5hmc Involved In The Change Of Mitochondrial Function After Cerebral Ischemia Injury

Part I The expression of 5hmc after cerebral ischemia injuryObjective: 5-hydroxymethylcytosine(5hmc),another DNA modification derived from 5mc through the Tet(Ten-Eleven-Translocation)enzyme,was found to widespreaded in mammalian DNA.The high level of 5hmc was not only found in the central nervous system,but also in other mammalian tissues,including liver and kidney.In this part,we will make mice brain middle cerebral artery model(MCAO),and observe the expression of 5hmc after ischemic reperfusion injury and the reason of this changes.Methods: The SPF male ICR mice were randomly separated into two groups,inculding Sham group and Ischemia-Reperfusion(I/R)group.After setting up the middle cerebral artery occlusion model for 45 minutes,ischemic tissues were collected at different time points after reperfusion.The expression of 5hmc and 5mc were detected by Dot blot at different time points of I/R.Results: Compared with Sham group,the level of 5hmc increased at 24 h after I/R injury,and significantly increased at 36h(p<0.05).With the development of time,the level of 5hmc reached the peak at 48h(p<0.05).While compared with Sham group,the expression of 5mc significantly increased at 36 h after I/R(p<0.05)and then gradually decreased with the extension of time.Conclusion: Our data demonstrated that the change of 5hmc and 5mc after I/R may be play an important role in the pathological process of cerebral ischemia injury.Part II 5hmc involved in the expression of mitochondrial gene after cerebral ischemia injuryObjective: As we all know,mitochondrial damage is one of pathological mechanism in cerebral ischemia injury,which could make mitochondrial injury more seriously.5hmc not only exists in many tissues and organs,also expresses in mitochondria.In this part,we will observe the expression of 5hmc in mitochondria and its effect on mitochondrial gene expression after I/R.Methods: The SPF male ICR mice were randomly separated into two groups,inculding Sham group and Ischemia-Reperfusion(I/R)group.After setting up the middle cerebral artery occlusion model for 45 minutes,ischemic tissues were collected at different time points after reperfusion.The expression of 5hmc and 5mc in mitochondria were detected by Dot blot at different time points of I/R.The m RNA and protein level of Tet2 were detected respectively by PCR and western blot.Mitochondrial gene was detected by q PCR.Results: Compared with Sham group,the level of mitochondrial 5hmc increased at 24 h after I/R injury,and with the development of time,the level of mitochondrial 5hmc declined slightly,but still significantly increased(p<0.05).While 5mc have no obvious change after I/R.The m RNA and protein level of Tet2 in mitochondria were both significantly increased at 24 h after I/R(p<0.05).The m RNA level of mitochondrial gene increased obviously inculding ND2,ND4 L,ND5,ND6 and COXIII(p<0.05),while COXI?COXII?ATP6?ATP8?Cytb?ND1?ND3 and ND4 had no changes.Conclusion: 5hmc level in mitochondria and mitochondrial gene increased obviously after I/R.These results indicated that 5hmc may be involved in the mitochondrial function after ischemia-reperfusion injury.