MicroRNA-33-3p Regulates Vein Endothelial Cell Apoptosis in Selenium-Deficient Broilers by Targeting E4F1.

Selenium (Se) is a type of nutrient element. The tissues of organisms can have pathological damage, including apoptosis, due to Se deficiency. Apoptosis is an important cell process and plays a key role in vascular disease and Se-deficient symptoms. In this study, the Se-deficient broiler model was duplicated, miR-33-3p in the vein was overexpressed in response to Se-deficiency, and miR-33-3p target gene E4F transcription factor 1 (E4F1) expression was also confirmed. We utilized ectopic miR-33-3p expression to validate its function for apoptosis. The results showed that miR-33-3p-targeted E4F1 are involved in the glucose-regulated protein 78- (GRP78-) induced endoplasmic reticulum stress (ERS) apoptosis pathway. We presumed that Se deficiency might trigger apoptosis via downregulating miR-33-3p. Interestingly, the miR-33-3p inhibitor and VER-155008 (GRP78 inhibitor) partly hindered the apoptosis caused by Se deficiency. Thus, the above information provides a new avenue toward understanding the mechanism of Se deficiency and reveals a novel apoptotic injury regulation model in vascular disease.

microRNA-33-3p involved in selenium deficiency-induced apoptosis via targeting ADAM10 in the chicken kidney.

Selenium (Se) deficiency induces typical clinical and pathological changes and causes various pathological responses at the molecular level in several different chicken organs; the kidney is one of the target organs of Se deficiency. To explore the mechanisms that underlie the effects of microRNA-33-3p (miR-33-3p) on Se deficiency-induced kidney apoptosis, 60 chickens were randomly divided into two groups (30 chickens per group). We found that Se deficiency increased the expression of miR-33-3p in the chicken kidney. A disintegrin and metalloprotease domain 10 (ADAM10) was verified to be a target of miR-33-3p in the chicken kidney. The overexpression of miR-33-3p decreased the expression levels of ß-catenin, cyclinD1, T-cell factor (TCF), c-myc, survivin, and Bcl-2; it increased the expression levels of E-cadherin, Bak, Bax, and caspase-3; and it increased the number of chicken kidney cells in the G0/G1 phase. In addition, Se deficiency caused the ultrastructure of the kidney to develop apoptotic characteristics. The results of flow cytometry analysis and AO/EB staining showed that the number of apoptotic chicken kidney cells increased in the miR-33-3p mimic group. All these results suggest that Se deficiency-induced cell cycle arrest and apoptosis in vivo and in vitro in the chicken kidney via the regulation of miR-33-3p, which targets ADAM10.

MicroRNA-193b-3p regulates hepatocyte apoptosis in selenium-deficient broilers by targeting MAML1.

Selenium (Se) is an important nutritional element in the diet. Apoptosis is one of the characteristic pathological changes in liver tissue resulting from Se deficiency. MicroRNA (miRNA) plays an important role in cell proliferation, differentiation, apoptosis and tumorigenesis. However, why apoptosis occurs during Se deficiency and how miRNA regulates hepatocyte apoptosis in broilers requires further study. We used a dual luciferase reporter assay system and quantitative real-time PCR (qPCR) to screen hepatocytes in Se-deficient broilers for the specificity of hepatocyte apoptosis miRNA and its target protein. We tested the apoptosis of Se-deficient broiler livers and microRNA-193b-transfected primary hepatocytes using qPCR, western blot (WB) and flow cytometry. Our studies revealed that Se deficiency led to microRNA-193b-3p (miR-193b-3p) overexpression and increased apoptosis-related gene expression, resulting in broiler hepatocyte apoptosis. Mastermind-like protein 1 (MAML1) was one of the miR-193b-3p targets, and its expression was down-regulated in miR-193b-3p-overexpressing hepatocytes. Further studies have shown that miR-193b-3p overexpression induced changes of apoptosis-related gene expression by inhibiting the release of MAML1. Interestingly, when we overexpressed miR-193b-3p, which was added to the Signal transducers and activators of transcription-1 (STAT1) inhibitor fludarabine (Flu), hepatocyte apoptosis was significantly reduced. When these results were combined, they indicated that miR-193b-3p is involved in broiler hepatocyte apoptosis in Se deficiency by regulating the target protein MAML1. This finding may provide new ideas for studying the mechanism of hepatocyte injury due to Se deficiency.

