Effect Of Selenium On NO-PGC1α Mediated Mitochondrial Biosynthesis Pathway Of Keshan Disease

Objective 1.To compare the expression of nitric oxide（NO）pathway molecules Arginase 2（Arg2）,nitric oxide synthase 1（NOS1）and peroxisome proliferator-activated receptor gamma coactivator-1α（PGC-1α）in peripheral blood derived from patients with Keshan Disease（KD）and healthy controls at the RNA and protein levels,thus providing clues to the mechanism of KD.2.H₂O₂ induced AC16 myocardial cells to establish oxidative injury cell model,explore the expression of NO pathway molecules in the injury model and the effect of different levels of selenium（Se）on it,so as to provide a theoretical basis for the prevention of KD.Materials and Methods 1.Total RNA and total protein from 16 KD-control samples was extracted,the expression of NOS1,Arg2 and PGC-1α were detected by q PCR and Western Blot.2.Se content in the whole blood of patients with KD and healthy controls was detected by inductively coupled plasma mass spectrometry（ICP-MS）.3.AC16（Homo sapiens）myocardial cells were cultured and randomly divided into five groups: Controls,H₂O₂（200μmol/L）,0.05μ mol/L Se+H₂O₂,0.5μmol/L Se+H₂O₂ and 2μmol/L Se+H₂O₂,cultured myocardial cells were observed under the optical inverted microscope.4.The contents of MDA,NO and ATP in AC16 myocardial cells of each group were detected by MDA-kit,NO-kit and ATP-kit.5.The expression of Arg2,NOS1 and PGC-1α in AC16 myocardial cells of each group was detected by Western Blot.6.The mitochondrial structure of cultured AC16 cells（Controls,H₂O₂,H₂O₂+2μmol/L Se）was observed under the transmission electron microscope（TEM）.7.Statistical analysis was performed using SPSS 23.0 software,the data were presented as mean ± standard deviation,and P<0.05 as significant difference.Results 1.Expression levels of Arg2,NOS1 and PGC-1α m RNA were not significantly different between patients with KD and healthy controls（P>0.05）,expression levels of Arg2,NOS1 and PGC-1α protein in patients with KD were significantly increased compared to healthy controls（P<0.05）.2.The Se content in the whole blood of patients with KD was 97.04±41.33μg/L,in healthy control was 123.89±44.49μg/L,the difference was not statistically significant（P>0.05）.3.AC16 myocardial cells grew adherently in vitro,and were mostly spindle,polygonal and irregular shape.The nucleus was clear,oval,and located in the center of the cytoplasm.4.H₂O₂ could cause damage to AC16 myocardial cells,inc reased the content of MDA and NO,and reduced the amount of ATP produced.After supplementation with Se（2μ mol/L）,the content of MDA and NO decrease d,and the amount of ATP produced increased significantly（P<0.05）.5.H₂O₂ could significantly increase the expression of Arg2 protein in AC16 myocardial cel s,and it can be significantly reduced after 2μmol/L Se supplementation（P<0.05）.6.H₂O₂ could cause ultrastructural changes in cultured AC16 myocardial cells,including mitochondrial swelling and deformation,mitochondrial ridges dissolved even disappeared,cell death or apoptosis.After supplementation with Se,the ultrastructural damage of AC16 myocardial cel s was reduced.Conclusions 1.The abnormal expression of NO signaling pathway related molecules Arg2,NOS1 and PGC-1α affects mitochondrial biosynthesis and may play an important role in the pathogenesis and development of KD.2.H₂O₂ could cause mitochondrial damage and increase the expression of Arg2 in AC16 myocardial cells,Se could effectively combat the damage and maintain the integrity of mitochondrial structure and function.