Selenium And Thiamine Alleviate Citreoviredin-Induced Autophagy-Dependent Apoptosis In Cardiomyocytes

Objective:Keshan disease?KD?is an endemic disease with pathological changes in myocardium.The main cause of KD includes low level-selenium and mycotoxin infection and so on.Citreoviridin?CIT?is one of the mycotoxin in KD region,from contained moldy cereals.CIT has much toxicity such as cardiotoxicity,genotoxicity,and it can induce apoptosis in cells.It is reported that CIT is associated with cardiac beriberi.Selenium?Se?is a trace element necessary for human.Se has many biological functions such as anti-oxidation,anti-inflammatory.Clinical practice shows that Se supplementation dramatically reduces KD incidence,but the mechanism is not clear.Vitamins B₁?VB1?also named Thiamine,is an essential water-soluble vitamin.VB1 deficiency?TD?is blamed for the pathogenesis of beriberi disease.Clinical practice proves VB1 protects myocardium,but the mechanism is not clear.Autophagy plays a significent role in maintaining cellular homeostasis,but excessive autophagy would cause many damages.Nowdays the mechanism of autophagy in Se and VB1protecting myocardium is not clear.In this study,we aim to investigate the potential preventive mechanism of Se and VB1 against CIT cardiotoxicity.Methods:In vivo,five-week-old male ICR mice were used as experimental subjects.Mice were injected intraperitoneally with 0 mg/kg bw,0.1 mg/kg bw,0.3mg/kg bw CIT?content 0.2%DMSO?in 20 ml/kg bw/day saline solution,the Se and VB1 groups were exposed to 0 mg/kg bw,0.3 mg/kg bw CIT?content 0.2%DMSO?meanwhile pretreated with drinking water contain Se?9 mg/L?and VB1?20 g/L?,for consecutive 6 weeks.We used the electronic microscopy to observe the autophagosomes in mice myocardium.The expression of autophagy biomarker microtubule-associated protein 1 light chain 3?LC3?-II protein,Sequestosome 1?P62?protein,mitochondirial apoptotic biomarker B cell lymphoma 2?Bcl-2?protein and Bcl-2 assaciated X?Bax?protein,and the level of Cathepsin B protein,biomarker of mitochondrial outer membrane permeability change Cytochrome c?Cyt c?protein in the mice myocardium were detected by Western blotting.We used the hematoxylin and eosin?HE?staining to observe the pathological changes of mice myocardium.The level of creatine kinase isoenzymes MB?CK-MB?myocardial enzymes,cardiac troponin T?cTnT?myocardial enzymes in serum was detected by enzyme-linked immunosorbent assay?ELISA?.In vitro,we treated H9c2 cells with the 8?M CIT at 12 h,24 h and 36 h.The autophagosomes in cells was observed by electron microscopy.The lysosomal membrane stability of cells was measured by acridine orange?AO?staining under a fluorescence microscope.The mitochondrial transmembrane potential???m?was observed by JC-1 staining with fluorescence microscopy.The expression of LC3-II protein,P62 protein,Bcl-2 protein and Bax protein in cells was evaluated by Western blot.Caspase-3 activity was determined using colorimetric method.Whether apoptosis occurred in cells was observed by TUNEL assay.The level of CK-MB myocardial enzymes,cTnT myocardial enzymes in medium was detected by ELISA.After pretreatment with Se,VB1 and autophagy inhibitor 3-MA,the H9c2 cells were treated with CIT in the same manner as above,we detected the changing of the experimental index measured by the methods mentioned above respectively.Results:In vivo,electron microscopy revealed that the number of autophagosomes was increased in the mice myocardium exposed to different concentrations of CIT?0.1 mg/kg bw,0.3 mg/kg bw?.Compared with the 0.3 mg/kg bw CIT group,the number of autophagosomes was obviously decreased in Se and VB1 groups.The expression of LC3-II protein in mice myocardium was increased with CIT treatment,while the change of P62 protein was opposite.Compared with 0.3mg/kg bw CIT group,the expression of LC3-II protein was decreased and the expression of P62 protein was increased in Se and VB1 groups.The level of Cathepsin B protein in the mice myocardium was increased with CIT,and the level of Cathepsin B protein in the Se and VB1 groups was lower than that in the 0.3 mg/kg bw CIT treated group.The level of Cyt c protein in the mice myocardium was increased with CIT treatment,and the level of Cyt c protein in the Se and VB1 groups was lower than that in the 0.3 mg/kg bw CIT treated group.The expression of Bax protein in mice myocardium was increased with CIT treatment,while the change of Bcl-2 protein was opposite.Compared with 0.3 mg/kg bw CIT group,the expression of Bax protein was decreased and the expression of Bcl-2 protein was increased in Se and VB1 groups.The level of CK-MB myocardial enzymes,cTnT myocardial enzymes in serum was increased with CIT treatment,and the level of CK-MB myocardial enzymes,cTnT myocardial enzymes in the Se and VB1 groups was lower than that in the 0.3 mg/kg bw CIT treated group.The significant pathological changes were found in CIT treated group,and there were no significant pathological changes in the Se and VB1 groups.In vitro,electron microscopy revealed that the number of autophagosomes was increased in H9c2 cells when exposed to 8?M CIT,but the number of autophagosomes was obviously decreased in the Se and VB1 groups.Cells were treated with 8?M CIT for 12 h,the expression of LC3-II protein in H9c2 was increased significantly with CIT,while the change of P62 protein was opposite.But in the Se and VB1 groups the expression level of the LC3-II protein was significantly lower than that in the 8?M CIT treated group,and the expression of P62 protein was increased.Cells were treated with 8?M CIT for 24 h,the lysosomal membrane permeabilization of H9c2 was increased by fluorescence microscopy.While compared with the 8?M CIT group,the lysosomal membrane permeabilization of cell was relieved after pretreatment with Se,VB1 and 3-MA.Cells were treated with 8?M CIT for 24 h,the mitochondrial membrane potentials of H9c2 was decreased under fluorescence microscopy.While compared with the 8?M CIT group,the mitochondrial membrane potentials of cell was relieved after pretreatment with Se,VB1 and 3-MA.Cells were treated with 8?M CIT for 24 h,the expression of Bax protein in H9c2 was increased significantly with CIT,while the change of Bcl-2protein was opposite.In the Se and VB1 pretreatment groups,the expression level of the Bax protein was significantly lower than that of the 8?M CIT treated group,and the expression of Bcl-2 protein was increased.The activity of cysteinyl aspartate specific proteinase 3?Caspase-3?was activated after treatment with 8?M CIT for 36h,and compared with the 8?M CIT group,it was relieved by 3-MA,Se and VB1.After treatment with 8?M CIT for 36 h,apoptosis was induced in H9c2 cells as shown in TUNEL assay.CIT induced apoptosis was relieved by pretreatment with 3-MA,Se and VB1.The level of CK-MB myocardial enzymes,cTnT myocardial enzymes in culture medium was increased with 8?M CIT for 36 h,and the level of the two myocardial enzymes in Se and VB1 pretreatment groups were lower than that in the CIT treated group.Conclusion:We demonstrated that CIT would induce excessive autophagy and activate apoptosis through the lysosomal-mitochondrial axis.Both Se and VB1 could alleviate CIT-induced autophagy-dependent apoptosis through inhibiting autophagy,thus relieve CIT-induced cardiomyopathy.