Calcium Overload and Ros Accumulation Induced by Selenium Deciency Promote Autophagy in Swine Intestine

and affects the content of elements in the intestine.


Introduction
Selenium is an indispensable nutrient element for human and animal organisms, which has physiological effects such as anti-in ammatory and antioxidant [1], anti-mutation [2] in immunity [3]. Selenium also plays an important role in immune function [4].Studies have shown that selenium de ciency can cause Keshan disease in humans [5], heart failure in swine [6], white muscle disease (WMD) of calves, lambs, ponies, and other animals, and vitamin E/selenium de ciency(VESD) syndrome of swine [7], even affect maternal thyroid metabolism and oxidative stress, leading to weight loss [8]. These have brought signi cant economic losses to the swine industry. Selenium has a signi cant effect on intestinal function in swine, such as diarrhea-induced colitis injury can be alleviated by selenium [9]. Moreover, the intestine is the main organ to absorb selenium [10], in short, the two are closely linked. Besides, swine can be used as a good model to study the potential risks and related mechanisms of human selenium intake [11]. Meanwhile, compared to rodent cell lines, porcine jejunal epithelial cells (IPEC-J2) plays an important role in the study of zoonotic infections, and is often used as an in vitro model for microbial research [12].
Regarding the intestinal tract, past studies have found that selenium de ciency can cause intestinal eosinophilic in ammation [13], but the speci c mechanism of selenium de ciency leading to damage is still unclear.
Oxidative stress is due to the imbalance between oxidation and anti-oxidation, which is more prone to oxidation and produces a large number of intermediate products. It is considered to be an important inducing factor leading to aging and disease. Selenium has a good antioxidant function who is considered as scavengers of free radicals and other reactive oxygen species (ROS) [14]. Therefore, selenium de ciency can contribute to oxidative stress and damage to various tissues [15]. As a stimulus point for oxidative stress, ROS is an intracellular chemical capable of triggering various biological responses [16]. Intestinal exposure to adverse environment triggers oxidative stress [17]. Under the condition of selenium de ciency, ROS can trigger the NF-kB in ammation signaling pathway and the intrinsic apoptosis pathway to cause the apoptosis of duodenal villi cells [18]. Moreover, ROS can mediate autophagy during nutrient de ciency [19]. ROS induces autophagic expression in the nucleus by triggering endoplasmic reticulum stress. Intracytoplasmic ROS may also in uence autophagy by modulating ATG4 activity [20]. Our previous experiments have demonstrated that selenium de ciency can activate the ROS mediated MAPK pathways to regulate autophagy [21].
Autophagy is a cellular process that occurs in eukaryotic cells, degrades cytoplasmic content by lysosomal phagocytosis, and recycles large molecules in the cytoplasm [22]. More studies have shown that differentially expressed genes caused by selenium de ciency can be enriched in the PI3K/AKT/mTOR signaling pathway [23], and the mTOR gene is closely related to the autophagy pathway. Autophagy plays a vital role in cell physiology, including adapting to metabolic stress, clearing dangerous goods, renewing during differentiation and development, and preventing damage to the genome [24]. It has been proved that selenium de ciency induces autophagy in cardiomyocytes [25]. Moreover, the change of calcium(Ca 2+ ) homeostasis is one of the main factors affecting autophagy [26]. The increase of free Ca 2+ activated by CAMKK-β and AMPK in the cytoplasm can be used as an effective inducer of autophagy and also become an ER target of Bcl-2 against autophagy [27]. In addition, as the Ca 2+ -ATPase pump, SERCA plays an important role in regulating cellular Ca 2+ homeostasis, while calpain is activated under the in uence of a high concentration of Ca 2+ [28]. In addition, studies have con rmed the interaction between calpain and SERCA through immunocoprecipitation [29]. Experimental results of cancer cell lines show that autophagy even apoptosis is induced by CAMKK-β and AMPK, which will cause [30], and this result has also been veri ed in carp [31]. Selenium de ciency causes symptoms in the digestive system of swine and triggers intestinal cell damage. However, it is unclear what role Ca 2+ homeostasis and autophagy play in selenium-de cient intestinal damage.
Consequently, we established an in vivo experiment of selenium de ciency in swine intestine and an in vitro experiment of selenium de ciency in IPEC-J2 of swine to explore the mechanism of the autophagycalcium pathway in selenium-de cient intestinal injury.

