An untargeted metabolomics study of cardiac pathology damage in rats caused by low selenium diet alone or in combination with T-2 toxin.

T-2 toxin is a highly cardiotoxic environmental contaminant. Selenium can uphold the cardiovascular system's functionality. Selenium insufficiency is common. The aim of this study was to elucidate the effects of low selenium diet alone or in combination with T-2 toxin on myocardial tissue damage. Thirty-two Sprague-Dawley rats of 3 weeks of age were randomized into control, low selenium diet, low selenium diet combined with T-2 toxin groups (at doses of 10 ng/g and 100 ng/g body weight) for 12-weeks intervention. Pathohistology and ultrastructural changes in cardiac tissue were observed. Changes in cardiac metabolites were analyzed using untargeted metabolomics. The findings demonstrated that cardiac tissue abnormalities, interstitial bleeding, inflammatory cell infiltration, and mitochondrial damage can be brought on by low selenium diet alone or in combination with the T-2 toxin. A low selenium diet alone or in combination with the T-2 toxin affected cardiac metabolic profiles and resulted in aberrant modifications in many metabolic pathways, including the metabolism of amino acids, cholesterol, and thiamine. Accordingly, low selenium diet and T-2 toxin may have a synergistic effect. Our findings provide fresh insights into the processes of cardiac injury by revealing the effects of low selenium diet and T-2 toxin on cardiac metabolism.

Low selenium and T-2 toxin may be involved in the pathogenesis of Kashin-Beck disease by affecting AMPK/mTOR/ULK1 pathway mediated autophagy.

Kashin-Beck disease (KBD) is an endemic, environmentally associated cartilage disease. Previous studies have shown that the environmental suspected pathogenic factors of KBD, T-2 toxin and low selenium, are involved in the regulation of inflammation, oxidative stress and autophagy in some tissues and organs. In cartilage diseases, the level of cellular autophagy determines the fate of the chondrocytes. However, whether autophagy is involved in KBD cartilage lesions, and the role of low selenium and T-2 toxins in KBD cartilage injury and autophagy are still unclear. This work took the classical AMPK/mTOR/ULK1 autophagy regulatory pathway as the entry point to clarify the relationship between the environmental suspected pathogenic factors and chondrocyte autophagy. Transmission electron microscopy was used to observe the autophagy of chondrocytes in KBD patients. qRT-PCR and western blot were used to analyze the expression of AMPK/mTOR/ULK1 pathway and autophagy markers. The rat model of KBD was established by low selenium and T-2 toxin, the autophagy in rat cartilage was detected after 4- and 12-week interventions. Chondrocyte autophagy was found in KBD, and the AMPK/mTOR/ULK1 pathway was down-regulated. In the rat model, the pathway showed an up-regulated trend when low selenium and T-2 toxin, were treated for a short time or low concentration, and autophagy level increased. However, when low selenium and T-2 toxin were treated for a long time or at high concentrations, the pathway showed a down-regulated trend, and the autophagy level was reduced and even defective. In conclusion, in the process of KBD cartilage lesion, chondrocyte autophagy level may increase in the early stage, and decrease in the late stage with the progression of lesion. Low selenium and T-2 toxins may affect autophagy by AMPK/mTOR/ULK1 pathway.

Low Selenium and Low Protein Exacerbate Myocardial Damage in Keshan Disease by Affecting the PINK1/Parkin-mediated Mitochondrial Autophagy Pathway.

OBJECTIVE: Keshan disease (KD) is a myocardial mitochondrial disease closely related to insufficient selenium (Se) and protein intake. PTEN induced putative kinase 1 (PINK1)/Parkin mediated mitochondrial autophagy regulates various physiological and pathological processes in the body. This study aimed to elucidate the relationship between PINK1/Parkin-regulated mitochondrial autophagy and KD-related myocardial injury. METHODS: A low Se and low protein animal model was established. One hundred Wistar rats were randomly divided into 5 groups (control group, low Se group, low protein group, low Se + low protein group, and corn from KD area group). The JC-1 method was used to detect the mitochondrial membrane potential (MMP). ELISA was used to detect serum creatine kinase MB (CK-MB), cardiac troponin I (cTnI), and mitochondrial-glutamicoxalacetic transaminase (M-GOT) levels. RT-PCR and Western blot analysis were used to detect the expression of PINK1, Parkin, sequestome 1 (P62), and microtubule-associated proteins1A/1B light chain 3B (MAP1LC3B). RESULTS: The MMP was significantly decreased and the activity of CK-MB, cTnI, and M-GOT significantly increased in each experimental group (low Se group, low protein group, low Se + low protein group and corn from KD area group) compared with the control group (P<0.05 for all). The mRNA and protein expression levels of PINK1, Parkin and MAP1LC3B were profoundly increased, and those of P62 markedly decreased in the experimental groups compared with the control group (P<0.05 for all). CONCLUSION: Low Se and low protein levels exacerbate myocardial damage in KD by affecting the PINK1/Parkin-mediated mitochondrial autophagy pathway.

