Metabonomics Study on the Mechanism of the Effect of Low Salt on

the Liver of Qinghai Lake Naked Carp

Yanfei Wu

1,2

, Jiang Qi Qu

, Yi Liu

, Yuxiang Cui

, Hongfang Qi

, Hong Zhang

, Yang Wang

and Qingjing Zhang

2,3,*

Zhejiang Ocean University, Zhoushan, 316022, China

Beijing Key Laboratory of fishery Biotechnology, Fisheries Research Institute, Beijing Academy of Agriculture and

Forestry Sciences, Beijing 100068, China

Qinghai Key Laboratory of Qinghai-Lake Naked Carps Breeding and Conservation, Rescue and Rehabilitation Center of

Naked Carps of Qinghai Lake, Xining 810016, China

Keywords: Low-Carbon Agriculture, Naked Carp Liver, Biology.

Abstract:

In order to explore the metabonomics study of the mechanism of low salt on the liver of Qinghai Lake

naked carp, this study investigated the liver enzyme activity, tissue structure and related immune genes

during the salinity change of Qinghai Lake naked carp. The role of naked carp in immunity can provide a

theoretical basis for the research on the adaptability of Qinghai Lake naked carp to changes in salinity. Two

experimental groups of Qinghai Lake naked carp with different salinities were established in the Emergency

Center (JH) and Qinghai Lake (QH). The rescue center is Freshwater, and the salinity of Qinghai Lake is

1.24‰. This experiment uses ultra-high performance liquid chromatography non-targeted metabonomics

technology, and the differential metabolites were screened according to the variable weight value (VIP) and

independent sample T test, and the KEGG pathway enrichment and annotation analysis were performed.

The results showed that compared with the JH group, in this study, 221 metabolites among the 1,525

differential metabolites identified received 90 KEGG annotations. We performed KEGG enrichment

analysis on the 65 differential proteins obtained, and the results showed that the differentially expressed

proteins mainly come from primary bile acid biosynthesis: M (Primary bile acid biosynthesis: M),

Parkinson's disease: (Parkinson disease: HD), Linoleic acid metabolism: M (Linoleic acid metabolism: M),

protein digestion and absorption: OS (Protein digestion and absorption: OS), cancer choline metabolism:

HD (Choline metabolism in cancer: HD) , This study clarified the metabonomics of the liver metabolism

mechanism of Qinghai Lake naked carp with different salinities, and the study of Qinghai Lake naked carp

liver function has a good guiding significance for molecular biology.

1 INTRODUCTION

Qinghai Lake naked carps belong to the genus

Cypriniformes, Cyprinidae, Schizoma subfamily,

and naked carps, commonly known as Huangyu. It

is the only commercial fish in Qinghai Lake and

occupies a very important position in the Qinghai

Lake ecosystem. (

CAO, WU, SHAO, 2010)

. Due to

its historical and natural reasons, Qinghai Lake

naked carp resources were once greatly destroyed.

In order to accelerate its resource recovery in

Qinghai Lake, the Qinghai Lake Naked Carp Rescue

Center has carried out artificial breeding and

breeding of Qinghai Lake naked carp resources in

the past 20 years, especially in recent years through

the establishment of a factory circulating water

breeding system to develop Qinghai Lake naked

carp Great progress has been made in breeding.

Qinghai Lake naked carp has the habit of

reproductive migration, has a strong adaptability to

changes in salinity, and can live in fresh water,

brackish water, and alkaline water (Walker, Dunn,

Edwards, Petr, Yang, 1995). Wang Ping et al.

(WANG, LAI, YAO, 2015) Metabonomics studies

and studies on the mechanism of the intestine of

Qinghai Lake naked carp under different salinity

environments have shown that there are gene

expression in many tissues such as Qinghai Lake

naked carp intestine and liver.

Salinity is one of the important factors affecting

the survival and growth of fish. Especially for

Wu, Y., Qu, J., Liu, Y., Cui, Y., Qi, H., Zhang, H., Wang, Y. and Zhang, Q.

Metabonomics Study on the Mechanism of the Effect of Low Salt on the Liver of Qinghai Lake Naked Carp.

