Sodium selenite inhibits deoxynivalenol-induced injury in GPX1-knockdown porcine splenic lymphocytes in culture
Sodium selenite inhibits deoxynivalenol-induced injury in GPX1-knockdown porcine splenic lymphocytes in culture
OPEN Published: xx xx xxxx Deoxynivalenol (DON) is a cytotoxic mycotoxin that can cause cell damages. The main effect is to inhibit protein synthesis. Oxidative stress is one of the effects of DON. Selenium (Se) can ameliorate the cell damage caused by DON-induced oxidative stress, but it is unclear whether through selenoprotein glutathione peroxidase 1 (GPX1). We established GPX1-knockdown porcine spleen lymphocytes, and treated them with DON and Se. Untransfected porcine splenic lymphocytes (group P) and transfected cells (group M, GPX1 knockdown) were treated with or without DON (0.824, 0.412, 0.206, or 0.103?g/mL, group D1-4), Se (Na2SeO3, 2 ?M, group Se), or both (group SD1-4) for 6, 12, or 24h. The cells were collected and the activities of SOD and CAT, levels of GSH, H2O2, malonaldehyde (MDA), total antioxidant capacity (T-AOC), and the inhibition of free hydroxyl radicals were determined. Levels of ROS were measured at 24 h. Compared with group P, the antioxidant capacity of group M was reduced. DON caused greater oxidative damage to the GPX1-knockdown porcine splenic lymphocytes than to the normal control cells. When Na2SeO3 was combined with DON, it reduced the damage in the GPX1-knockdown porcine splenic lymphocytes, but less effectively than in the normal porcine splenic lymphocytes.
GPX1 was the first antioxidant enzyme shown to reduce H2O2 in red blood cells via GSH19. It is the most
important antioxidant enzyme in the body and is widely expressed during major cell division. It can remove free
radicals and peroxide from cells, and together with other antioxidant enzymes (catalase [CAT] and superoxide
dismutase [SOD]), constitutes the endogenous antioxidant defence system20,21. An appropriate increase in GPX1
expression can enhance the antioxidant capacity of cells22,23.
Our laboratory has shown that Se can reduce the damage to porcine spleen lymphocytes caused by
DON-induced oxidative stress19, and can prevent the concomitant changes in cytokines induced in porcine
spleen lymphocytes24. However, it remains unclear whether it antagonizes DON toxicity through the
selenoprotein GPX1. In this study, we established GPX1-knockdown porcine spleen lymphocytes and treated them sodium
selenite (Na2SeO3) and DON, singly or combined, in a culture system. We then measured the intracellular
antioxidant index and the ROS content of the GPX1-knockdown porcine spleen lymphocytes to determine the
protective effects of sodium selenite on DON-induced oxidative damage in these cells and whether Se acts through the
selenoprotein GPX1 in antagonizing the toxicity of DON.
Transfection efficiency of GPX1-directed small interfering RNA (siRNA) in porcine spleen lym
phocytes. The transfection efficiency of GPX1-directed siRNA in porcine spleen lymphocytes is shown in
Fig.?1. Transfection efficiency of GPX1-directed siRNA in porcine splenic lymphocytes. the blank control shown
in A. In B, C, D, E shown the different transfection efficiency of combination. Combination E has the best
transfection effect, we selected combination E for the subsequent experiment.
Expression of GPX1 mRNA after siRNA transfection. The relative expression of GPX1 mRNA after
siRNA transfection is shown in Table?1. The expression of GPX1 in the group of cells treated with the control
GPX1-directed siRNA was 28.4% of that in the control group, This suggests that there was nonspecific gene
Expression of GPX1 protein in porcine spleen lymphocytes after siRNA transfection. The
expression of the GPX1 protein in porcine spleen lymphocytes after siRNA transfection is shown in Fig.?2. That
GPX1-knockdown cells expressed only 36.9% of the GPX1 expressed by the normal group. Therefore, the
knockdown efficiency was 63.1%.
