Effect of Boron on Thymic Cytokine Expression, Hormone Secretion

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Effect of boron on thymic cytokine expression, hormone secretion, antioxidant functions, cell proliferation, and apoptosis potential via the ERK1/2 signaling pathway Erhui Jin, Man Ren, Wenwen Liu, Shuang Liang, Qianqian Hu, Youfang Gu, and Shenghe Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04069 • Publication Date (Web): 14 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Journal of Agricultural and Food Chemistry

Effect of boron on thymic cytokine expression, hormone secretion, antioxidant functions, cell proliferation, and apoptosis potential via the ERK1/2 signaling pathway

Erhui Jin, Man Ren, Wenwen Liu, Shuang Liang, Qianqian Hu, Youfang Gu, Shenghe Li* Affiliations: College of Animal Science, Anhui Science and Technology University Address: College of Animal Science, Anhui Science and Technology University, No.9 Donghua Road, Fengyang County, Anhui Province, 233100 (postal code), P. R. China

Corresponding author: Prof. Shenghe Li, China-Phone: +86 550 6732 373. Fax: +86 550 6733 165. Email: [email protected]. 1 ACS Paragon Plus Environment


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Abstract: Boron is an essential trace element in animals. Appropriate boron

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supplementation can promote thymus development; however, a high dose of boron can lead

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to adverse effects and cause toxicity. The influencing mechanism of boron on the animal

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body remains unclear. In this study, we examined the effect of boron on cytokine expression,

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thymosin and thymopoietin secretion, antioxidant function, cell proliferation and apoptosis,

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and the ERK1/2 pathway in the thymus of rats. We found that supplementation with 10 and

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20 mg/L of boron to the drinking water significantly elevated levels of IL-2, IFN-γ, IL-4,

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and thymosin α1 in the thymus of rats (P < 0.05), increased the number of PCNA+ cells and

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concentrations of GSH-Px and p-ERK (P < 0.05), and promoted mRNA expression of

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PCNA and ERK1/2 in thymocytes (P < 0.05). However, the number of Caspase-3+ cells and

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the expression level of Caspase-3 mRNA were reduced (P < 0.05). Supplementation with 40,

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80, and 160 mg/L of boron had no apparent effect on many of the above indicators. In

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contrast, supplementation with 480 and 640 mg/L of boron had the opposite effect on the

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above indicators in rats and elevated levels of pro-inflammatory cytokines such as IL-6,

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IL-1β, and TNF-α (P < 0.05). Our study showed that supplementation of various doses of

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boron to the drinking water had a U-shaped dose-effect relationship with thymic cytokine

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expression, hormone secretion, antioxidant function, cell proliferation, and apoptosis.

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Specifically, supplementation with 10 and 20 mg/L of boron promoted thymocyte

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proliferation and enhanced thymic functions. However, supplementation with 480 and 640

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mg/L of boron inhibited thymic functions and increased the number of apoptotic thymocytes,

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suggesting that the effects of boron on thymic functions may be caused via the ERK1/2

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signaling pathway.

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Keywords: Boron; thymus; cytokine; antioxidant; proliferation and apoptosis; ERK1/2 2 ACS Paragon Plus Environment

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signaling pathway

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INTRODUCTION

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Boron is a nonmetallic element with metallic properties. It is naturally deposited in the

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form of boric acid or borate within soil, water, air, and rock1. Due to its unique physical and

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chemical properties2, boron is widely used in food processing, agriculture, manufacturing,

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industry, medicine, and healthcare. Consequently, the concentration of boron in the

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environment and its consumption by humans and animals has also significantly increased.

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The effect of boron on human and animal health has now become a focus for many

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researchers.

