Effect of Boron on Thymic Cytokine Expression, Hormone Secretion

Oct 14, 2017 - Mouse anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (catalog number 2586S) was obtained from Cell Signaling Techno...
0 downloads 24 Views 2MB Size
Subscriber access provided by Northwestern Univ. Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

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

Journal of Agricultural and Food Chemistry

1

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

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

signaling pathway.

23

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

Page 2 of 40

Page 3 of 40

Journal of Agricultural and Food Chemistry

24

signaling pathway

25

INTRODUCTION

26

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

27

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

28

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

29

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

30

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

31

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

32

researchers.

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

Journal of Agricultural and Food Chemistry

47

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

48

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

49

mesenchymal stem cells and promote osteoblast and chondrocyte differentiation from

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

either beneficial or harmful effects within the body.

63

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

64

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

65

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

66

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

67

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

68

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

69

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

Page 4 of 40

Page 5 of 40

Journal of Agricultural and Food Chemistry

70

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

71

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

72

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

73

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

74

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

75

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

76

thymic functions as well as the involved pathways.

77

MATERIALS AND METHODS

78

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

79

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

80

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

81

Provincial Experimental Animal Management Committee, and all experimental procedures

82

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

83

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

84

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

85

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

86

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

87

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

88

prior to use.

89

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

90

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

91

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

92

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

Journal of Agricultural and Food Chemistry

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

Bioengineering Institute (Nanjing, Jiangsu, China).

114

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

115

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

Page 6 of 40

Page 7 of 40

Journal of Agricultural and Food Chemistry

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

curves were 0.9998, 0.9983, and 0.9981, respectively.

137

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

138

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

Journal of Agricultural and Food Chemistry

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

analysis software Image Pro Plus 6.0.

157

Determination of antioxidative function. After thawing thymus homogenates at room

158

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

159

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

160

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

161

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

Page 8 of 40

Page 9 of 40

Journal of Agricultural and Food Chemistry

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

2-ΔΔCt.

173

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

174

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

175

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

176

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

177

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

178

statistically significant difference.

179

RESULTS

180

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

181

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

182

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

183

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

184

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

Journal of Agricultural and Food Chemistry

Page 10 of 40

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

209

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

210

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

211

control group (P > 0.05).

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

supplementation (Figure 4).

224

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

225

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

226

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

227

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

228

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

229

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

230

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

Journal of Agricultural and Food Chemistry

231

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

232

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

(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

Page 12 of 40

Page 13 of 40

Journal of Agricultural and Food Chemistry

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

399

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

400

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

401

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

402

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

403

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

404

suggesting that appropriate boron supplementation could promote thymocyte proliferation,

405

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

406

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

407

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

408

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

409

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

410

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

411

increased cell apoptosis.

412

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

413

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

414

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

Journal of Agricultural and Food Chemistry

Page 20 of 40

415

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

416

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

417

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

418

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

419

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

420

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

421

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

422

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

423

expression and promotes mouse spermatogonial stem cells into proliferation and

424

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

425

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

426

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

427

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

428

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

429

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

430

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

431

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

432

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

433

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

434

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

435

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

436

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

437

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

Page 21 of 40

Journal of Agricultural and Food Chemistry

438

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

439

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

440

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

441

concentration and ERK1/2 mRNA expression were significantly increased after

442

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

443

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

444

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

445

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

446

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

447

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

448

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

449

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

450

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

Journal of Agricultural and Food Chemistry

Page 22 of 40

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

22 ACS Paragon Plus Environment

breathing

system;

IHC:

Page 23 of 40

Journal of Agricultural and Food Chemistry

484

REFERENCES

485

(1) Kabu, M.; Akosman, M. Biological effects of boron. Rev. Environ. Contam. Toxicol.

486 487 488 489 490

2013, 225, 57-75. (2) Geyikoglu, F.; Turkez, H. Acute toxicity of boric acid on energy metabolism of the breast muscle in broiler chickens. Biologia. 2007, 62, 112-117. (3) Scorei, R. Is boron a prebiotic element? A mini-review of the essentiality of boron for the appearance of life on earth. Origins. Life. Evol. Biospheres. 2012, 42, 3-17.

491

(4) Orenay Boyacioglu, S.; Korkmaz, M.; Kahraman, E.; Yildirim, H.; Bora, S.; Ataman, O.

492

Y. Biological effects of tolerable level chronic boron intake on transcription factors. J.

493

Trace Elem. Med. Biol. 2017, 39, 30-35.