[Evaluation of dietary diversity of rural residents in Arun Banner, a typical area of low selenium belt in China].

OBJECTIVE: To understand the dietary diversity and food sources of different groups in Arun Banner. Comparing and analyzing the changes of dietary diversity of local residents in different periods. METHODS: In October 2019, according to the differences of Kashin-Beck disease conditions and land use patterns of Arun Banner, Hulun Buir, 78 rural residents were randomly selected from four villages to investigate the frequency of daily food consumption by using self-made food frequency questionnaire. Dietary diversity score(DDS) was used to evaluate the dietary diversity of different populations. RESULTS: The average DDS of the investigated residents was 9. 76±2. 32. DDS of the residents of Minzu and Tieshan villages(10. 14±2. 66) in the West was higher than that of Longtoushan and Hongqi villages(9. 42±1. 95) in the East. However, the difference was not statistically significant(Z=-1. 875, P& gt; 0. 05). DDS in males(10. 37±2. 47) was significantly higher than that in females(9. 36±2. 14)(P& lt; 0. 05). There was no significant difference in DDS(9. 52±2. 29) between Kashin-Beck disease affected patients and healthy residents(9. 85±2. 34)(Z=-0. 601, P& gt; 0. 05). There was no significant difference in DDS among different age groups. The average of DDS was higher in those younger than 60 years old(9. 90±2. 35). Residents mainly eat rice and flour, and the proportion of purchasing food from other places reaches 96% and 95%, respectively. CONCLUSION: The staple food in Arun Banner was mainly rice and flour purchased from other places. The dietary structure of residents tends to be diversified. Residents reduced dependence on low selenium natural environment is an important factor for local selenium-susceptible endemic diseases to be stable and gradually controlled.

Selenium Nutritional Status of Rural Residents and Its Correlation with Dietary Intake Patterns in a Typical Low-Selenium Area in China.

China is recognized as a selenium-deficient country, and nutritional selenium intake has always been a concern. To clarify the current inhabitants' selenium nutrition status and the characteristics of dietary consumption in low-selenium areas, samples of human hair and grains were collected, and food frequency questionnaires were administered in Binxian County, Shaanxi Province, a typical low-selenium area in the Loess Plateau. The subject number of the study is 85, and the age range is from 11 to 81 years, with an average of 60. The results showed that the average hair selenium content of the residents was 231.7 µg/kg, and 62.4% of the participants had levels higher than the selenium deficiency threshold (200 µg/kg). There was a significant positive correlation between the hair selenium content and the food consumption score after adjusting for rice outsourcing. Three different dietary patterns were noted according to hierarchical cluster analysis. This study provides a tool for assessing the selenium nutrition of inhabitants in low-selenium areas and has considerable significance for improving the dietary pattern of residents.

The effects of long-term low selenium diet on the expression of CHST-3, CHST-12 and UST in knee cartilage of growing rats.

OBJECTIVES: To investigate the effect of low selenium diet on rat´s knee cartilage and expression of chondroitin sulfate (CS) sulfated enzymes in articular and epiphyseal-plate cartilage of rats' femur and tibia. METHODS: Twenty-four SD rats were randomly divided into two groups with six female and six male in each group: control group (selenium 0.18 mg/kg), and low selenium group (selenium 0.02 mg/kg). After 109 days, the rats were sacrificed. The ultrastructural changes in chondrocytes of rat knee cartilage were observed by transmission electron microscopy (TEM). The morphology and pathology changes of knee cartilage were examined by hematoxylin-eosin (HE) and toluidine blue (TB) staining. The localization and expression of enzymes involved in CS sulfation, including chondroitin 6-O-sulfotransferase 1 (CHST-3), chondroitin 4-O-sulfotransferase 2 (CHST-12) and uronyl 2-O-sulfotransferase (UST) were examined by immunohistochemical staining and semi-quantitative analysis. RESULTS: In low selenium group, ultrastructural changes of chondrocytes were observed in articular cartilage of femur (AF), articular cartilage of tibia (AT), epiphyseal-plate cartilage of femur (EF) and epiphyseal-plate cartilage of tibia (ET); however, no significant changes in chondrocytes number were observed in the above AF, AT, EF or ET. Moreover, reduced thickness of cartilage layer in AF, EF and ET was detected along with reduced staining areas of sulfated glycosaminoglycan in EF and ET in low selenium group. In addition, positive staining rate of CHST-3 was lower in AF, AT and EF, while positive staining rates of CHST-12 and UST were lower in AF, AT, EF and ET in low selenium group when compared with control group. CONCLUSIONS: Low selenium undermines the ultrastructure of chondrocytes, inhibits the normal development of cartilage and the expression of CS sulfated enzymes.