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Many studies have shown that boron may be an essential trace element for humans and

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animals3, and that it plays an important regulatory role for the mineral metabolism, hormone

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secretion, cell membrane function, enzymatic reaction, embryonal development, bone

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growth, immune function, and mental activity in animals4,5. Boron deprivation or

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insufficient intake can significantly inhibit teeth and bone growth in animals, reduce bone

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volume fraction, and impair normal bone development6. Furthermore, lack of boron can also

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damage immune and reproductive functions in animals, leading to exacerbated adjuvant

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arthritis, testicular and ovarian atrophy, decreased sperm count, increased sperm deformity,

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blocked oocyte maturation in rats, and impaired mouse embryo development7,8. In addition,

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reduced plasma steroids, alkaline phosphatase, and calcium and magnesium ion

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concentrations have been reported to be caused by boron insufficiency9,10. Appropriate

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boron supplementation can increase the mRNA levels of bone growth-related genes in

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mouse osteoblasts, such as type I collagen and osteocalcin6, increase the plasma levels of

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total cholesterol, triglycerides, low-density lipoprotein, glucose, insulin, and non-esterified 3 ACS Paragon Plus Environment


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fatty acids in pregnant cows and improve the metabolism during the perinatal phase11.

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Furthermore, sufficient boron levels can enhance the activity of long-term cryopreserved

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mesenchymal stem cells and promote osteoblast and chondrocyte differentiation from

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human tooth germ stem cells12, increase animal growth performance and feed conversion,

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improve egg quality13, increase the level of antibodies in rats injected with human typhoid

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vaccine, and reduce the number of circulating natural killer (NK) cells and CD8a+/CD4-

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cells14,15. However, supplementation with high-doses of boron or high levels of boron

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exposure can result in significant injuries and even toxicity in animals, increase prenatal

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fetal mortality, reduce fetal weight and organ weight, and damage cardiovascular and central

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nervous systems as well as the bone development of fetuses16. Moreover, supplementation

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with high-doses of boron has been shown to induce testicular atrophy, seminiferous tubule

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abnormalities, spermatogenic cell loss, and block sperm development and discharge17, as

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well as causing the formation of binucleated and micronucleated lymphocytes and acute

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leukemia cells in vitro, leading to mitochondrial swelling and an increase of the number of

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apoptotic cells18. All of these studies demonstrated that different doses of boron can induce

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either beneficial or harmful effects within the body.

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As a key central immune organ, the main function of the thymus is to generate and

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further develop T lymphocytes that then participate in all cell-mediated immune responses

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of the body. Normal thymic development and function are important for maintaining a

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cellular immune response and even for the entire immunity of the body. One of our previous

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studies reported that an appropriate amount of boron could promote thymic development

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and improve tissue structure in broiler chickens, while high boron levels were inhibitory to

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thymic development and resulted in structural tissue damage19. However, the relationship 4 ACS Paragon Plus Environment

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between the specific amount of boron supplementation and the immune function of the

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thymus in mammals, as well as the underlying mechanisms of the boron-induced effect on

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the immune function remain unclear. In this study, we examined the effect of different boron

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doses on cytokine secretion, hormone synthesis, antioxidant function, as well as cell

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proliferation and apoptosis in the thymus of rats, and furthermore analyzed the changes in

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the mRNA and protein expression of ERK1/2 in thymocytes to reveal the effect of boron on

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thymic functions as well as the involved pathways.

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MATERIALS AND METHODS

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Animal management. Healthy specific pathogen free (SPF) Sprague Dawley (SD) rats

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were purchased from the Qinglongshan animal breeding farm of the Jiangning district,

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Nanjing, China (lot: 2015-03-15). Animal use was reviewed and approved by the Anhui

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Provincial Experimental Animal Management Committee, and all experimental procedures

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were conducted in strict accordance with the provincial “Guide for the Care and Use of

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Laboratory Animals” and the “National Guide for the Care and Use of Laboratory Animals”.

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Animals were housed in individual ventilated cages in the SPF experimental animal center

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of the Anhui Science and Technology University (license: SYXK (Wan) 2013-007), at a

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room temperature of 22-25°C, 50-60% humidity, and a 14/10 light/dark cycle. Animals had

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access to food and water ad libitum. All cages, covers, and water bottles were sterilized

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prior to use.

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Experimental design and animal diet. After one week of acclimatization, 120 male SD

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rats (23 days old, 53 ± 2 g) were randomly divided into ten groups with one control group

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and nine experimental groups (n = 12 each). Rats in the control group received sterilized

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distilled water, rats in the nine experimental groups (I-IX) received drinking water 5 ACS Paragon Plus Environment


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supplemented with 5, 10, 20, 40, 80, 160, 320, 480, and 640 mg/L of boron for a total of 60

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days, respectively. Rat feed was purchased from the Qinglongshan animal breeding farm

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(Catalogue No.: GB14921.1-2001), and mainly comprised of ≤ 10% water, ≥ 18.15% crude

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protein, ≥ 4.03% crude fat, ≥ 5.12% crude fiber, ≥ 7.94% crude ash, and 1.96 mg/kg boron.

