Comparative Transcriptomics Reveals the Role of the Toll-Like

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Omics Technologies Applied to Agriculture and Food

Comparative transcriptomics reveals the role of the Toll-like receptor signaling pathway in fluoride-induced cardiotoxicity tong wang, xiao yan yan, Nisha Dong, Xianhui Hao, Yangang Xing, Xiaolin Tian, Jing Feng, Jiaxin Xie, Yi Lv, Cailing Wei, Yi Gao, and Yulan Qiu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00312 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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Journal of Agricultural and Food Chemistry

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Comparative transcriptomics reveals the role of the Toll-like receptor signaling

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pathway in fluoride-induced cardiotoxicity

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Author names and affiliations

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Xiaoyan Yan†, Nisha Dong†, Xianhui Hao‡, Yangang Xing§, Xiaolin Tian#, Jing Feng#,

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Jiaxin Xie†, Yi Lv†, Cailing Wei†, Yi Gao†, Yulan Qiu†, Tong Wang*†.

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†

School of Public Health, Shanxi Medical University, Shanxi 030001, China

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‡

Medical Faculty, Kunming University of Science and Technology, Kunming 650000,

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China

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§

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Shanxi 030200, China

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#

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Shanxi Medical University, Taiyuan, Shanxi 030001, China

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Corresponding author

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Tong Wang*

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*

Gujiao City Animal Husbandry and Veterinary Technical Service Center, Taiyuan,

Shanxi Key Laboratory of Experimental Animal and Human Disease Animal Models,

Tel: 0351-4135397; Fax: 86-0351-4135397. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Many studies have shown that fluorosis due to long-term fluoride intake, has

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damaging effects on the heart. However, the mechanisms underlying cardiac fluorosis

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have not been illuminated in detail. We performed High-throughput transcriptome

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sequencing (RNA-Seq) on rat cardiac tissue to explore the molecular effects of NaF

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exposure. In total, 372 and 254 differentially expressed genes (DEGs) were identified

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between a group given 30 mg/L NaF and control, and between a group given 90 mg/L

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NaF and control, respectively. The transcript levels of most of these genes were

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significantly downregulated and many were distributed in the Toll-like receptor

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signaling pathway. Transcriptome analysis revealed that herpes simplex infection,

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ECM-receptor interaction, influenza A, cytokine-cytokine receptor interaction,

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Apoptosis and Toll-like receptor signaling pathway were significantly affected. IL-6

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and IL-10 may play a crucial role in the cardiac damage caused by NaF as external

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stimuli according to protein-protein interaction (PPI) network analysis. The results of

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qRT-PCR and western blotting showed markedly decreased mRNA and protein levels

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of IL-1, IL-6, and IL-10 in the low concentration fluoride (LF) and high concentration

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fluoride (HF) groups, which was in agreement with RNA-Seq results. This is the first

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study to investigate NaF-induced cardiotoxicity at a transcriptome level.

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Key words: fluoride; cardiac injury; inflammatory injury; RNA-seq; Toll-like pathway

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INTRODUCTION Fluorine, a highly active element that exists abundantly in the environment,

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combines with other elements to produce compounds known as fluorides

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accumulation of NaF in the human body can not only cause the damage of bone tissue,

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but also cause the damage of non-bone tissue, including the cardiovascular system (2).

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According to numerous studies, ingestion of a large amount of fluoride causes

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electrocardiac changes and heart enlargement in humans

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dysfunction (4), peripheral vascular disease (5), and carotid atherosclerosis (6) have also

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been observed. The effects of fluoride on the cardiovascular system and the underlying

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mechanism are still unknown and further research is needed. According to our previous

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study, NaF induces oxidative stress in heart tissue via the Bcl-2/Ba signaling pathway,

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and enhances the levels of H9c2 cell apoptosis mediated by directly increasing

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intracellular reactive oxygen species (ROS), and downregulating ΔΨm (7, 8).

(3).

(1).

The

Ventricular diastolic

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Inflammation, which has been widely accepted to play a pivotal role in the

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pathological mechanisms of cardiovascular diseases (9, 10), also plays an important role

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in the mechanism of the cardiovascular toxicity of fluoride (11). The Toll-like receptor

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(TLR) pathway is one of the mechanisms by which the innate immune system perceives

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invasion of pathogenic microorganisms. It can help against invading pathogens in early

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host defense, and activate innate immunity in damaged myocardium (12, 13). TLR4, with

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the highest mRNA relative expression level in cardiac TLRs, plays a crucial part in

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myocardial inflammation in myocarditis, myocardial infarction, and I/R injury

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heart failure

(12),

and hypertension

(15).

