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Overexpression and lowexpression of selenoprotein S impact ochratoxin A-induced porcine cytotoxicity and apoptosis in vitro Fang Gan, Zhihua Hu, Yajiao Zhou, and Kehe Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02115 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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Journal of Agricultural and Food Chemistry

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Overexpression and lowexpression of selenoprotein S impact

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ochratoxin A-induced porcine cytotoxicity and apoptosis in vitro

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Fang Gana,b, Zhihua Hua,b, Yajiao Zhoua,b, Kehe Huanga,b∗

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a

5

Jiangsu Province, China

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b

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Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095,

Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls,

8 9 10

∗

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Tel: +86-25-84395507

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Fax: +86-25-84398669

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E-mail address: [email protected]

Correspondence to: Prof. Kehe Huang

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ACS Paragon Plus Environment


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Abstract:

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Our previous study demonstrated that selenium could alleviate ochratoxin A

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(OTA)-induced nephrotoxicity in PK15 cells. Selenoprotein S (SelS) has antioxidant

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activities, but it is unclear whether SelS plays a role in the alleviating effects of

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selenium on OTA-induced nephrotoxicity. We previously have stably transfected pig

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pCDNA3.1-SelS to PK15 cells to over-express SelS. Here, we demonstrated that SelS

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overexpression alleviated OTA-induced cytotoxicity and apoptosis as demonstrated by

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cell viabilities, LDH activities, Annexin V-bing, caspase 3 activities and apoptotic

25

nuclei. SelS overexpression increased glutathione (GSH) levels and decreased

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reactive oxygen species (ROS) and malondialdehyde levels in PK15 cells, regardless

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of OTA treatment. SelS overexpression inhibited OTA-induced p38 phosphorylation.

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Adding buthionine sulfoximine reversed all of the above SelS-induced changes. In

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addition, Knockdown of SelS by SelS-specific siRNA decreased GSH levels,

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increased ROS levels, and aggravated OTA-induced p38 phosphorylation.

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Knockdown of SelS aggravated OTA-induced cytotoxicity and apoptosis in PK15

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cells. These data indicate that pig SelS overexpression and lowexpression impact

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OTA-induced cytotoxicity and apoptosis by modulating the oxidative stress and p38

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phosphorylation. Our work provides new insights into the relationship between SelS

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and OTA-induced cytotoxicity and apoptosis and describes an antitoxic mechanism of

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action for Se.

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Key words: Selenoprotein S; ochratoxin A; cytotoxicity; apoptosis; oxidative stress;

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p38 signal pathway; PK15 cells

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Introduction

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Ochratoxins are secondary metabolic products of several species of Aspergillus

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and Penicillium (1). Ochratoxin A (OTA) shows the highest toxicity among

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ochratoxins, and is a world-wide mycotoxin that naturally occurs in food and feeds

43

such as corn silage, barley, oats, rye, wheat, and other plant products (2). As

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widespread presence in food and feeds, animals and humans are frequently exposed to

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OTA. It has been demonstrated that OTA has been identified as a nephrotoxin in

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animals and humans (3, 4), and the mechanisms underlying OTA-induced

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nephrotoxicity are associated with the ability of OTA to generate the reactive oxygen

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species (ROS), disturb antioxidant enzymes (5, 6) and p38 mitogen-activated protein

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kinase (MAPK) signal transduction pathways (7), one MAPK that is involved in the

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regulation of cell proliferation, differentiation, and apoptosis (8, 9). In addition, our

51

previous study demonstrated that selenium (Se), an antioxidant trace element, has

52

been

53

concentration-dependent manner (10). However, it is still not clear how Se

54

supplementation alleviates OTA-induced toxicity.

shown

to

alleviate

OTA-induced

toxicity

in

PK-15

cells

in

a

55

Selenium (Se), an antioxidant trace element for humans and animals, plays a key

56

role in redox regulation and antioxidant defense (11). The biological effects of Se are

57

due to its incorporation into the selenocysteine and further into the selenoproteins

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such as glutathione peroxidase (GPx), thioredoxin reductases (TRs), and

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endoplasmic-reticulum selenoproteins (12, 13). We previously reported that GPx1

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knockdown aggravated OTA-induced nephrotoxicity and reversed the ability of Se to

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alleviate OTA-induced nephrotoxicity (10). However, further work is needed to

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determine the roles of other selenoproteins in OTA-induced toxicity.

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Selenoprotein S (SelS), an important selenoprotein, is expressed in a pancreatic β

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cell line, human endothelial cells (ECs), and porcine liver, kidney, and muscle (14, 15).