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Antibodies and reagents. Mouse anti-PCNA monoclonal antibody (Cat: #2586S) was

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obtained from Cell Signaling Technology (Danvers, MA, USA), DAB (Diaminobenzidine,

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Cat: D5637-5G) was obtained from Sigma-Aldrich (St. Louis, MO, USA); rabbit

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anti-Caspase-3 polyclonal antibody (Cat: 19677-1-AP), biotinylated goat anti-rat IgG (Cat:

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SA00004-2), and HRP (horse radish peroxidase)-labelled streptavidin (Cat: SA00001-0)

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were obtained from the Proteintech Group, Inc (Chicago, IL, USA); biotinylated horse

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anti-mouse IgG (Cat: ZB-2020) was obtained from ZSGB-Bio (Xicheng District, Beijing,

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China); IL-2, TNF-α, IL-4, IL-6, IL-1β, and IFN-γ ELISA detection kits were obtained from

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Wuhan USCN Business Co., Ltd. (Wuhan, Hubei, China); thymosin α1, TMPO

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(thymopoietin) and phosphorylated ERK (p-ERK) ELISA detection kits were obtained from

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R&D System, Inc (Minneapolis, MN, USA); SYBR Premix Ex TaqTM (Cat: RR420A) was

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obtained from Takara Biomedical Technology (Dalian) Co., Ltd. (Dalian, Liaoning, China);

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RNAprep pure Tissue Kit (Cat: DP431) was obtained from Tiangen Biotech (Beijing) Co.,

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Ltd. (Haidian District, Beijing, China); the RevertAid First Strand cDNA Synthesis Kit (Cat:

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#K1622) was obtained from Thermo Fisher Scientific (Waltham, MA, USA); and SOD,

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GSH-Px, MDA, and T-AOC detection kits were obtained from the Nanjing Jiancheng

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Bioengineering Institute (Nanjing, Jiangsu, China).

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Sample collection and processing. Ten rats per group were anesthetized with the anesthetic

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breathing system (ABS) (4-5% isoflurane to induce anesthesia and 1-2% isoflurane to 6 ACS Paragon Plus Environment

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maintain anesthesia) at the end of the experiment. Animals were bled to death via cardiac

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puncture, and the thymus was immediately harvested. One portion of the thymus was fixed

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in 4% formaldehyde phosphoric acid buffer for staining via immunohistochemistry (IHC),

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while the other portion was stored in liquid nitrogen for cytokine detection, antioxidative

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function detection, and total RNA extraction. After 72 h of fixation in 4% formaldehyde, the

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thymus tissue was dehydrated in an ethanol gradient, cleared in xylene, paraffin-embedded,

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and cut into 6 μm thick consecutive transverse sections using a microtome (RM2235, Leica,

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Wetzlar, Hessen, Germany). Two of every 10 thymus sections were mounted on a

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polylysine-coated slide, and were stored in 37°C for IHC staining. Total thymic RNA was

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extracted using the RNAprep pure Tissue Kit as per the manufacturer’s instructions.

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ELISA. The thawed thymus tissue was weighed on an electronic balance (accuracy up to

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one thousandth of a gram), homogenized in an ice bath, and centrifuged at 3000 rpm/min for

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15 min at 4°C. The supernatants were obtained and stored at -80°C for later use. The levels

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of IL-2, TNF-α, IL-4, IL-6, IL-1β, and IFN-γ, and concentrations of thymosin α1,

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thymopoietin (TMPO), and p-ERK in the thymus were measured with the corresponding

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ELISA detection kits as per the manufacturer’s instructions, and absorbance was determined

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with a full-wavelength microplate reader (Multiskan GO, Thermo Fisher Scientific,

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Waltham, MA, USA). The correlation coefficients of the IL-2, TNF-α, IL-4, IL-6, IL-1β,

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and IFN-γ standard curves were 0.9988, 0.9994, 0.9983, 0.9993, 0.9976, and 0.9995,

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respectively. The correlation coefficients of the thymosin, TMPO, and p-ERK standard

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curves were 0.9998, 0.9983, and 0.9981, respectively.