(14),

After Toll-like receptors are stimulated by



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microbial products, signaling pathways are activated, resulting in the induction of

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antimicrobial genes and inflammatory cytokines such as interferons (IFNs),

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interleukins (ILs), and tumor necrosis factor (TNF)

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induced cardiomyocyte apoptosis caused by NaF has been studied in animal and cell

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models in previous researches

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understood. In this study, RNA-seq was used to explore the mechanism of NaF-induced

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rat heart injury, investigating the role of the genes involved in Toll-like signaling

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pathways in inflammatory mechanisms of chronic fluorosis.

(7, 8),

(16).

Although oxidative stress

fluoride-induced cardiotoxicity is not yet fully

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We systematically identified and comprehensively analyzed mRNA profiles of

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cardiac tissue from experimental rats exposed to drinking water with NaF for six

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months, and compared them with control rats. Differentially expressed genes were

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identified, and subjected to gene ontology (GO) ,Kyoto Encyclopedia of Genes and

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Genomes database (KEGG), cluster, and protein-protein interaction (PPI) analyses to

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identify critical genes and pathways. Our results may provide useful insights into the

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transcriptomic dynamics of hearts exposed to NaF, as well as into the mechanisms

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underlying chronic fluorosis in rat hearts.

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MATERIALS AND METHODS

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Establishment and treatment of animal models

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Thirty-two SPF-grade male SD rats (aged 3-4 weeks; 85-105 g) were purchased

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from the Experimental Animal Center of the Chinese People's Liberation Army

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Academy. They were kept in a spacious animal house and fed a standard mouse diet


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and water ad libitum. The feed and ultrapure water were kept for 1 week, and after

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weighing, the rats were randomly divided into 3 groups of 10-12 each: a control group,

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which drank distilled water, and two groups given fluoride in the water. This was at a

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low (30 mg/L) or high (90 mg/L) level of NaF (Fengxian Fengcheng Reagent Factory,

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Shanghai, China). The doses were based on the basis of previous research of

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cardiovascular fluoride toxicity (7). To obtain a rat model of chronic fluorosis, rats were

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treated with NaF for six months. During this period, we observed and recorded their

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weight and other indicators of their growth and development. The study design was

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approved by the Institutional Animal Care and Use Committee of China.

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Histopathological examination

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After treatment for six months, all rats were anesthetized with 20% urethane (0.7

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ml/100 g B.W.)( Tianjin guangfu fine chemical research institute, Tianjin, China). The

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hearts were quickly removed to observe their morphology by HE staining, and their

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ultrastructure by transmission electron microscopy (TEM). Freshly prepared rat heart

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tissue washed with saline was fixed with 4% paraformaldehyde (Tianjin guangfu fine

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chemical research institute, Tianjin, China) and then embedded. They were then sliced,

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spread, dried, dewaxed, stained, placed in a neutral resin (Shanghai yiyang instrument

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co., LTD, Shanghai, China), and allowed to dry naturally. Finally, the rat heart tissue

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sections were observed under an optical microscope (Olympus BX53, Tokyo, Japan).

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The left ventricle was excised, cut into small pieces of 1 mm3, and fixed for 2 h at 4 ℃

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in 2.5% glutaraldehyde (Shanxi Medical University Electron Microscope Laboratory,



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Taiyuan, Shanxi, China) for electron microscopy. After pouring off the fixative,

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absolute ethanol, pure acetone and embedding agent were used for embedding. The

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embedded tissue was then trimmed, sliced, and treated with a staining agent, then

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observed under a JEM-1011 TEM. The fixative, absolute ethanol, pure acetone,

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embedding agent and staining agent were purchased from Shanxi Medical University

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Electron Microscope Laboratory (Taiyuan, Shanxi, China).

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Activity assays of heart-related enzymes

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We assayed heart-related enzymes in myocardial tissue. These included Glutamic-

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oxal(o)acetic transaminase (GOT), creatine kinase (CK), Lactic dehydrogenase (LDH),

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and creatinine (CRE). Their activity levels were determined using enzyme kits

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(Jiancheng Biochemisty Institute, Nanjing, Jiangsu, China).

