Introduction

Pyrethroids, as a new generation of compounds in recent years, have gradually replaced traditional insecticides, which is inseparable from its low persistence, easy metabolism, high efficacy, and low toxicity to mammals and birds [1, 2]. Deltamethrin ([(S)-cyano-(3-phenoxyphenyl)methyl](1R,3R)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropane-1-carboxylate, DLM), an α-cyano type-II synthetic pyrethroid, is one of the most important pyrethroids first marketed in 1977 [2, 3]. DLM has been widely used within the agriculture and aquaculture based on its ability to effectively control and prevent pests such as mites, aphids, sea lice, grubs, beetles, and weevils [4,5,6]. Although DLM was initially thought to be less toxic, studies have demonstrated the toxicity of DLM on nontarget organisms, such as earthworm, lobster, zebrafish, and even humans [3, 7,8,9]. This is closely related to the residues of DLM to soil and water sources in the process of widespread use. Exposure to DLM may cause the damage of multiple tissue and organ toxicity, such as cardiac, neuro, hepatopancreas, cardiovascular, and testicular, and induce varying degrees of histopathological changes [10,11,12,13]. Hence, clarifying the key mechanism of DLM-induced potential injury may lead to beneficial therapeutic targets for DLM poisoning.

One of the main pathophysiological mechanisms by which DLM mediates organ injury is oxidative stress [14]. Studies have confirmed DLM-induced oxidative damage and biochemical alterations in organs and cells including hepatopancreas, embryos, and oocytes [15,16,17,18]. The cerebrum is an organ with high oxygen consumption and rich lipid, which might be vulnerable to oxidative stress caused by pesticides [19]. For instance, glutamate changed oxidative stress indices in adult albino rat cerebrum, leading potential oxidative damage [20]. Accordingly, we speculate that DLM may cause a certain degree of toxic effect on the cerebral antioxidant system, and the key regulatory factors of cerebral toxicity induced by DLM may still largely unknown. Therefore, it is necessary to elucidate the potential mechanism of DLM-triggered cerebrum injury, and a safe and effective antioxidant for treating DLM-induced organism damage is urgent to be found.

Selenium (Se), a nutritionally essential trace element for humans, acts on mediating varieties of biological protection mechanisms, such as anti-cancer, anti-apoptosis, anti-inflammatory, and improvement of autophagy dysfunction, especially antioxidation [21,22,23,24,25]. Se functionally incorporates into the selenocysteine and then exerts its biological activity through forming various selenoproteins [26, 27]. Also worth noting is that the avian genome encodes 24 selenoproteins [28]. Glutathione peroxidase (GPX) is one of the pivotal antioxidative selenoproteins widely existing in organisms. Concretely, the active center of GPX is selenocysteine, and its activity well reflects the Se level of the body [29]. In homeostasis, GPX composes a component of the antioxidative defense of glutathione (GSH) and plays a fundamental role in eliminating excessive reactive oxygen species (ROS) [30]. During the cellular detoxification of ROS, GPX catalyzes the conversion of GSH to oxidized state-disulfide glutathione (GSSG), and this process involves the decomposition of hydrogen peroxide (carbon dioxide and water as the final products), so as to protect the structure and function of cell membrane from the interference and damage of oxide [31]. GPX4 (phospholipid hydroperoxide GPX) is the key selenoenzyme in GPX family, and each subunit contains one selenocysteine [32]. GPX4 can directly reduce phospholipid and cholesterol hydroperoxides [27]. Previous studies have shown that Se supplementation effectively improved GSH activity, enhanced GPX4 expression, and reduced ROS levels [33]. These results positively provide a basis for the effect of Se as the active center on the activity of GPXs.