DOI: 10.5220/0011196600003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 225-230

ISBN: 978-989-758-595-1

225

euryhalinic fish, the salinity changes in the water

environment in which they live will cause the

adaptation of enzyme activities, tissue structure and

gene expression in the fish. Sexual changes (Yong

Zhong); (Wang, 2018); (Zhang, Wen, Zhang, 2018),

studying the growth, survival, metabolism and other

physiological activities of fish under low-salt

conditions (Lian, 2012) can help understand the

anti-low-salt mechanism of fish and guide the

healthy breeding of fish. Although predecessors

performed correlation analyses on liver tissues at

different salinities to explain the differences in gene

transcription level expression of Qinghai Lake

naked carp, but due to the regulation of gene

expression and translation level, Qinghai Lake

naked carp cannot perform liver transcriptomics

research without saline environment. Fully explain

the types of products involved in the synthesis of

liver metabolites.

Non-targeted metabolomics can perform

qualitative and relative quantitative analysis of small

molecular metabolites in biological systems for

specific physiological conditions, and reflect the

total metabolite information to the greatest extent

(Wen, 2019); (Yang, 2020). Non-targeted

metabolomics can provide the maximum amount of

information about the metabolism of the central

carbon cycle, reflecting the body's metabolism after

being subjected to environmental stress through

biological processes such as gene expression,

transcription, post-transcriptional regulation, protein

translation and modification, etc. Changes in the

final product. In this part of the research, we used

the naked carp of Qinghai Lake (JH) and Qinghai

Lake (QH) as the research object, and used

non-targeted metabolomics to study the metabolic

changes of Qinghai Lake naked carp in response to

low-salt stress, hoping to reveal Qinghai Lake naked

carp. Carp provides metabolite data and basic

information on the regulation mechanism of low-salt

stress.

2 MATERIALS AND METHODS

2.1 Materials

The test materials were naked carps in the rescue

center (JH) and Qinghai Lake (QH) in September

2020. All naked carps were fresh and live fish

species identified by the staff of Qinghai Lake

Naked Carp Rescue Center. 10 samples of each were

collected. The collected naked carp was washed and

deplaned, and the liver was collected, and

immediately subjected to biological reaction

inactivation treatment (liquid nitrogen freezing), and

stored in a refrigerator at -80°C.

2.2 Sample Pretreatment

The pretreatment method of the liver tissue sample

was prepared according to the method of He et al.

(He, An, Huang, 2019). Accurately weigh 50 mg of

the liver tissue sample and transfer it to a 1.5 mL EP

tube; add 400 µl of extraction solution (methanol:

water=4:1) to the sample at low temperature ,

High-throughput tissue disrupter (-20°C, 50HZ,

6min); vortex (30s) to mix, then low-temperature

ultrasonic extraction for 30min (5°C, 40KHz); place

the sample at -20°C, 30min; centrifuge Centrifuge

the sample at 13000 rpm and 4°C for 15 minutes,

aspirate the supernatant, and transfer it to an LC-MS

vial for analysis. The quality control sample (QC) is

prepared by mixing equal volumes of all sample

extracts, each The QC volume is the same as the

sample. All extraction reagents are pre-cooled at -20

℃ before use.

2.3 LC-MS Detection

The instrument platform for this LC-MS analysis is

the UPLC-TripleTOF system of AB SCIEX. The

chromatographic conditions are: The

chromatographic column is a BEH C18 column (100

mm × 2.1 mm id, 1.7 µm; Waters, Milford, USA);

the mobile phase A is water (containing 0.1% formic

acid), and the mobile phase B is

acetonitrile/isopropanol (1/1) (containing 0.1%

formic acid); the mobile phase elution gradient

program is as follows: 0 min, 5% B; 3 min, 20% B;

9 min, 95% B; 13.0 min, 95% B; 13.1 min, 5% B;

16min, 5%B. The flow rate is 0.40mL/min, the

injection volume is 10μL, and the column

temperature is 40°C.

The sample mass spectrum signal acquisition

adopts positive and negative ion scanning mode and

ion spray voltage. The mass spectrometry

parameters are as follows: spray gas 50V; auxiliary

heating gas 50V; curtain gas 30V; ion source

heating temperature 500℃; ionization voltage

(positive) 5000V, ionization voltage (negative)

-4000V; interface heating on; declustering voltage

80V, The collision energy is 20-60(rolling)V.

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2.4 Quality Control

Quality control samples (QC) are prepared by

mixing the extracts of all samples in equal volumes.

The volume of each QC is the same as that of the

sample. It is processed and tested in the same way as

the analysis sample. In the process of instrument

analysis, every 10 analysis Insert a QC sample into

the sample to examine the repeatability of the entire

analysis process.

3 RESULTS AND ANALYSIS

3.1 Principal Component Analysis

Principal component analysis is used for pattern

recognition analysis of multivariate variables.