494

(5) Pan, H.B.; Zhao, X.L.; Zhang, X.; Zhang, K.B.; Li, L.C.; Li, Z.Y.; Lam,W.M.; Lu, W.W.;

495

Wang, D.P.; Huang, W.H.; Lin, K.L.; Chang, J. Strontium borate glass: Potential

496

biomaterial for bone regeneration. J. R. Soc. Interface. 2010, 7, 1025-1031.

497

(6) Hakki, S.S.; Dundar, N.; Kayis, S.A.; Hakki, E.E.; Hamurcu, M.; Kerimoglu, U.;

498

Baspinar, N.; Basoglu, A.; Nielsen, F.H. Boron enhances strength and alters mineral

499

composition of bone in rabbits fed a high energy diet. J. Trace Elem. Med. Biol. 2013,

500

27, 148-153.

501

(7) Lanoue, L.; Strong, P.L.; Keen, C.L. Adverse effects of a low boron environment on the

502

preimplantation development of mouse embryos in vitro. J. Trace. Elem. Exp. Med.

503

1999, 12, 235-250.

504 505 506

(8) Nielsen, F.H. The emergence of boron as nutritionally important throughout the life cycle. Nutrition. 2000, 16, 512-514. (9) Stucki, U.; Schmid, J.; Hämmerle, C.F.; Lang, N.P. Temporal and local appearance of 23 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

507

alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive

508

histochemical study in humans. Clin. Oral. Implants. Res. 2001, 12, 121-127.

509

(10)Alliot-Licht, B.; Bluteau, G.; Magne, D.; Lopez-Cazaux, S.; Lieubeau, B.; Daculsi, G.;

510

Guicheux, J. Dexamethasone stimulates differentiation of odontoblast-like cells in

511

human dental pulp cultures. Cell. Tissue. Res. 2005, 321, 391-400.

512

(11) Kabu, M.; Civelek, T. Effects of propylene glycol, methionine and sodium borate on

513

metabolic profile in dairy cattle during periparturient period. Rev. Med. Vet. 2012, 163,

514

419-430.

515

(12)Demirci, S.; Dogan, A.; Sisli, B.; Sahin, F. Boron increases the cell viability of

516

mesenchymal stem cells after long-term cryopreservation. Cryobiology. 2014, 68,

517

139-146.

518

(13)Mızrak, C.; Yenice, E.; Can, M.; Yıldırım, U.; Atik, Z. Effects of dietary boron on

519

performance, egg production, egg quality and some bone parameters in layer hens. S.

520

Afr. J. Anim. Sci. 2010, 40, 257-264.

521

(14)Bai, Y.; Hunt, C.D.; Newman, S.M. Jr. Dietary boron increases serum antibody (IgG

522

and IgM) concentrations in rats immunized with human typhoid vaccine. Proc. N. D.

523

Acad. Sci. 1997, 51, 181.

524

(15)Hunt, C.D.; Idso, J.P. Dietary boron as a physiological regulator of the normal

525

inflammatory response: A review and current research progress. J. Trace Elem. Exp.

526

Med. 1999, 12, 221-233.

527

(16)Price, C.J.; Marr, M.C.; Myers, C.B.; Seely, J.C.; Heindel, J.J.; Schwetz, B.A. The

528

developmental toxicity of boric acid in rabbits. Fundam. Appl. Toxicol. 1996, 34,

529

176-187. 24 ACS Paragon Plus Environment

Page 24 of 40

Page 25 of 40

530 531 532 533

Journal of Agricultural and Food Chemistry

(17)Fail, P.A.; Chapin, R.E.; Price, C.J.; Heindel, J.J. General, reproductive, developmental, and endocrine toxicity of boronated compounds. Reprod. Toxicol. 1998, 12, 1-18. (18)Canturk, Z.; Tunali, Y.; Korkmaz, S.; Gulbaş, Z. Cytotoxic and apoptotic effects of boron compounds on leukemia cell line. Cytotechnology. 2016, 68, 87-93.

534

(19)Jin, E.; Gu, Y.; Wang, J.; Jin, G.; Li, S. Effect of supplementation of drinking water with

535

different levels of boron on performance and immune organ parameters of broilers. Ital.