In 1957, Se is categorized into the following forms by Schwarz and Foltz: elemental Se, inorganic Se, and organic Se [34]. Among them, elemental Se is poor to be absorbed by organisms [35]. Inorganic Se and organic Se are two sources of dietary Se in animal husbandry [36]. It is worth mentioning that organic Se forms have higher absorption and bioavailability, and more significant biochemical and physiological benefits in animal tissues compared with inorganic Se [37]. This is because inorganic Se may form insoluble complexes in the intestine by recombing with other nutrients, thus reducing the absorption of Se, whereas organic Se can be absorbed faster and higher [38]. Study reported that organic Se (feed with corn–soy-based diets added with 0.15 mg/kg of Se from selenomethionine or Se-enriched yeast) dramatically increased (p < 0.05) cytoplasmic thioredoxin reductase activity in both the kidney and the liver than inorganic Se (sodium selenite), thereby enhancing the body’s antioxidant capacity [39]. Organic Se was approved as feed supplement by the US Food and Drug Administration in 2000 [40]. According to reports, the maximum amount of organic Se added to diets for poultry feeding in North America is 0.3 mg/kg, and that is 0.5 mg/kg in Europe [41, 42].

Se yeast (SY) is a common and effective organic form of Se supplement, which is famous for its high selenomethionine content [43]. As described previously, SY may increase the content of GPX, and the activity of catalase and superoxide dismutase (SOD) simultaneously decreases malondialdehyde (MDA) level, thereby removing free radicals and suppressing lipid peroxidation [44, 45]. SY has been shown to protect against oxidative stress-induced intestinal mucosa disruption [46]. Moreover, SY combined with gum Arabic can effectively ameliorate liver inflammation via inhibiting toll-like receptor 4 (TLR4) [44]. However, it is uncertain whether organic Se (from SY) exerts a protective function against DLM-induced cerebral toxicity.

Accordingly, to verify our hypothesis, the current study was performed to interrogate the therapeutic potential and underlying mechanism of dietary SY on DLM-induced cerebrum damage in quails.

Materials and Methods

Chemicals and Reagents

DLM (25 mg mL−1) was acquired from Nanjing Red Sun Co., Ltd (Nanjing, China). SY (Se content ≥ 3 g/kg) was provided by Bosar Biology Group (Yunnan, China). Assay kits (Jiancheng Bioengineering Institute, Nanjing, China) for GPX, MDA, GSH, and SOD were acquired for evaluating redox homeostasis. RIPA buffer and bicinchoninic acid kits were from Beyotime Biotechnology (Jiangsu, China). Trizol was obtained from Ambion (Foster City, CA, USA). SYBR Green RT-qPCR SuperMix and cDNA synthesis kits were bought from Vazyme Biotech Co., Ltd (Nanjing, China), and 2 × PCR Taq Plus Master Mix with dye were from Applied Biological Materials (Vancouver, Canada). Nuclear extraction kits were acquired from Beyotime Biotechnology (Shanghai, China). DNA marker was acquired from Tiangen Biotech Co., Ltd (Beijing, China). Antibodies against nuclear factor-kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were obtained from Abcam (Cambridge, UK). Antibody against GPX4 was from Beyotime Biotechnology (Shanghai, China). Antibodies against tumor necrosis factor-alpha (TNF-α) and Lamin B were from Bioss Biotechnology (Beijing, China). The antibody of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was acquired from Ambion (USA). All secondary antibodies were from ZSGB-BIO (Beijing, China).

Animals

Healthy male quails from Wanjia poultry farm (Harbin, China), 3 weeks age, 85 ± 10 g, were acclimated for 1 week prior to official experiment. Quails were housed under the specific conditions (12-h interval light/dark cycle, 22 ± 2 °C, 55 ± 5% relative humidity) with standard diet and water (ad libitum). The animal protocol was performed under the approbation of the Ethical Committee for Animal Experiments (Northeast Agricultural University, Grant Number: 20190928).