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IHC. Thymus tissue sections were dewaxed in xylene, rehydrated in an ethanol gradient and

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distilled water, washed thrice with 0.01 mol/L PBS (pH 7.4) for 5 min each, and immersed 7 ACS Paragon Plus Environment


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in 0.1 mol/L citrate buffer (pH 6.0). Antigen was retrieved via the microwave antigen

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retrieval method for 15 min, and the section was then cooled to room temperature. The

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tissue sections were thrice washed with 0.01 mol/L PBS (5 min each), and incubated with

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10% normal horse serum or goat serum (in 0.01 mol/L PBS) at room temperature for 30 min.

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After serum removal, the sections were incubated with PCNA or Caspase-3 antibody

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overnight at 4°C. On the following day, tissue sections were warmed to room temperature

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for 30 min, washed thrice with 0.01 mol/L PBS (5 min each), and incubated with

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biotinylated horse anti-mouse or goat anti-rabbit IgG for 2 h at 37°C. Sections were washed

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thrice with 0.01 mol/L PBS (5 min each), incubated with HRP-labelled streptavidin for 2 h

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at 37°C, followed by two washes in 0.01 mol/L PBS (5 min each). Finally, tissue sections

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were preincubated with DAB preincubation liquid for 30 min and DAB coloration for 5 min,

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re-dyed with hematoxylin for 3 min, dehydrated in an ethanol gradient, cleared in xylene,

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and mounted in neutral gum. Tissue sections were examined and imaged using the

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OLYMPUS BX51+DP73 upright fluorescence microscope and micrograph system (Tokyo,

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Japan). Fifteen thymus transverse sections were selected for IHC staining per rat in each

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experimental group, and five even views per section were selected for photograph. The

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number of positive cells on the IHC staining was determined via the microscopic imaging

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analysis software Image Pro Plus 6.0.

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Determination of antioxidative function. After thawing thymus homogenates at room

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temperature, SOD and T-AOC activities, and GSH-Px and MDA concentrations were

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determined with the antioxidant assay kit as per the manufacturer’s instructions.

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Real-time Quantitative PCR. The concentration and quality of total thymic RNA were

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verified on a Multiskan GO (Thermo Scientific, Waltham, MA, USA). cDNA was 8 ACS Paragon Plus Environment

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synthesized from 1 μg total RNA using the RevertAid First Strand cDNA Synthesis Kit as

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per the manufacturer’s instructions. All real-time quantitative PCR (qPCR) primers were

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synthesized via the Primer Premier 5 software (Supporting Information Table 1). qPCR

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amplification was performed in the Roche LightCycler® 480II (Basel, Kanton Basel-Stadt,

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Switzerland) as per the instructions of the SYBR Premix Ex TaqTM Kit. Amplification

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conditions: 95°C for 30 s, 95°C for 5 s, and 59°C for 30 s for 40 cycles, followed by 60°C

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for 1 min and 40°C for 30 s. Melting curve analyses were performed to confirm the

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specificity of each amplification, and the size of the amplicon was determined via agarose

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gel electrophoresis. Each sample was measured thrice. GAPDH was used reference gene to

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ensure precision of the qPCR data. Relative expression of the target gene was calculated via

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2-ΔΔCt.

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Statistical analysis. One-way ANOVA was performed on the obtained data using the SPSS

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22.0 statistical software. Homogeneity of variance, normality, and significant differences of

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the data were determined with the Levene’s test, the Kolmogorov-Smirnov test, and the

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Dunnett’s test, respectively. All data are expressed as means ± SD. P < 0.05 was considered

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as a statistically significant difference, while P < 0.01 was considered as an extremely

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statistically significant difference.