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High SelS levels protected, and SelS knockdown increased human ECs from

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H2O2-induced oxidative injury in human endothelial cells (16) and vascular smooth

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muscle cells (17). In addition, SelS-siRNA increased LPS-induced production of ROS

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in hepatoma HepG2 cells (18). These reports indicate that SelS has antioxidation in

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humans. In addition, our previous study indicated that we successfully constructed

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PK15 cell lines that overexpress SelS (19). However, whether pig SelS

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overexpression and lowexpression affects OTA-induced cytotoxicity and apoptosis is

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unknown.

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The objective of this study was to investigate the effects of pig SelS

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overexpression and lowexpression on OTA-induced cytotoxicity and apoptosis in

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PK15 cells.

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Materials and methods

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Cell culture

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The porcine kidney epithelial 15 (PK15) cells were obtained from the China

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Institute of Veterinary Drug Control, and maintained in Dulbecco’s minimal Eagle’s

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medium (DMEM, Invitrogen, USA) supplemented with heat-inactivated 8% fetal

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bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a

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humidified atmosphere containing 5% CO2. Ochratoxin A (OTA) stock solution (2

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mg/mL) used in the experiments was prepared by dissolving OTA in DMSO (100%).

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Final concentrations of OTA were obtained by dilution in the culture medium. DMSO

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was added to cells without OTA treatment in the final concentration of 0.2%.

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Construction of the PK 15 cell lines with over-expression of SelS

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PK 15 cell lines with over-expression of SelS were constructed as described in

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our previous study (19). Briefly, SelS over-expression plasmid (pc-SelS) was

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constructed by using pcDNA3.1 eukaryotic expression vector, then pc-SelS was

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transfected using X-tremeGENE transfection reagent (Roche) into PK15 cells cultured

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in DMEM supplemented with 8% FBS. To select stable transfectants, cells were

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grown in complete medium supplemented with 400 mg/ml Geneticin G418 antibiotics

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(Invitrogen). Control cells were prepared by transfecting PK15 cells with the empty

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pCDNA3.1 construct and then selecting resistant clones as above. Positive and stably

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transfected PK15 cells in DMEM with 8% FBS were analyzed for porcine SelS

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mRNA levels by real-time PCR and for SelS protein expression by western-blot.

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Assessment of cell viability by MTT in PK15 cells

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PK15 cells were cultured for 72 h in 96-well plates and subjected to the colorimetric

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

(MTT)

100

assay (Sigma, USA). Absorbance was measured at 490 nm with a secondary

101

wavelength of 650 nm. All tests were performed with four replicates. Cell viability

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was calculated as % of control cells.

103

Assessment of lactate dehydrogenase (LDH) release from PK15 cells

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PK15 cells were cultured in 12-well plates at a density of 8 × 104 cells/well with

5



105

corresponding treatment. After the treatment, the culture medium was collected in

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1.5-mL Eppendorf tubes and centrifuged at 12,000 rpm for 15 min at 4 °C. The

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supernatants were stored at –20 °C for the assessment of LDH activity. LDH activity

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was determined by using commercially available kits according to the manufacturer's

109

instructions (Jiancheng, China). Absorbance was measured at a wavelength of 450 nm.

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The data were expressed as percentage of the control values.

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Assessment of caspase-3 activity of PK15 cells

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PK15 cells were cultured in 6-well plates at a density of 2 × 105 cells/well with

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corresponding treatment. At the end of the treatment, caspase-3 activity in PK15 cells

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was assessed using the colorimetric assay kit (KeyGEN, China) according to the

115

manufacturer’s instructions as described previously (10). Caspase-3 activity was

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calculated as OD (inducer)/OD (negative control) and expressed as percentage of

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control values.

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Assessment of morphological changes and annexin V binding of PK15 cells

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PK15 cells were cultured at a density of 2 × 105 cells/well in 6-well plates with

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corresponding treatment. Morphology of apoptotic cell nuclei was detected by

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staining with the DNA binding fluorochrome Hoechst 33258 (bisbenzimide).

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Apoptosis was monitored by annexin V/PI (BD Pharmingen™) method as described

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previously (20) with minor modification. Briefly, after removing the culture medium,

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cells were washed two times with PBS, then resuspended in 100 µL of 1× binding

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buffer, and incubated with 5 µL of annexin V and 5 µL of PI at 25°C in the dark for 15

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min. For flow cytometric analysis, the reaction volume was raised to 500 µL by

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adding binding buffer, cells then were analyzed using FACS Calibur flow cytometry

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(BD Biosciences).

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Assessment of intracellular ROS levels by flow cytometry

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corresponding treatment and the intracellular ROS was measured as described

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previously using the oxidation-sensitive dye 2′,7′-dichlorofluorescein diacetate

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(DCFH-DA) (21). Briefly, after removing the culture medium, cells were washed

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three times with serum-free DMEM and incubated with 10 µM of DCFH-DA for 30

135

min at 37°C. Subsequently, the cells were washed three times with serum-free DMEM

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and re-suspended in PBS. Intracellular ROS level was expressed as % of the control

137

values.