Treatments and Sample Collection

A model of SY intervention DLM-induced cerebrum injury in quails was established. Forty quails were distributed into 4 groups randomly: control, DLM, DLM + SY, SY groups (n = 10 per group). The weight of quails before treatment was 101.6 ± 1.497 (mean ± SEM). Control quails were intragastrically injected with physiological saline (0.9%, w/v) and provided with standard diet normally. The DLM group was intragastrically injected with 45 mg kg−1 body weight DLM solution once a day with standard diet normally. The DLM + SY group was intragastrically injected with 45 mg kg−1 body weight DLM solution once a day and provided standard diet with SY (0.4 mg kg−1 SY added in daily standard diet). The SY group was provided standard diet with SY (0.4 mg kg−1, added in standard diet). The composition of the standard diet was listed in Table 1 of the supplementary material. DLM was diluted using 0.9% normal saline as a vehicle. The dosage and mode of administration of DLM and SY are based on previous literature and our pre-experimental results [47, 48]. The treatment period was 12 weeks according to our pervious study [48]. All quails were administered with ether anesthesia 24 h after the last treatment. The blood samples were promptly taken out from vein and collected in a vacuum tube (containing heparin sodium anticoagulant) for hematology analysis [49]. Subsequently, quails were dissected, and cerebrum tissue was frozen in liquid nitrogen and stored at − 80 °C for further assay.

Table 1 Primers sequences for qRT-PCR
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Determination of Blood Index

The blood samples of each quail from 4 groups were collected from jugular vein for measuring white blood cells (WBC) count and red blood cells (RBC) number with a cell counting plate [50].

Determination of Se Concentrations

Five cerebrum samples (about 0.1 g) from each group were collected. And the inductively coupled plasma mass spectrometry (ICP-MS) (Thermo, USA) was carried out for analyzing Se concentrations. According to the conventional method described previously [51, 52], cerebrums were added to 5 mL HNO3 for digestion procedure in a microwave system (Anton Paar MW3000, Anton Paar GmbH). Specifically, the samples were digested at 800 W with 190 °C for 5 min, followed by 1400 W with 190 °C for 20 min under 38 MKPa. Then the clarified and transparent filtrates were diluted to 10 mL with deionized water finally used for ICP-MS analysis. The measurement of Se was analyzed based on a standard curve.

Oxidative Stress Bio-marker Measurement

Cerebrum samples (about 0.1 g) from four groups (8 quails per group) were homogenized in 0.9% normal saline on a high-throughput tissue grinder (Scientz-48L, Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China) at 60 Hz for 3 min [53]. After a centrifugation at 3000 r/min for 15 min at 4 °C, the supernatant was extracted for GPX, SOD, MDA, and GSH test in cerebrum with the corresponding commercial kits, respectively [54]. Assays were performed following the manufacturer’s instructions.

Organ Histopathology

Cerebrum samples were fixed in 10% formalin and cut into small pieces of 1–2 mm in thickness and 1 cm in length and width. Then sections were washed for 9 h and dehydrated through graded alcohol series [55]. Subsequently, the slices were embedded in paraffin, cut into 3–4 µm thickness, and put on the slides. After drying for 24 h and dewaxing by a series of steps, the slides were stained with hematoxylin and eosin (H&E). Finally, morphology was scanned by a light microscope (BX-FM, Olympus Corp, Tokyo, Japan) [56].

TUNEL Assay

Cerebrum cellular apoptosis (n = 5) was assayed with a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit. After dewaxed with xylene and graded alcohol series, the paraffin sections were penetrated with protease K for 15 min at room temperature. After washed with PBS, the slices were blocked with 3% H2O2-carbinol and rinsed with PBS. Then the slices were treated with TUNEL reaction mixture at 37 °C for 1 h, followed by 3 times PBS washing. The slices were incubated with converter-POD and rinsed again with PBS. Finally, the slices were stained with 3,3’-Diaminobenzidine for 30 min at room temperature, rinsed with PBS, and followed by mounted under glass coverslip [57]. Finally, the samples were evaluated by a light microscope.

Quantitative Reverse Transcription PCR Assay

Total RNA (n = 6) was isolated from cerebrum tissues with Trizol reagent. The concentration of RNA was determined with a spectrophotometry at 260 nm [58]. High-Capacity cDNA Reverse Transcription kits were used to synthesize cDNA following the manufacturer’s instructions. The primer sequences were designed by Sangon Biotech (Shanghai, China) and shown in Table 1. The PCR was performed with a 20 μL reaction system. Then the melting curve analysis was performed. Relative mRNA levels were normalized compared to β-actin expression. The PCR procedure was performed as previously described [59]. Relative RNA levels were calculated by the 2−ΔΔCt method. Meanwhile, heatmaps of mRNA expression were performed (https://www.omicstudio.cn/tool/4).