Antioxidant indices and ROS levels. The activities of SOD and CAT, the levels of GSH, hydrogen
peroxide (H2O2), and malonaldehyde (MDA), the total antioxidant capacity (T-AOC), and the ability to inhibit free
hydroxyl radicals are shown in Tables?2?8. The activities of SOD and CAT and the ability to inhibit free hydroxyl
radicals were significantly lower in group M than in group P at most time points (P < 0.01); the level of GSH was
significantly lower in group M than in group P at each time point, except at 6 h; and the levels of H2O2 and MDA
were significantly higher in group M than in group P at each time point. Treatment with DON (0.824?0.103 ?g/mL)
alone reduced the activities of SOD and CAT, the levels of GSH and T-AOC, and the ability to inhibit free
hydroxyl radicals significantly more strongly in group M than in the groups D1?4 at most time points; and the
levels of H2O2 and MDA were significantly higher in the groups D1?4 than in group M. Treatment with Na2SeO3
(2 ?mol/L) alone significantly increased the activities of SOD and CAT, T-AOC, and free hydroxyl radical
inhibition relative to those in group M, except for GSH at 6 h. The levels of H2O2 and MDA were significantly
lower in the groups D1?4 than in group M. When the lymphocytes were treated with both DON and Na2SeO3,
the activities of SOD and CAT, the levels of GSH and T-AOC, and the inhibition of free hydroxyl radicals were
significantly higher in group SD1?4 than in group D1?4 at most time points.
The rates of change in SOD, CAT, GSH, H2O2, MDA, T-AOC, and the inhibition of free hydroxyl radicals are
shown in Tables?9?15. Except in a few cases, most KG-An(the rates of change in the GPX1-knockdown porcine
splenic lymphocytes with knockdown group An) are less than NG-An(the change rates of our early achievements
were normal group An). The levels of ROS are shown in Table?16. The level of ROS was lowest in group P, whereas
group D1 had the highest ROS content. The ROS content was significantly higher in group M than in group P. The
ROS content was significantly higher in groups D1?4 than in group M, except for group D4. The ROS content was
significantly lower in group Se than that in group M. When the cells were treated with both DON and Na2SeO3,
the ROS content was significantly lower in the groups SD1?4 than in the groups D1?4, except for group SD1.
Oxidative stress occurs when the concentration of ROS exceeds the antioxidant capacity of the cell. When cells
cultured in vitro are subjected to oxidative stress, they are mainly protected by the enzymes of their own
antioxidant system, predominantly SOD, CAT, and GPX. GPX1 is the main GPX in spleen lymphocytes, and plays an
important role in protecting the cells against oxidative stress. Using GSH as its substrate, GPX1 participates in the
reduction of toxic peroxides, promotes the decomposition of H2O2, and thus protects the cell membrane. Yan25
knocked down the expression of GPX1 in ATDC5 cells with small hairpin RNA (shRNA), and found that the
antioxidant capacity of the cells decreased. Our results are similar insofar as after GPX1 expression was reduced,
the H2O2 content in group M increased as the incubation time increased, relative to that in group P, even at the
beginning of silence that the SOD and CAT might compensate. The MDA and ROS content of group M was
significantly higher than that of group P throughout the whole experiment (P < 0.01), whereas the GSH, SOD and
CAT activities, T-AOC, and the capacity of the cells to inhibit hydroxyl radicals were significantly lower in group
M. After GPX1 expression was knocked down in the porcine splenic lymphocytes, the antioxidant capacity of the
cells decreased compared with that in group P, and the oxidative stress in the cells caused them more damage.
The presence of large amounts of lipids in cells makes them highly susceptible to peroxide and the damage
caused by oxidative stress. The many lipid peroxidation products generated also have a toxic effect on the cells,
causing further damage. The cellular levels of important lipid peroxidation products, including MDA, indicate
the degree of lipid peroxidation and the amounts of free oxygen radicals in the cells, and can be used to indirectly
determine the degree of oxidative damage to them26. When Kouadio et al.27 added 5?40 ?M DON to the Caco-2
cell line, there was a significant increase in the MDA content after 24h. Li28 also showed a significant increase in
the MDA content after adding 100?2000 ng/mL DON to a chicken embryo fibroblasts (DF-1 cells) for 6?48h. In
the present study, the content of MDA in the GPX1-knockdown porcine splenic lymphocytes increased as the
DON concentration and the culture period increased. Therefore, our results are similar to those of the studies
described above. We compared the results of this experiment with the results of our experiment with prophase
cells19. After treatment with DON, the MDA content in the GPX1-knockdown porcine splenic lymphocytes was
significantly higher than in the normal porcine splenic lymphocytes, indicating that lipid peroxidation increased
in the cells after GPX1 knockdown. After the addition of DON, the content of H2O2 was significantly higher in
the GPX1-knockdown porcine splenic lymphocytes than in the normal porcine splenic lymphocytes because
GPX1 decomposes H2O2 and thus reduces DON-induced oxidative stress. T-AOC reflects the overall antioxidant
capacity of the cells and is a comprehensive indicator of the cells antioxidant system. In this study, the T-AOC
of the GPX1-knockdown porcine splenic lymphocytes decreased as the DON concentration and the incubation
time increased, and was significantly lower than that in the normal porcine splenic lymphocytes treated with the
same concentrations of DON for the same culture periods (P < 0.01). The results of this study are similar to those
of Hao et al.29. who added AFB1 to lymphocytes from the spleens of pigs in which GPX1 was knocked down.