Principal component analysis (PCA) is a statistic

that converts a set of observed possibly related data

into linear uncorrelated data (ie principal

components) through orthogonal

transformationmethod. As an unsupervised analysis

method, it can automatically analyze the income

without default grouping arrangement. Model

construction of sample metabolism data, using a

small amount of principal components to reduce

data dimensions, represent data information, and

reveal The internal structure of the data. Among

them, 35.5% of the data in Figure 1aPCA

participated in the model construction; in the P-LCA

model established in Figure 1b, 35.3% of the data

participated in the model construction. The closer

R2Y and Q2 are to 1, the more stable and reliable

the model is. Generally, the model with Q2 greater

than 0.5 is stable and reliable. As shown in the table,

the tested model is reliable regardless of the cation

or anion mode. The metabolites identified on this

basis are Believed to be reliable.

a.PCAScore graph

b.PLS-DAScore graph

Figure 1: Principal component analysis diagram.

3.2 Screening of Differential

Metabolites

Obtain more accurate labeled compounds, and

further conduct biomarker mining and functional

analysis of compounds with complete secondary

information detected in the positive and negative ion

mode. The differential metabolite identification

standard used in this study was P<0.05 by paired

t-test, and it also satisfies log 2 Fold change<-1 or

log 2 Fold change>1. See the volcano map for the

screening results of differential metabolites (Figure

2). Each point in the volcano map represents a

metabolite, the abscissa represents the fold change of

each compound compared to the Qinghai Lake naked

carp and the rescue center control (take the logarithm

to the base 2), and the ordinate represents the P value

of the paired t test (Take the negative logarithm to

the base 10). The scattered colors represent the final

screening results. There are a total of 901 differential

metabolisms, of which 124 are up-regulated and 97

are down-regulated.

Figure 2: Volcano map of differential metabolites.

Metabonomics Study on the Mechanism of the Effect of Low Salt on the Liver of Qinghai Lake Naked Carp

227

3.3 KEGG Pathway Annotation and

Enrichment Analysis

In this study, out of the 1,525 differential

metabolites identified, 221 metabolites received 90

KEGG annotations. In order to further reveal the

overall pathway enrichment characteristics of all

differential metabolites, we performed an

enrichment analysis on the KEGG annotation results

of the differential metabolites. The KEGG

annotation analysis of the differential proteins is

shown in Figure 3a. The results showed that under

high altitude stress conditions, the liver metabolites

of Qinghai Lake naked carp were significantly

enriched in the sensory system, Nervous system,

Immune system, Endocrine system, and digestive

system. Digestive system, Nucleotide metabolism,

Metabolism of other amino acids, Metabolism of

cofactors and vitamins, Lipid metabolism, Energy

metabolism, carbohydrate metabolism (Biosynthesis

of other secondary metabolism), amino acid

metabolism (substance dependence),

neurodegenerative diseases, cancer: specific types,

cancer: overview, transport (Translation), signaling

molecules and interactions (Signaling molecules and

interaction), signal transduction, membrane

transport and catabolism.

a.KEGG annotation analysis of differentialproteins b.KEGG enrichment analysis of differential proteins

Figure 3: Annotation diagram of KEGG pathway enrichment of differential proteins.

We performed KEGG enrichment analysis on the

65 differential proteins obtained. The analysis

results are shown in Figure 3band table 1. The

results indicate that the differentially expressed

proteins mainly come from Primary bile acid

biosynthesis: M, Parkinson disease: HD, Linoleic

acid metabolism: M, Protein digestion and

absorption: OS, cancer choline metabolism: HD.

Table 1: Enrichment analysis of KEGG pathway in the liver of Qinghai Lake naked carp.

Metabolite Metab ID Formula

VIP_pred_OP

LS-DA

FC(JH/Q

P_valu

mode

Taurine metab_7226

C2H7NO

2.727 2.869 <0.001 neg

3a,7a,12a-Trihydroxy-5b-cholestan-26-al metab_106

C27H46O

7.024 0.127 0.0165 pos

Taurocholic acid metab_610

C26H45N

O7S

1.290 0.013 <0.001 pos

5b-Cyprinol sulfate metab_2325

C27H48O

7.908 0.110 0.025 pos

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9,10-DiHOME metab_6109

C18H34O

1.628 10.651 <0.001

neg

PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/1

8:2(9Z,12Z))

metab_9591

C48H80N

O8P

5.434 7.685 <0.001 neg

PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/2

2:6(4Z,7Z,10Z,13Z,16Z,19Z))

metab_9633

C52H80N

O8P

1.479 1.704 0.008 neg

9-OxoODE metab_2646

C18H30O

2.090 0.108 <0.001 pos

L-Isoleucine metab_5783

C6H13N

4.023 0.447 <0.001 pos

Piperidine metab_1437 C5H11N 1.843 0.422 <0.001 pos

L-Tyrosine metab_1454

C9H11N

1.047 0.528 <0.001 pos

4 DISCUSSION

Salinity is one of the important environmental

factors that affect the survival of fish. Changes in

salinity will cause changes in enzyme activities,

tissue structure and gene expression levels in fish.