Material And Method
All procedures used in this study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University (SRM-11).

Swine and diets
A total of 24 healthy and similar-weights pure line big white emasculated swine (chosen from 12 nests, 2 heads/nests) were weaned for one week. Then the swine were randomly assigned to 2 groups of 12 (adopting the full sibling pairing test): the Se-de cient group and the control group. The swine were maintained on either a Se-de cient diet (the Se-de cient group) containing 0.007 mg/kg Se or a normal selenite diet (the control group) containing 0.300 mg/kg Se for 16 weeks. The detailed feed ingredients are shown in Table 1. All the swine were housed under the same conditions from the start of the experiment, and the swine were fed in swine pens. The experimental swine were fed three times each day and provided with free access to drinking water. Intestine tissue samples were collected and promptly frozen at the 16th week of the experiment, part of the clean tissue was sliced and immersed in 10% neutral buffered formalin solution and electron microscopy solution at 4 ° C Store and take part of the intestinal tissue frozen at -80℃ for future use. Note: Trace elements are provided per kilogram of diet: copper (5mg a , 4mg b) , iodine (0.14mg a, b ), iron (100mg a , 60mg b ), manganese (3mg a , 2mg b ), Zinc (80mg a , 60mg b ); vitamins: VA (1750IU a , 1300IU b ), VD3 (200IU a , 150IU b ), VE (11IU a, b ), VK3 (0.5mg a, b ), biotin (0.05mg a, b ), choline (0.4g a , 0.3g b ), folic acid (0.3mg a, b ), nicotinic acid (30mg a, b ), pantothenic acid (9mg a , 8mg b ), ribo avin (3mg a , 2.5mg b ), thiamine (1mg a, b ), VB6 (3mg a , 1mg b ), VB12 (15µg a , 10µg b ).

Cell culture and treatment
The IPEC-J2 cell line is obtained from the College of animal science, Northeast Agricultural University and cultured using DMEM/High Glucose (GIBCO, NY, USA) medium as a liquid environment which containing 10% FBS (GIBCO, NY, USA), and 1% penicillin-streptomycin (GIBCO, NY, USA). DMEM/High Glucose medium, FBS and penicillin-streptomycin were all sterilized through a 0.22 µm millipore lter to remove any contaminants. Cells were fed once a day and subcultured once every 2-3 days until the cell density reaches 70-80%. When passaging, rst inoculated the cells in a culture ask, and incubated for 12 hours to ensure that the cells are attached to the culture ask. Then discarded the original medium and cultured the cells in other mediums. For the Se-de cient group, the cells were cultured in DMEM/High Glucose medium with 1% FBS, 1% penicillin-streptomycin, 10 µg/ml insulin, and 5 µg/ml transferrin resulting in selenium depletion at least for 5 days [32]. IPEC-J2 cells in the Se-de cient group needs to be changed every day to remove dead cells. But IPEC-J2 cells in the control group were still cultured on the normal medium, passaged, and allowed to grow for 5 days. The cells were cultured at 37 °C and 5% CO 2 and collected for analysis after ve days.

Morphological examination of swine intestine and IPEC-J2 cells
The technique adopted to observe ultrastructural changes was similar to that of our previous study: The jejunum tissues IPEC-J2 cells were xed in 2.5% glutaraldehyde phosphate-buffered saline, post-xed with 1% osmium tetroxide, stained with 4.8% uranyl acetate, and nally dehydrated in a graded ethanol series. The ultra-thin sections were cut, incubated with uranyl acetate and lead citrate. The intestine specimens were visualized using the transmission electron microscopy (GEM-1200ES, Japan).
The treatment method of IPEC-J2 cells electron microscope observation was the same as that of tissue.