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RESULTS

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Thymic cytokine expression. To determine the effect of different boron doses on immune

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functions, the concentrations of IL-2, IFN-γ, IL-4, IL-6, TNF-α, and IL-1β in the thymus

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were measured via ELISA (Figure 1). Compared to the control group, the IL-2 concentration

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was significantly increased in experimental groups II (20.36%, P < 0.05) and III (47.09%, P

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< 0.01); however, it was significantly decreased in experimental groups VII (18.30%, P < 9 ACS Paragon Plus Environment


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0.05), VIII (27.63%, P < 0.01), and IX (38.62%, P < 0.01). The IFN-γ concentration was

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significantly increased in the experimental groups II (22.82%), III (19.98%), IV (20.69%),

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V (19.85%), and VI (19.61%) (all P < 0.05); however, they were markedly decreased in

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experimental groups VII (21.29%), VIII (21.43%), and IX (22.42%) (all P < 0.05) compared

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to the control group. The IL-4 concentration was significantly increased in experimental

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groups II (20.24%, P < 0.05) and III (24.20%, P < 0.01); however, it was significantly

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decreased in experimental groups VIII (19.42%, P < 0.05) and IX (26.40%, P < 0.01)

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compared to the control group. The IL-6 concentration was significantly increased in the

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experimental groups VI (50.32%), VII (58.61%), and VIII (69.24%) (all P < 0.01); however,

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it was significantly decreased in the experimental group IX (27.03%, P < 0.05) compared to

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the control group. The IL-1β concentration was significantly increased in experimental

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groups VII (32.40%, P < 0.05) and VIII (46.76%, P < 0.01) compared to the control group.

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The TNF-α concentration was significantly decreased in experimental groups II (25.83, P
0.05). In

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addition, while the TMPO concentration was significantly decreased in experimental group

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IX (41.42%, P < 0.05), it remained similar between all other experimental groups and the

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control group (P > 0.05).

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Localization of PCNA+ and Caspase-3+ cells in the thymus. To determine the effect of

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different boron doses on thymocyte proliferation and apoptosis, both the localization and

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expression of PCNA and Caspase-3 were first examined via immunohistochemistry (Figure

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3-4), while the mRNA expression of PCNA and Caspase-3 were measured via quantitative

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PCR (Figure 5). As shown in Figures 3-4, PCNA+ cells were stained with a brownish yellow

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color in the nuclei, and Caspase-3+ cells were stained brownish black in their cytoplasm.

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These two types of cells were differently distributed between the thymic cortex and the

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medulla. PCNA+ cells were mainly found in the thymic medulla and the cortex near the

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medulla, and first increased, followed by a decrease as the amount of boron supplementation

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increased (Figure 3). However, Caspase-3+ cells were mainly found in the thymic medulla,

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and first decreased followed by an increase with increasing amount of boron

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supplementation (Figure 4).

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The number of PCNA+ cells per unit area in the thymus (Figure 3L) was significantly

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increased in experimental groups II (28.98%) and III (58.93%) (both P < 0.01); however,

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the cell number was significantly decreased in experimental groups VII (38.22%), VIII

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(33.42%), and IX (58.16%) (all P < 0.01) compared to the control group. However, no

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significant changes were found in the number of PCNA+ cells between all other

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experimental groups and the control group (P > 0.05). In contrast, the number of Caspase-3+

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cells per unit area in the thymus (Figure 4L) was significantly decreased in experimental 11 ACS Paragon Plus Environment


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groups II (30.17%) and III (35.95%) (both P < 0.05); however, it was significantly increased

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in experimental groups VI (29.66%, P < 0.05), VII (137.98%, P < 0.01), VIII (147.10%, P
0.05).

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The expression of PCNA and Caspase-3 mRNA in the thymus. As shown in Figure 5, the

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level of PCNA mRNA expression in the thymus was significantly increased in experimental

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groups II (21.05%, P < 0.05) and III (25.00%, P < 0.01); however, it was significantly

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decreased in experimental groups VIII (17.11%) and IX (14.47%) (both P < 0.05) compared

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to the control group. Furthermore, the level of Caspase-3 mRNA expression in the thymus

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was significantly decreased in experimental group III (24.14%, P < 0.05); however, it was

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significantly increased in experimental groups VI (51.11%, P < 0.01), VII (26.58%, P
0.05).