138

Assessment of GSH and MDA levels

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140

corresponding treatment and GSH and MAD levels was measured as described

141

previously (22) using commercially available kits (Jiancheng, China) according to the

142

manufacturer's

143

(SonicsVCX105, USA) in ice-cold PBS and centrifuged at 12,000 rpm for 20 min to

144

remove debris. The supernatant fluid was collected and GSH levels determined at 412

145

nm by reaction with 5, 5’-dithiobis (2-nitrobenzoicacid). Malondialdehyde (MDA)

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levels were measured spectrophotometrically at 532 nm using the thiobarbituric acid

147

reaction method as described previously (23) by using commercially available kits

148

(Jiancheng, China). Total protein concentration was determined using a BCA protein

instructions.

Cell

extracts

were

prepared

7


by

sonication


149

assay kit (Beyotime, China). The data were expressed as nanomoles of GSH or MDA

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per milligram of protein.

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Determination of mRNA levels by real-time PCR

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SYBR green real-time PCR was performed to determine the levels of SelS

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mRNA in PK15 cells. The primer sequences for SelS target gene and β-actin (a

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control reference gene) were obtained from a published article (19, 24). Total RNA

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was extracted using the RNAiso Plus kit (TaKaRa, China) according to the

156

manufacturer's protocols. Potential DNA contamination of the extract was eliminated

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using the DNA-Free kit (TaKaRa) and the RNA quality was assessed indirectly from

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the ratio OD260/OD280. First-strand cDNA was synthesized and PCR was carried out

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using the ABI Prism Step One Plus detection system (Applied Biosystems, USA) as

160

described previously (11). The relative mRNA levels of target genes were determined

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using the ∆ cycle threshold (∆Ct) method with β-actin serving as a reference gene

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(22).

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Determination of protein expression by western blot

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For the assessment of protein expression, PK15 cells were cultured at a density

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of 2 × 105 cells/well in 6-well plates with corresponding treatment. After the treatment,

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cells were collected in 80 µl lysis buffer containing protease inhibitors (Beyotime,

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China) and were sonicated (SonicsVCX105, USA). The lysate was centrifuged at

168

12,000 rpm for 20 min at 4 °C and the supernatant was immediately collected for use.

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Protein concentration was determined using the BCA kit (Beyotime, China). Fifty µg

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of protein was diluted in sample loading buffer and heated at 95°C for 5 min. The

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denatured proteins were resolved by 12% sodium dodecyl sulphate-polyacrylamide

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gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The

173

membranes were incubated for 2 h at room temperature (RT) in Tris-buffered saline

174

(TBS) containing 5% milk (for SelS) or BSA (for β-actin, p38, p-p38, ERK1/2,

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p-ERK1/2, AKT and p-AKT), and 0.1% Tween 20 (TBST), followed by overnight

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incubation at 4°C in specific primary antibodies (anti-SelS from Santa Cruz

177

Biotechnology, diluted 1/500; anti-β-actin, anti-p38, anti-p-p38, anti-ERK1/2,

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anti-p-ERK1/2, anti-AKT and anti-p-AKT from Cell Signaling, diluted 1/1000). The

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membranes were washed and incubated in polyclonal anti-rabbit-horseradish

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peroxidase conjugated secondary antibody (Sigma) at RT for 1h. The blots were

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visualized and analyzed by a Luminescent Image Analyzer (FUJIFILM LAS-4000)

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and normalized to the control group.

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Small interfering RNA (siRNA) transfection

184

Three SelS-specific siRNAs were designed using the sequence of Sus scrofa SelS

185

mRNA (GenBank Accession No. NM_001164113) and Invitrogen BlockiT RNAi

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designer). Control siRNA sequences were obtained from a published paper (25), and

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SelS-specific siRNA sequences was 5’-GCUUUAGCAGCAGCUCGUUtt-3’ as

188

published in our previous study (19). The two double-stranded RNAs were

189

synthesized by Invitrogen. Duplexes were re-suspended in RNA-free water to obtain

190

20µM solutions before use. The duplexes were transiently transfected into PK15 cells

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via liposomes using X-tremeGENE transfection reagent (Roche). Briefly, PK15 cells

192

in DMEM with 8% FBS without antibiotics were cultured overnight at 37°C. When

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193

cells were 30–50% confluent, siRNA was introduced using the X-tremeGene siRNA

194

transfection reagent according to the manufacturer’s protocol. Transfection reagent

195

and siRNA (5:1) were added to each well and incubated for 5h. The cells were then

196

washed with DMEM and transferred to DMEM with 4% FBS.

