Effect Of ZNRF2 On Cerebral Ischemia/Reperfusion Injury In Rats And Ints Mechansim

Ischemic stroke is a kind of cerebrovascular disease and one of the leading causes of death and disability in the world,threatening the physical and mental health of humans.The effective treatment is thrombolysis or surgical treatment to achieve blood flow reperfusion in the ischemic area in clinic currently,but it will cause"cerebral ischemia/reperfusion injury(CIRI).The pathogenesis of CIRI is complex.Therefore,exploring latent regulatory mechanisms in CIRI injury may help overcome these limitations and treatment side effects to some extent.Autophagy is a type of cell self-protection way under external stimuli.However,more and more studies have shown that autophagy is a“double-edged sword"in CIRI which involves in the regulation of neuronal death.ZNRF2 is highly expressed in the central nervous system and participated in the establishment of neuronal synapses and the maintenance of synaptic plasticity.However,there is little research about ZNRF2 in nervous system diseases.In this study,middle cerebral artery occlusion/reperfusion(MCAO/R)in rats and oxygen-glucose deprivation/reoxygenation(OGD/R)are established to explore the role of ZNRF2 in I/R injury and to determine whether the effect of ZNRF2 on CIRI relates to the regulation of neuronal autophagy.The study may provide a novel target for neuroprotective drugs and new strategy for the clinical treatment of stroke.PartⅠ Expression of ZNRF2 in the Blood of Patients with Ischemic StrokeObjective:To measure the expression of ZNRF2 mRNA in the blood of control and patients with ischemic stroke and to explore the relation between ZNRF2 and ischemic stroke.Methods:Admission/exclusion criteria of patients with ischemic stroke were formulated according to the criteria of diagnosing ischemic stroke.The peripheral blood samples of patients with ischemic stroke and control were collected in the Dianjiang People’s Hospital of Chongqing.q RT-PCR was used to measure the expression of ZNRF2 mRNA in the blood.Results:A total of 81 subjects were recruited including 40 stroke patients and 41 control.1.There was no significant difference in the age,gender,diabetes,TC,TG,and LDL-C between control and patients with ischemic stroke;while the numbers of patients with hypertension in stroke patients was more than that in the control(62.5%vs.26.8%,P<0.01),and the level of HDL-C in patients was significantly lower than that in the control(1.25±0.29 mmol/m L vs.1.46±0.37 mmol/m L,P<0.01).2.Compared with control,ZNRF2 mRNA in the blood of patients with ischemic stroke was significantly reduced(P<0.05);the expression of ZNRF2 mRNA in the blood of patients with ischemic stroke was negatively correlated with NIHSS score(R~2=0.1054,p<0.05).Conclusion:1.ZNRF2 mRNA was decreased significantly in the blood of patients with ischemic stroke.2.Expression of ZNRF2 in the blood of patients with ischemic stroke was negatively correlated with NIHSS score3.ZNRF2 may relate to the pathogenesis and progression of ischemic stroke.PartⅡ Effect of ZNRF2 on Cerebral Ischemia/Reperfusion Injury in RatsObjective:To observe the expression of ZNRF2 in plasma and cortex of MCAO/R-treated rats;to observe the effect of ZNRF2 on CIRI;to observe the effect of ZNRF2 on the MCAO/R-induced autophagy.Methods:1.To establishe the MCAO/R modelMale sprague-dawley rats were anesthetized with 3%sodium pentobarbital(1 m L/kg)though intraperitoneal injection.Laser doppler flowmetry was performed to measure the dynamic process of blood and CCA,ECA and ICA were exposed carefully.ECA was produced by permanent ligation and a nylon monofilament suture was inserted from the CCA into ICA until the blood was reduced 70%.After 1.5 h of occlusion,the reperfusion was performed.The time of reperfusion was different according to aim.2.Groups and treatments(1)To measure the expressions of ZNRF2 mRNA and protein in blood and cortex,animals were grouped including normal group,sham group,MCAO/R 6 h group,MCAO/R 12 h group,MCAO/R 24 h group,MCAO/R48 h group,MCAO/R 72 h group,n=4.