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Antioxidant function. Changes in the activities of SOD and T-AOC and the contents of

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GSH-Px and MDA were measured to determine the effect of different boron doses on the

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antioxidant function of the thymus (Figure 6). SOD activity in the thymus was significantly

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enhanced in experimental groups II (34.12%, P < 0.05), III (49.28%, P < 0.01), and IV

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(38.40%, P < 0.05); however, it was significantly decreased in experimental groups VIII

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(34.10%) and IX (37.78%) (both P < 0.05) compared to the control group. GSH-Px content

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in the thymus was significantly increased in experimental groups II (43.32%, P < 0.01), III

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(31.83%, P < 0.05), IV (30.94%, P < 0.05), V (36.04%, P < 0.05), and VI (45.51%, P < 12 ACS Paragon Plus Environment

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0.01); however, it was significantly decreased in experimental groups VIII (30.42%) and IX

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(35.46%) (both P < 0.05) compared to the control group. T-AOC activity in the thymus was

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significantly increased in experimental group I (28.82%, P < 0.05), II (33.63%, P < 0.05),

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and III (45.44%, P < 0.01); however, it was significantly decreased in experimental groups

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VII (32.03%, P < 0.05), VIII (51.53%, P < 0.01), and IX (62.60%, P < 0.01) compared to

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the control group. The MDA content in the thymus was significantly decreased in

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experimental groups II (30.28%) and III (28.49%) (both P < 0.05); however, it was

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significantly increased in experimental groups VII (29.24%, P < 0.05), VIII (89.36%, P
160 mg/L resulted in reduction of

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spleen antioxidative function. Similarly, our study demonstrated that supplementation of 10,

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20, and 40 mg/L of boron in the drinking water significantly increased SOD activities and

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GSH-Px content. Furthermore, supplementation of 10 and 20 mg/L of boron in the drinking

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water also significantly increased T-AOC activities and reduced MDA content in the thymus

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of rats. These effects were particularly prominent in the 10 and 20 mg/L boron groups,

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suggesting that appropriate boron supplementation could promote thymocyte proliferation,

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while inhibiting apoptosis via enhancing the antioxidative function of the thymus. However,

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supplementation with 480 and 640 mg/L of boron significantly reduced SOD and T-AOC

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activities, decreased GSH-Px content, and increased MDA content. Supplementation with

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320 mg/L of boron also significantly reduced T-AOC activities and increased MDA content,

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demonstrating that high boron doses could damage the antioxidative system of thymocytes,

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reduce the antioxidative function of the thymus, induce oxidative stress, and result in

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increased cell apoptosis.

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Effect of boron on ERK signaling in the thymus of rats. Extracellular signal-regulated

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kinase (ERK) is a member of the mitogen-activated protein kinase (MAPK) family. It

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mainly mediates the transmission of an extracellular stimulus signal from the membrane 19 ACS Paragon Plus Environment


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surface receptor to the cell nucleus, and then regulates various cellular physiological

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processes43. The ERK family consists of five members, namely ERK1-ERK5. In particular,

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ERK1 and ERK2 are the most widely distributed members of the ERK family, and the

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signaling pathways they mediate are important for the regulation of cell growth, division,

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proliferation, and apoptosis44. He et al.45 reported that cell proliferation and apoptosis

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induced by numerous extracellular stress factors are closely associated to the ERK1/2

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signaling pathway. For example, neurotrophic factors can activate the ERK1/2 signaling

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pathway by inducing ERK1/2 phosphorylation, which then upregulates c-Fos protein

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expression and promotes mouse spermatogonial stem cells into proliferation and

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differentiation. Huang et al.46 reported that leptin induced the progression of vascular

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smooth muscle cells from the G1 phase to the S phase, and promoted cell division and

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proliferation by activating the ERK1/2 signaling pathway. Addition of a suitable amount of

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zinc has been reported to promote cell proliferation via activation of the ERK signaling

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pathway in mouse myoblasts C2C1247. Furthermore, activation of the ERK1/2 signaling

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pathway can induce cell apoptosis and cell death via cellular DNA damage, IFN-γ secretion,

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and Fas expression48. For example, oxidative stress in the mouse pulmonary fibroblasts

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L929 (induced by exogenous H2O2) can activate the ERK1/2 signaling pathway and thus

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significantly increase the number of apoptotic cells49. Vitamin E succinic acid can induce

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apoptosis of human gastric cancer cells SGC-7901 by activating the ERK1/2 signaling

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pathway, thus inhibiting the deterioration of the tumor50. ERK1/2 signaling-mediated

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proliferation and apoptosis are critical for the maturation and differentiation of thymocytes

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during thymus development. A study in humans demonstrated that ERK1/2 activation not