197

Statistical analysis

198

One-way analysis of variance followed by Duncan’s multiple range tests were

199

used to determine differences between means using the SPSS computer program for

200

Windows (version 17.0). Results are expressed as the mean ± standard error (SE).

201

P-values of less than 0.05 were considered statistically significant.

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Results

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Overexpression of pig SelS increases SelS mRNA and protein levels in PK15 cells

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We have constructed the PK15 cell lines with overexpression of SelS. As shown

205

in Figure 1, the mRNA (Figure 1A) and protein (Figure 1B) levels of SelS were

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significantly increased in PK15 cells with overexpression of SelS as compared to that

207

in control and pcDNA3.1 vector-transfected cells.

208

SelS overexpression increases antioxidant capacity of PK15 cells

209

To determine whether SelS overexpression increased antioxidant capacity, GSH,

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ROS and MDA levels were measured in PK15 cells. As shown in Figure 2, viability

211

and LDH activity were similar in PK15 cells, vector-PK15 cells, and SelS-PK15 cells

212

(Figure 2A, B). SelS overexpression increased GSH levels (Figure 2C), and decreased

213

ROS and MDA levels (Figure 2D, E) compared to the control and empty vector

214

groups. These results suggest that SelS overexpression increases antioxidant capacity

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of PK15 cells.

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SelS overexpression increases cell viability after OTA treatment in PK15 cells

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To determine whether SelS overexpression could increase cell viability after

218

OTA treatment in PK15 cells, cell viability and LDH activity were measured. As

219

shown in Figure 3, OTA at 2.0 and 4.0 µg/ml significantly decreased cell viability and

220

increased LDH activity in PK15 cells and Vector-PK15 cells compared with OTA at

221

0.0 µg/ml. Overexpression of SelS in PK15 cells reversed the decrease of cell viability

222

(Figure 3A) and increase of LDH activity (Figure 3B) induced by OTA at 2.0 and 4.0

223

µg/ml. These results suggest that SelS overexpression increases cell viability after

224

OTA treatment in PK15 cells.

225 226 227

SelS overexpression alleviates OTA-induced cytotoxicity and apoptosis in PK15 cells To determine whether SelS overexpression could alleviate OTA-induced

228

cytotoxicity and apoptosis in PK15 cells, cell viability, LDH activity, caspase 3

229

activity, annexin V-bing and apoptotic nuclei were measured. As shown in Figure 4,

230

OTA at 2.0 µg/ml significantly decreased cell viability (Figure 4A), increased LDH

231

activity (Figure 4B), caspase 3 activity (Figure 4C), annexin V-bing (Figure 4D) and

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apoptotic nuclei (Figure 4E) in Vector-PK15 cells compared with OTA at 0.0 µg/ml.

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Overexpression of SelS in PK15 cells reversed the decrease of cell viability and

234

increase of LDH activity, caspase 3 activity, annexin V-bing and apoptotic nuclei

235

induced by OTA at 2.0 µg/ml. These results suggest that SelS overexpression

236

alleviates OTA-induced cytotoxicity and apoptosis in PK15 cells.

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237

SelS overexpression decreases OTA-induced oxidative stress in PK15 cells

238

To understand whether SelS overexpression could decrease OTA-induced

239

oxidative stress, GSH, ROS and MDA levels were measured in PK15 cells. As shown

240

in Figure 5, OTA treatments significantly decreased GSH levels (Figure 5A), and

241

increased ROS (Figure 5B), and MDA levels (Figure 5C) in Vector-PK15 cells

242

compared with OTA at 0.0 µg/ml. Overexpression of SelS in PK15 cells reversed the

243

parameters changes induced by OTA at 2.0 µg/ml. These results suggest that SelS

244

overexpression decreases OTA-induced oxidative stress in PK15 cells.

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SelS overexpression inhibits OTA-induced p38 phosphorylation in PK15 cells

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Next, we investigated the role of p38, ERK1/2 and AKT in SelS overexpression

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alleviating OTA-induced cytotoxicity in PK15 cells. As shown in Figure 6, OTA

248

treatments significantly increased p38, ERK1/2 and AKT phosphorylation in

249

Vector-PK15 cells compared without OTA. However, overexpression of SelS in PK15

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cells reversed the increases of p38 phosphorylation instead of ERK1/2 and AKT

251

phosphorylation induced by OTA. These results suggest that the alleviating effects of

252

SelS overexpression on OTA-induced cytotoxicity and apoptosis in PK15 cells may

253

be through p38 MAPK signal pathway.