Cell autophagy detection
Autophagic staining was measured using a cell autophagy detection assay kit (Beijing Solarbio Science & Technology, Beijing, China). The 10 µM MDC staining agent (Dansylcadaverine) was added to the medium containing enterocytes, which incubated in a constant temperature incubator (37 °C) for 25 min.
Discarding the medium and washing the cells with PBS (37℃ preheat) three times. Finally, cells were collected using a uorescence microscope at an excitation wavelength of 355 nm and an emission wavelength of 512 nm for observation of uorescence.

ROS activities detection
ROS activities were measured using the ROS assay kit (Nanjing Jiancheng Bioengineering Institute, China). Add 10 µM DCFH-DA (2,7-dichloro uorescin diacetate) in the culture medium, where there are cell samples to be tested, and incubate in constant temperature incubator (37℃) for 45 min, discard the medium and use PBS (37℃ preheat) wash the cells three times, nally, collect the cells for detected the activities of ROS in excitation wavelength 500 ± 15 nm and emission wavelength 530 ± 20 nm. Enterocytes were visualized using uorescence microscopy.

Intracellular Ca 2+ concentration detection
The Fluo-3 AM assay kit (Beijing Solarbio Science & Technology Co., Ltd) was used to detect the intracellular Ca 2+ concentration. After 4 days of treatment, the cells cultured in 6-well plates were digested with collagenase-(0.1 g %), then the cells were resuspended and plated in 12-well plates for 24 h. The Flour-3am mother liquor was diluted with PBS until the concentration reached 1 µM for use. The cells were washed with PBS before they were covered with the diluted working uid. After incubating at 37 °C for 40 minutes, the cells were cleaned and observed under a uorescence microscope. 7. Total RNA extraction and determination of the mRNA expression of the autophagy-calcium homeostasis related genes Total RNA was isolated from intestine tissues and enterocyte using Trizol reagent according to the manufacturer's instructions (Invitrogen, Shanghai China). The dried RNA pellets were resuspended in 50 µL of diethyl-pyrocarbon-ate-treated water. The concentration and purity of the total RNA were determined by a spectrophotometer. cDNA was synthesized from 5 µg of the total RNA using oligo dT primers and Superscript II reverse transcriptase according to the manufacturer's instructions (Promega, Beijing, China), and cDNA was stored at − 80 °C [33].

Detection of selenoproteins
Detection of selenoproteins content in intestine tissue and IPEC-J2 cells was performed by qRT-PCR, whose method was the same as the detection of autophagy-calcium target genes, and the speci c primers for selenoproteins genes ( Table 2) were designed based on known sequences using Primer-BLAST at the NCBI. 9. Total protein extraction and determination of the protein expression of autophagy-calcium homeostasis related genes Total protein was extracted from intestine tissues and enterocytes by lysis buffer for Western blotting with phenylmethanesulfonyl uoride (PMSF) (100 mM). These extracts were subjected to SDSpolyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% methanol at 4 °C. The membranes were blocked with 5% skim milk for 2 h and incubated overnight with diluted primary antibodies against Beclin1

Statistical analysis
Each group consisted of 6 single observation replications (n = 6) and two parallel experiments were performed to ensure the accuracy of the experimental data. The data are expressed as the mean ± standard deviation (mean ± SD), and GraphPad Prism v8.0 software was used for all the statistical analyses and the Multiple t-tests showed that the data were normally distributed. The data were compared using a t-test analysis of variance to determine the difference between the control group and the Se-de cient group. * Signi cant difference from the corresponding control (P < 0.05).

Ultrastructural observation of autophagosomes in swine intestine
The ultrastructure of the swine intestine tissue (Fig. 1A) of the control group and the Se-de cient group was observed with a transmission electron microscope (TEM). In the control group, normal mitochondria and a few lysosomes were observed. However, a large number of autophagic vesicles with a double-layer membrane structure and seldom lysosomes appeared in the Se-de cient group.