Western Blot Assay

Cerebrum samples (about 50 mg) were separated and adequately lysed in RIPA solution (1 mL, including PMSF) for 30 min on ice. Then the Eppendorf tubes were placed in a centrifugal machine to collect the supernatants at 12,000 rpm. The concentration of protein was tested by a BCA kit (Beyotime Biotechnology, Jiangsu, China) [60]. Equal aliquot of the total proteins from cerebrum issues (4 quails per group) were extracted with the appropriate protein extraction kits following the manufacturer’s instructions. The concentrations of proteins were tested using the bicinchoninic acid method. Samples (5 μL) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes [61]. Subsequently, all membranes were blocked in 5% nonfat milk plus Tris-buffered saline and 20% Tween 20 (TBST) and incubated with primary antibodies overnight at 4 °C. After this, the blots were washed with TBST, incubated with horseradish peroxidase-conjugated secondary antibodies at 37 °C for 30 min, and finally washed 6 times (10 min each time) with TBST. The bands were then visualized with an enhanced chemiluminescence kit [62]. The internal control is designed as GAPDH and Lamin B.

PPI Analysis

Protein–protein interaction (PPI) network of relative genes was constructed and analyzed using STRING database. The functional interaction networks of Gallus gallus and Mus musculus were based on the interrelationships of target genes in the database. The networks for above two species may better account for the relationship among related genes in this study [63].

Statistical Analysis

One-way analysis of variance and two-way Student’s t-test were used to analyze the data. Data represented mean ± standard error of mean (SEM) and analyzed by SPSS 19.0 software (SPSS, Chicago, IL, USA). A p < 0.05 was considered statistically significant.

Results

SY Attenuated DLM-Induced Changes of Hematological Indexes in Quail

According to the result of hematologic test, the number of RBC was decreased under DLM treatment with no significant changes (Fig. 1A, p > 0.05), while SY reversed this result to a certain extent (Fig. 1A, p > 0.05). Meanwhile, DLM significantly decreased WBC count; however, the suppressive effect of DLM was reversed by SY administration (Fig. 1B, p < 0.05). Furthermore, there were no mortalities in quails from control or experimental groups.

Fig. 1
figure 1

Effect of SY on hematological changes and Se content induced by DLM in quails. A RBC count (n = 10). B WBC count (n = 10). C Content of Se in quail cerebrum (n = 5). Values are presented as mean ± SEM. *Significantly different (p < 0.05) vs. control group; #significantly different (p < 0.05) vs. DLM-treated group

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SY Attenuated DLM-Induced Changes of Se Content in Quail Cerebrum

As displayed in Fig. 1C, the Se content in the DLM group was decreased, but the difference was not significant compared to the control group (Fig. 1C, p > 0.05), while SY treatment effectively attenuated the decrease of Se content induced by DLM in DLM + SY group (Fig. 1C, p < 0.05).

SY Attenuated DLM-Induced Changes of Pathology in Quail Cerebrum

To visually observe the protective effect of SY on DLM-treated cerebrum, we evaluated the pathological changes according to the H&E staining (Fig. 2). As shown in Fig. 2, the control and SY groups showed normal cerebrum tissue structures (Fig. 2A and D). DLM-treated cerebrum tissues showed disordered cell arrangement with increased spacing of cells (Fig. 2B). Furthermore, karyopyknosis of nerve cells could be observed (Fig. 2B). However, DLM-induced pathological changes were attenuated under the condition of SY treatment (Fig. 2C).

