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 alread....

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 ....

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