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Oxidative stress plays a role in the alleviating effects of SelS overexpression on

255

OTA-induced cytotoxicity and apoptosis in PK15 cells

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SelS has antioxidation (16), and the present work indicates that SelS

257

overexpression increases antioxidant activity in PK15. We investigated whether SelS

258

overexpression in PK15 cells alleviated OTA-induced cytotoxicity and apoptosis by

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inhibiting oxidative stress. To address this question, we assessed the effects of 50 µM

260

buthionine sulfoximine (BSO) on SelS overexpression alleviating OTA-induced

261

cytotoxicity and apoptosis and oxidative stress. As shown in Figure 7, BSO abrogated

262

the protective effects of SelS overexpression against OTA-induced cytotoxicity and

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apoptosis as demonstrated by decreasing cell viability (Figure 7A), increasing LDH

264

activity (Figure 7B), Annexin V-bing (Figure 7C) and caspase 3 activity (Figure 7D)

265

compared with SelS group. SelS overexpression alleviated OTA-induced decreases of

266

GSH levels (Figure 7E) and increases of ROS levels (Figure 7F) in PK15 cells, and

267

BSO reversed these changes. These results indicate that SelS overexpression

268

alleviates OTA-induced cytotoxicity and apoptosis by inhibiting oxidative stress.

269

Effects of SelS-siRNA on SelS expression in vector-PK15 cells

270

To evaluate the extent of SelS knockdown, Vector-PK15 cells were cultured

271

overnight and then transfected with SelS-specific siRNA or control siRNA. As shown

272

in Figure 8, transfection of PK15 cells with SelS-specific siRNA resulted in

273

significant reduction in SelS mRNA (Figure 8A) and protein levels (Figure 8B).

274

SelS-siRNA increases OTA-induced oxidative stress and p38 phosphorylation in

275

vector-PK15 cells

276

To evaluate whether SelS-siRNA could increase OTA-induced oxidative stress

277

and p38 phosphorylation, Vector-PK15 cells were cultured overnight and then

278

transfected with a control-siRNA or SelS-siRNA. After 5 h of transfection treatment,

279

the medium was removed and fresh basal medium was added, and cells were then

280

incubated with 2.0 µg/mL of OTA for an additional 48 h. Results are shown in Figure

13



281

9, OTA at 2.0 µg/ml decreased GSH levels (Figure 9A), increased ROS levels (Figure

282

9B), and p38 phosphorylation (Figure 9C). SelS knockdown significantly aggravates

283

these parameters changes induced by OTA (Figure 9).

284

SelS-siRNA aggravates OTA-induced cyotoxicity and apoptosis in PK15 cells

285

Next, we used SelS-siRNA to confirm that SelS overexpression alleviates

286

OTA-induced cyotoxicity and apoptosis. Vector-PK15 cells were cultured overnight

287

and then transfected with a control-siRNA or SelS-siRNA. After 5 h of transfection

288

treatment, the medium was removed and fresh basal medium was added, and cells

289

were then incubated with 2.0 µg/mL of OTA for an additional 48 h. Results are shown

290

in Figure 10, OTA at 2.0 µg/ml induced cytotoxicity and apoptosis by decreasing the

291

cell viability (Figure 10A), increasing LDH activity (Figure 10B), caspase-3 activity

292

(Figure 10C), annexin V-binding (Figure 10D) and apoptotic nuclei (Figure 10E).

293

SelS overexpression (pc-SelS+OTA group) alleviated OTA-induced cytotoxicity and

294

apoptosis compared to the control-siRNA+OTA group (Figure 10). In contrst, SelS

295

knockdown significantly aggravated the OTA-induced cytotoxicity and apoptosis

296

compared to the control-siRNA+OTA group (Figure 10). These results suggest that

297

SelS knockdown aggravates OTA-induced cytotoxicity and apoptosis in PK15 cells.

298

Discussion

299

It has been reported that approximately twenty-five known selenoproteins are

300

characterized (26), and have function in catalyzing redox reactions and defending

301

cells against oxidative stress (27). In addition, some studies reported that some

302

selenoproteins may regulate mycotoxin-induced toxicity. For example, GPx1

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knockdown increased OTA-induced cytotoxicity (10), SelS-siRNA or GPx1-siRNA

304

increased AFB1-induced immune toxicity in primary porcine splenocytes (28).

305

However, little is known concerning the relationship between SelS and OTA-induced

306

toxicity.

307

SelS, a new selenoprotein, has been developed to study its antioxidant functions

308

among the known selenoproteins (16, 29). Our previous study successfully

309

constructed a pig SelS-plasmid and PK15 cell lines overexpressing SelS and

310

demonstrated that pig SelS also has antioxidation (19). Here, we demonstrated that

311

SelS overexpression alleviated OTA-induced cytotoxicity and apoptosis as

312

demonstrated by increasing cell viability, decreasing LDH activity, caspase-3 activity,

313

annexin V-binding and apoptotic nuclei. In contrast, knock-down of SelS by

314

its-specific siRNA aggravated OTA-induced cytotoxicity and apoptosis. These results

315

suggest that SelS overexpression alleviates OTA-induced cytotoxicity and apoptosis

316

in PK15 cells.