Fig. 2
figure 2

Effect of SY on cerebrum pathological changes induced by DLM in quails. A Control group. B DLM group. C DLM + SY group. D SY group. Yellow stars, disordered cell arrangement. Green stars, increased cell spacing. Red arrows, karyopyknosis of nerve cells. (H&E staining, magnification 200 ×)

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SY Attenuated DLM-Induced Ultrastructure Alteration in Quail Cerebrum

Transmission electron microscope was further used to assess the pathological changes of cerebrum induced by DLM (Fig. 3). The control and SY groups showed normal mitochondria structure (Fig. 3A and D). In the DLM group, severe pathological changes could be frequently observed including mitochondrial swelling, fragmented mitochondrial cristae, and vacuolization of mitochondria (Fig. 3B). Markedly, these pathological changes in the DLM + SY group were less severe than those in the DLM group (Fig. 3C).

Fig. 3
figure 3

Effect of SY on cerebrum ultrastructural changes induced by DLM in quails. A Control group. B DLM group. C DLM + SY group. D SY group. Red arrows, swollen mitochondria. Green arrows, fragmented mitochondrial cristae. Yellow arrows, vacuolization of mitochondria. (Scale bar = 1 μm, magnification 20,000 ×)

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SY Changed Redox Homeostasis in Quail Cerebrum Treated by DLM

To further evaluate the redox homeostasis interfered by SY, oxidative stress indices including GPX, SOD, GSH, and MDA were tested, respectively. The decreased GPX and SOD activities were shown in DLM-treated group. While in DLM plus SY cotreatment group, the activities of these two enzymes were increased (p < 0.05, Fig. 4A and B). MDA concentration, which is considered as the standard to monitor oxidative stress, was significantly increased in cerebrum exposed to DLM compared with control (p < 0.05, Fig. 4C). But compared to the DLM group, MDA level was markedly decreased in DLM plus SY treatment group (p < 0.05, Fig. 4C). Similarly, GSH concentration was markedly decreased in DLM-treated groups and treated with SY significantly improved this decrease compared with the control (p < 0.05, Fig. 4D).

Fig. 4
figure 4

Effect of SY on oxidative stress indices in quail cerebrum induced by DLM. A GPX activity. B SOD activity. C MDA level. D GSH concentration. Values are presented as mean ± SEM, n = 8. *Significantly different (p < 0.05) vs. control group; #significantly different (p < 0.05) vs. DLM group

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SY Alleviated GPX4 and Nrf2-Related Redox Homeostasis in DLM-Treated Quail Cerebrum

GPX4 is a member of GPX family and plays a key role in protecting against oxidative stress. The protein level and the mRNA expression of GPX4 were measured, respectively. From Fig. 5A, the protein level of GPX4 was found to be suppressed by DLM, while SY upregulated its level (p < 0.05, Fig. 5A). Similarly, the mRNA level change of GPX4 was consistent with GPX4 protein level (p < 0.05, Fig. 5B).

Fig. 5
figure 5

Effect of SY on GPX4 and Nrf2-related oxidative stress in quail cerebrum induced by DLM. A The oxidative stress-related protein level of GPX4 and Nrf2 (nuclear) (n = 4). B The oxidative stress-related mRNA expression of GPX4, Nrf2, NQO1, and HO-1 in cerebrum tissues (n = 6). Values are presented as mean ± SEM. *Significantly different (p < 0.05) vs. control group; #significantly different (p < 0.05) vs. DLM group

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To further evaluate whether SY may ameliorate the degree of oxidative stress in quail cerebrum, the levels of Nrf2-related proteins and mRNA expression were examined. The results showed that compared to the control group, the protein level of nuclear Nrf2 and mRNA expression of Nrf2 was significantly downregulated under DLM exposure, but SY supplementation significantly reversed these results (p < 0.05, Fig. 5). The downstream mRNA expression of nicotinamide adenine dinucleotide phosphatase: quinone acceptor 1 (NQO1) and heme oxygenase-1 (HO-1) was consistent with the results of Nrf2 (p < 0.05, Fig. 5B).

SY Alleviated TLR4-Associated Inflammation in Quail Cerebrum Induced by DLM

The mRNA levels of inflammation-related genes were detected by qRT-PCR. The mRNA levels of TLR4, NF-κB, TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were significantly upregulated in DLM-treated group compared with control (p < 0.05, Fig. 6B), but co-treated with SY significantly downregulated these changes (p < 0.05, Fig. 6B). Moreover, the results of NF-κB and TNF-α protein expression levels were completely consistent with qRT-PCR results (p < 0.05, Fig. 6A).