536

J. Anim. Sci. 2014, 13, 205-214.

537

(20)Huang, H.B.; Xiao, K.; Lu, S.; Yang, K.L.; Ansari, A.R.; Khaliq, H.; Song, H.; Zhong,

538

J.; Liu, H.Z.; Peng K.M. Increased thymic cell turnover under boron stress may bypass

539

TLR3/4 pathway in African ostrich. PloS. One. 2015, 10, 0129596.

540 541

(21)Yang-Snyder, J.A.; Rothenberg, E.V. Developmental and anatomical patterns of IL-2 gene expression in vivo in the murine thymus. Dev. Immunol. 1993, 3, 85-102.

542

(22)Peng, X.; Cui, H.; Yuan, J.; Cui, W.; Fang, J.; Zuo, Z.; Deng, J.; Pan, K.; Zhou, Y.; Lai,

543

W. Low-selenium diet induces cell cycle arrest of thymocytes and alters serum IL-2

544

content in chickens. Biol. Trace Elem. Res. 2011, 144, 688-694.

545 546

(23)Carding, S.R.; Bottomly, K. IL-4 (B cell stimulatory factor 1) exhibits thymocyte growth factor activity in the presence of IL-2. J. Immunol. 1988, 140, 1519-1526.

547

(24)Plum, J.; De Smedt, M.; Leclercq, G.; Tison, B. Inhibitory effect of murine recombinant

548

IL-4 on thymocyte development in fetal thymus organ cultures.J. Immunol. 1990, 145,

549

1066-1073.

550

(25)Sempowski, G.D.; Hale, L.P.; Sundy, J.S.; Massey, J.M.; Koup, R.A.; Douek, D.C.;

551

Patel, D.D.; Haynes, B.F. Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell

552

factor mRNA expression in human thymus increases with age and is associated with 25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

553

thymic atrophy. J. Immunol. 2000, 164, 2180-2187.

554

(26)Guevara Patiño, J.A.; Ivanov, V.N.; Lacy, E.; Elkon, K.B.; Marino, M.W.; Nikoliczugić,

555

J. TNF-alpha is the critical mediator of the cyclic AMP-induced apoptosis of

556

CD8+4+double-positive thymocytes. J. Immunol. 2000, 164, 1689-1694.

557 558

(27)Brigelius-Flohé, R.; Banning, A.; Kny, M.; Bol, G.F. Redox events in interleukin-1 signaling. Arch. Biochem. Biophys. 2004, 423, 66-73.

559

(28)Yeh, H.Y.; Winslow, B.J.; Junker, D.E.; Sharma, J.M. In vitro effects of recombinant

560

chicken interferon-gamma on immune cells. J. Interferon. Cytokine. Res. 1999, 19,

561

687-691.

562

(29)Yamaguchi, E.; de Vries, J.; Yssel, H. Differentiation of human single-positive fetal

563

thymocytes in vitro into IL-4- and/or IFN-γ-producing CD4+ and CD8+ T cells. Int.

564

Immunol. 1999, 11, 593-603.

565 566 567 568

(30)Siemion, I.Z.; Kluczyk, A.; Cebrat, M. The peptide molecular links between the central nervous and the immune systems. Amino. Acids. 2005, 29, 161-176. (31)Lunin, S.M.; Novoselova, E.G. Thymus hormones as prospective anti-inflammatory agents. Expert Opin. Ther. Targets. 2010, 14, 775-786.

569

(32) Zhang, L.; Wang, G.; Chen, S.; Ding, J.; Ju, S.; Cao, H.; Tian, H. Depletion of

570

thymopoietin inhibits proliferation and induces cell cycle arrest/apoptosis in

571

glioblastoma cells. World. J. Surg. Oncol. 2016, 14, 267-275.

572 573

(33)Uddin, M.N.; Nishio, N.; Ito, S.; Suzuki, H.; Isobe, K. Autophagic activity in thymus and liver during aging. Age. 2012, 34, 75-85.

574

(34)Sidler, C.; Wóycicki, R.; Ilnytskyy, Y.; Metz, G.; Kovalchuk, I.; Kovalchuk, O.

575

Immunosenescence is associated with altered gene expression and epigenetic regulation 26 ACS Paragon Plus Environment

Page 26 of 40

Page 27 of 40

576 577 578 579 580 581 582 583 584

Journal of Agricultural and Food Chemistry

in primary and secondary immune organs. Front. Genet. 2013, 4, 1-16. (35)Savino, W. The thymus is a common target organ in infectious diseases. PLoS. Pathog. 2006, 2, e62. (36)Hale, J.S.; Boursalian, T.E.; Turk, G.L.; Fink, P.J. Thymic output in aged mice. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 8447-8452. (37)Kannan, K.; Jain, S.K. Oxidative stress and apoptosis. Pathophysiology. 2000, 7, 153-163. (38)Suzuki, Y.J.; Forman, H.J.; Sevanian, A. Oxidants as stimulators of signal transduction. Free. Radical. Biol. Med. 1997, 22, 269-285.