317

What is the mechanism of SelS overexpression alleviating OTA-induced

318

cytotoxicity and apoptosis? Several previous studies showed that OTA-induced

319

cytotoxicity is associated with oxidative stress (30-32). In addition, it has been

320

reported that SelS overexpression increases antioxidant activity (16, 19, 33). We

321

speculate that SelS overexpression alleviates OTA-induced cytotoxicity by reducing

322

oxidative stress. In the present study, SelS overexpression reversed OTA-induced

323

decreases in GSH levels, as well as OTA-induced increases in ROS levels and MDA

324

levels in PK15 cells. SelS lowexpression has the opposite effects on OTA-induced

15



325

oxidative stress. In addition, we used BSO, a specific inhibitor of glutamate-cysteine

326

ligase which causes oxidative stress (34, 35), to confirm the function of SelS in the

327

present work. BSO decreased GSH levels, increased ROS production, and eliminated

328

the alleviating effects of SelS overexpression on OTA-induced cytotoxicity. These

329

results strongly support the hypothesis that SelS overexpression alleviates

330

OTA-induced cytotoxicity by inhibiting oxidative stress.

331

Further, we investigated the signal pathway mechanism in the alleviating effects

332

of SelS overexpression on OTA-induced cytotoxicity and apoptosis. It has been

333

reported that Se regulates MAPK signaling pathways. Se supplementation inhibited

334

p38, ERK and JNK phosphorylation (36). In contrast, Se deficiency increased p38,

335

JNK, and ERK protein phosphorylation in chickens (37). p38 phosphorylation was

336

also increased in GPx1-knockdown mice (38). In addition, our previous work

337

indicated that OTA induced cytotoxicity and apoptosis by activating the p38 signaling

338

pathways in PK15 cells (7). Thus, we propose that the alleviating effects of SelS

339

overexpression on OTA-induced cytotoxicity and apoptosis are due in part to

340

inhibition of the p38 signaling pathways. The present results show that 2.0 µg/ml of

341

OTA induced p38 phosphorylation. SelS overexpression in turn inhibited p38

342

phosphorylation induced by OTA in PK15 cells. In contrast, SelS knockdown

343

aggravated OTA-induced p38 phosphorylation. The results suggest that SelS

344

overexpression alleviates OTA-induced cytotoxicity and apoptosis through p38 signal

345

pathway.

346

In conclusion, pig SelS overexpression alleviated OTA-induced cytotoxicity and

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apoptosis, and SelS knockdown had the opposite effects. In addition, the alleviating

348

effects of SelS overexpreesion were due to its ability to inhibit oxidative stress and

349

p38 signaling pathway activation. Our work provides new insights into the

350

relationship between SelS and OTA-induced cytotoxicity and apoptosis and describes

351

an antitoxic mechanism of action for Se.

352

Authors' contributions Study concept and design: KH. Acquisition of data: FG, HH, ZJ, KH. Analysis

353 354

and interpretation of data: FG, HH. Drafting of the manuscript: FG, KH. Critical

355

revision of the manuscript for important intellectual content: KH, FG. Statistical

356

analysis: FG, KH. Obtained funding: KH and FG. All authors read and approved the

357

final manuscript.

358

Acknowledgments

359

This work was funded by the National Natural Science Foundation of China

360

(31472253, 31602123), National Key R & D Program (2016YFD0501203), Natural

361

Science Foundation of Jiangsu Province (BK20160736), Fundamental Research

362

Funds for the Central Universities (KJQN201710), and the Priority Academic

363

Program Development of Jiangsu Higher Education Institutions (Jiangsu, China).

364

Author Disclosure Statement We do not have any commercial associations that might create a conflict of

365 366

interest in connection with this article.

367

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495 496 497 498 499 500

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501

Figure legends

502

Figure 1. SelS mRNA and protein levels in PK 15 cells with overexpression of SelS.

503

SelS mRNA (A) and protein (B) levels in PK15 cells with overexpression of SelS

504

were determined using real-time PCR and western blotting as described in Materials

505

and Methods. Data are presented as means ± SE. *P < 0.05 and **P < 0.01 vs. control.

506

#

507

Figure 2. SelS overexpression increased antioxidant capacity of PK15 cells.

508

SelS-overexpressing PK15 cells were incubated for 72 h in DMEM. The cell viability

509

(A), LDH activity (B), GSH levels (C), ROS levels (D) and MDA levels (E) were

510

assayed as described in the Materials and Methods. Data are presented as means ± SE.