Ultrastructural observation of autophagosomes in IPEC-J2 cells
Observing the ultrastructure of the control group and Se-de cient group of IPEC-J2 cells (Fig. 1B) by the transmission electron microscope. In the control group of IPEC-J2 cells, a large number of normal mitochondria could be observed. There were a small number of autophagy lysosomal vesicles characterized by incomplete boundary membrane, intact intima, and amorphous substances. There were also a small number of autophagic vesicles that do not contain substances. In the Se-de cient group, the cytoplasm was mainly autophagic lysosome vesicles, which contained mitochondria. There were also a large number of uncovered secondary lysosomes and autophagic vesicles, as well as a small number of mitochondria with normal morphology.
2. Effects of selenium de ciency on the selenium content of swine intestine tissue and IPEC-J2 cells In intestine tissue and IPEC-J2 cells, the expression of 22 selenoproteins in the control group was higher than that in the Se-de ciency group. It should be noticed that the expression of GPX2 in the tissue selenium de ciency group was slightly higher than that of the control group.
3. Effects of selenium de ciency on ROS viability in IPEC-J2 cells Affected by the lack of selenium nutrition, the ROS activity of the Se-de cient group had a signi cant increase compared to the control group (p < 0.01). The activities of ROS increased signi cantly (p < 0.01) in the Se-de cient group.

Effects of selenium de ciency on autophagy in IPEC-J2 cells
Dansylcadaverine-MDC is a uorescent pigment that stains normal cells into a uniform yellowish-green, and autophagy becomes bright green. The IPEC-J2 cells in the control group were yellowish-green with few green highlights. Compared with the control group, the bright green spot of the Se-de ciency group was dense, which meant that the Se-de cient group had a more autophagosome accumulation (p < 0.01).

Effects of selenium de ciency on the concentration of Ca 2+ in IPEC-J2 cells
The lack of selenium nutrition caused an increase in the cytoplasmic Ca 2+ concentration of the Sede ciency group. Compared with the control group, the Se-de ciency group had more bright green spots, and calcium overload occurred signi cantly (p < 0.05).
6. Protein and mRNA expression of autophagy-Ca 2+ related genes in swine intestine tissues Affected by selenium de ciency, protein and mRNA expression abundance of autophagy-related genes in intestine tissues are shown in Fig. 4.
qPCR results revealed that mRNA expression of autophagy-related genes (Beclin1, LC3-1, LC3-2, ATG5, ATG12, ATG16, mTOR) signi cantly increased (p < 0.05) in the Se-de cient group, compared with the control group. Similarly, the mRNA expression of Ca 2+ pathway-related genes (CAMKK, SERCA, and calpain) in the Se-de ciency group also increased signi cantly compared to the control group. But the mRNA expression of LC3-1 and AMPK increased slightly, did not show signi cant differences(p > 0.05). Meanwhile, the protein expression of LC3-1, LC3-2, AMPK, CAMKK-β signi cantly increased (p < 0.05) in the Se-de cient group compared with the control group respectively. Compared with the control group, the protein expression of Beclin1 in the Se-de ciency group increased slightly, and there was no signi cant difference. The protein expression of SERCA and mTOR in the Se-de ciency group was signi cantly lower than that in the control group (p < 0.01). The expression results of the above genes indicated that selenium de ciency caused Ca 2+ overload and autophagy accumulation in the swine intestine.
7. Protein and mRNA expression of autophagy-Ca 2+ related genes in IPEC-J2 cells Affected by selenium de ciency, protein and mRNA expression abundance of autophagy-related genes in IPEC-J2 cells are shown in Fig. 5.
Similar to the expression in intestine tissue, compared with the control group, autophagy-related genes (LC3-1, LC3-2, ATG5, ATG12, ATG16, mTOR) were signi cantly increased in the mRNA expression of Sede cient group IPEC-J2 (Fig. 5A). The mRNA expression of Beclin1 was not signi cantly different in the two groups. The mRNA expression of Ca 2+ related genes (CAMKK, SERCA, calpain, AMPK) had the same trend as that of the main autophagy-related genes, and the expression in the Se-de ciency group was signi cantly higher than that in the control group (p < 0.01).
The change in protein expressions of autophagy-Ca 2+ related genes was shown in Fig. 5B. As detected, compared with the control group, the protein expressions of Beclin1, LC3-1, LC3-2, and ATG16 were signi cantly increased. However, the protein expression of SERCA in the Se-de ciency group was signi cantly lower than that in the control group. The results of gene expression in IPEC-J2 cells are the same as in vivo, showing that selenium de ciency can lead to Ca 2+ overload and autophagy accumulation in cells.

