Objective:To exploring the effect of selenium on tumor necrosis factor-α (TNF-α) induced inflammatory injury of AC16 cardiomyocytes.Methods:AC16 cardiomyocytes cultured in vitro were divided into control group, selenium pretreatment group (100 μg/L sodium selenite pretreatment for 4 h), 50 μg/L TNF-α group, selenium pretreatment + 50 μg/L TNF-α group, 100 μg/L TNF-α group, and selenium pretreatment + 100 μg/L TNF-α group. After 24 h of culture, the cell culture medium or cells were collected for detection. Griess method was used to detect nitric oxide (NO) level in cell culture medium. Hoechst 33342 nuclear staining was detected by fluorescence inverted microscope and karyopyknosis ratio was analyzed. The mRNA expression levels of B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2 associated X protein (Bax) and inducible nitric oxide synthase (iNOS) were detected by real-time fluorescence quantitative PCR. The protein expression levels of nuclear factor-kappa B (NF-κB) p65 and inhibitory κB-α (IκB-α) were detected by Western blotting. Results:The levels of NO in the control group, selenium pretreatment group, 50 μg/L TNF-α group, selenium pretreatment + 50 μg/L TNF-α group, 100 μg/L TNF-α group and selenium pretreatment + 100 μg/L TNF-α group were (10.58 ± 2.32), (9.07 ± 0.73), (15.53 ± 3.97), (12.05 ± 1.11), (30.65 ± 4.16) and (19.02 ± 3.72) μmol/L, respectively. Selenium and TNF-α had main effects on NO level ( F = 37.71, 99.07, P < 0.001) and interaction effect ( F = 11.80, P < 0.001). Selenium and TNF-α had main effects on karyopyknosis ratio and Bcl-2 and Bax mRNA expression levels ( F = 56.37, 462.81, 18.04, 32.85, 18.38, 170.77, P < 0.05). Selenium and TNF-α had an interactive effect on both karyopyknosis ratio and Bax mRNA expression level ( F = 21.48, 19.96, P < 0.001). Selenium and TNF-α had main effects on iNOS mRNA expression level ( F = 129.98, 1 051.76, P < 0.001) and interaction effect ( F = 53.28, P < 0.001). Selenium and TNF-α had main effects on the protein expression levels of NF-κB p65 and IκB-α ( F = 73.65, 145.49, 710.20, 105.66, P < 0.001) and interaction effects ( F = 26.73, 197.59, P < 0.001). Conclusion:Selenium may inhibits iNOS expression through NF-κB signal system and protects cardiomyocytes from TNF-α induced inflammatory injury.

Objective:To investigate the relationship between single nucleotide polymorphisms of transforming growth factor-β2 (TGFβ2) gene and Keshan disease (KD) in Han population of Shaanxi Province.Methods:KD region in Huangling County, Yan'an City, Shaanxi Province was selected as the investigation site in this study. Using the method of cluster random sampling, 52 families with KD in 6 administrative villages in Huangling County (Duanjiawan Village, Taoqu Village, Yaoping Village, Jianzhuang Village, Anjiao Village in Yaoping Town, and Houziping Village in Diantou Town) were selected for epidemiological investigation. According to the "Diagnosis of Keshan Disease" (WS/T 210-2011), 285 subjects were identified, including 79 patients with KD (case group) and 206 healthy controls (control group). Genomic DNA was extracted from the peripheral venous blood. The polymorphism of genetic variation of TGFβ2 gene rs6658835 was genotyped by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Chi-square (χ 2) test and t-test were used to analyze the baseline data, and binary logistic regression model was used to analyze the influencing factors of KD, all samples were tested for Hardy-Weinberg equilibrium using goodness-of-fit χ 2 test, differences in genotype and allele frequencies between case and control groups were compared by χ 2 test, and logistic regression analysis was used to compare the genotype frequencies between two groups after adjusting for confounding factors. Results:Epidemiological investigation showed that there were significant differences in age and heart murmur between case group and control group ( t = 7.03, χ 2 = 9.66, P < 0.05). The analysis of binary logistic regression model showed that the influence of age on KD was statistically significant (χ 2 = 20.72, P < 0.001). The gene frequency distribution of TGFβ2 gene rs6658835 in case group and control group conformed to the Hardy-Weinberg equilibrium (χ 2 = 0.02, P = 0.900). Correlation analysis results: the difference of genotype frequency of TGFβ2 gene rs6658835 in case group (GG, GA, AA: 6.3%, 38.0%, 55.7%) and control group (GG, GA, AA: 10.7%, 43.7%, 45.6%) was not statistically significant (χ 2 = 2.78, P = 0.249). After adjustment by age, the difference of genotype frequency and dominant model of TGFβ2 gene rs6658835 in case group and control group was statistically significant (χ 2adj = 5.43, 4.86, P < 0.05), the difference of recessive model of TGFβ2 gene rs6658835 in case group and control group was not statistically significant (χ 2adj = 2.12, P = 0.145). Conclusion:TGFβ2 gene rs6658835 is associated with KD in Han population of Shaanxi Province.

