Hyperhomocysteinemia Induced Myocardial Injury In ApoE^-/- Mice Via Mitochondrial Damage And P53-dependent Apoptosis Pathway

Objective： To observe the effects of hyperhomocysteinemia（HHcy） onmyocardial injury by detecting the changes of ROS、 casepas9、 caspase3、 p53.Makeit clear that the effect of HHcy to mitochondrial damage and P53regulation ofapoptosis in myocardial tissue of ApoE^-/-mice,and find a key target ofHcy-induced myocardial injure, and provide new views and theoretical basis to theprevention and treatment of cardiovascular disease due to hyperhomocysteinemiaMethods:5weeks old healthy male mice （SPF grade C57BL/6J） werechosen as the normal control group （fed with normal diet）. Meanwhile,5weeksold male homozygous ApoE^-/-mice （SPF grade inbred C57BL/6J） were randomlydivided into3groups: ApoE^-/- control group （fed with normaldiet）,Hyperhomocysteinemia （HHcy） group （fed with high-methionine diet）,Intervention group （fed with high-methionine and folic acid and Vitamin B12diet）,with12mice in each group. After14weeks, blood samples were collected to assayserum Hcy, serum lipid levels and the changes of myocardial enzymes. Thereactive oxygen species （ROS）、 casepas3、 caspase9、 p53、 protein levels weredetected by ELISA and the mitochondrial DNA （mtDNA）、 p53mRNA expressionwas assayed by real-ime PCR.Results:1.After14weeks，serum Hcy levels were detected in each group. The serum Hcy in HHcy group was about2.5times higher than the normal control group （P<0.01）.Compared with the HHcy group, Hcy were decreased by20%in the Interventiongroup （P<0.01）.2. After14weeks， the serum enzyme activities of Myocardial zymogram weredetected in each group. CK, LDH and HBDH in HHcy group were2.72（P<0.01）,3.48（P<0.01） and3.12-fold（P<0.01） higher than the normal control group, whichindicated that HHcy might induce the most severe cardiac injury. Compared withthe HHcy group, CK and CK-MB were decreased by55.8%,42.8%in theIntervention group （P<0.05）.3Myocardial fibers of the normal control group no significant degeneration,necrosis, myocardial interstitial there is no obvious change. The ApoE^-/- controlgroup can be observed myocardial degeneration, local necrosis. The HHcy groupwere observed in cardiac muscle fibers atrophy, fracture pathological changes.Myocardial fibers of the intervention group there was a slight degeneration,can beseen the local cytoplasm homogenized.4.After the formation of HHcy, compared with normal control group, ROSconcentrations were1.41and1.73higher than normal control group （P<0.01）respectively in the ApoE^-/-control group and HHcy group; After the intervention offolic acid and vitamin B12, ROS concentration decreased by0.7-fold （P<0.01） inHHcy group.5.After the formation of HHcy, compared with normal control group, set ofmtDNA expression were63.40%and57.19%in the ApoE^-/-control group and theHHcy group respectively （P<0.05）. Compared with the ApoE^-/-control group, themtDNA expression level was1.29times higher than HHcy group, which suggestedHHcy promoted mtDNA damage.6. The activities of apoptosis-associated proteins caspase-9、 caspase-3were detected.Our results showed that the activity of caspase-9in ApoE^-/-control groupand HHcy group and intervention group were2.26,2.47and2.13-fold higher thanthe N-control group respectively （P<0.05, P<0.01）. However, the activity ofcaspase-3in ApoE^-/-control group and HHcy group were1.12and1.20timeshigher than N-control group （P<0.01）.7. The levels of p53in HHcy and invention groups were much higher than theN-control group, were2.57,2.44-fold higher than the N-control group, meanwhile,the mRNA and protein levels of p53decreased significantly in the interventiongroup （P<0.01）. The protein levels of Bax increased significantly in the HHcygroup compared with the normal control group. On the other hand, compared withN-control group, the Bax/Bcl-2ratio in HHcy group was2.27-fold higher thanN-control group （P<0.01）. Meanwhile, the protein levels of NOXA were2.25times higher than the normal control group （P<0.01）.ConclusionExcessive ROS were produced by high concentrations Hcy in the HHcy blood, andcaused an aggregation of reactive oxygen species in cells and mitochondrial,resulting in damage to the mitochondria and myocardial cells; This processactivates the p53protein and induced p53-dependent mitochondrial apoptosissignaling pathway, eventually leading to myocardial cells damage, and promotethe development of cardiovascular diseases.