Therefore, DON causes the levels of MDA and H2O2 to increase and the cellular T-AOC to decrease more severely
in GPX1-knockdown porcine splenic lymphocytes than in control cells. Our results also show that, compared
with the normal porcine splenic lymphocytes, the capacity of the GPX1-knockdown cells to inhibit hydroxyl
radicals decreased more dramatically as the DON concentration increased and the incubation time increased,
resulting in a greater accumulation of free radicals, a greater degree of oxidative stress, a greater reduction in
T-AOC, and therefore more-severe oxidative damage.
Oxidative damage occurs when the intracellular reactive oxygen concentration exceeds the cell?s antioxidant
capacity. ROS mainly include superoxide anions (?O2?), H2O2, and the hydroxyl radical (?OH). Cells scavenge
ROS through both enzymatic and non-enzymatic pathways. The enzymatic pathways consist of antioxidant
enzymes such as SOD, CAT, and GPX, and the non-enzymatic pathways involve GSH, Se, vitamin C, vitamin
E, and ?-carotene30. SOD uses the superoxide anion (?O2?) produced in cells as its substrate, producing reduced
SOD (SOD?) and O2, and then SOD? reacts with ?O2? to produce SOD and H2O2. H2O2 is then catalysed by CAT
and GPX to generate H2O and O231, thus protecting the cell membrane from damage. CAT is a terminal oxidase
that catalyses the decomposition of H2O2 into H2O and O2. GSH is a co-substrate of GPX, which catalyses it to
GSSG, thus reducing a toxic peroxide to a nontoxic hydroxyl compounds, and at the same time promoting the
decomposition of H2O2. This protects the cell membrane structure and function are safe from the oxide
interference and damage. Studies have shown that at lower GSH contents can result in decreased GPx1 activity32.
Therefore, after reactive oxygen is produced in cells, SOD acts as the first line of defence and CAT and GPX as the
second line of defence, acting together in the process of scavenging intracellular reactive oxygen. Our results show
that DON caused the activities of SOD and CAT to increase, and reduced the levels of GSH as the DON
concentration and incubation time increased, demonstrating the time and concentration dependence of its effects. The
results of this study are similar to those of Gan et al.33, who showed that when the expression of the GPX1 protein
was knocked down, the GSH content of the cells decreased significantly after ochratoxins (OTA) were added.
When the results of the present study were compared with the results of our study of prophase cells19, the SOD
and CAT activities and the levels of GSH were significantly lower in the GPX1-knockdown porcine splenic
lymphocytes when same concentrations of DON were added and the cells were cultured for the same time. This may
be because the cells themselves had a lower antioxidant capacity after GPX1 knockdown, and the intracellular
accumulation of ROS and the consumption of antioxidant enzymes and GSH were increased by the cytotoxicity
of DON and DON-induced oxidative stress.
Selenium is a necessary trace element in the diet of mammals because it plays an important role in many
organ systems and life activities. The antioxidant effects of Se have always been a research hotspot, and it mainly
occurs in selenocysteine and selenomethionine in selenoproteins, where it plays its antioxidant role. GPX is the
main selenium antioxidant enzyme in cells34. GPX has at least four isoenzymes, and GPX1 is the most strongly
expressed GPX in porcine splenic lymphocytes, where it plays an important role in ameliorating oxidative stress.
In this study, after knocking down GPX1 expression, we added Na2SeO3 to the group M cells, and showed that the
levels of MDA and H2O2 are significantly lower, and the activities of SOD and CAT, the levels of GSH and T-AOC,
and the capacity to inhibit hydroxyl radicals were significantly higher than group M. These results are similar to
the results of Tang35, who showed that Na2SeO3 had an antagonistic effect on GPX1-knockdown-induced
oxidative damage to porcine splenic lymphocytes.
A large number of studies have shown that Se prevents the oxidative stress induced by some mycotoxins36?38.
