| Part One Effect of 1,25-dihydroxyvitamin D3 preconditioning on myocardial injury in mice with myocardial infarction in vivoObjective:1.To investigate the changes of cardiomyocytes autophagy in mice with acute myocardial infarction.2.To determine the effect of 1,25-dihydroxyvitamin D3 on the myocardium in acute myocardial infarction.Methods:30 healthy male C57/BL6 J mice were randomly divided into 3 groups with 10 mice in each group.The myocardial infarction model was established by permanent ligation of the left anterior descending branch,and the control group was only thread without ligation.Mice in the 1,25VD3 pretreatment group(referred to as VD3 group)was subcutaneously injected at 150ng/kg/d per mouse for 1 week.Myocardial injury markers,such as creatine Kinase(CK),Isoenzyme(CKMB),cardiac troponin I(c Tn I),brain natriuretic peptide(BNP)and inflammatory markers such as interleukin-6(IL-6),the tumour necrosis factor-?(TNF-?)were determined by ELISA.Cardiac ultrasonography was performed 24 hours after surgery.The following indicators need to be measured: the left ventricular end-diastolic dimension(LVEDD)Diameter(LVESD): left ventricular anterior wall thickness at end Diastolic(LVAWD)and ejection fraction Fraction,EF)and Left ventricular short axis shortening rate(FS).Immunohistochemical staining of macrophages was performed to understand the inflammatory infiltration of myocardium;TUNEL staining was used to understand the myocardial apoptosis.Western blotting was used to detect the pro-apoptotic proteins such as caspase-3,caspase-9 and Bax,the anti-apoptotic proteins such as Bcl-2 and the autophagy markers such as autophagy microtubule associated protein light chain 3-II(LC3-II),beclin-1,soluble p62 and insoluble p62.5.HE staining was used to observe the morphological changes of myocardial tissue in mice.Transmission electron microscopy was used to observe the autophagy of myocardial cells in mice.Results:1.Compared with the control group,LVEDD and LVESD increased significantly,while LVEDD,EF and FS decreased significantly in mice of AMI group,which showed cardiac dysfunction occurred in AMI guroup.In the VD3 treatment group,the above indexes were significantly improved.2.Cardiac injury Markers which include CK,CKMB,c Tn I and BNP,and inflammatory markers which include IL-6 and TNF-? increased significantly in AMI group.While the above indexes in VD3 group were significantly lower than AMI group.Myocardial infarct size was the largest in mice of the AMI group and pathological damage could be found by HE staining.Compared with the AMI group,the above situation was significantly improved in VD3 group.3.Compared with the control group,the number of myocardial cell apoptosis was significantly increased in AMI group,the pro-apoptotic proteins Caspase-3 Caspase-9 and Bax were significantly up-regulated,and the anti-apoptotic protein Bcl-2 was down-regulated.The above trends were improved in the VD3 group.4.The levels of LC3 II and Beclin-1 in AMI group were higher than those in control group,and the levels of soluble p62 in AMI group were lower than those in control group.The trend was further enhanced in VD3 group.Autophagosomes could be seen in myocardial cells of AMI group under color transmission electron microscope,and autophagosomes were increased in VD3 group.Conclusions:1.1,25VD3 preconditioning can reduce myocardial infarction area,relieve inflammatory cell infiltration and apoptosis,which can improve cardiac function ultimately in AMI mice.2.Autophagy was enhanced in AMI group and further enhanced in VD3 group.Part Two Study on the protective effect of 1,25-dihydroxyvitamin D3 on myocardial cells in mice with acute myocardial infarctionObjective: To investigate the possible mechanism of 1,25VD3 enhancing autophagy in myocardial protection,namely whether it participates in autophagy through PI3K/ Akt /m TOR pathway.Methods: AMI model was established in vivo and divided into control group,AMI group and VD3 group,with 10 mice in each group.Mice in VD3 group were subcutaneously injected with 1,25-dihydroxyvitamin D3 at 150ng/kg/d for one week.The AMI model was established by ligation of the left anterior descending branch.Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the m RNA expressions of PI3 K,Akt and m TOR in myocardium.Western blotting was used to detect the expressions of PI3K/ Akt /m TOR pathway markers,namely p-PI3 K,p-Akt and p-m TOR.The above results were further validated in an in vitro model of hypoxia of mouse cardiomyocytes.The cells were divided into three groups: hypoxia group,VD3 group and VD3 combined with 3-MA group,in which the 1,25VD3 concentration of VD3 group was 1u M.In the VD3 combined with 3-MA group,750ug/ml 3-methyladenine(3-MA,a specific inhibitor of class III PI3 K blocking autophagy activation)and 1,25VD3(1u M)were combined.P-PI3 K p-Akt and p-m TOR were selected as markers of PI3K/ Akt /m TOR pathway,and autophagy markers LC3 II Beclin-1 and p62 were selected to reflect the changes of autophagy.Expression levels of the above proteins were measured by Western blotting.Results:1.Transcription and protein expression levels of PI3 K,Akt,and m TOR were decreased in AMI group,and the above levels were further decreased in VD3 group.2.Compared with hypoxia group,LC3-II and Beclin-1 increased while soluble p62 decreased in VD3 group,which show that the 1,25VD3 could enhance autophagy.3.P-PI3 K,p-Akt and p-m TOR decreased in VD3 group compared with hypoxia group,suggesting that VD3 group could inhibit the expression of PI3K/ Akt /m TOR pathway.Conclusions:1.The PI3K/ Akt /m TOR pathway can negatively regulate autophagy.2.1,25VD3 can enhance myocardial autophagy by negatively regulating the PI3K/ Akt /m TOR pathway.In conclusion,the results of this study confirm that 1,25VD3 can improve cardiac function in acute myocardial infarction by reducing myocardial injury,reducing myocardial inflammatory cell infiltration and weakening myocardial cell apoptosis.The protective effect on AMI was realized by negatively regulating the PI3K/ Akt /m TOR pathway to enhance autophagy.Part Three To explore the significance of serum 25 hydroxyvitamin D in patients with acute myocardial infarctionObjective: To investigate the changes of serum 25(OH)D [25(OH)D] levels in patients with acute myocardial infarction and its clinical significance.Methods: The acute myocardial infarction patients with less than 6 hours from onset of symptoms to presentation were selected from our hospital between October 2019 and October 2020.A total of 50 patients with acute myocardial infarction were enrolled at all,and 50 healthy subjects in the same period were enrolled as the control group during the same time.Blood samples were collected to test the complete blood count and C-reactive protein(CRP),and fasting blood samples were taken to determine 25(OH)D concentration in the next morning.Fasting blood samples were collected from healthy subjects for the determination of the above indexes.Patients with acute myocardial infarction were further divided into groups according to the number of coronary vascular lesions and Grace score,and the 25(OH)D status among groups was compared.Results:1.The level of serum 25(OH)D in acute myocardial infarction group was significantly lower than that in the control group.WBC and CRP levels were significantly higher than those in the control group.2.Compared with patients with a single vascular lesion,the serum 25(OH)D levels were significantly lower in patients with two vessel lesions or multiple vessel lesions,but there was no difference between the later groups.When compared with low-risk AMI patients,serum 25(OH)D levels were significantly lower in the high-risk groups,but no difference was observed between the low-risk and medium-risk group or the medium-risk and high-risk group.Conclusions: The detection of serum 25(OH)D concentration in patients with acute myocardial infarction can help to make early diagnosis,and further provide a certain degree of basis for risk stratification in diagnosis,which is worthy of extensive clinical application. |
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