MiR-128-1-5p regulates tight junction induced by selenium deficiency via targeting cell adhesion molecule 1 in broilers vein endothelial cells.

Vein endothelial cells (VECs) constitute an important barrier for macromolecules and circulating cells from the blood to the tissues, stabilizing the colloid osmotic pressure of the blood, regulating the vascular tone, and rapidly changing the intercellular connection, and maintaining normal physiological function. Tight junction has been discovered as an important structural basis of intercellular connection and may play a key role in intercellular connection injuries or vascular diseases and selenium (Se) deficiency symptoms. Hence, we replicated the Se-deficient broilers model and detected the specific microRNA in response to Se-deficient vein by using quantitative real time-PCR (qRT-PCR) analysis. Also, we selected miR-128-1-5p based on differential expression in vein tissue and confirmed its target gene cell adhesion molecule 1 (CADM1) by the dual luciferase reporter assay and qRT-PCR in VECs. We made the ectopic miR-128-1-5p expression for the purpose of validating its function on tight junction. The result showed that miR-128-1-5p and CADM1 were involved in the ZO-1-mediated tight junction, increased paracellular permeability, and arrested cell cycle. We presumed that miR-128-1-5p and Se deficiency might trigger tight junction. Interestingly, miR-128-1-5p inhibitor and fasudil in part hinder the destruction of the intercellular structure caused by Se deficiency. The miR-128-1-5p/CADM1/tight junction axis provides a new avenue toward understanding the mechanism of Se deficiency, revealing a novel regulation model of tight junction injury in vascular diseases.

Effect of Low-Selenium/High-Fat Diet on Pig Peripheral Blood Lymphocytes: Perspectives from Selenoproteins, Heat Shock Proteins, and Cytokines.

The aim of the present study was to clarify the effect of low selenium (Se)/high fat on the mRNA expression of selenoproteins, heat shock proteins (HSPs) and cytokines in pig peripheral blood lymphocytes. Forty crossbred boar piglets with healthy lean body weights of 10 kg were randomly divided into four treatment groups (group C, group L-Se, group H-fat, and group L-Se-H-fat) (n = 10/group) and fed with the corresponding diet for 16 weeks. The pig peripheral blood lymphocytes were extracted, and the mRNA expression of selenoproteins, HSPs, and cytokines was measured. Most mRNA levels for selenoproteins decreased in group L-Se, group H-fat, and group L-Se-H-fat, except Gpx1, Gpx2, Selt, and Selm, which were elevated in group H-fat. At the same time, low-Se/high-fat diet increased the expression of HSPs (HSP40, HSP60, HSP70, and HSP90) and inflammatory cytokines (IL-1α, IL-1ß, IL-6, IL-8, IL-9, iNOS, COX-2, NF-κB, and TNF-α) in group L-Se, group H-fat, and group L-Se-H-fat, and genes in group L-Se-H-fat showed greater increases. Also, low-Se/high-fat diet inhibits the expression of TGF-ß1 and IFN-γ. In summary, a low-Se/high-fat diet can cause relevant selenoprotein expression changes and promote the expression of pro-inflammatory factors and HSPs, and low Se enhances the expression of HSPs and inflammation factors induced by high fat. This information is helpful for understanding the effects of low-Se and high-fat diet on pig peripheral blood lymphocytes.

Lower Selenoprotein T Expression and Immune Response in the Immune Organs of Broilers with Exudative Diathesis Due to Selenium Deficiency.

