Expression Profile Analysis of Selenium-Related Genes in Peripheral Blood Mononuclear Cells of Patients with Keshan Disease.

Keshan disease (KD) is an endemic cardiomyopathy, which mainly occurs in China. Selenium deficiency is believed to play an important role in the pathogenesis of KD, but the molecular mechanism of selenium-induced damage remains unclear. To identify the key genes involved in selenium-induced damage, we compared the expression profiles of selenium-related genes between patients with KD and normal controls. Total RNA was isolated, amplified, labeled, and hybridized to Agilent human 4 × 44 K whole genome microarrays. Selenium-related genes were screened using the Comparative Toxicogenomics Database. The microarray data were subjected to single-gene and gene ontology (GO) expression analysis using R Studio and Gene Set Enrichment Analysis (GSEA) software. Quantitative real-time PCR was conducted to validate the microarray results. We identified 16 upregulated and 11 downregulated selenium-related genes in patients. These genes are involved in apoptosis, metabolism, transcription regulation, ion transport, and growth and development. Of the significantly enriched GO categories in KD patients, we identified four apoptosis-related, two metabolism-related, four growth and development-related, and four ion transport-related GOs. Based on our results, we suggest that selenium might contribute to the development of KD through dysfunction of selenium-related genes involved in apoptosis, metabolism, ion transport, and growth and development in the myocardium.

The Functional Analysis of Selenium-Related Genes and Magnesium-Related Genes in the Gene Expression Profile Microarray in the Peripheral Blood Mononuclear Cells of Keshan Disease.

Keshan disease (KD) is an endemic cardiomyopathy with high mortality. Selenium (Se) deficiency is closely related to KD, while magnesium (Mg) plays many critical roles in the cardiovascular function. The molecular mechanism of KD pathogenesis is still unclear. Until now, we have not found any studies investigating the association between Se- or Mg-related genes and KD. In this study, oligonucleotide microarray analysis was used to identify the differentially expressed genes in the peripheral blood mononuclear cells between KD patients and normal controls. Next, human metabolome database (HMDB) was used to screen Se- and Mg-related genes. Function classification, gene pathway, and interaction network of Se- and Mg-related genes in KD peripheral blood mononuclear cells were defined by FunRich (functional enrichment analysis tool). Among 83 differentially expressed genes, five Se-related (DIO2, GPX1, GPX2, GPX4, and GPX7) and five Mg-related (ACSL6, EYA4, IDH2, PPM1A, and STK11) genes were recognized from HMDB. Two significant biological processes (energy pathways and metabolism), one molecular function (peroxidase activity), one biological pathway (glutathione redox reactions I), and one gene interaction network were constituted from Se-related and Mg-related genes. Se-related gene DIO2 and Mg-related genes STK11 and IDH2 may have key roles in the myocardial dysfunction of KD. However, we still have not obtained any interaction between Se-related gene and Mg-related gene. The interactions between RPS6KB1, PTEN, ATM, HSP90AA1, SNRK, PRKAA2, SMARCA4, HSPA1A, and STK11 may play important roles in the abnormal cardiac function of KD.

Efficacy of Long-term Selenium Supplementation in the Treatment of Chronic Keshan Disease with Congestive Heart Failure.

Few effective treatments for chronic Keshan disease have been available till now. The efficacy of long-term selenium supplementation in the treatment of chronic Keshan disease with congestive heart failure is inconclusive. This study aimed to determine whether selenium supplementation is associated with a decreased risk of cardiac death in chronic Keshan disease with congestive heart failure by ten years of follow-up. A retrospective long-term follow-up analysis was performed on a monitored cohort consisting of 302 chronic Keshan disease patients with a mean age of 40.8±11.4 years. Of the 302 chronic Keshan disease patients, 170 (56.3%) were given selenium supplementation until the end point of follow-up. Cox proportional hazards regression models were used to identify the independent predictors of cardiac events. Our results showed that during the follow-up, there were 101 deaths of patients with chronic Keshan disease in the selenium supplementation group (101/170, 59.4%) and 98 in non-selenium supplementation group (98/132, 74.2%). Multivariate analyses suggested that selenium supplementation was associated with a decreased risk of cardiac death (HR 0.39, 95% CI 0.28-0.53) after adjustment for baseline age, sex, cigarette smoking, family history of Keshan disease, body mass index (BMI), heart rate, electrocardiogram (ECG) abnormalities, blood pressure, initial cardiothoracic ratio, left ventricular ejection fractions (LVEF) and whole-blood selenium concentration. Our ten-year follow-up analysis indicated that selenium supplementation, specifically combined with the use of angiotensin-converting enzyme inhibitor and beta blocker therapy, improved the survival of patients with chronic Keshan disease with congestive heart failure. BMI, selenium deficiency, male, combined ECG abnormalities, LVEF, and fast heart rate increased the risk of cardiac events.

Network Analysis of Se-and Zn-related Proteins in the Serum Proteomics Expression Profile of the Endemic Dilated Cardiomyopathy Keshan Disease.

