing Jiancheng Bioengineering Institute	A001-3-1
TBA	Nanjing Jiancheng Bioengineering Institute	E003-2-1
T-CHO	Nanjing Jiancheng Bioengineering Institute	A111-1-1
TG	Nanjing Jiancheng Bioengineering Institute	A110-1-1
TGR5 Polyclonal Antibody	ImmunoWay Biotechnology Company	YT4636
TNF-α	Elabscience Biotechnology Co., Ltd	E-EL-H0109c
Trypsin solution (0.25%)	Yeasen Biotechnology (Shanghai) Co., Ltd	40126ES60
UDCA	Chengdu Lemetian Medical Technology Co., Ltd	DSTDX007601
β-Actin Rabbit mAb	Wuhan ABclonal Biotechnology Co., Ltd.	AC026