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.

Effects of selenium on myocardial apoptosis by modifying the activity of mitochondrial STAT3 and regulating potassium channel expression.

The present study investigated the effects of myocardial mitochondrial signal transduction and activator of transcription 3 (STAT3), succinate dehydrogenase activity and changes of potassium channel expression on cardiomyocyte apoptosis under low selenium conditions. Primary cultured cardiomyocytes from neonatal mice were divided into the non-toxic control group (0.1 µM sodium selenite) and low selenium treatment group (0.05 µM sodium selenite) according to different selenium concentrations. The expression of mitochondrial STAT3, p-STAT3, p-Kv1.2 potassium channel and apoptosis-related proteins, Bax and Bcl-2, were assessed by immunoblotting. Succinate dehydrogenase activity was measured by spectrophotometry. Flow cytometry was used to detect cardiomyocyte apoptosis. Low selenium treatment reduced the expression of p-STAT3, but did not affect the expression of STAT3. In addition, low selenium treatment reduced the activity of mitochondrial STAT3 and succinate dehydrogenase in cardiomyocytes, leading to injury of myocardial mitochondria. Compared with the control group, low selenium conditions reduced the activity of p-Kv1.2 and reduced the normal electrophysiological function of cardiomyocytes. In the low selenium-treated group, the expression of Bax protein increased, whereas the expression of Bcl-2 protein decreased. The apoptotic rate increased. In conclusion, selenium deficiency in cardiomyocytes leads to decreased potassium channel expression and decreased mitochondrial STAT3 activity and mitochondrial function, which in turn promotes the apoptosis of cardiomyocytes.

Long-Term Selenium-Deficient Diet Induces Liver Damage by Altering Hepatocyte Ultrastructure and MMP1/3 and TIMP1/3 Expression in Growing Rats.

The effects of selenium (Se)-deficient diet on the liver were evaluated by using growing rats which were fed with normal and Se-deficient diets, respectively, for 109 days. The results showed that rats fed with Se-deficient diet led to a decrease in Se concentration in the liver, particularly among male rats from the low-Se group. This causes alterations to the ultrastructure of hepatocytes with condensed chromatin and swelling mitochondria observed after low Se intake. Meanwhile, pathological changes and increased fibrosis in hepatic periportal were detected by hematoxylin and eosin and Masson's trichrome staining in low-Se group. Furthermore, through immunohistochemistry (IHC) staining, higher expressions of metalloproteinases (MMP1/3) and their tissue inhibitors of metalloproteinases (TIMP1/3) were observed in the hepatic periportal of rats from the low-Se group. However, higher expressions of MMP1/3 and lower expressions of TIMP1/3 were detected in hepatic central vein and hepatic sinusoid. In addition, upregulated expressions of MMP1/3 and downregulated expressions of TIMP1/3 at the messenger RNA (mRNA) and protein levels also appeared to be relevant to low Se intake. In conclusion, Se-deficient diet could cause low Se concentration in the liver, alterations of hepatocyte ultrastructure, differential expressions of MMP1/3 and TIMP1/3 as well as fibrosis in the liver hepatic periportal.

Oxidative damage induced by T-2 toxin toxication in articular cartilage of rats under selenium deficiency / 中华地方病学杂志