Results of detection and analysis of multiple elements in intestine tissue
The detection results of 23 elements in swine intestine tissue are shown in Fig. 6. It can be roughly divided into three groups of main elements, majorelements (Na, Mg, K, Ca), essential trace elements (Fe, Zn, B, Cu, Mn, Ni, Ba, Sb, Se, Ti, V), and toxic trace elements (Al, Li, As, Cd, Pd, Sn, Sr). The results showed that selenium de ciency caused an increase in the major element Ca (p < 0.05), while the levels of other major elements were basically not affected (p > 0.05). The content of essential trace elements B, Cu, Ni, V was signi cantly reduced (p < 0.05), and other essential trace elements were not affected (p > 0.05). At the same time, selenium de ciency led to a decrease in the content of toxic trace element Al (p < 0.05). The results showed that the lack of selenium caused the imbalance of the ion level in the swine intestine tissue, the compensation of Ca increased, and the loss of B, Cu, Ni, V, and Al.

Discussion
Selenium is an indispensable trace element in the body and affects the body's health. Selenium protein is involved in the regulation of cellular redox homeostasis, protection of oxidative stress [35]. Selenium de ciency can cause swine mulberry hearts and has a particularly signi cant effect on the digestive system of swine [36], which can lead to enteritis [37] and even death. Although different forms of selenium supplements are a convenient method to treat these diseases, such as selenium can be supplemented to the host by adding a daily diet. The fact has also proved that this method can indeed effectively reduce the incidence and mortality of mouse models and human colon cancer [38]. However, there are still many factors that affect its effectiveness, such as the chemical form of selenium additives and the health of animals [39]. Moreover, some studies have shown that dietary selenium affects the host's intestinal ora balance and gastrointestinal colonization, thereby affecting the host's selenium status and the expression of selenoprotein [40]. Besides, the study has shown that selenium de ciency can change the distribution and steady-state of other minerals [32], which has been con rmed again in our experiment. In this study, we established the selenium de ciency model in swine and the selenium de ciency model in IPEC-J2 cell in vitro, in which results showed that selenium de ciency caused a downward trend in the overall selenoprotein expression and disrupted the balance of intestinal trace elements. Selenium de ciency induced the occurrence of oxidative stress, the imbalance of Ca 2+ homeostasis in the intestine and in vitro, and ultimately promotes autophagy.
Selenium is involved in regulating ROS levels and redox balance in all tissues [41], a large number of studies have shown that ROS is an important target for tissue damage caused by selenium de ciency.
Gao et al. found that selenium de ciency could stimulate ROS-induced in ammation [42]. Selenium can be used as a ROS scavenger to exert additive effects on the proliferation and paracrine of human amniotic uid-derived mesenchymal stem cells with bFGF (Basic Fiber Growth Factor) [43]. Due to the combined effect of selenium de ciency and low protein intake, the levels of ROS in the serum and myocardial tissue defects of rats are signi cantly increased. Eventually, it causes myocardial oxidative stress and induces apoptosis through mitochondrial-mediated pathways [44]. The imbalance between ROS production and the elimination of protective mechanisms may lead to chronic in ammation [45].
Besides, our previous research also showed that selenium de ciency did cause in ammation of swine intestinal tissue and IPEC-J2 cells [46]. In our present experiment, it was observed that ROS production in IPEC-J2 cells increased due to selenium de ciency stimulation. Similar to the speci c expansion of myeloid cells that causes excessive ROS and promotes intestinal pathology [47], we believe that the swine intestinal damage caused by selenium de ciency is also closely related to the accumulation of ROS. Also, studies have shown that the addition of trace element chelating agents can improve the antioxidant capacity and immune function [48]. But excessive cadmium can promote oxidative stress [49] [50]. Therefore, we speculate that the destruction of the homeostasis of trace elements in our experiment may also be one of the reasons for the accumulation of ROS.