In this study, the levels of MDA and H2O2 were significantly lower in the groups SD1?4 than in the groups D1?4
(P < 0.05 or P < 0.01), and the activities of SOD and CAT, the levels of GSH, T-AOC, and the capacity of the
cells to inhibit hydroxyl radicals were higher in the groups SD1?4 than in the groups D1?4 mostly (P < 0.05
or P < 0.01). The rates of these changes in GPX1 knockdown porcine splenic lymphocytes were greater than in
the normal porcine splenic lymphocytes, note the elevated ratio of the activities of SOD and CAT, the levels of
GSH, T-AOC and the capacity of cells to inhibit hydroxyl radicals, the reduced ratio of the levels of MDA and
H2O2 of GPx1 knockdown porcine splenic lymphocytes are lower than porcine splenic lymphocytes. These data
indicate that the protective effects of Na2SeO3 against DON-induced oxidative damage were reduced by GPX1
In summary, our results demonstrate that the knockdown the GPX1 in porcine splenic lymphocytes reduces
their anti-oxidative capacity, and the cells? own oxidative stress causes them more damage than is caused in
normal cells?. DON caused greater oxidative damage in GPX1-knockdown porcine splenic lymphocytes than in
normal control cells. When combined with DON, Na2SeO3 ameliorated the DON-induced oxidative damage to
GPX1-knockdown porcine splenic lymphocytes, but its protective effects were less marked than in normal cells.
In the future, we will overexpress the GPX1 gene to in-depth study its effects, or to study spleen lymphocyte
organelles. These studies are required to understand the molecular mechanisms underlying these phenomena. Our
results also suggest that improved nutrition may be a novel approach to mitigating mycotoxin contamination in
Materials and Methods
Reagents. Foetal bovine serum was purchased from Gibco/Life Technologies (California, USA). Na2SeO3
powder was purchased from Xiya Reagent (Chengdu, China). DON was obtained from Sigma-Aldrich (USA).
RPMI-1640 medium was obtained from Boster Biological Technology Co., Ltd (Wuhan, China). Hank?s solution
and lymphocyte separation medium were obtained from Tianjin Hao Yang Biological Institute (China). Kits for
testing glutathione(GSH), malonaldehyde (MDA), total antioxidant capacity (T-AOC), glutathione peroxidase
(GPx), superoxide dismutase (SOD), catalase (CAT), hydrogen peroxide (H2O2), Hydroxyl Free Radical, were
obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Reactive Oxygen Species
Assay Kit was obtained from Beyotime Biotechnology (Shanghai, China). TRIzol Reagent was purchased from
Invitrogen Biotechnology Co., Ltd (Shanghai, China). PrimeScript? RT Reagent Kit and SYBR? Premix Ex Taq?
II were purchased from Takara (Shiga, Japan). The anti-GPX1 primary antibody (ab50427) and the rabbit
antigoat IgG H& L secondary antibody (ab6741) were from abcam.
Production and treatment of porcine splenic lymphocytes and the establishment of
GPX1-knockdown porcine spleen lymphocytes. For a description of the production of the porcine
spleen lymphocytes, refer to our earlier paper24. All study procedures were approved by the Institutional Animal
Care and Use Committee of Sichuan Agricultural University. All experiments were performed in
accordance with relevant guidelines and regulations. Based on a published sequence of porcine GPX1 mRNA
(GenBank NM-214201.0), siRNA was designed using Block-iTTM siRNA RNAi Designer (Thermo Fisher
Scientific). The sequence with the highest score was selected, which had the control siRNA sequence
5?-GGGACUACACCCAGAUGAATT-3?. The scrambled siRNA was synthesized by Thermo Fisher Scientific,
with the sequence 5?-UUCGUAUCUGGGUGUACCCTT-3?. The control siRNA sequence was confirmed to be
consistent with that reported by Gan et al.33. The FAM fluorescent marker was added to the siRNA as required.
RFectPM small nucleotide transfection agents was used for the transfection. To determine the optimal
concentration of siRNA and transfection reagent, we tested four combinations, according to the reagent instructions.
The specific information is shown in Table?17. The cells with the highest transfection efficiency were used for the
subsequent experiments. The expression of GPX1 mRNA was detected with quantitative real-time PCR (qPCR)
and the expression of GPX1 protein was detected 48 h after transfection with western blotting.
The prepared porcine spleen lymphocytes and GPX1-knockdown lymphocytes were cultured in triplicate in
six-well tissue culture plates at 3 ? 106 cells/mL. To determine the effects of DON and Se14,24, 11 groups of both
types of cells were treated with medium only or with DON and/or Se in the following combinations: Group
P (porcine splenic lymphocytes), group M (GPX1-knockdown porcine splenic lymphocytes), D1 (824 ng/mL
DON), D2 (412 ng/mL DON), D3 (206 ng/mL DON), D4 (103 ng/mL DON), Se (2 ?mol/L Na2SeO3),
SD1 (2 ?mol/L Na2SeO3 + 824 ng/mL DON), SD2 (2 ?mol/L Na2SeO3 + 412 ng/mL DON), SD3 (2 ?mol/L
Na2SeO3 + 206 ng/mL DON), and SD4 (2 ?mol/L Na2SeO3 + 103 ng/mL DON). The cells were incubated for 6,
12, or 24 h. The concentration of DON and Se and the time of cells were incubated have been determined in the
early stage of the laboratory. And the antioxidant indices were determined at each time point. The levels of ROS
were detected at 24 h.
