Sodium selenite inhibits deoxynivalenol-induced injury in GPX1-knockdown porcine splenic lymphocytes in culture

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 μg/mL, group D1-4), Se (Na2SeO3, 2 μM, group Se), or both (group SD1–4) for 6, 12, or 24 h. 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 H 2 O 2 in red blood cells via GSH 19 . 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 system 20,21 . An appropriate increase in GPX1 expression can enhance the antioxidant capacity of cells 22,23 .
Our laboratory has shown that Se can reduce the damage to porcine spleen lymphocytes caused by DON-induced oxidative stress 19 , and can prevent the concomitant changes in cytokines induced in porcine spleen lymphocytes 24 . 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 (Na 2 SeO 3 ) 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. , the transfection efficiency of combination was 51.1% + 0.8% shown in (B), the transfection efficiency of combination was 71.3% + 1.3% shown in (C), the transfection efficiency of combination was 80.6% + 1.7% shown in (D), and the transfection efficiency of combination was 92.9% + 2% shown in (E). Therefore, we selected combination E for the subsequent experiment.

Scrambled siRNA Control
Relative expression (%) 28  Relative expression of GPX1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) proteins. Its show that there was no significant difference in the expression of GAPDH between the normal and knockdown groups. However, the expression of GPX1 differed significantly between the normal control and knockdown groups, and that GPX1-knockdown cells expressed only 36.9% of the GPX1 expressed by the normal group. Therefore, the knockdown efficiency was 63.1%. ## P < 0.01. 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, whereas the expression of GPX1 in the scrambled siRNA group was 99.4% of that in the control group. This suggests that there was no nonspecific gene silencing.

Results
Transfection efficiency of GPX1-directed small interfering RNA (siRNA) in porcine spleen lymphocytes. 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 knockdown.
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 (H 2 O 2 ), 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 H 2 O 2 and MDA were significantly higher in group M than in group P at each time point. Treatment with DON (0.824-0.103 μg/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 H 2 O 2 and MDA were significantly higher in the groups D1-4 than in group M. Treatment with Na 2 SeO 3 (2 μmol/L) alone significantly increased the activities of SOD and CAT, T-AOC, and free hydroxyl radical  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 Na 2 SeO 3 , the ROS content was significantly lower in the groups SD1-4 than in the groups D1-4, except for group SD1.

Discussion
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 H 2 O 2 , and thus protects the cell membrane. Yan 25 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 H 2 O 2 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 them 26 . When Kouadio et al. 27 added 5-40 μM DON to the Caco-2 cell line, there was a significant increase in the MDA content after 24 h. Li 28 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 6-48 h. 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 cells 19 . 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 H 2 O 2 was significantly higher in the GPX1-knockdown porcine splenic lymphocytes than in the normal porcine splenic lymphocytes because GPX1 decomposes H 2 O 2 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 H 2 O 2 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.  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 H 2 O 2 . 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 activity 32 . 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 cells 19 , 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  34 . 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 Na 2 SeO 3 to the group M cells, and showed that the levels of MDA and H 2 O 2 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 Tang 35 , who showed that Na 2 SeO 3 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 mycotoxins [36][37][38] . In this study, the levels of MDA and H 2 O 2 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 H 2 O 2 of GPx1 knockdown porcine splenic lymphocytes are lower than porcine splenic lymphocytes. These data indicate that the protective effects of Na 2 SeO 3 against DON-induced oxidative damage were reduced by GPX1 knockdown.
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, Na 2 SeO 3 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 animal production.  Table 9. Rates of change in the H 2 O 2 contents of GPX1-knockdown porcine splenic lymphocytes and normal porcine splenic lymphocytes at 6, 12, and 24 h after treatment.   Table 10. Rates of change in the MDA content of GPX1-knockdown porcine splenic lymphocytes and normal porcine splenic lymphocytes at 6, 12, and 24 h after treatment.

Groups
The change rates of SOD(%)  Table 11. Rates of change in the SOD activity of GPX1-knockdown porcine splenic lymphocytes and normal porcine splenic lymphocytes at 6, 12, and 24 h after treatment.

Groups
The change rates of CAT(%) 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 paper 24 . 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-iT TM 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. RFect PM 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 × 10 6 cells/mL. To determine the effects of DON and Se 14,24  Reverse transcription (RT)-qPCR analysis of GPX1 mRNA expression after siRNA transfection. For a description of the RT-qPCR performed, see the paper by Wang 26 . The primer sequencing for GPx1: (F-TGGGGAGATCCTGAATTG, R-GATAAACTTGGGGTCGGT) β-Actin was used as reference gence: (F-CTGCGGCATCCACGAAACT, R-AGGGCCGTGATCTCCTTCTG).

Detection of GPX1 protein with western blotting after transfection. The cells were washed twice
with precooled PBS and suspended in 300 μL 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 μg) 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 image analysis.

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 H 2 O 2 , 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 Na 2 SeO 3 was added were calculated by comparing the absolute values of KG-An with NG-An, to determine whether Na 2 SeO 3 was antagonistic to the porcine spleen lymphocyte by GPX1.

Data Availability
All data generated or analysed are valid during this study, included in this published article (and its Supplementary Information files).