| Part one The effect of oxidative stress on the expression of sonic hedgehog in cardiomyocytesObjective:Hedgehog family is a kind of important signal protein for directing patterning events during embryogenesis,and dysfunction of such pathway has related with cancer and diabetic complications such as nerve damage and wound healing delay. The hedgehog family consists of sonic hedgehog (Shh), desert hedgehog (Dhh) and Indian hedgehog (Ihh). The signaling coded by these three genes activates the same signaling cascade. Two kinds of acceptors are related to hedgehog signal transmission, one is called patched(Ptc), a 12-span transmembrane protein,which can be directly combined to ligand, and can negatively regulate hedgehog signal, the other one is named smoothened(Smo), a 7-span transmembrane protein,which is the essential receptor for hedgehog signal transmission.Ptc relieves the inhibitory action to Smo when combined with Hh,which can precipitate Gli to form molecule compound with Costal2,Fused, and inhibiting factor of Fused. The compound can make the Gli go into cell nucleus and activate transcription of downstream of target gene. Shh is the most common and the best documented of the three. A lot of previous studies suggested that Shh was related to angiogenesis factors, which could promote the expression of VEGF and participate in the growth of blood vessel, and also had effect on such cardiovascular diseases as acute and chronic myocardial ischemia and myocardial ischemia-reperfusion-induced injury. Data also showed that hedgehog was dramatically decreased in vessels,corpus cavernosum penis and nerves of the diabetic animal models, some famous research institutions have already reported that administration of Shh plasmid or Shh protein could play an import role in dealing with the nerves and its vasa nervorum and skin intention. Our lab have comfirmed that Shh was indeed decreased in the myocardium of diabetic mice, but the reason was really unknown. So we hypothesized that oxidative stress affected Shh expression in cardiomyocytes because of the accepted fact of diabetic induced-oxidative stress.Methods:The study was performed with primary cultured neonatal rat cardiac myocytes. Expression of Shh mRNA was determined by RT-PCR. Expression of Shh protein was determined by western blot.Results:1. Shh mRNA levels in cultured cardiomyocytes treated with xo (0.5u/L)/x(0.5mM) and xo (1u/L)/x(0.5mM) for 24h were decreased according to different concentration (by 40.1 % ,59.2 % (all P<0.05)) compared to normal group. After incubation of SOD homologue tempol (0.1mM,0.5mM) and xo (1u/L)/x(0.5mM) for 24h, Shh mRNA levels were increased according to different concentration (by 7.4%(P>0.05),45.2%(P<0.05)) compared to purely oxidative stress group.2. Shh protein levels in cultured cardiomyocytess treated with xo (0.5u/L)/x(0.5mM) and xo (1u/L)/x(0.5mM) for 24h were decreased according to different concentration (by 19%,38.6%(all P<0.05)) compared to normal group. After incubation of SOD homologue tempol (0.1mM,0.5mM) and xo (1u/L)/x(0.5mM) for 24h, Shh protein levels were increased according to different concentration (by 0.08%(P>0.05), 24.4%(P<0.05)) compared to purely oxidative stress group. Conclusion:Administration of oxidative stress system(xanthinoxidase/xanthine) to cardiomyocytess could lead to decreased expression of Shh mRNA and protein, and SOD homologue tempol could retard oxidative-stress-induced decreased expression of Shh mRNA and protein. Therefore, oxidative stress indeed affected the expression of Shh in cardiomyocytes.Part two: The preparation of acute myocardial infarction in mice.Objective:Many clinical researches showed that diabetes was tightly associated with heart failure. A lot of research institution reported that diabetes was more liable to have heart failure than non-diabetes, and those who simultaneously contracted diabete and myocardial infarction were more difficult to recover.Some authoritative reseached have comfirmed that sonic hedgehog was increased in the myocardial infarction model of mice, and administration of Shh plasmid or Shh protein also had effect on such cardiovascular diseases as acute and chronic myocardial ischemia and myocardial ischemia-reperfusion-induced injury. Our lab have comfirmed that Shh was indeed decreased in the myocardium of diabetic mice. In order to evaluate the value of decreased expression of Shh, we should firstly prepare the acute myocardial infarction model in mice.Methods:Ligation of left anterior descending (LAD) coronary to make the myocardial infarction (MI) model in mice. Two weeks after MI, we comfirmed the successful model through morphologic and echocardiographic assessment,which included infarct size, left ventricular end-systolic dimension (LVID;s), left ventricular end-diastolic dimension (LVID;d), fractional shortening (FS%), ejectional fraction (EF%), left ventricular anterior wall thickness in systolic phrase (LVAW;s), left ventricular posterior wall thickness in systolic phrase (LVPW;s), left ventricular anterior wall thickness in diastolic phrase (LVAW;d),left ventricular posterior wall thickness in diastolic phrase (LVPW;d).Results:1. Compared to normal group, myocardial infarction group of two weeks after operation had thin and pale anterior wall, and its left ventricular cavity has significantly enlarged.2. Application of real-time ultrasonography systerm Vevo770 (Visualsonics) to normal and myocardial infarction group two weeks after operation, M- Echocardiography showed the latter group had thinner anterior wall and enlarged left ventricular cavity; Moreover, compared to normal group, LVAW;s, LVAW;d, EF%, FS% and CO in the latter group were all decreased by 70%, 53.8%, 65.2%, 71.1%, 47.1% (P<0.05); LVID;s and LVID;d were all increased by 67.5%, 15.6% (P<0.05).ConclusionData from morphology and ECG showed that following the method established in our lab indeed effectively prepared the acute myocardial infarction model, which will be beneficial for further investigation in the meaning of decreased Shh in diabetic heart. |
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