511

*

512

Figure 3. SelS overexpression increased cell viability after OTA treatment in PK15

513

cells. PK15 cells were cultured for 24 h and then treated for an additional 48 h with

514

OTA at 0.0, 1.0, 2.0 and 4.0 µg/ml. Cells were assayed for cell viability (A) and LDH

515

activity (B). Data are presented as means ± SE. *P < 0.05 and **P < 0.01 vs. control

516

(without OTA). #P < 0.05 and ##P < 0.01 vs. control cells. $P < 0.05 and $$P < 0.01 vs.

517

Vector cells.

518

Figure 4. SelS overexpression alleviated OTA-induced cytotoxicity and apoptosis in

519

PK15 cells. PK15 cells overexpressing vector or SelS were cultured for 24 h and then

520

treated for an additional 48 h with or without OTA at 2.0 µg/ml. Cells were assayed

521

for cell viability (A), LDH activity (B), caspase 3 activity (C), annexin V-bing (D) and

522

apoptotic nuclei (E). Data are presented as means ± SE. *P < 0.05 and **P < 0.01.

P < 0.05 and ##P < 0.01 vs. vector control.

P < 0.05 and **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. vector control.

24


Page 24 of 37

Page 25 of 37


523

Figure 5. SelS overexpression decreased OTA-induced oxidative stress in PK15 cells.

524

PK15 cells overexpressing vector or SelS were cultured for 24 h and then treated for

525

an additional 48 h with or without OTA at 2.0 µg/ml. Cells were assayed for GSH

526

levels (A), ROS levels (B), and MDA levels (C). Data are presented as means ± SE.

527

*

528

Figure 6. SelS overexpression inhibited OTA-induced p38 phosphorylation in PK15

529

cells. PK15 cells overexpressing vector or SelS were cultured for 24 h and then

530

treated for an additional 48 h with or without OTA at 2.0 µg/ml. Cells were assayed

531

for p38, p-p38, ERK1/2, p-ERK1/2, AKT and p-AKT protein levels. Data are

532

presented as means ± SE. *P < 0.05 and **P < 0.01 vs. vector cells (without OTA). #P

533

< 0.05 and ##P < 0.01 vs. vector cells (with OTA).

534

Figure 7. Oxidative stress plays a role in the alleviating effects of SelS

535

overexpression on OTA-induced cytotoxicity and apoptosis in PK15 cells. PK15 cells

536

overexpressing vector or SelS were cultured with or without 50 µM BSO for 24 h and

537

then treated for an additional 48 h with or without OTA at 2.0 µg/ml. Cells were

538

assayed for cell viability (A), LDH activity (B), caspase 3 activity (C), annexin V-ding

539

(D), GSH levels (E) and ROS levels (F). Data are presented as means ± SE.

540

Significance compared with control, *P < 0.05 and **P < 0.01. Significance compared

541

with cells with OTA treatment, #P < 0.05 and ##P < 0.01. Significance compared with

542

cells with OTA and SelS overexpression, $P < 0.05 and $$P < 0.01.

543

Figure 8. Effect of SelS-siRNA on SelS expression in vector-PK15 cells. SelS mRNA

544

levels (A) and SelS protein levels (B) were assayed. Data are presented as mean ± SE.

P < 0.05 and **P < 0.01.

25



Page 26 of 37

545

Significance compared with control, *P < 0.05 and **P < 0.01.

546

Figure

547

phosphorylation in vector-PK15 cells. GSH levels (A), ROS levels (B), and p-p38

548

protein levels (C) were assayed. Data are presented as mean ± SE. Significance

549

compared with control, *P < 0.05 and

550

treatment, #P < 0.05 and ##P < 0.01.

551

Figure 10. Effects of SelS-siRNA on OTA-induced cytotoxicity and apoptosis in

552

vector-PK15 cells. Cell viability (A), LDH activity (B), caspase-3 activity (C) and

553

annexin V-binding (D) and apoptotic nuclei (E) were assayed. Data are presented as

554

mean ± SE. Significance compared with control, *P < 0.05 and

555

Significance compared with OTA treatment, #P < 0.05 and ##P < 0.01.

9.

SelS-siRNA increased

OTA-induced

oxidative

stress

and

p38

**

P < 0.01. Significance compared with OTA

26


**

P < 0.01.