The objective of the present study was to investigate whether dietary selenium (Se) deficiency would affect the expression of selenoprotein T (SelT) and immune response in the immune organs of broilers. Changes in expression of inflammatory cytokines and oxidative stress response caused by Se deficiency can lead to organism damage, which in turn leads to immune response. Sixty (1-day-old) broilers were divided into the control group and Se-deficiency group. Animal models with exudative diathesis were duplicated in the broilers by feeding them Se-deficient diet for 20 days. After the Se-deficient group exhibited symptoms of exudative diathesis, all the broilers were euthanized, and their immune organs were taken for analysis. The tissues including spleen, bursa of Fabricius, and thymus were treated to determine the pathological changes (including microscopic and ultramicroscopic), the messenger RNA (mRNA) expression levels of SelT and its synthetase (SecS and SPS1), cytokine mRNA expression levels, and antioxidant status. The microscopic and ultramicroscopic analyses showed that immune tissues were obviously injured in the Se-deficient group. The mRNA expression of SelT was decreased compared with that in the control group. Meanwhile, the mRNA expression levels of SecS and SPS1 were downregulated. In the Se-deficient group, the mRNA expression levels of IL-1R and IL-1ß were higher than those of three control organs. Additionally, the IL-2 and INF-γ mRNA expression levels were lower than those of the control group. The activity of CAT was decreased, and the contents of H2O2 and •OH were increased due to Se deficiency. Pearson method analysis showed that the expression of SelT had a positive correlation with IL-2, INF-γ, SecS, and SPS1 and a negative correlation with IL-1R and IL-1ß. In summary, these data indicated that Se-deficient diet decreased the SelT expression and its regulation of oxidative stress, and it inhibited a pleiotropic mechanism of the immune response.

Down-regulation of microRNA-155 promotes selenium deficiency-induced apoptosis by tumor necrosis factor receptor superfamily member 1B in the broiler spleen.

The aim of this work was to explore the microRNA profile and the effect of microRNA-155 on apoptosis in the spleen of selenium-deficient broilers. We replicated the splenic-apoptotic model in selenium-deficient broilers. In vitro, microRNA-155 oligonucleotides were transfected into lymphocytes and subsequently treated with H2O2. We observed that selenium deficiency altered the microRNA profile and decreased the expression of microRNA-155 in the broiler spleens. Tumor necrosis factor receptor superfamily member 1B was verified as a target of microRNA-155 in the splenocytes. Morphological changes, increased levels of tumor necrosis factor receptor superfamily member 1B, c-Jun N-terminal kinase, Bak, Bax, Cyt-c, caspase9 and caspase3 and decreased levels of Bcl-2 demonstrated that selenium deficiency induced apoptosis in the spleen tissues. In vitro, microRNA-155 m inhibited the levels of ROS and reduced apoptosis compared with microRNA-155i in the lymphocytes. These results suggested that the reduced levels of microRNA-155 due to selenium deficiency could promote oxidative stress-induced apoptosis by increased tumor necrosis factor receptor superfamily member 1B in splenic cells.

Antagonistic effects of selenium on cadmium-induced apoptosis by restoring the mitochondrial dynamic equilibrium and energy metabolism in chicken spleens.

The aim of this study was to investigate the mechanism of cadmium-induced apoptosis in chicken spleens and the antagonistic effects of selenium. We duplicated the selenium-cadmium interaction model and examined the expression of apoptosis-, immune-, mitochondrial dynamics- and energy metabolism-related genes. The results demonstrated that after treatment with cadmium, the frequency of apoptosis was significantly increased, and the morphological characteristics of apoptosis were observed. The expression of pro-apoptotic genes was increased, and that of anti-apoptotic genes was decreased. The mRNA levels of tumor necrosis factor-α and interlenkin-1ß were observably increased, but the interlenkin-2 and interferon-γ levels were markedly decreased. Furthermore, the mRNA and protein levels of dynamin-related protein 1 and mitochondrial fission factor were significantly enhanced, whereas mitofusin 1, mitofusin 2, and optic atrophy 1 were markedly decreased. The expression of hexokinase 1, hexokinase 2, aconitase 2, lactate dehydrogenase A, lactate dehydrogenase B, succinatedehydrogenase B, pyruvate kinase and phosphofructokinase were also reduced. Selenium supplements remarkably attenuated cadmium-induced effects (p < 0.05). Based on the above results, conclude that the cadmium treatment promoted a mitochondrial dynamic imbalance and reduced energy metabolism, leading to apoptosis and immune dysfunction in chicken spleens, and selenium had an antagonistic effect on Cd-induced apoptosis.

Selenium accelerates chicken dendritic cells differentiation and affects selenoproteins expression.