Moreover, a large number of reports indicate that ROS is an early inducer of autophagy during nutritional de ciencies [51]. NOX2 production of ROS is the key to phagocytic cells to kill microorganisms, and LC3 needs to recruit to phagosomes [52]. ROS can induce autophagy by activating the MCOLN1-lysosome Ca 2+ -TFEB pathway to eliminate damaged mitochondria and excess ROS [53]. Our previous research found that selenium de ciency could inhibit the expression of TNNT2, thereby activating the channel for Ca 2+ to ow outside the cell, and inhibiting the channel for Ca 2+ to ow inward [54]. We note that AMPK is an important signaling molecule for Ca 2+ -dependent autophagy activation [55]. AgNP induces ER stress and autophagy occurs through the Ca 2+ /CAMKK-beta/AMPK/mTOR pathway. The speci c mechanism is that CAMKK-β and AMPK are activated by the increase of Ca 2+ levels in the cytoplasm after being stimulated by AgNP, which leads to the downregulation of mTOR and the upregulation of Beclin1, eventually activating autophagy [56]. In this experiment, selenium de ciency also caused the accumulation of autophagy in swine intestine by increasing the expression of CAMKK-β and AMPK in the Ca 2+ /CAMKK-β/AMPK/mTOR pathway, and decreasing the expression of mTOR. However, it is worth noting that the mRNA expression of Beclin1 in our experimental results decreased slightly in vitro. We believe that there may be other pathway genes that are stimulated by selenium de ciency at the mRNA level and inhibit the expression of Beclin1. For example, the anti-apoptotic protein, Bcl-2, which interacts with Beclin1 to regulate autophagy, keeps cells alive rather than dead [57].
Autophagy can prevent cell damage and promote cell survival in the absence of nutrition, and can also respond to cytotoxic damage [58]. Therefore, autophagy is sensitive to the lack of dietary selenium.
Selenium de ciency can cause increased expression of autophagy in the chicken spleen, bursa, and thymus and cause damage to chicken immune organs [59]. Our previous studies on selenium nutrition have shown that selenium de ciency can cause autophagy in cardiomyocytes [60]. Autophagy can antagonize apoptosis mechanisms in cardiomyocytes knocked out of GPX3 gene, a selenoprotein [61]. Autophagy plays a key role in regulating the interaction between the gut microbiota and innate and adaptive immunity and the host's defense against intestinal pathogens, maintaining intestinal homeostasis [62]. In this experiment, the key genes of autophagy (LC3, Beclin1) were up-regulated in swine intestinal tissues and IPEC-J2 cells treated with selenium de ciency, and the ultrastructure also showed autophagic vacuoles and encapsulated mitochondria. The above results suggest that due to the stimulation of selenium de ciency, homeostasis of Ca 2+ and ROS are destroyed and overload occurs, leading to increased autophagy. To maintain normal cell function and protect the intestine from selenium de ciency, autophagy plays a role in resisting damage.
However, it is too early to say that autophagy effectively protects swine intestine injury caused by selenium de ciency. Because we observed the apoptotic bodies such as the formation of apoptotic bodies and the shrinkage of the nucleus while observing the Se-de cient group autophagic vesicles, we suspect that the lack of selenium nutrition may also trigger apoptosis of intestinal. It may be due to excessive autophagy or damage that has exceeded cell death caused by autophagic protection. Studies have shown that inducing autophagy in tumor cells lacking nutrition can maintain cell metabolism and vitality during periods of nutritional de ciencies, but long-term lack of nutrition can lead to cell death [63].
Attention should also be paid to the destruction of element balance. In this experiment, the contents of B, V and Cu were reduced by selenium de ciency, which could increase the risk of dementia, autism and depression [66]. Decreased levels of Ni and Al affect fasting blood glucose [67]. Increased Ca content can exacerbate the risk of diabetes, and even accumulation of Ca 2+ in mitochondria will promote apoptosis [68]. Calcium accumulation is often regarded as one of the signs of injury [69]. Our results show that selenium de ciency disrupts the balance of elements in the intestines of swine and increases the risk of suffering from various diseases.

