Protective Effect of Gegen Hawthorn Ginseng Granules on Alcoholic

Liver Injury in Mice

Yang Li

, Zhenglong Li

, Liying Chang

, Shujing Zheng

, Maoying Yan

and Shumin Wang

College of Pharmacy, Changchun University of Chinese Medicine, Changchun, Jilin, China

wangsm@ccucm.edu.cn

Keywords: Gegen Hawthorn Ginseng Granules, Alcoholic Liver Injury, Oxidative Stress, Inflammation, Mechanism of

Action.

Abstract: Gegen hawthorn ginseng granules (GHGG) is composed of 6 Chinese medicinal materials including

pueraria lobata, hawthorn and ginseng. This study aims to explore the protective effect of GHGG on

alcoholic liver injury, and the correlation between oxidative stress, lipid metabolism, inflammation and

alcoholic liver disease. The mice were randomly divided into control group and model group, diammonium

glycyrrhizinate positive drug group, and GHGG low and high dose groups (GHGG-L and GHGG-H). Oral

alcohol was used to establish an acute alcoholic liver injury model. Determination of liver function index

ALT and AST levels after administration; Determination of superoxide dismutase (SOD), malondialdehyde

(MDA) and reduced glutathione in liver tissue (GSH), TNF-α, IL-1β, IL-6 and other biochemical indicators;

HE staining to analyze the morphological changes of tissue sections. Experimental results show that GHGG

can significantly reduce the levels of ALT, AST, and MDA in mice; increase the level of liver tissue The

activity of SOD and GSH, and GHGG can significantly inhibit the levels of biochemical indicators such as

pro-inflammatory factors TNF-α, IL-1β, IL-6. The degree of pathological changes in the liver tissue of mice

in the GHGG group was significantly reduced. In summary, GHGG can improve ALD by anti-oxidation,

inhibiting inflammation, and promoting liver cell regeneration.

1 INTRODUCTION

In recent years, the global incidence of alcoholic liver

disease (ALD) has been increasing year by year, and

it has become a major disease that seriously

endangers human health. The main clinical

symptoms are hepatitis and liver fibrosis, and severe

cases can lead to liver cirrhosis and liver cancer

(Kong 2019). Treatment is mainly through drug

intervention and liver transplantation (Bloom Patricia

2021). So far, there have been many researches and

developments of alcoholic liver disease drugs at

home and abroad, but they have not yet been able to

meet the clinical needs. Its pathogenesis is complex

https://orcid.org/0000-0003-4579-939X

https://orcid.org/0000-0003-4352-3023

https://orcid.org/0000-0001-7719-8367

https://orcid.org/0000-0003-3509-8362

https://orcid.org/0000-0001-7243-1342

https://orcid.org/0000-0002-0730-4475

and diverse, among which oxidative stress, abnormal

lipid metabolism, disturbance of intestinal flora, and

inflammation are currently considered to be the main

causes of ALD (Kong 2019, Natalia 2017,

Woodhouse 2018). In this experiment, by studying

the effects of GHGG on the antioxidant enzyme

activity, liver function index levels in mice with

acute alcoholic liver injury and detecting the level of

related inflammation in mice with acute alcoholic

liver injury, the GHGG can relieve alcohol The liver-

protecting effect and its mechanism of action are

preliminarily discussed to provide experimental

evidence for its clinical application.

146

Li, Y., Li, Z., Chang, L., Zheng, S., Yan, M. and Wang, S.

Protective Effect of Gegen Hawthorn Ginseng Granules on Alcoholic Liver Injury in Mice.

DOI: 10.5220/0011192300003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 146-151

ISBN: 978-989-758-595-1

2 MATERIALS AND METHODS

2.1 Materials

2.1.1 Medicines and Reagents

GHGG (Homemade in the laboratory); diammonium

glycyrrhizinate (China Zhengda Tianqing

Pharmaceutical Group Co., Ltd.); alanine

aminotransferase (ALT), aspartate aminotransferase

(AST), malondialdehyde (MDA), Superoxide

dismutase (SOD), reduced glutathione (GSH)

detection kit (Nanjing Jiancheng Institute of

Biological Engineering). Tumor Necrosis Factor

(TNF-α), Interleukin-6 (IL-6), IL-1β (Jiangsu

Enzyme Industry Co., Ltd.); BCA protein

concentration determination kit (Shanghai Biyuntian

Biotechnology Limited company).

2.1.2 Animals

50 SPF KM mice, provided by (Liaoning

Changsheng Biotechnology Co., Ltd., production

license number: SCXK (Liao) 2020-0001; certificate

number: 210726200100461748). The animals are

maintained under a 12h/12h light/dark cycle at

25±3℃ and a relative humidity of 50±20%. This

study was conducted in accordance with the

Declaration of Helsinki, and the protocol has been

approved by the Animal Health and Welfare

Committee of Changchun University of Chinese

Medicine (20190123). The procedures involving

animals and their care comply with the institutional

guidelines of national and international laws and

policies.

