The Role Of ACE2 In Brain Ischemia Injury And The Protective Action Of Scutellarin On The Hypoxia Injury

Stroke represents the first leading cause of death in China and the most important cause of chronic disability.Cerebral ischemia is caused by a deficiency in blood supply to a part of brain,which in turn triggers various pathophysiological changes.It has been accepted recently that ischemic cell injury arises from complex interactions between multiple electrophysiological, hemodynamic,and biochemical cascades,which include disturbances in energy metabolism,modifications in synaptic transmission,production of reactive oxygen species,stimulation of the inflammatory process,endothelial dysfunction, et al.All neuroprotective agents so far targeting a specific pathway in ischemic cascade failed to demonstrate clinical efficacy.Therefore,the search for novel mechanism and therapeutic approaches is even more critical.There are two parts in this study:one is to investigate the role of ACE2 in the brain ischemia injury, the other is to study the protective effect of scutellarin on the hypoxia-induced injury in PC12 cells.Partâ… :The role of ACE2 in rat brain ischemia injuryThe renin-angiotensin system(RAS) is a very important regulator for the physiological function and angiotensinâ…¡(Angâ…¡) is the major component of RAS.It is reported that Angâ…¡acts important roles in brain ischemia injury and inhibiting Angâ…¡ is becoming an new approach in stroke therapy.In 2000,angiotensin converting enzyme 2(ACE2) was found.ACE2 can catelyze Angâ…¡to Ang(1-7) which has the opposite actions of Angâ…¡.It is believed that ACE2 may act as a negative regulator in RAS through two ways:one is inactivating Angâ…¡and the other is producing Ang(1-7).Since both ACE2 and Ang(1-7) are distributed in brain and the action of Angâ…¡in brain ischemia injury is proved,we assume that ACE2 may acts important functions in brain ischemia injury.In this study,we studied the role of ACE2 in brain ischemia injury both in vivo and in vitro.Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO).The rats were divided into 5 groups randomly:sham,ischemia 6h, ischemia 12h,ischemia 6h + 5mg/kg losartan,ischemia 6h + 5mg/kg captopril. The behavioral tests and the infarct area by 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the brain injury.RT-PCR was used to examine the ACE2 mRNA expression and immunohistochemistry staining was used to evaluate the ACE2 protein level.Comparing with the sham group,the obvious neurological disorders and the enlarged infarction areas were found in the ischemia group.Both ACE2 mRNA and ACE2 protein were increased after brain ischemia injury.Compared with the ischemia group,both losartan and captopril improved the neurological functions,decreased the infarction area and increased the expression of ACE2 in both mRNA and the protein level.These results show that the brain ischemia injury can increase ACE2 expression.Both the AT1 receptor blocker losartan and angiotensin converting enzyme inhibitor captopril can increase ACE2 expression and manage the protective action against brain ischemia injury.It point out that the high expression of ACE2 perhaps has relation to the anti-ischeimic action in brain and Angâ…¡may be involved in the expression of ACE2. To further verify the reasoning,several experiments were carried in vitro.Using the cultured rat cortical astrocyte,the hypoxia(95%N₂+5%CO₂) induced cell injury was examined.Cells were divided into 6 groups:control,hypoxia 2h, hypoxia 4h,hypoxia 8h,hypoxia 4h + 10^-7mol/L losartan,hypoxia 4h + 10^-7mol/L PD123319.Cell viability was assessed by methylthiazol tetrazolium(MTT) assay and cell injury by the rate of lactate dehydrogenase(LDH) release and adenosine triphosphate(ATP) production.RT-PCR was used to examine the mRNA expression of ACE2 and western blot analysis was used to evaluate the protein level of ACE2.Comparing with the control group,decrease of cell viability and ATP concentration and increase of LDH release were found in the hypoxia groups. Losartan increased the cell viability and ATP concentration and decreased the LDH release.PD123319 had no effect on cell viability and cell injury.Both the mRNA and the protein of ACE2 were increased after hypoxia injury.Losartan increased the expression of ACE2 in both mRNA and the protein level,while PD123319 had no effect on ACE2 expression.These results show that the hypoxia injury can increase ACE2 expression and the AT1 receptor,not the AT2 receptor is involved in the expression of ACE2.It further reminder that the high expression of ACE2 has relation to the anti-hypoxic injury.To further confirm the the action of high expression of ACE2 on the hypoxic injury,we transfected the rat brain astrocytes with a plasmid pcDNA-ACE2 which highly expressed ACE2.The efficiency of transfection was examined by RT-PCR and western blot analysis.Cells were divided into 