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Total RNA extraction

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RNA was extracted using the Trizol Reagent (Takara, Dalian, Liaoning, China) for

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RNA-Seq and qRT-PCR. DNase I (Takara, Dalian, Liaoning, China) was treated to

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remove DNA contamination. The RNA samples were taken and the concentration,

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A260/280

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spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, USA). We then

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carried out conventional 0.8% agarose gel electrophoresis to detect the quality of the

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extracted RNA. Total RNA from 10 rats per group were mixed for qRT-PCR.

and

A260/230

ratios

were

determined

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using

Nanodrop-2000

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Library preparation for RNA sequencing

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Six cDNA libraries were constructed using heart tissues from the control (C_1, 2),

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LF (L_1, 2), and HF (H_1, 2) groups. After extracting total RNA from the sample and

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digesting the DNA with DNase I, we enriched the mRNA with magnetic beads with

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Oligo (dT). We then added the interrupting reagent (Life Technologies, Carlsbad, CA,

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USA) to break the mRNA into short fragments at a suitable temperature in the

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Thermomixer. The constructed library was qualified by Agilent 2100 Bioanalyzer

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(Agilent Technologies, Palo Alto, CA, USA) and ABI StepOnePlus Real-Time PCR

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System (Applied Biosystems, Foster, CA, USA), and then sequenced using Illumina

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HiSeqTM 2000. The above experiments were completed by Shenzhen bgi technology

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service co. LTD (Shenzhen, Guangdong, China).

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Analysis of RNA-Seq data

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By removing reads containing more than 10% unidentified bases, adapter-

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containing reads, and low-quality reads, raw reads became clean reads and were then

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mapped to the reference genome and reference gene set using BWA and Bowtie

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software. (https://www.ncbi.nlm.nih.gov/genome/?term=Rattus_norvegicus). Expression of

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genes and transcripts was quantified using the RNA Seq by Expectation Maximization

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(RSEM) tool. The clustering results are displayed by javaTreeview. We used GO and

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KEGG analyze to functionally annotate differentially expressed genes. Protein

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interaction network analysis was used to integrate the composition of BIND, BioGrid,

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HPRD, and other interactive network databases. The results file was displayed by



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Medusa software.

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Quantitative Real Time RT-PCR

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The 2 μl RNA were added to the 2μl Prime ScriptTM RT Master Mix(Takara,

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Dalian, Liaoning, China) and 8μl RNase Free dH2O(Takara, Dalian, Liaoning, China).

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Specific primers of 11 genes were originated by the software of Primer 3.0 Plus

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(Applied Biosystem, Foster, CA, USA). Gene names, primer sequences, PCR product

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lengths, and gene accession numbers are shown in Table 1. A two-step SYBR® QRT-

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PCR kit (Takara, Dalian, Liaoning, China), PCR primers and RNase Free dH2O (Takara,

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Dalian, Liaoning, China) were used on a Quant-Studio™ 7 Flex Real-Time PCR

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System (Life Technologies, Carlsbad, CA, USA). The experimental conditions were

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set as follows: 45 cycles at 95 ℃ for 20 s, 55 ℃ for 20 s, and 72 ℃ for 20 s. All genes

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were repeated 3 times and averaged. The mRNA expression of target genes were

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calculated with the 2−ΔΔCT method and Glyceraldehyde-3-phosphate dehydrogenase

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(GAPDH) was selected as a reference gene.

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Western blot

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Based on the results of DEGs and PPI, we selected three genes IL-1, IL-6 and IL-

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10 for protein quantification. The total protein was obtained by tissue homogenization,

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action of RIPA lysate (BiYunTian, Shanghai, China), and centrifugation. The

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concentration was determined using a BCA kit (BiYunTian, Shanghai, China).

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Subsequently, the target protein were separated by agarose gel electrophoresis (SDS-


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PAGE), and transferred to an nitrocellulose (NC) film (Soulebao, Beijing, China).

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Primary antibodies (Santa Cruz Biotechnology, Delaware Ave Santa Cruz, CA, USA

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and Abcam, Cambridge, Cambridgeshire, UK) were used to combine target protein IL-

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1, IL-6, and IL-10. Membranes were placed in a Fluor Chem Q multi-function imaging

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system (ProteinSimple, Silicon Valley, CA, USA) for optical density analysis of protein

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bands.

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Statistical analysis

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All data were analyzed by GraphPad Prism 5 and rendered as means ± SEM. One-

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way ANNOVA followed by Dunnett-t Test was performed to analyze significance

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between pairs. And statistically significant difference was considered when p

Comparative Transcriptomics Reveals the Role of the Toll-Like

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