Fig. 6
figure 6

Effect of SY on TLR4-related inflammation in quail cerebrum induced by DLM. A The inflammation-related protein levels of NF-κB and TNF-α in quail cerebrums (n = 4). B The inflammation-related mRNA expression of TLR4, NF-κB, TNF-α, iNOS, and COX-2 in cerebrum (n = 6). Values are presented as mean ± SEM. *Significantly different (p < 0.05) vs. control group; #significantly different (p < 0.05) vs. DLM group

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SY Alleviated Apoptosis in Quail Cerebrum Induced by DLM

Imaged and quantified results of TUNEL staining revealed the therapeutic effect of SY on DLM-induced neuronal apoptosis. DLM exposure dramatically increased the ratio of neuronal apoptosis compared with control (p < 0.05, Fig. 7A and B). Notably, SY supplementation reversed this growing tendency in the DLM + SY group, indicating that SY significantly restrained DLM-induced apoptosis (p < 0.05, Fig. 7B).

Fig. 7
figure 7

Effect of SY on DLM-induced apoptosis in quail cerebrum. A TUNEL staining (400 × magnification; bars = 25 μm). B Values of quantitative analysis (n = 5). C The inflammation-related protein levels of Bcl-2, caspase 3, and Bax in cerebrum of quails. Values are presented as mean ± SEM (n = 4). D The apoptosis-related mRNA expression of JNK3, caspase 3, Bax, and Bcl-2, in cerebrum of quails (n = 6). *Significantly different (p < 0.05) vs. control group; #significantly different (p < 0.05) vs. DLM group

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To further demonstrate the effect of SY on apoptosis induced by DLM, apoptosis-related proteins and mRNA expression were, respectively, measured by western blot and qRT-PCR. From data, DLM treatment led to a markedly elevation in the mRNA levels of Jun N-terminal kinas e3 (JNK3), caspase 3, and B cell lymphoma gene 2-associated X protein (Bax), but these results were reversed by SY (p < 0.05, Fig. 7D). Oppositely, the mRNA expression of B cell lymphoma gene 2 (Bcl-2) was just contrary to the expression results of the above indicators (p < 0.05, Fig. 7D). The trend of western blot results including caspase 3, Bax, and Bcl-2 was the same as those of qRT-PCR (p < 0.05, Fig. 7C).

Bioinformatics Analysis

The clustering analysis heatmaps and PPI network of genes associated with this study were revealed to demonstrate our hypothesis (Fig. 8A and B). The expression levels of all genes in each group were shown in Fig. 8A, indicating that the oxidative stress (GPX4, Nrf2, HO-1, and NQO1), inflammation (TLR4, NF-κB, TNF-α, iNOS, and COX-2), and apoptosis (JNK3, Caspase 3, Bax, and Bcl-2) related genes were affected by DLM and/or SY administration. As shown in Fig. 8B, the dynamic clusters including GPX1, GPX4, Nrf2, HO-1, NQO1, Keap1, TLR4, COX-2, TNF-α, Caspase 3, JNK3, and Bcl-2 were indicated on the diagram. The functional interaction network revealed the close relationship of the above genes.

Fig. 8
figure 8

Bioinformatics analysis of related genes expression. A Heatmaps of related gene expression. B PPI analysis. Protein network of proteins regulated between oxidative stress inflammation-related genes and apoptosis-related genes expressed in the Gallus gallus and Mus musculus species. GPX1 (Gpx1), GPX4 (Gpx4), Nrf2 (NFE2L2, Nfe2l2), HO-1 (HMOX1, Hmox1), NQO1 (Nqo1), TLR4 (Tlr4), TNF-α (Tnf), COX-2 (Ptgs2), JNK3 (MAPK10, Mapk10), caspase 3 (CASP3, Casp3), Bcl-2 (BCL2, Bcl2)