Conclusions
In conclusion, our research showed that the lack of selenium nutrition could destroy the Ca 2+ homeostasis of the swine intestine and trigger the overload of ROS, which ultimately leads to the accumulation of autophagy. The key mechanism for promoting autophagy is the Ca 2+ -CAMKK-β-AMPK-mTOR pathway. Selenium de ciency also destroys the balance of other elements, increasing the risk of physical and even psychological diseases. However, the speci c relationship and mechanism of crosstalk between autophagy and apoptosis will be explored in the next experiments.

Declarations
Availability of data and materials The datasets analyzed during the current study are available from the corresponding author upon request. Figure 1 Ultrastructural observation of swine intestine tissues and IPEC-J2 cells. A (swine intestine tissues) and B
Page 26/34 Results were expressed as mean ± SD (n=6).   Detection of autophagy-Ca2+ homeostasis-related genes in intestine tissue. The expression of autophagy-Ca2+ homeostasis related genes at mRNA (A) and protein levels (B) in intestine tissue control and selenium-de cient groups, respectively. * indicate that there were signi cant differences (P < 0.05) between the two groups and the data are presented as the mean ± SD (n=6).

Figure 4
Detection of autophagy-Ca2+ homeostasis-related genes in intestine tissue. The expression of autophagy-Ca2+ homeostasis related genes at mRNA (A) and protein levels (B) in intestine tissue control and selenium-de cient groups, respectively. * indicate that there were signi cant differences (P < 0.05) between the two groups and the data are presented as the mean ± SD (n=6).

Figure 5
Detection of autophagy-Ca2+ homeostasis-related genes in IPEC-J2 cell. The expression of autophagy-Ca2+ homeostasis related genes at mRNA (A) and protein levels (B) in IPEC-J2 cell control and seleniumde cient groups, respectively. * indicate that there were signi cant differences (P < 0.05) between the two groups and the data are presented as the mean ± SD (n=6).

Figure 5
Detection of autophagy-Ca2+ homeostasis-related genes in IPEC-J2 cell. The expression of autophagy-Ca2+ homeostasis related genes at mRNA (A) and protein levels (B) in IPEC-J2 cell control and seleniumde cient groups, respectively. * indicate that there were signi cant differences (P < 0.05) between the two groups and the data are presented as the mean ± SD (n=6).

Figure 6
Using ICP-MS technology detected 23 kinds of trace element ion levels, roughly divided into three groups of main elements, macroelements (Na, Mg, K, Ca), essential trace elements (Fe, Zn, B, Cu, Mn, Ni, Ba, Sb, Se, Ti, V), and toxic trace elements (Al, Li, As, Cd, Pd, Sn, Sr). * indicate that there were signi cant differences (P < 0.05) between the two groups and the data are presented as the mean ± SD (n=6).

Figure 6
Using ICP-MS technology detected 23 kinds of trace element ion levels, roughly divided into three groups of main elements, macroelements (Na, Mg, K, Ca), essential trace elements (Fe, Zn, B, Cu, Mn, Ni, Ba, Sb, Se, Ti, V), and toxic trace elements (Al, Li, As, Cd, Pd, Sn, Sr). * indicate that there were signi cant differences (P < 0.05) between the two groups and the data are presented as the mean ± SD (n=6).