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only affects thymocyte maturation, but also determines the positive and negative selections 20 ACS Paragon Plus Environment

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of thymocytes51. Furthermore, activation of the ERK1/2 signaling pathway can counteract

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receptor-mediated death signals in mature T cells, inhibit Caspase-dependent cell apoptosis,

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and promote cell survival, differentiation, and proliferation52. Our study showed that p-ERK

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concentration and ERK1/2 mRNA expression were significantly increased after

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supplementation with 10 and 20 mg/L of boron; however, expressions were significantly

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reduced after supplementation with 480 and 640 mg/L of boron, suggesting that appropriate

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boron supplementation could induce the activation of the ERK1/2 signaling pathway,

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increase PCNA expression in thymocytes, enhance antioxidative functions of the thymus,

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and consequently promote thymocyte proliferation, and increase Th2-type cytokine and

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thymosin α1 levels. In contrast, high boron doses inhibited activation of the ERK1/2

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signaling pathway, increased Caspase-3 expression in thymocytes, elevated the

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concentrations of MDA and proinflammatory cytokines in the thymus (except for

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concentrations of IL-6 and IL-1β after supplementation with the 640 mg/L boron), reduced

451

the concentration of thymosin α1, and thus weakened thymic immune functions and

452

suppressed thymus development. In contrast to our findings, Huang et al. (2015) reported

453

that supplementation with 640 mg/L of boron can promote thymocyte death in a 90 d ostrich

454

via activation of the TLR3/4 pathway, with no significant changes of ERK concentration.

455

This inconsistency may be due to the varying expression levels of ERK family members in

456

the thymocytes of animals at different ages, as well as different sensitivities towards high

457

boron doses in different species. Consequently, further studies will be required to elucidate

458

the reasons behinds these observations.

459

In conclusion, this study demonstrated that appropriate supplementation with boron

460

(10-20 mg/L) in drinking water can elevate IL-2, IFN-γ, and IL-4 levels in the thymus of 21 ACS Paragon Plus Environment


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461

rats, increase thymosin α1 and GSH-Px concentrations, increase the number of PCNA+ cells,

462

enhance SOD and T-AOC activities, reduce TNF-α and MDA levels, reduce the number of

463

Caspase-3+ cells (except for supplementation with 10 mg/L of boron), and hence promote

464

thymocyte proliferation, inhibit apoptosis, and enhance thymus immune functions. However,

465

high boron doses (480-640 mg/L) have the opposite effect on the above indicators, and these

466

effects may be mediated through the ERK1/2 signaling pathway in thymocytes.

467

ABBREVIATIONS USED

468

ERK1/2: extracellular signal-regulated kinase 1/2; GSH-Px: glutathione peroxidase; SOD:

469

superoxide dismutase; MDA: malondialdehyde; PCNA: proliferating cell nuclear antigen;

470

SPF:

471

immunohistochemistry; TMPO: thymopoietin; DAB: 3-3 diaminobenzidine; T-AOC: total

472

antioxidant capacity; NK cell: natural killer cell; cAMP: cyclic adenosine monophosphate;

473

ROS: reactive oxygen species; MAPK: mitogen-activated protein kinase.

474

ACKNOWLEDGMENTS

475

We thank the workers of the SPF Experimental Animal Center of the Anhui Science and

476

Technology University for rat feeding and management. We appreciate the help of Dr.

477

Jinxing Zhou with the use of the Roche LightCycler® 480II.

478

SUPPORTING INFORMATION DESCRIPTION

479

The Primer Sequences for qPCR in experiment (PDF)

480

NOTES

481

The authors declare no competing financial interest.

specific-pathogen

free;

ABS:

anesthetic

482 483

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breathing

system;

IHC:

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645

FIGURE CAPTIONS

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Figure 1. Concentration of IL-2(A), IFN-γ(B), IL-4(C), IL-6(D), IL-1β(E), and TNF-α (F)

647

in the thymus of rats after supplementation with different doses of boron to the drinking

648

water for a duration of 60 days, determined via ELISA. Values are presented as the mean ±

649

SD, n = 10 rats per treatment. Mean values with asterisk (*) differed significantly (P < 0.05),

650

and mean values with two asterisks (**) indicate extremely significant differences (P

Effect of Boron on Thymic Cytokine Expression, Hormone Secretion

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