Effects And Mechanisms Of ANNAO Tablets On Cerebral Ischemia-reperfusion Injuries

Ischemic stroke is one of the leading causes of disability and mortality worldwide,which exerts a profoundly negative impact on both the patients and society.The current stroke treatment drugs mainly include thrombolytic drugs,anticoagulants and antiplatelet drugs represented by rt-PA.Since the cerebral ischemia-reperfusion imposes multiple insults to the cerebral microcirculation and microcirculatory disturbances also involve multiple,coordinated events,which makes single-target therapy overshadowed.A remedy consisting of multiple compositions may be more competent for the complicated disease.ANNAO tablets are a traditional Chinese medicine for Angong Niuhuang,which reduces the toxicity and cost of Angong Niuhuang while ensuring the efficacy.ANNAO tablets are mainly used for the treatment of neurological diseases in the clinical practices,but its therapeutic mechanism for cerebral ischemia-reperfusion is still unclear.The purpose of this study was to use the rat middle cerebral artery occlusion(MCAO)for 2 h and then reperfusion to cause acute cerebral ischemia in vivo.The reperfusion model was used to observe the protective effect of ANNAO tablets against cerebral ischemia-reperfusion injury,and to explore the mechanism of action of ANNAO tablets from the multi-level of tissue level and molecular level,and provide a more favorable theoretical basis for its clinical application.Part I:Effects of ANNAO tablets on cerebral ischemia-reperfusion injuries1.In MCAO rats,ANNAO tablets treatment for 1 day or 7 days remarkably decreased neurological deficit score(P<0.001);2.Results of TTC staining showed that infarct volume was significantly increased in MCAO rats(P<0.001).The ANNAO tablets administration significantly reduced the infarct volume in acute phase MCAO rats(P<0.001);3.H&E staining further demonstrated that infarct volume was significantly increased in MCAO rats(P<0.01).The ANNAO tablets treatment significantly reduced the of cerebral infarct volume in acute phase rats(P<0.05);4.Immunohistochemistry analysis indicated that ischemic reperfusion injury significantly decreased the number of NeuN positive cells in the ischemic cortex(P<0.001),and treatment with ANNAO tablets obviously ameliorated the loss of NeuN-positive neurons(P<0.001).Part ?:Studies on the mechanisms of ANNAO tablets on cerebral ischemia-reperfusion injuries1.Mitochondrial fission and fusion related protein(Drpl and OPA1)increased in infarct region one day after ischemic reperfusion(P<0.05,P<0.05).ANNAO tablets treatment significantly decreased these two proteins,which may facilitate to rectify the mitochondrial fission-fusion dysfunction.2.In ischemic cortex of MCAO rats,the western blot results showed that mitochondrial fission and fusion protein(Drpl and OPA1)decreased at 1 d after ischemia reperfusion(P<0.05,P<0.05).ANNAO tablets treatment remarkably elevated these two proteins and defended the mitochondrial fission-fusion cleavage acute stress.3.In ischemic cortex and infarction of MCAO rats,the western blot results showed that mitochondrial fission and fusion protein(Drpl and OPA1)reduced at 7 d after ischemia reperfusion(P<0.05,P<0.05).ANNAO tablets remarkably raised these two proteins and resisted the mitochondrial fission-fusion dysfunction.4.Results also indicated that mitophagy in ischemic cortex was inhibited in MCAO rats.The expression of PINK1 and Parkin evidently decreasd(P<0.05).Treatment with ANNAO tablets promoted mitophagy via activating the PINK1/Parkin pathway.5.Meanwhile,he Bcl-2/Bax ratio observably declined after ischemia reperfusion injury(P<0.05)and ANNAO tablets markedly enhanced the Bcl-2/Bax ration both in infarction and cortex(P<0.05,P<0.001).Part III:Effect of ANNAO Tablets on phenotypic transition of glia cells1.The number of astrocytes and microglia in the injured penumbra,infarct area was analyzed.The results showed that in the acute phase of cerebral ischemia,the number of astrocytes and microglia increased significantly in the penumbra tissue(P<0.001);in the infarcted tissue,the number of astrocytes decreased(P<0.001),the number of microglia was significantly increased(P<0.001),and the number of microglia in the cortex of rats in the 1 day group was significantly increased.In the long-term injury of cerebral ischemia,in the penumbra tissue The number of astrocytes and microglia increased significantly(P<0.001);the number of astrocytes decreased(P<0.01)and the number of microglia increased significantly(P<0.001).There was no significant difference in the number of astrocytes in the Anzhen tablets administration group,and there was no significant difference between the rats in the 7-day group and the model group.2.Astrocyte phenotype was analyzed in injured lateral cortex of rats.Results showed that the number of A1 astrocytes increased significantly after 1 day and 7 days of MCAO injury(P<0.05,P<0.001),The number of A2 astrocytes was significantly decreased(P<0.05).ANNAO tablets administration group significantly reduced the number of A1 astrocytes(P<0.05,P<0.01)and significantly increased the number of A2 astrocytes(P<0.01,P<0.05).3.The microglia phenotype was analyzed in injured lateral cortex of rats.Results showed that the number of Ml type microglia increase significantly after 1 day and 7 days of MCAO injury(P<0.05).The number of M2 type microglia was significantly reduced(P<0.05).ANNAO tablets administration significantly reduced the number of M1 microglia(P<0.01,P<0.001)and significantly increased the number of M2 microglia(P<0.01,P<0.05).4.To further investigate the interaction between neurons and astrocytes,an OGD/R model was established on rat primary cortical neurons and astrocytes.Neurons were cultured in astrocyte conditioned medium at different time points after modeling for 24 h and then measure the survival rate of normal and injured neurons.The results of IOD values showed that astrocyte conditioned medium significantly increase neuronal activity and neuroprotective effect of 72 h conditioned medium reach the highest.5.Astrocytes were cultured in neuron conditioned medium at different time points after modeling for 24 h,and then the survival rate of normal and injured astrocytes was measured.The results showed that the neuron conditioned medium had no significant effect on astrocyte activity.In summary,we found that ANNAO tablets show a good neuroprotective effect on acute and long-term injury models of cerebral ischemia-reperfusion,which can significantly improve ischemia-reperfusion induced-mitochondrial dysfunction by reducing mitochondrial damage and maintaining mitochondrial quality control,meanwhile inhibiting neuronal apoptosis.Moreover,ANNAO tablets can regulate the changes of astrocyte and microglial activation after cerebral ischemia.The present research provides experimental basis and theoretical basis for clinical application in cerebral ischemia-reperfusion treatment.