Page 27 of 37


Figure 1. SelS mRNA and protein levels in PK 15 cells with overexpression of SelS. SelS mRNA (A) and protein (B) levels in PK15 cells with overexpression of SelS were determined using real-time PCR and western blotting as described in Materials and Methods. Data are presented as means ± SE. *P < 0.05 and **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. vector control. 127x62mm (300 x 300 DPI)



Figure 2. SelS overexpression increased antioxidant capacity of PK15 cells. SelS-overexpressing PK15 cells were incubated for 72 h in DMEM. The cell viability (A), LDH activity (B), GSH levels (C), ROS levels (D) and MDA levels (E) were assayed as described in the Materials and Methods. Data are presented as means ± SE. *p < 0.05 and **p < 0.01 vs. control. #p < 0.05 and ##p < 0.01 vs. vector control. 136x69mm (300 x 300 DPI)


Page 28 of 37

Page 29 of 37


Figure 3. SelS overexpression increased cell viability after OTA treatment in PK15 cells. PK15 cells were cultured for 24 h and then treated for an additional 48 h with OTA at 0.0, 1.0, 2.0 and 4.0 µg/ml. Cells were assayed for cell viability (A) and LDH activity (B). Data are presented as means ± SE. *p < 0.05 and **p < 0.01 vs. control (without OTA). #p < 0.05 and ##p < 0.01 vs. control cells. $p < 0.05 and $$p < 0.01 vs. Vector cells. 90x32mm (300 x 300 DPI)



Figure 4. SelS overexpression alleviated OTA-induced cytotoxicity and apoptosis in PK15 cells. PK15 cells overexpressing vector or SelS were cultured for 24 h and then treated for an additional 48 h with or without OTA at 2.0 µg/ml. Cells were assayed for cell viability (A), LDH activity (B), caspase 3 activity (C), annexin V-bing (D) and apoptotic nuclei (E). Data are presented as means ± SE. *p < 0.05 and **p < 0.01. 185x130mm (300 x 300 DPI)


Page 30 of 37

Page 31 of 37


Figure 5. SelS overexpression decreased OTA-induced oxidative stress in PK15 cells. PK15 cells overexpressing vector or SelS were cultured for 24 h and then treated for an additional 48 h with or without OTA at 2.0 µg/ml. Cells were assayed for GSH levels (A), ROS levels (B), and MDA levels (C). Data are presented as means ± SE. *p < 0.05 and **p < 0.01. 181x130mm (300 x 300 DPI)



Figure 6. SelS overexpression inhibited OTA-induced p38 phosphorylation in PK15 cells. PK15 cells overexpressing vector or SelS were cultured for 24 h and then treated for an additional 48 h with or without OTA at 2.0 µg/ml. Cells were assayed for p38, p-p38, ERK1/2, p-ERK1/2, AKT and p-AKT protein levels. Data are presented as means ± SE. *P < 0.05 and **P < 0.01 vs. vector cells (without OTA). #P < 0.05 and ##P < 0.01 vs. vector cells (with OTA). 208x159mm (300 x 300 DPI)


Page 32 of 37

Page 33 of 37


Figure 7. Oxidative stress plays a role in the alleviating effects of SelS overexpression on OTA-induced cytotoxicity and apoptosis in PK15 cells. PK15 cells overexpressing vector or SelS were cultured with or without 50 µM BSO for 24 h and then treated for an additional 48 h with or without OTA at 2.0 µg/ml. Cells were assayed for cell viability (A), LDH activity (B), caspase 3 activity (C), annexin V-ding (D), GSH levels (E) and ROS levels (F). Data are presented as means ± SE. Significance compared with control, *p < 0.05 and **p < 0.01. Significance compared with cells with OTA treatment, #p < 0.05 and ##p < 0.01. Significance compared with cells with OTA and SelS overexpression, $p < 0.05 and $$p < 0.01. 256x357mm (300 x 300 DPI)



Figure 8. Effect of SelS-siRNA on SelS expression in vector-PK15 cells. SelS mRNA levels (A) and SelS protein levels (B) were assayed. Data are presented as mean ± SE. Significance compared with control, *P < 0.05 and **P < 0.01. 138x80mm (300 x 300 DPI)


Page 34 of 37

Page 35 of 37


Figure 9. SelS-siRNA increased OTA-induced oxidative stress and p38 phosphorylation in vector-PK15 cells. GSH levels (A), ROS levels (B), and p-p38 protein levels (C) were assayed. Data are presented as mean ± SE. Significance compared with control, *P < 0.05 and **P < 0.01. Significance compared with OTA treatment, #P < 0.05 and ##P < 0.01. 187x153mm (300 x 300 DPI)



Figure 10. Effects of SelS-siRNA on OTA-induced cytotoxicity and apoptosis in vector-PK15 cells. Cell viability (A), LDH activity (B), caspase-3 activity (C) and annexin V-binding (D) and apoptotic nuclei (E) were assayed. Data are presented as mean ± SE. Significance compared with control, *P < 0.05 and **P < 0.01. Significance compared with OTA treatment, # < 0.05 and ##P < 0.01. 207x209mm (300 x 300 DPI)


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