Selenium (Se) promotes immune cell differentiation and improves immune response. Antigen-presenting cells such as dendritic cells (DCs) play an important role in immune system, however, the impact of Se on DCs is still unclear. In this study, we successfully induced and cultured chicken DCs from peripheral blood mononuclear cells by incubating mononuclear cells with 50 ng/mL recombinant chicken granulocyte-macrophage colony stimulating factor and 10 ng/mL recombinant chicken interleukin-4 for total 9 days. In + Se group, we added 10-7 mol/L sodium selenite from the first day of cell culture. The results showed that Se supplementation expedited and increased the expression of cell surface markers including CD11c, CD40, CD86, and MHC II. Principal component analysis showed that the expression of selenoproteins SelW, SelK, Dio3, GPX1, GPX2, SelN, SelS, SelH in chicken DCs was highly correlated, and SelW had highest correlation with the cell surface markers MHC II and CD11c. In conclusion, Se accelerates the differentiation and maturation of chicken DCs. Se regulates the differentiation and maturation of chicken DCs by selenoproteins. Selenoproteins has closely correlated to surface markers of chicken DCs.

Cadmium-induced endoplasmic reticulum stress in chicken neutrophils is alleviated by selenium.

Cadmium (Cd) decreases immune function and induces apoptosis of immune cells. Selenium (Se) can antagonize some metal element toxicity including Cd. To evaluate the cytotoxicity of Cd and the chemoprotective role of Se on bird neutrophils in vitro, we incubated chicken neutrophils cells with Cadmium chloride (CdCl2) (10-6M), Sodium selenite (Na2SeO3) (10-7M), and with a mixture of Na2SeO3 (10-7M) and CdCl2 (10-6M) for 12, 24, 36, and 48h. We found that Interleukin 1ß (IL-1ß), Interleukin 10 (IL-10), and interferon gamma (IFN-γ) increased and interleukin 17 (IL-17), interleukin 4 (IL-4) decreased significantly in the chicken neutrophils of the Cd treatment groups. Cd significantly increased the mRNA expression levels of nuclear factor kappaB (NF-κB), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) and the nitric oxide (NO) content. In addition, we demonstrated that Cd induced the apoptosis of chicken neutrophils and increased mRNA level of Bak, Cysteine-aspartic protease (Caspase)-3, Caspase-9, Caspase-12, glucose-regulated protein 78 (GRP78) and activating transcription factor 6 (ATF6), decreased mRNA level of Bcl-xl, and Ca/calmodulin-dependent protein (CaM). Moreover, the expression of NF-κB and Caspase-12 protein increased significantly in the Cd treatment groups. Se pretreatment significantly protected neutrophils against Cd-caused alterations. Our work suggested that Cd-induced immune suppression, inflammatory response, and apoptosis via endoplasmic reticulum stress (ERS). Moreover, these factors played critical roles in Se-mediated chemoprevention against Cd-induced immunotoxicity.

Selenium Deficiency Induces Autophagy in Immune Organs of Chickens.

The aim of the present study was to investigate the effects of selenium (Se) deficiency on autophagy-related genes and on ultrastructural changes in the spleen, bursa of Fabricius, and thymus of chickens. The Se deficiency group was fed a basal diet containing Se at 0.033 mg/kg and the control group was fed the same basal diet containing Se at 0.15 mg/kg. The messenger RNA (mRNA) levels of the autophagy genes microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin 1, dynein, autophagy associated gene 5 (ATG5), and target of rapamycin complex 1 (TORC1) were assessed using real-time qPCR. The protein levels of LC3-II, Beclin 1, and dynein were investigated using western blot analysis. Furthermore, the ultrastructure was observed using an electron microscope. The results indicated that spleen mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of LC3-II, Beclin 1, and dynein were increased in the Se deficiency group compared with the control group. In the bursa of Fabricius, the mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of Beclin 1 and dynein were increased; furthermore, the protein level of LC3-II was decreased in the Se deficiency group compared to the control group. In the thymus, the mRNA levels of LC3-I, Beclin 1, and ATG5 increased; the levels of LC3-II, dynein, and TORC1 were decreased; the protein level of Beclin 1 increased; and the levels of LC3-II and dynein decreased in the Se deficiency group compared to those in the control group. Further cellular morphological changes, such as autophagy vacuoles, autolysosomes, and lysosomal degradation, were observed in the spleen, bursa of Fabricius, and thymus of the Se-deficiency group. In summary, Se deficiency caused changes in autophagy-related genes, which increased the autophagic process and also caused structural damages to the immune organs of chickens.