3 groups:pcDNA3.1(control), pcDNA3.1+hypoxia 4h,pcDNA-ACE2+hypoxia 4h.Cell viability was assessed by MTT assay and cell injury by the rate of LDH release and ATP production. Comparing with the control group,decrease of cell viability and ATP concentration and increase of LDH release were found in the hypoxia groups. Compared with the hypoxia group,both the cell viability and ATP production were increased and the LDH release was decreased in pcDNA-ACE2 group.These results show that the high expression of ACE2 can protect against the hypoxia injury in rat brain astrocytes.It confirmed the relation between the high ACE2 expression and the anti-hypoxic ation.To further explore the regulation of ACE2 expression,the Angâ…¡-induced brain astrocyte injury was examined.Cells were divided into 6 groups:control,10^-8mol/L Angâ…¡,10^-7mol/L Angâ…¡,10^-6mol/L Angâ…¡,10^-7mol/L Angâ…¡+10^-7mol/L losartan, 10^-7mol/L Angâ…¡+10^-7mol/L PD123319.Cell viability was assessed by MTT assay and cell injury by the rate LDH release and ATP production.RT-PCR was used to examine the mRNA expression of ACE2 and western blot analysis was used to evaluate the protein level of ACE2.Comparing with the control group,decrease of cell viability and ATP production and increase of LDH release were found in the hypoxia groups.Losartan increased the cell viability and ATP concentration and decreased the LDH release,but PD123319 had no effect on it.Both the mRNA and the protein of ACE2 was decreased after Angâ…¡injury.Losartan increased the expression of ACE2 in both mRNA and the protein level,but PD123319 has no effect on the expression of ACE2.These results show that Angâ…¡can inhibit the ACE2 expression via AT1 receptor.In conclusion,ACE2 expression was increased after the brain ischemia injury in vivo and the hypoxia injury in vitro.Angâ…¡can inhibit the expression of ACE2,while losartan can antagonized its function through an AT1 pathway.All the results provide a deep understanding on RAS in the brain ichemia injury.Partâ…¡:Effects of scutellarin on apoptosis induced by CoCl₂ in PC12 cellsThe mechanisms of ischemia cerebral vascular disease(ICVD) are complex,and it can result in the injury of neurons.Recent research suggests that the apoptosis of neurons play a major role in ICVD,and hypoxia is one of the important factors which can induce apoptosis.Many researchs suggested that scutellarin has the ability of anti-oxidation.This investigation was preformed to study the detailed characterization of PC12 cells responses to the presence of cobalt chloride(CoCl₂) and evaluate the protective effects of scutellarin on the hypoxia-induced apoptosis in PC12 cells.PC12 cells were cultured and passaged in DMEM.Experiments were performed with cells from the same passage.As grown to 80%confluence in DMEM with 10% FBS,PC12 cells were divided into 5 groups:control group,500μmol/L CoCl₂ group and scutellarin 0.1,1,10μmol/L groups.The drug-treated groups were pre-incubated with scutellarin for 2 hours.Other groups with the exception of the normal one were stimulated by CoCl₂(500μmol/L) simultaneously for 24 hours.All the above steps were carried out under sterile conditions.The viability of cells was detected by MTT assay. Morphological changes of PC12 cells were visualized under light and electron microscopes.Flow cytometry was employed to observe the occurrence of apoptosis. Both the reactive oxygen species(ROS) generation and Caspase-3 activity were quantified by their reagents and the absorbance was assessed by fluorescence spectrophotometer.The expression of Bax,Bcl-2,Bcl-X_L,p38 and P-p38 was examined by western blot.The results show that CoCl₂ triggers neuronal PC12 cells apoptosis in a dose- and time-dependent manner,and the best concentration and time of CoCl₂-induced PC12 apoptosis are 500μmol/L and 24 h,respectively.Incubation of PC12 cells with 500μmol/L CoCl₂ for 24 h resulted in significant apoptosis as evaluated by the MTT assay,electron microscopy and flow cytometry assays.The increase of Caspase-3 activity,decrease of Bcl-X_L expression,phosphorylation of p38 mitogen-activated protein kinase(MAPK) and accumulation of intracellular ROS were also seen in CoCl₂-treated PC12 cells.Scutellarin at 0.1,1 and 10μmol/L significantly protected against the apoptotic cell death induced by CoCl₂.Scutellarin decreased Caspase-3 activity,increased Bcl-X_L expression,inhibited p38 phosphorylation and attenuated ROS production.These results demonstrate that scutellarin can protect PC12 cells from CoCl₂ induced apoptosis by scavenging ROS,inhibiting p38 phosphorylation, up-regulating Bcl-X_L expression and decreasing Caspase-3 activity,and may reduce the cellular damage in pathological conditions associated with hypoxia-mediated neuronal injuryThis research shows that scutellarin can protect PC12 cells from CoCl₂-induced apoptosis which indicates scutellarin has remarkable ability on rescuing ICVD.