The liver is an important metabolism-based organ in

the fish body. It plays a vital role in the synthesis

and catabolism of carbohydrates, fats and proteins.

Therefore, the strength of the liver can be used as

the ability of fish to resist external environmental

stress. An important indicator of size. We mainly

focus on Primary bile acid biosynthesis: M, Linoleic

acid metabolism: M, Protein digestion and

absorption: OS, several major The differential

metabolism of the KEGG pathway was analyzed.

The liver occupies an important position in the

metabolism of bile acids, which is closely related to

the synthesis, secretion, and conversion of bile

acids. The changes of bile acids are also known as

metabonomic markers of liver injury (Liu, Jiang,

Shen, 2016), the primary bile acid is directly

synthesized by hepatocytes using cholesterol as a

raw material. It is secreted from the liver and enters

the intestinal lumen, where it becomes a secondary

bile acid under the metabolism of some enzymes

and bacteria (MA, XIE, WANG, 2019). Li Xiulong

et al. (LI, HU, LI, 2020) found that the acute liver

injury induced by acetaminophen in rats may be

related to glycerophospholipid metabolism,

sphingolipid metabolism, and primary bile acid

biosynthesis and other metabolic pathways. related.

Jin Wenjie (Jin, Li, Ran, 2021) et al. conducted a

transcriptome analysis of copper toxicology in

naked carp, and the results showed that several

genes involved in oxidative stress in the gill and

liver were up-regulated. Up-regulation of these

genes indicates that copper treatment causes

oxidative stress, which may cause ribosome damage.

The metabolism of linoleic acid is the main

component of the cell membrane. The increase and

decrease of its content may be related to the necrosis

and apoptosis of hematopoietic stem cells. Liu Teng

et al. (LIU, XU, LU, 2020) studied the intervention

effect of Astragalus injection on leukopenia model

mice based on LC-MS metabonomics and found that

Astragalus injection can increase the white blood

cells, lymphocytes and neutral of leukopenia model

mice. Granulocyte and monocyte count; its effect of

increasing white blood cells may be related to the

metabolism of linoleic acid, the biosynthesis of

phenylalanine, tyrosine and tryptophan, and the

metabolism of phenylalanine. Protein digestion and

absorption: OSis a component of the animal body

and participates in various life activities of the

Qinghai Lake naked carp (Deng, 2020), Li et al. (Li,

Wang, Xu, 2015) used transcriptomics to study hens

before and after laying eggs In liver tissue, a large

number of differential genes were found to

participate in amino acid metabolism pathways.

5 CONCLUSION

In this paper, a non-targeted metabolomics study

was conducted on 30 cases of naked carp liver tissue

samples using LC-MS analysis method. The main

conclusions can be summarized as follows: (1)

Obtain the metabolite list and data matrix, combine

the T test and VIP(OPLS-DA) to screen out the

different metabolites, (2) Among the 1,525

differential metabolites identified, there are 221 90

KEGG annotations were obtained for metabolites

(3) The 65 differentially expressed proteins

identified were mainly derived from primary bile

acid biosynthesis: M, linoleic acid metabolism: M,

protein digestion and absorption: OS. In future

work, Progenesis QI (Waters Corporation, Milford,

USA) software is used for metabolite annotation,

data preprocessing, etc., to carry out metabonomics

research on the mechanism of low-salt impact on the

Metabonomics Study on the Mechanism of the Effect of Low Salt on the Liver of Qinghai Lake Naked Carp

229

liver of Qinghai Lake naked carp, and to improve

the use of pathway analysis, Advanced analysis,

such as association analysis and cluster analysis,

mine the biological information of differential

metabolism.

ACKNOWLEDGMENTS

This research was funded by funds from the

National Key R&D Program of China

(2020YFD0900103); Beijing Academy of

Agriculture and Forestry Sciences young scholar

fund (QNJJ202020); Beijing modern agricultural

industrial technology system project.

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