Objective To investigate the relationship between single nucleotide polymorphisms of interleukin 23 receptor (IL-23R) gene and Keshan disease (KD) in Northwest Chinese Han population.Methods A total of 285 Chinese Han subjects from Huangling,Shaanxi,including 79 KD patients (case group) and 206 control subjects (control group) were involved in this study.Genomic DNA was extracted from peripheral venous blood.The polymorphism of genetic variation was genotyped by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF).All sample groups were tested for Hardy-Weinberg equilibrium using goodness-of-fit x2 test.Differences in genotype distribution between two groups were compared by x2 test.Logistic regression analysis was applied to detect association using age as a confounding factor.Results The gene frequency distribution of IL-23R gene rs10889677 in case group and control group conformed to the Hardy-Weinberg equilibrium (x2 =0.254,P ＞ 0.05).Correlation analysis results:the difference of genotype frequency of IL-23R gene rs10889677 in case group (CC,CA,AA were 6.3％,36.7％,57.0％,respectively) and control group (CC,CA,AA were 5.3％,43.2％,51.5％,respectively) was not statistically significant (x2 =1.008,P ＞ 0.05).After age adjustment,there was no significant difference in genotype frequency of IL-23R gene rs10889677 (x2sdj =0.669,P ＞ 0.05) between two groups.Conclusion There is no correlation between IL-23R gene rs10889677 and KD in Northwest Chinese Han population.

Objective To investigate the protective effects of selenium on nitric oxide(NO)-mediated myocardial apoptosis.Methods The AC16 cardiomyocyte cultured in vitro were divided into control group,selenium treatmentgroup,sodium nitroprusside(SNP) treatment group and selenium + SNP treatment group,SNP was the exogenous NO donor.There was no intervention in the control group,and an equal volume of the culture solution was added to the treatment groups.The selenium treatment group added a dose of 100 μg/L of selenium,the SNP treatment group added a dose of 1.0 mmol/L of SNP,and the selenium + SNP treatment group was pretreated by 100 μg/L selenium for 4 h followed by 1.0 mmol/L SNP;the cells or supernatants were collected after 24 h of culture.The content of NO was detected by Griess method in supernatants.The level of cell reactive oxygen species was detected by flow cytometry.The changes of cell mitochondrial membrane potential and apoptosis were observed under fluorescence microscope.The real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of apoptosis-related genes B-cell lymphoma-2 (Bcl-2) associated X protein (Bax) and Bcl-2,respectively.Results The NO content in the control group,selenium treatment group,SNP treatment group and selenium + SNP treatment group were (10.3 ± 1.8),(9.2 ± 2.1),(15.2 ± 3.5),(14.3 ± 2.6) μmmol/L,respectively;SNP had a main effect on NO content (F =23.33,P ＜ 0.05).The cell reactive oxygen species were 31.63 ± 1.40,29.52 ± 2.86,60.62 ± 4.83,50.08 ± 2.41,respectively;selenium and SNP had main effects on reactive oxygen species (F =12.19,187.20,P ＜ 0.05),selenium combined with SNP had an interactive effect on reactive oxygen species (F =5.42,P ＜ 0.05).The cell mitochondrial membrane potential levels were 0.42 ± 0.11,0.37 ± 0.07,7.25 ± 1.91,and 5.21 ± 1.59,respectively;selenium and SNP had main effects on cell mitochondrial membrane potential levels (F =14.21,440.01,P ＜ 0.05),selenium combined with SNP had an interactive effect on cell mitochondrial membrane potential levels (F =12.89,P ＜ 0.05).Selenium had main effects on nuclear pyknosis ratio,Bcl-2 mRNA and Bax protein expressions (F =9.52,10.84,22.17,P ＜ 0.05);SNP had main effects on nuclear pyknosis ratio,Bax and Bcl-2 mRNA expressions,and Bcl-2 protein expression (F =192.86,21.90,16.09,18.39,P ＜ 0.05);selenium combined with SNP had an interactive effect on Bax,Bcl-2 mRNA and protein expressions (F =20.51,7.59,15.38,11.97,P ＜ 0.05).Conclusion The SNP can induce apoptosis of AC16 cardiomyocyte;selenium combined with SNP has an interactive effect on AC16 cardiomyocyte,indicating that selenium has protective effect on NO modiated myocardial apoptosis.