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Discussion

Pesticide residues in the environment caused by excessive use of DLM have gradually become the focus of ecotoxicology research, so it is necessary to explore the exact mechanism of its potential toxicity. DLM can be absorbed by organisms through the respiratory tract and digestive tract or via the dermal route causing toxicity to the organism [64,65,66]. Although studies have interrogated DLM toxicity in some organs, the toxic mechanism of DLM-induced cerebrum damage is poorly understood, and there is a lack of effective therapeutic drugs. Se, a micro-mineral, is a central component of the important active ingredient of selenoprotein GPX, playing a key role in protecting against oxidative stress [28]. The present study firstly reveals that Se from SY effectively attenuates cerebral injury triggered by chronic DLM treatment.

In this study, the negative influence of DLM on the change of histopathology including disordered cell arrangement, increased the spacing of cells spacing, and karyopyknosis of neuron could be visibly observed. Strikingly, SY administration relieved cerebrum injury exposed to DLM. Moreover, SY treatment effectively elevated the Se content in the organisms. And the hematological toxicity mediated by DLM can be reversed by in the presence of SY, which further suggests the possibility of SY as an effective dietary supplement to alleviate cerebrum injury.

Oxidative stress is one of the pivotal mechanisms of nontarget biological damage caused by DLM [14]. Although cells produce ROS under normal metabolism, the over-production of ROS can weaken antioxidant defense system [67]. DLM can promote the accumulation of ROS, thereby leading to the damage of normal oxidative metabolism [18, 66]. GPX and SOD are both the pivotal antioxidant enzymes catalyzing the breakdown of ROS [68]. Notably, SY contains the pivotal component of GPX, Se, acted on catalyzing the conversion of reduced GSH into oxidized GSH–GSSG and converting toxic peroxides and organic hydroperoxide into non-toxic hydroxyl compounds, in order to detoxify lipid peroxides and defense against ROS-induced cellular and subcellular oxidative damage, thereby reducing the damage of peroxide to cells [69]. Meanwhile, SOD acts on catalyzing the dismutation of free superoxide to hydrogen peroxide, which was further decomposed to water and GSSG by GPX [31]. In present study, the suppression of GPX and SOD activities suggested that antioxidant defense system failed to counteract the influx of ROS production induced by chronic DLM exposure. Subsequently, excessive production of ROS attacked the cell membranes and led to lipid peroxidation generating the main products of lipid peroxidation MDA [70]. By contrast, when under the intervention of Se (from SY), the above oxidative stress-related indexes were reversed to a certain extent. In this study, we observed the elevation of GPX and SOD activities, the upregulated level of GSH concentration, and the reduction of MDA in the DLM-treated cerebrum. Meanwhile, we found that the GPX4 expression level was elevated under SY administration, indicating that SY enhances the antioxidant enzyme defense system and contributes to correct the imbalance between oxidation and antioxidation, thereby protecting cerebrum against DLM. Further analysis revealed the possibility of GPX4 as a target for the treatment of DLM-induced cerebral injury.

Nrf2 is a key redox sensitive transcription factor, which plays a vital role in defending against various kinds of toxic-induced oxidative stress damage, and is widely distributed in various tissues and organs [71,72,73]. Nrf2 binds to Kelch-like ECH-associated protein 1 (Keap1) and remains inactive status in the cytoplasm under physiological conditions. Subsequently, Nrf2 is stimulated to separate from Keap1 and transferred to the nucleus to bind with Maf [59]. The Nrf2-Maf heterodimers then bind to antioxidant response elements in the promoters of key antioxidant genes inducing the regulation of a series of antioxidant genes and phase II detoxification enzymes (including HO-1 and NQO1) to maintain intracellular redox homeostasis [47, 59, 74]. Moreover, Reszka et al. (2015) confirmed that Keap1 and Nrf2 change with the concentration of Se in plasma, and they are negatively correlated and positively correlated, respectively [75]. Our data further verify the antioxidant activity of SY and demonstrate that SY protects quail cerebrum from oxidative stress injury induced by DLM, that is, by elevating the expression of Nrf2 and its target gene products.