2.2 Methods

2.2.1 Design of Animal Experiment

Fifty mice were randomly divided into 5 groups,

namely control group, model group, diammonium

glycyrrhizinate positive drug group (150 mg/kg bw)

(Zhang 2020) and GHGG low and high dose groups

(800, 2000mg/kg bw), each group has 10 animals.

Gavage is given once a day for 10 consecutive days.

Four hours after the last administration, except the

control group, the mice in each group were given

56% ethanol (12mL/kg bw) to establish an acute

alcoholic liver injury model. The blank control

group mice were given an equal volume of distilled

water.

2.2.2 Measurement of Liver Index

The liver of the mice was aseptically removed, the

final body mass and organ mass of the mice were

weighed, and the organ coefficient was calculated

according to the ratio of the organ mass to the body

mass.And weighed to measured organ index

according to thefollowing formula:

Organ index (%) = organ mass/final body

mass × 100%

(1)

2.2.3 Determination of Biochemical

Indicators of Liver Function

Blood was collected from the eyeballs of mice. After

clotting at 4℃ for 1 hour, the blood was centrifuged

at 4000 r/min for 15 minutes. The supernatant was

collected as serum. The ALT and AST levels were

determined according to the kit instructions.

2.2.4 Determination of Oxidative Stress

Indicators

Take the liver, place it in a glass tissue homogenizer,

add appropriate amount of physiological saline,

prepare a 10% (m:V) tissue homogenate, centrifuge

at 4000r/min for 15 minutes, collect the supernatant,

and determine SOD, MDA and GSH according to

the kit instructions level.

2.2.5 Determination of Hepatic

Proinflammatory Cytokines

Take the liver, place it in a glass tissue homogenizer,

add an appropriate amount of physiological saline,

prepare a 10% (m:V) tissue homogenate, centrifuge

at 4000r/min for 15 minutes, collect the supernatant,

and according to the kit instructions to determine the

levels of IL-6, IL-1β and TNF-α.

2.2.6 Histological Investigation of Liver

Samples of liver were separated from each mouse

and fixed in formalin solution (10%) for 24 h, and

after that dehydrated using graded alcohol and

xylene, and implanted in paraffin. Paraffin-

embedded sections, stained with hematoxylin and

eosin (H&E) for histological investigation.

2.2.7 Statistical Analysis

The data of the animal experiment was evaluated by

using Graph Pad Prism version 5 (La Jolla, CA,

USA). One-way analysis of variance (ANOVA) was

used to compare variations between groups,

Protective Effect of Gegen Hawthorn Ginseng Granules on Alcoholic Liver Injury in Mice

147

followed by Duncan’s multiple range test.

Differences between groups were found statistically

significant at p < 0.01 or p < 0.05 and the data was

expressed as mean ± SD.

3 RESULTS

3.1 Effect of GHGG on Body Weight

and Organ Index in ALD Mice

The food intake of each group of mice was recorded,

and the calculation formula was intake g/day. The

results in Table 1 show that compared with the

normal group, the food utilization rate of the model

group was significantly reduced (p<0.05), and the

feed intake of GHGG-H was significantly improved

(P<0.05). The liver index results are shown in Table

1. The liver index of the model group was

significantly higher than that of the control group

(p<0.05). However, compared with the model group,

giving different doses of GHGG (GHGG-L and

GHGG-H) can reduce liver swelling. There was no

significant difference between GHGG-L and

GHGG-H groups (p>0.05).

Table 1: Body weight and organ index in ALD mice.

Control Model Positive drug GHGG-L GHGG-H

Food intake

g/day

6.30±0.25 4.94±0.25

5.67±044

5.24±0.18 5.47±0.40

Liver index

4.01±0.27 6.07±0.33

4.60±0.17

4.31±0.43

4.46±0.53

<0.05 represents compared with the control group, and p

<0.05represents compared with the model group.

3.2 Effects of GHGG on Liver

Function Indexes of ALD Mice

AST and ALT are biochemical markers for clinical

evaluation of liver function. When liver function is

impaired, the levels of AST and ALT increase

sharply compared with normal physiological

conditions (Katrine 2021, Sun 2021). Figure 1-2

shows the effect of GHGG on serum AST and ALT

in ALD mice. The levels of AST and ALT in the

model group were significantly higher than those in

the blank group (P<0.05), indicating that the liver

was damaged after a large amount of alcohol intake,

which led to a significant increase in serum ALT and

AST. GHGG in different dose groups can

significantly reduce the serum content level and

achieve the effect of protecting the liver.

Figure 1: AST levels in serum of ALD mice.

Figure 2: ALT levels in serum of ALD mice.

3.3 Effects of GHGG on Antioxidant

Capacity of Liver Tissue in ALD

Mice

Oxidative stress is one of the main mechanisms of

the pathogenesis of alcoholic liver disease, and it has

an important impact on the initial liver fibrosis and

hepatocellular carcinoma (Zhang 2018, NWFLALD

2018, Xia 2017). MDA, SOD and GSH play an

important role in protecting alcohol-induced liver

damage and oxidative stress. Figure 3-5 shows the

effect of GHGG on liver antioxidant enzymes MDA,

SOD and GSH. Compared with the control group,

the liver MDA level of the model group was

significantly increased (p<0.05). Compared with the

model group, the MDA content of the GHGG

treatment group (GHGG-L and GHGG-H) was

significantly reduced (p<0.05). In addition,

compared with the control group, the liver SOD and

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

148

GSH levels in the model group were significantly

lower (p<0.05), and the GHGG administration group

(GHGG-L and GHGG-H) could increase their

levels. The experimental results show that GHGG

can reduce the content of MDA and increase the

activity of antioxidant enzymes SOD and GSH to

protect the liver in mice with alcoholic liver injury.