Objective To study the change of rats serum malondialdehyde (MDA) and the expression levels of 4-hydroxy acid nonene (4-HNE) and 8-hydroxy uridine (8-OHdG) of articular cartilage under low selenium (Se) and T-2 toxin poisoning,to explore oxidative damage of articular cartilage in rats.Methods Thirtytwo healthy male SD rats were divided into two groups by weight which were normal diet group and Se-deficiency group,16 rats in each group.Rats in normal diet group was fed with selenium 101.5 μg/kg diet,and rats in Sedeficiency group was fed with selenium 1.1 μg/kg diet for 30 d.Normal diet group was divided into control group and T-2 toxin group,and low selenium diet group was randomly divided into Se-deficiency group and Se-deficiency plus T-2 toxin group,8 rats in each group.After that,rats in T-2 toxin and Se-deficiency plus T-2 toxin groups were administrated intragastrically with T-2 toxin (100 mg/kg) everyday for 30 d.Rats were put to death,the left knee was taken and stained with hematoxylin-eosin and Safranin-Fast green,pathological changes of rat's knee joint cartilage were observed under light microscopy,expression levels of 8-OHdG and 4-HNE in rat's articular cartilage cells were determined by immunohistochemical method and rat's MDA content was determined by glucosinolates barbituric acid method.Results Chondronecrosis in deep zone of articular cartilage of knee joint stained with hematoxylin-eosin was seen in Se-deficient plus T-2 toxin diet group under light microscope.Significantly less Safranin-Fast green staining was observed in the cartilage of knee joints in the Se-deficient plus T-2 toxin diet group compared to the control group.Compared with control group [(3.41 ± 2.48)％,(2.28 ± 1.74)％],8-OHdG and 4-HNE in Se-deficient plus T-2 toxin group [(62.61 + 10.97)％,(75.03 ± 7.92) ％] positive expression rate increased significantly (F =16.24,18.61,all P ＜ 0.05).Comparison of serum MDA content in each group,the difference was statistically significant (F =4.32,P ＜ 0.05).The Se-deficiency group [(2.803 ± 0.163) μmol/L] was compared with control group [(1.873 ± 0.475) μmol/L] that the contents of serum MDA were increased.The T-2 toxin group [(2.890 ± 0.453) μmol/L] was compared with control group [(1.873 ± 0.475) μmol/L] that the content of serum MDA was increased (P ＜ 0.05).The Se-deficiency plus T-2 toxin group [(3.521 ± 0.292) μmol/L] was compared with Sedeficiency group and control group that the contents of serum MDA were increased (all P ＜ 0.05).Conclusions The marker of peroxidation products are increased in articular cartilage of SD rats under the condition of Sedeficiency and T-2 toxin poisoning.The cartilage damage and chondronecrosis due to Se-deficiency and T-2 toxin poisoning are related to oxidative damage.

EFFECT OF LOW SELENIUM ON CHONDROCYTE DIFFERENTIATION AND DIFFERENTIAL EXPRESSION OF COLLAGEN TYPES Ⅰ , Ⅱ AND X IN ARTICULAR CARTILAGE FROM MINI-PIGS / 药物分析学报

Objective According to the distribution of low selenium areas, low nutrition state of the residents and the affecting cartilage growth and articular cartilage of Kashin-Beck Disease(KBD),the chondrocyte differentia- tion and differential expression of collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage from Chinese mini-pigs treated with low selenium were investigated in order to gain insight into the effects of these conditions on chondrocyte differ- entiation in KBD cartilage. Mothods Eleven male juvenile mini-pigs, aged from 4 weeks to 6 weeks after birth, were divided into 3 groups. The Se content in the diet of the “low Se” group was 0. 035mg/kg diet, and 0. 175 mg/kg diet in the control. For Se-supplemented group 0. 390mg /kg diet was added. The content of Se in blood was assayed at the beginning and at the end of each experiment. Samples of articular cartilage were taken from the right femur condylus, and collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage were analyzed by immunohistochemistry and in situ hybridization. Results①All cartilage samples from juvenile mini-pigs fed with low selenium diet revealed a re- duction in type Ⅹ collagen mRNA expression in the hypertrophic chondrocytes as shown by in situ hybridization, and reduced type Ⅹ collagen deposition in the lower hypertrophic zone as shown by immunohistochemistry. ②Addition of selenium to the diet restored the type Ⅹ collagen to normal level. ③Type Ⅱ collagen was evenly distributed over the entire articular cartilage in all experimental and control groups. Type Ⅱ collagen mRNA signals were most prominent in the upper articular layer as well as in the hypertrophic zone in all groups. Type Ⅱ collagen expression was restrict- ed to the zone of endochondral ossification in all experimental groups and the control. Conclusion Low selenium has an down-regulatory role on the synthesis and deposition of collagen type Ⅹ in hypertrophic chondrocytes in articular cartilage of mini-pigs. Supplement of the low Se diet with additional Se restored the signals of collagen type Ⅹ to nor- mal levels. These findings indicate that selenium deficiency may disturb chondrocyte differentiation to hypertrophic cells in the growth plate,and worthy to be investigated further.