TLR4 is one of the pattern recognition receptors triggering different inflammatory responses [18]. TLR4 can be suppressed by Nrf2 to alleviate inflammation-associated lung injury, indicating that oxidative stress along with inflammation is closely related to the development of the disease [76]. The activation of TLR4 signaling works depended on the adaptor protein MyD88, further enhancing the signal cascades that stimulate inflammatory-inducing factor NF-κB gene expression [77]. Subsequently, NF-κB is activated and acts on a variety of pro-inflammatory cytokines including TNF-α, COX-2, and iNOS [78,79,80]. TNF-α is a major cytokine participated in systemic inflammatory responses [81, 82]. Meanwhile, COX-2 is well considered as a precipitating factor of neuroinflammation in the brain, often used to evaluate the level of brain inflammation [83]. COX-2 and iNOS are both inflammatory proteins that are expressed when microglia are stimulated and activated, suggesting inflammatory responses [84]. The anti-inflammatory mechanism of Se is mainly based on its signal transduction regulation effect on target cells. In vivo experiments have demonstrated that the increase of Se level can suppress NF-κB activation through the catalysis of GPX [85]. Se supplementation can reduce the gene expression of the main pro-inflammatory factors TNF-α and COX-2 via inhibiting the mitogen-activated protein kinase signal pathway [86, 87]. In our study, the alterations in inflammation-related genes and proteins in quails indicate that SY treatment significantly promotes Nrf2 expression and then inhibits the activation of TLR4 and further downregulates inflammatory factor TNF-α, COX-2, and iNOS. Moreover, the increasing trend of WBC was dramatically reversed under SY administration, revealing the protective effect of SY in immune and defense system of organisms. Therefore, our study suggests that SY may effectively protect the quail cerebrum against DLM exposure-induced inflammatory damage to a certain extent via regulating TLR4/NF-κB pathway.

The excessive production of ROS caused by oxidative stress is also one of the mediators of apoptosis pathway [88]. Pervious study announced that TLR-related pathways can be triggered by ROS to lead to cardiomyocyte apoptosis [89]. Of note, Se has been demonstrated to attenuate high glucose-induced apoptosis of rat cardiomyocytes via suppressing the ROS/TLR4 pathway [90]. JNK3, a member of the JNK family, has been identified to be significantly active and highly expressed in the brain [91]. Under pathological conditions, TLR4 activates JNK3 and then causes the imbalance of apoptosis regulation, which is manifested by downregulating anti-apoptotic protein Bcl-2 and upregulating pro-apoptotic protein Bax [92,93,94]. Bax enhances the permeability of mitochondrial outer membrane, thereby leading to excessive release of cytochrome c and caspase 3 activation [72, 95]. The activation of caspase 3 subsequently participates in various apoptotic pathway, finally resulting in cell apoptosis [96]. Hence, our study suggests that exogenous addition of SY effectively alleviates DLM-induced apoptosis in quail cerebrum involved in the activation of TLR4 pathway. Taken together, the organic Se supplement through SY raises hopes that the amelioration in DLM-induced cerebral toxicity may be possible, and the positive regulation of GPX4 on antioxidative defense is considered as the main therapeutic target of Se administration for treating cerebrum injury. In summary, this study suggests that activation of the GPX4/TLR4 is primarily involved in the cerebrum injury induced by chronic DLM exposure (Fig. 9).

Fig. 9
figure 9

Schematic diagram of the mechanism of SY attenuating DLM-induced cerebral injury. SY attenuates DLM-induced cerebral injury in quails via activation of the GPX4/TLR4 signaling pathway

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Conclusion

Herein, our study demonstrates that SY attenuates DLM-induced cerebrum injury in quails via activation of the GPX4/TLR4 signaling pathway. GPX4 may be an effective therapeutic target for the treatment of DLM-induced cerebral injury. Furthermore, this study not only provides a novel insight for elucidating DLM-induced cerebrum toxicity, but also a practical foundation for the treatment of DLM-induced damage by dietary supplement of SY.