Objective To investigate the relationship between single nucleotide polymorphisms of Bcl-2 related anti apoptotic protein 3 (BAG3) gene and Keshan disease (KD) in north Chinese Han population.Methods In 2002 a total of 285 Chinese Han subjects,including 79 KD patients and 206 control subjects were involved in this study.Genomic DNA was extracted from the peripheral venous blood sample.Blood samples were provided by the Institute of Endemic Disease Prevention,Xi'an Jiaotong University,and stored at 80 ℃.The polymorphism of genetic variation was genotyped by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF).The data was analyzed using TYPER 4.0 or SPSS16.0 software.All sample groups were tested for Hardy-Weinberg equilibrium using goodness-of-fit x2 test.Differences in genotype distribution and allele frequencies between case and control were compared by x2 test.Logistic regression analysis was applied to detect association using age as a confounding factor.Results All sample group passed the Hardy-Weinberg equilibrium test (P ＞ 0.05).Significant differences were not observed in genotype distribution between cases (rs2234962:CC,CT,TT were 0.0％,0.0％ and 100.0％,respectively;rs196295:GG,GA,AA were 22.8％,54.4％ and 22.8％,respectively;rs3858339:GG,GT,TT were 5.1％,38.0％ and 56.9％,respectively;rs3858340:TT,TC,CC were 5.1％,38.0％ and 56.9％,respectively) and controls (rs2234962:CC,CT,TT were 0.0％,1.0％ and 99.0％,respectively;rs196295:GG,GA,AA were 21.4％,51.5％ and 26.2％,respectively;rs3858339:GG,GT,TT were 5.8％,34.5％ and 59.7％,respectively;rs3858340:TT,TC,CC were 5.8％,34.5％ and 59.7％,respectively) for rs2234962,rs3858339,rs196295 and rs3858340 on BAG3 gene (x2 =0.685,0.408,0.330,0.330,P ＞ 0.05).Significant differences were not observed in genotype after agecorrecting between cases and controls for 4 SNPs on BAG3 gene (x2 =0.001,0.019,1.009,0.019,P ＞ 0.05).Conclusion The results suggest that the BAG3 gene might not be a susceptibility gene of KD in north Chinese Han population.

Objective To investigate the clinical diagnostic value and pathogenesis of serum protein identification in Keshan disease (KD).Methods A total of 65 chronic KD patients were selected as the patient group in KD endemic areas,while 29 cases of dilated cardiomyopathy (the DCM group),62 healthy cases from KD endemic areas (control 1 group) and 28 healthy cases from non-endemic areas (control 2 group) were selected as controls.Liquid chip time of flight mass spectrometry (ClinProtTM MALDI-TOF-MS) was used to determine the expression of proteins/peptide peaks.ClinProTools 2.2 software was used to analyze the protein profiles to determine differentially expressed proteins/peptide peaks.The Genetic Algorithm (GA),QuickClassifer Algorithm (QC) and Supervised Neural Network Algorithm (SNN) methods were used to screen marker proteins.Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry technique (MALDI-TOF/TOF) was also used as a secondary mass spectrometry to identify differentially expressed peptides.Results Between the KD and control 1 groups,34 differentially expressed proteins/peptides and 5 marker proteins were identified,while 52 differentially expressed proteins/peptides and 5 marker proteins were identified between the KD and control 2 groups,and there were 67 differentially expressed proteins/peptides and 5 marker proteins between the KD and DCM groups.During secondary mass spectrometry,two peptides for mass-to-charge ratio (m/z) 2 079 and 1 465 were obtained,peptide of matching β-globin showed low expression while peptide of matching fibrinogen showed high expression in the KD patients.Conclusions Serum marker proteins can be used as biomarkers for diagnosis and differentiation of KD.β-globin and fibrinogen play an important role in the development of KD myocardial injury.

[ ABSTRACT] AIM:To observe the effect of high glucose on the protein expression of calreticulin ( CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes.METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+CRT siRNA group and isotonic con-trol group.The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed.RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain.The protein expression of CRT was significantly increased in high glucose group.However, compared with high glucose group, high glucose+CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes.CONCLUSION:High glucose brings about CRT over-expression to induce mito-chondrial injury, thus increasing myocardial apoptosis.