Keshan disease (KD) is an endemic cardiomyopathy with high mortality. Selenium (Se) and zinc (Zn) deficiencies are closely related to KD. The molecular mechanism of KD pathogenesis is still unclear. There are only few studies on the interaction of trace elements and proteins associated with the pathogenesis of KD. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) technique analysis was used to analyze the differential expression of proteins from serum samples. Comparative Toxicogenomics Database (CTD) was used to screen Se- and Zn-associated proteins. Then, pathway and network analyses of Se- and Zn-associated proteins were constituted by Cytoscape ClueGO and GeneMANIA plugins. One hundred and five differentially expressed proteins were obtained by 2DLC-MS/MS, among them 19 Se- and 3 Zn-associated proteins. Fifty-two pathways were identified from ClueGO and 1 network from GeneMANIA analyses. The results showed that Se-associated proteins STAT3 and MAPK1 and Zn-associated proteins HIF1A and PARP1, the proteins involved in HIF-1 signaling pathway and apoptosis pathway, may play significant roles in the pathogenesis of KD. The approach of this study would be also beneficial for further dissecting molecular mechanism of other trace element-associated disease.

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.

[Progressive Risks of Latent Keshan Disease: a Long Term Follow-up Study].

OBJECTIVE: To observe ten-year prognosis of patients with latent Keshan disease (KD) and to determine its associated risk factors. METHODS: A total of 448 patients with newly diagnosed latent KD were monitored and followed up for 10 years. Their ECG abnormalities were classified as major or minor using the Minnesota Code. COX proportional hazards regression models were established to identify risk factors associated with the development of chronic KD. RESULTS: A final sample of 414 cases was included in analyses, with an average of (112.9 ± 17.5) months of follow-up. At the end of follow-up, 92 (22. 2%) patients developed chronic KD. Older age (> 15 years), male, family history of KD, smoking, lower level of blood selenium (< 60 µg/L), major ECG abnormalities, and 18.5 kg/m² ≤ body mass index (BMI) 23.9 kg/m² were associated with higher cumulative incidence of chronic KD. The COX regression models showed that major ECG abnormalities, BMI, selenium deficiency, hypertension, and ventricular premature complex (VPC) abnormalities contributed to increased risk of chronic KD. A positive linear correlation (r = 0.719, P < 0.01) between GPx activity and blood selenium concentration was found. CONCLUSION: Major ECG abnormalities, BMI, selenium deficiency, hypertension and VPC abnormalities are associated with the development of chronic KD.

Selenium deficiency induced damages and altered expressions of metalloproteinases and their inhibitors (MMP1/3, TIMP1/3) in the kidneys of growing rats.

Selenium is an essential trace element for the maintenance of structures and functions of kidney. To evaluate the effects of low selenium on the kidneys of growing rats, newborn rats were fed with selenium deficient and normal diets respectively for 109 days. As a result, rats fed with low selenium diets resulted in a decline in the body weight and the concentration of selenium in the kidney, especially the male rats from the low selenium groups. Moreover, the ultrastructure of glomerulus and tubules were damaged in low selenium group: the glomeruli were observed with hyperplasia of mesangial cells, fusion of podocyte foot processes and thickening of basement membrane; and the tubules were observed with vacuolar degenerated epithelial cells, increased edema fluid or protein solution between cells, microvilli edema, increased cell gaps and decreased cell links. Furthermore, the pathological changes in selenium deficient group included the increase of fibers around renal hilum aorta and in the renal collecting duct, and shed of cells in the proximal convoluted tubules. In addition, up-regulated expressions of matrix metalloproteinases (MMP1/3) and down-regulated expressions of their inhibitors (TIMP1/3) at the mRNA and protein levels were also appeared to be relevant to low selenium. The results suggested that low selenium in diet may cause low selenium concentration in the kidney of growing rat and lead to damages of the ultrastructure and extracellular matrix (ECM) of kidney.

Effect of Selenium Deficiency on Phosphorylation of the AMPK Pathway in Rats.

Selenium is an important trace element for human health. Previous studies have raised concern that dietary selenium intake may change energy metabolism. AMP-activated protein kinase (AMPK) is a sensor of energy status that controls cellular energy homeostasis. We aimed to determine the effect of selenium on the phosphorylation of AMPK pathway between Se-deficient and normal Sprague-Dawley rats. Twenty-four weaning rats were fed either a Se-deficient diet (0.02 mg Se/kg) or a standard diet (0.18 mg Se/kg). After 109 days, total serum levels of non-esterified fatty acid and total amino acids were significantly higher and the serum insulin concentration was significantly lower in Se-deficient rats than in healthy controls. Selenium concentration and the activity of glutathione peroxidase (GPx) in myocardial tissue were significantly lower in Se-deficient rats. Importantly, mRNA levels of acetyl-CoA carboxylase beta (ACACB), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and protein levels of p-AMPKα were increased in the Se-deficient group compared to normal controls (p < 0.05). In conclusion, our results suggest that selenium deficiency induces changes in metabolic and molecular parameters involved in energy metabolism in the AMPK pathway.

[Effect of selenium on the protection of myocardial cells from injuries induced by overloaded reactive oxygen species, and on the expression of actin in myocardial cells].