Figure 3: MDA levels in tissues of ALD mice.

Figure 4: SOD levels in tissues of ALD mice.

Figure 5: GSH levels in tissues of ALD mice.

3.4 Effects of GHGG on the Level of

Pro-inflammatory Cytokines in the

Liver of ALD Mice

The results of pro-inflammatory cytokines such as

IL-6, IL-1β and TNF-α in mouse liver tissue are

shown in Figure 6-8. Compared with the blank

group, the liver TNF-α, IL-1β and IL-6 of the mice

in the model group all increased significantly

(p<0.05). Compared with the model group, the

GHGG administration group (GHGG-L and GHGG-

H) can significantly reduce the levels of TNF-α, IL-

1β and IL-6 (p<0.05). The above results indicate that

GHGG can protect liver cells by reducing the

occurrence of alcohol-induced liver inflammation.

Figure 6: TNF-α levels in tissues of ALD mice.

Figure 7: IL-1β levels in tissues of ALD mice.

Figure 8: IL-6 levels in tissues of ALD mice.

3.5 Effects of GHGG on

Histopathological Characteristics of

Liver in ALD Mice

The H&E staining method was used to study the

pathological changes of liver tissue in ALD mice. As

shown in Figure 9, the structure of the liver lobules

in the control group was complete and clear, the

liver cells were arranged in an orderly manner, and

the nuclei were complete and clear. The liver tissue

sections of mice in the model group showed obvious

inflammatory cell infiltration, fatty vacuoles and

hepatocyte enlargement. Compared with the model

group, the GHGG administration group (GHGG-L

and GHGG-H) had less edema, fatty vacuoles and

inflammatory cell infiltration.

Protective Effect of Gegen Hawthorn Ginseng Granules on Alcoholic Liver Injury in Mice

149

Figure 9: Liver tissue slices of mice in the control group

Figure 10: Liver tissue slices of mice in the model group

Figure 11: Liver tissue slices of mice in the positive drug

group

Figure 12: Liver tissue slices of mice in the GHGG-L

group

Figure 13: Liver tissue slices of mice in the GHGG-H

group

4 DISCUSSION

The liver is one of the main organs for alcohol

metabolism and the largest gland in the human body.

It has very important physiological functions (Li

2019). When the human body consumes excessive

alcohol, a large amount of metabolic waste cannot

be excreted in time, which will cause degeneration

and necrosis of liver cells, which will cause

disorders of related metabolic pathways in the cells,

cause liver cells to produce inflammation and

oxidative stress, fibrosis and lead to liver lipids. and

its can induce liver cell damage (Xie 2021, Tu 2019,

TESCHKE 2018). For oxidative stress, excessive

alcohol intake leads to the weakening of SOD's

antioxidant capacity, and the body's oxidation and

antioxidant balance is disrupted, causing oxidative

stress in the body, leading to a chain reaction of lipid

peroxidation, and damage to mitochondrial function.

Liver damage caused by endoplasmic reticulum

stress and immune inflammatory response (Zhang

2021). MDA is an important product of lipid

peroxidation, and its content can reflect the degree

of lipid peroxidation in the body (Rani 2016), GSH

is an important antioxidant substance in the body. It

is a substrate of two enzymes, GSH-Px and GSH-

ST. It is a low-molecular scavenger that can remove

O2, H2O2, so the level of GSH content is a measure

of the body’s antioxidant capacity (Wu 2021). The

balance of pro-inflammatory cytokines and anti-

inflammatory cell levels is essential for maintaining

human health (Sun 2021). In this experiment, we

tested the levels of three inflammatory cytokines,

TNF-α, IL-1β and IL-6. TNF-α is significantly

increased during alcoholic liver injury compared to

normal physiological levels (Shen 2018). It can be

activated by binding to caspase3 to induce apoptosis

of hepatocytes (Dalia 2019). At the same time, it

also activates the NF-κB signaling pathway, triggers

the secretion of inflammatory factors such as IL-1β

or IL-6, further aggravates liver inflammation and

induces the occurrence of alcoholic liver disease

(Liu 2017).

5 CONCLUSIONS

In summary, GHGG can effectively reduce liver

lipid peroxidation levels by inhibiting the increase of

liver function indicators in mice, regulate liver

metabolic disorders, and improve the antioxidant

capacity of mice, thus playing a protective role on

alcohol-induced acute liver injury in mice. This

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

150

research lays an experimental foundation for the in-

depth development of drugs for the treatment of

alcoholic liver injury, provides a basis for clinical

medication, and also broadens the use of medicinal

materials for both food and medicine.

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