OBJECTIVETo observe the effect of angiotensin II (Ang II) on calreticulin (CRT) expression and its association with mitochondrial dysfunction in cardiomyocytes.

METHODSPrimary neonatal rat cardiomyocytes were randomly divided into CRT siRNA group, control siRNA group, control group, Ang II+ CRT siRNA group, Ang II+ control siRNA group and Ang II group. The cell surface area, protein synthesis rate, mitochondrial membrane potential level, enzyme activities, and CRT expression were observed.

RESULTSCompared with those in the control group, the cell surface area and protein synthesis rate were both increased and mitochondrial membrane potential level and enzyme activities decreased in Ang II groups. CRT expression was significantly down-regulated in Ang II+ CRT siRNA group with increased cell surface area, protein synthesis rate, mitochondrial membrane potential level and enzyme activities as compared with those in Ang II+ control siRNA group.

CONCLUSIONAng II up-regulates CRT expression to induce mitochondrial injury, which may be an important mechanism of myocardial hypertrophy.

Angiotensin II ; pharmacology ; Animals ; Calreticulin ; metabolism ; Cardiomegaly ; Cells, Cultured ; Membrane Potential, Mitochondrial ; Mitochondria ; pathology ; Myocytes, Cardiac ; pathology ; Protein Biosynthesis ; RNA, Small Interfering ; Rats

BACKGROUNDKeshan disease (KD) is an endemic cardiomyopathy in China. The etiology of KD is still under debate and there is no effective approach to preventing and curing this disease. Young women of child-bearing age are the most frequent victims in rural areas. The aim of this study was to determine the differences between molecular pathogenic mechanisms in male and female KD sufferers.

METHODSWe extracted RNA from the peripheral blood mononuclear cells of KD patients (12 women and 4 men) and controls (12 women and 4 men). Then the isolated RNA was amplified, labeled and hybridized to Agilent human 4×44k whole genome microarrays. Gene expression was examined using oligonucleotide microarray analysis. A quantitative polymerase chain reaction assay was also performed to validate our microarray results.

RESULTSAmong the genes differentially expressed in female KD patients we identified: HLA-DOA, HLA-DRA, and HLA-DQA1 associated with spontaneous autoimmunity; BMP5 and BMP7, involved in cardiomyocyte differentiation defect; and ADAMTS 8, CCL23, and TNFSF15, implicated in anti-angiogenic activities. These genes are involved in the canonical pathways and networks recognized for the female KD sufferers and might be related to the pathogenic mechanism of KD.

CONCLUSIONOur results might help to explain the higher susceptibility of women to this disease.

ADAM Proteins ; genetics ; ADAMTS Proteins ; Adult ; Autoimmunity ; genetics ; physiology ; Bone Morphogenetic Protein 5 ; genetics ; Bone Morphogenetic Protein 7 ; genetics ; Cardiomyopathies ; genetics ; pathology ; Cell Differentiation ; genetics ; physiology ; Chemokines, CC ; genetics ; Enterovirus Infections ; genetics ; pathology ; Female ; Gene Expression Profiling ; HLA-D Antigens ; genetics ; HLA-DQ alpha-Chains ; genetics ; HLA-DR alpha-Chains ; genetics ; Humans ; Male ; Middle Aged ; Myocytes, Cardiac ; cytology ; metabolism ; Oligonucleotide Array Sequence Analysis ; Sex Factors ; Tumor Necrosis Factor Ligand Superfamily Member 15 ; genetics

Objective To select an optimal non-specific antigen blocking method by using immuno-infiltration assay so as to suit protein chip preparation. Methods Human papillomavirus type 16 L1 protein expressed by insect-baculovirus espressin system was incubated with skimmed milk powder, calf serum, bovine serum albumin (BSA) combinations of five kinds of methods to block the non-specific antigen. PBS was used as control. The effect of eliminating non-specific stain was detected by immuno-infiltration assay. Results After repeated tests, the results showed that the stability and repeatability of blocking effects were poor for the fixing up antigen first and then blocking method, and the blank control was prone to false positive. The infiltration rate of NC membrane would be affected by using skimmed milk powder as a blocking agent because the pore of NC membrane was easily plugged by milk powder particles. The use of calf serum as a blocking agent made it very difficult to determine the result because the calf serum absorbed by NC membrane produced the background; however, when 20g/L BSA was used to blocking before fixing up antibody, the results became satisfactory. Conclusion Fixing up antibody after blocking in immuno-infiltration assay showed that the blocking effect against non-specific antigen was satisfactory, stable and repeatable, indicating this method is a novel optimal blocking method compared with others.