OBJECTIVE: To investigate the effect of selenium on the protection of myocardial cells from injuries induced by H2O2 and on the expression of alpha-actin and beta-actin in myocardial cells. METHOD: Myocardial cells of suckling mice in the culture were divided into six groups: Controls group (without H2O2 or Se), H2O2 group, Se 0.05 micromol/ L group, Se 0.5 micromol/L group, Se 1.0 micromol/L group and Se 5.0 micromol/L group. The ultrastructure of myocardial cells was observed by transmission electron microscope (TEM), and the LDH and MDA contents in the culture media were determined by colorimetry. The expression of alpha-actin and beta-actin in myocardial cells was detected by Western blot. RESULTS: The injury of myocardial cells observed under TEM was attenuated in the 0.5 micromol/L Se group. The LDH and MDA contents in the culture media of the Se groups was higher than the control group (P < 0.05), but lower than the H2O2 group (P < 0.05). The LDH and MDA contents in the 0.5 micromol/L Se group were the lowest in all Se groups. The expression level of alpha-actin and beta-actin in the 0.5 micromol/L Se group is higher than that in the H2O2 group, even higher than the control group. CONCLUSION: The protective effect of Se on myocardial cells damaged by H2O2 was better in the 0.5 micromol/ LSe group, which could maintain the expression of alpha-actin and beta-actin, even induce the remolding of cytoskeleton proteins.

[Effect of a traditional Chinese medicine compound on chronic heart failure in guinea-pigs].

OBJECTIVE: To investigate the effect of a traditional Chinese medicine compound (CTMC) on chronic heart failure (CHF) in guinea-pigs. METHODS: The CHF of guinea-pigs were induced by repeated injection of hyodemic isoproterenol. The hemodynamics, organ (heart, lung, liver and kidney)/body weight ratio, pathological changes, and serum cTn-I and CK-MB were measured to determine the effectiveness of the traditional Chinese medicine treatement. RESULTS: The LVDP and LVEDP were decreased and the absolute value of + dp/dt(max) and - dp/ dt(max) were increased by the administration of 10 mg/kg, 20 mg/kg and 30 mg/kg of the compound tablets. The effect increased with doses. The traditional Chinese medicine also decreased the area of myocardial necrosis and the degree of injury to myocardiacyte. The intervention group had lower serum cTn-I and CK-MB levels than the controls. CONCLUSION: The compound tablets can improve the left ventricular diastolic function of CHF and reduce the myocardial damage in a dose-dependent manner.

[The change of certain biochemical indexes and apotosis protein (Fas/FasL) expression in rat liver of selenium and iodine deficiency].

OBJECTIVE: To investigate the effect of selenium (Se) and iodine (I) deficiency on certain biochemical indexes and apotosis protein (Fas/FasL) expression. METHOD: Healthy SD rats were divided into 4 groups at random, including control group, Se deficient group, I deficient group and both Se and I deficient group.The rats in each group were given different man-made feeds containing different levels of Se and I to breed. The second generation rats were chosen as research subjects. The GPX-Px activity and MDA and NO contents and expression of apoptosis protein (Fas/FasL) in liver were determined by chromometry and western blot. RESULT: GPX-Px of rat liver in Se deficient group significantly reduced, MDA and NO content s and expression of apoptosis of Fas/FasL increased. There were no apparent change in GPX-Px activities and NO contents of rat liver in I deficient group, while the contents of MDA and Fas/FasL expressions were much higher. The changes of GSH-Px activities and contents of MDA, NO as well as the Fas/FasL expressions in both Se and I deficiency groups are much higher than those of Selenium and Iodine deficiency groups. CONCLUSION: Se deficiency can cause the decrease of GPX-Px activities of rat liver and the increase of contents of MDA and NO. It can also lead to over expression of Fas/FasL. It seems that I deficiency can enhance the effect of selenium deficiency.

Analysis of iodine and selenium trace elements in umbilical cord blood in cretinous regions in northwest China in 1999.

Shaanxi Province located at Midwest inland of China was a typical iodine deficient disorders region. To investigate iodine and selenium levels of neonates in the Shaanxi sub-clinical cretinism region of China after supplement of iodine salt for nearly twenty years. We collected 56 umbilical cord blood samples from cretinous regions of Yijun County (a selenium deficient region) north of Shaanxi Province and Ziyang County (a selenium-enriched region) south of the province and from Lintong in Xi'an (a non-cretinous region for control). Among these samples 17 were collected from Ziyang, 20 from Lintong and 19 from Yijun. Seven trace elements of iodine, selenium, zinc, copper, iron, calcium and magnesium in the umbilical cord blood samples were measured and the results were processed statistically. There were no significant differences in the levels of iodine among all three counties. However, the level of selenium in Ziyang was the highest and in Yijun it was the lowest. The other trace elements such as Cu Zn Fe and Mg showed no significant difference among the three counties except for the Ca level which was lower in Yijun.The regression equation was established with the backward method of multiple regression was: Se=0.180+0.00006654 Fe-0.006 Cu-0.005956 Mg+0.1.