585

(39)Benammar, C.; Hichami, A.; Yessoufou, A.; Simonin, A.; Belarbi, M.; Allali, H.; Khan,

586

N. Zizyphus lotus, L. (Desf.) modulates antioxidant activity and human T-cell

587

proliferation. BMC Complement. Altern. Med. 2010, 10, 1-9.

588

(40)Puthpongsiriporn, U.; Scheideler, S.E.; Sell, J.L.; Beck, M.M. Effects of vitamin E and

589

C supplementation on performance, in vitro lymphocyte proliferation, and antioxidant

590

status of laying hens during heat stress. Poult. Sci. 2001, 80, 1190-1200.

591 592 593 594

(41)Sinha, K.; Das, J.; Pal, P.B.; Sil, P.C. Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch. Toxicol. 2013, 87, 1157-1180. (42)Hu, Q.; Li, S.; Qiao, E.; Tang, Z.; Jin, E.; Jin, G.; Gu, Y. Effects of boron on structure and antioxidative activities of spleen in rats. Biol. Trace Elem. Res. 2014, 158, 73-80.

595

(43)Marquis, M.; Daudelin, J.F.; Boulet, S.; Siroisa, J.; Craina, K.; Mathiene, S.; Turgeone,

596

B.; Rousseaue, J.; Melochec, S.; Labrecquea, N. The catalytic activity of the MAP

597

kinase ERK3 is required to sustain CD4+CD8+ thymocyte survival. Mol. Cell. Biol.

598

2014, 34, 3374-3387. 27 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

599 600

(44)Mebratu, Y.; Tesfaigzi, Y. How ERK1/2 activation controls cell proliferation and cell death: Is subcellular localization the answer?. Cell. Cycle. 2009, 8, 1168-1175.

601

(45)He, Z.; Jiang, J.; Kokkinaki, M.; Golestaneh, N.; Hofmann, M.C.; Dym, M. Gdnf

602

upregulates c-Fos transcription via the Ras/Erk1/2 pathway to promote mouse

603

spermatogonial stem cell proliferation. Stem. Cells. 2008, 26, 266-278.

604

(46)Huang, F.; Xiong, X.; Wang, H.; You, S.; Zeng H. Leptin-induced vascular smooth

605

muscle cell proliferation via regulating cell cycle, activating ERK1/2 and NF-κB. Acta.

606

Biochim. Biophys. Sin. 2010, 42, 325-331.

607

(47)Ohashi, K.; Nagata, Y.; Wada, E.; Zammit, P.S.; Shiozuka, M.; Matsuda, R. Zinc

608

promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK

609

signaling cascade. Exp. Cell. Res. 2015, 333, 228-237.

610 611

(48)Lu, Z.; Xu, S. ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life. 2006, 58, 621-631.

612

(49)Lee, Y.J.; Cho, H.N.; Soh, J.W.; Jhon, G.J.; Cho, C.K.; Chung, H.Y.; Bae, S.; Lee, S.J.;

613

Lee, Y.S. Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation.

614

Exp. Cell. Res. 2003, 291, 251-266.

615

(50)Zhao, Y.; Wu, K.; Yu, Y.; Li, G. Roles of ERK1/2 MAPK in vitamin E

616

succinate-induced apoptosis in human gastric cancer SGC-7901 cells. Wei Sheng Yan

617

Jiu. 2003, 32, 573-575.

618

(51)Sirois, J.; Daudelin, J.F.; Boulet, S.; Marquis, M.; Meloche, S.; Labrecque, N. The

619

atypical MAPK ERK3 controls positive selection of thymocytes. Immunology. 2015,

620

145, 161-169.

621

(52)Smirnova, I.S.; Chang, S.; Forsthuber, T.G. Prosurvival and proapoptotic functions of 28 ACS Paragon Plus Environment

Page 28 of 40

Page 29 of 40

622

Journal of Agricultural and Food Chemistry

ERK1/2 activation in murine thymocytes in vitro. Cell. Immunol. 2010, 261, 29-36.

623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644

29 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 40

645

FIGURE CAPTIONS

646

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