Flow-cytometric determination of positive siRNA transfection efficiency. After transfection for
24 h, the cells were collected with centrifugation at 1800 r/min for 5 min at 4 ?C. The supernatant, was discarded
and the cells were washed twice with phosphate-buffered saline (PBS) at 4 ?C. The PBS cell suspension (100?L)
was precooled to 4 ?C and filtered through a 300 mesh filter. The cells were then analysed with flow cytometry.
Reverse transcription (RT)?qPCR analysis of GPX1 mRNA expression after siRNA transfec
tion. For a description of the RT?qPCR performed, see the paper by Wang26. The primer sequencing for GPx1:
(F- TGGGGAGATCCTGAATTG, R- GATAAACTTGGGGTCGGT) ?-Actin was used as reference gence:
(FCTGCGGCATCCACGAAACT, R- AGGGCCGTGATCTCCTTCTG).
Detection of GPX1 protein with western blotting after transfection. The cells were washed twice
with precooled PBS and suspended in 300 ?L of PBS. The cells were collected and homogenized on ice, and
phenylmethanesulfonyl fluoride was added to the protein lysates. After 40 min on ice, the lysates were centrifuged
at 12,000 rpm for 40 min at 4 ?C and the supernatants collected. The cellular protein was quantified with the
BCA method using bovine serum albumin as the standard. Samples of protein (50 ?g) were diluted in sample
loading buffer and heated at 95 ?C for 5 min. The denatured proteins were separated with 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membrane,
and placed in closed liquid for 1 h at 37 ?C. The primary antibody was added and the membranes incubated at
4 ?C. At the same time, another membrane was incubated without antibody in Tris-buffered saline containing
Tween 20 (TBS-T) as the negative control. After repeated washes, the membrane was incubated with a rabbit
anti-goat IgG H&L secondary antibody with gentle shaking for 1 h at room temperature. After the membrane
was washed, used western blot mark to observe, the absorbance (A) values were quantitatively analysed with an
Determination of antioxidant indices and levels of ROS. The antioxidant indices and the levels of
ROS in the cell preparations (supernatants, cell lysates, and cells) were measured according to the protocols of
the corresponding kits.
Statistical analysis. The test results are expressed as means standard? deviations. Excel was used to
preliminarily test and collate the results. The statistical software SPSS ver. 22 was used for later statistical analyses, and
Duncan?s method was used for multiple comparisons. The rates of change in some antioxidant indices, including
H2O2, MDA, SOD, CAT, GSH, T-AOC, and the inhibition of hydroxyl radicals, were calculated in the SD and Group
D1?4s (as follows). To express the rates of change in the GPX1-knockdown porcine splenic lymphocytes with
knockdown group An(KG-An), the change rates of our early achievements were normal group An(NG-An). The
changes in the antioxidant indices after Na2SeO3 was added were calculated by comparing the absolute values of
KG-An with NG-An, to determine whether Na2SeO3 was antagonistic to the porcine spleen lymphocyte by GPX1.
An = (SD1 ? 4/D1 ? 4 ? 1) ? 100% (n = 4)
All data generated or analysed are valid during this study, included in this published article (and its
Supplementary Information files).
This study was supported by the National Natural Science Foundation of China (General Program, grant no.
31402269). We thank XG Du for his assistance with the experiments and to D Li for experimental material.
We thank Janine Miller, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the
English text of a draft of this manuscript.
Z.H. Ren and Z.C. Zuo conceived the study. Y. Fan and Z. Zhang wrote the manuscript. C.X. Chen, X.M. Wang
and Z.W. Xu conducted the real-time P.C.R. experiments, S.Z. Cao, X.P. Ma and L.H. Shen analysed the results,
C.H. Chen and Y.C. Hu prepared the figures and tables, Z.Y. Zhou assisted with the RNA extractions. G.N. Peng,
S.M. Yu, Z.J. Zhong and J.L. Deng conducted the western blotting experiments, and determined the antioxidant
indices. All the authors have reviewed the manuscript.
Competing Interests: The authors declare no competing interests.
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Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36149-x.