(2)To explore the effect of ZNRF2 on cerebral ischemia/reperfusion injury in rats,animals were divided randomly into 4 groups:sham group,model group(MCAO/R),model group+empty virus group(MCAO/R+LV-NC),model group+ZNRF2 overexpression lentivirus group(MCAO/R+LV-ZNRF2),n=10.MCAO/R+LV-NC group and MCAO/R+LV-ZNRF2 were given empty virus and ZNRF2 overexpression virus respectively in the lateral ventricle 14 days before MCAO/R.3.Detection indexes:Laser doppler flowmetry was used to measure the dynamic change of cerebral blood flow;zea-longa score was used to detect the neurological deficits of rats to observe the neurobehavioral changes;cerebral infarct volume of rats was detected by TTC staining;pathological changes of cortex were examined by HE staining;transmission electron microscope was used to measure the autophagosomes in cortical neurons;WB was applied to observed the proteins expressions of ZNRF2,m TOR,p-m TOR(ser2448),LC3II/LC3I,p62,Beclin1,Bax,and Bcl-2 in the cortex of rats.Immunofluorescence was used to observe the expressions of LC3B and p62 in cortical neurons of rats.Results:1.Cerebral blood flow was reduced 70%compared to the base line.Compared with sham group,the expression of ZNRF2 mRNA in the plasma in the MCAO/R group was reduced(P<0.001);ZNRF2 mRNA and protein in the cortex of rats after different reperfusion time showed first increase in6h and 12h,then significant downregulation(P<0.05)in 24h,48h and 72h;the neurological deficit score(P<0.001)and cerebral infarction volume significantly increased(P<0.001);pathological injury of the cortex of MCAO/R-induced rats was anabatic and the number of dead cells were increased(P<0.001);the number of autophagosomes in cortical neurons increased(P<0.01);ZNRF2(P<0.05),the relative ratio of p-m TOR(ser2448)/m TOR(P<0.001),p62(P<0.01)and Bcl-2(P<0.01)proteins were significantly reduced,Beclin1(P<0.01),Bax(P<0.01&P<0.05)proteins and LC3II/LC3I(P<0.01)expression increased significantly;2.Compared with the MCAO/R+LV-NC group,in MCAO/R+LV-ZNRF2 group,the neurological deficit score(P<0.01)and cerebral infarction volume were significantly reduced(P<0.01);the nucleus pyknosis and vacuolation in the cortex were significantly improved(P<0.001);the number of autophagosomes in cortical neurons was reduced(P<0.05);ZNRF2(P<0.01),the relative ratio of p-m TOR(ser2448)/m TOR(P<0.001),p62(P<0.01)and Bcl-2(P<0.01)proteins were significantly up-regulated,and Beclin1(P<0.01),Bax(P<0.05)proteins and LC3II/LC3I(P<0.05)expression were significantly decreased.Conclusion:1.Expressions of ZNRF2 mRNA and protein were significant downregulated in plasma and cortex of MCAO/R-treated rats.2.Autophagy was over-activated in cortex of MCAO/R-treated rats and brain was injuryed.3.ZNRF2 plays a protective role in CIRI of rats.4.Protection of ZNRF2 in MCAO/R-treated rats may be related to inhibit neuronal autophagy.PartⅢ Effect of ZNRF2 on the PC12 Cells Injury Induced by OGD/RObjective:To observe the expressions of ZNRF2 mRNA and protein in PC12 cells induced by OGD/R;to observe the effect of ZNRF2 on OGD/R-treated PC12 cell injury;to explore the relation between ZNRF2 and OGD/R-induced autophagy in PC12 cells.Methods:1.To establishe the OGD/R model in PC12 cells:OGD/R model was established by oxygen-glucose deprivation for 2 hours and reoxygenation for24 hours.2.Groups and treatments:(1)PC12 cells were divided into normal group and model group(OGD/R)to observe the expression of ZNRF2 in PC12 cells,n=3.(2)In order to explore the role of ZNRF2 in OGD/R-treated PC12 cell injury,the cells were grouped into:normal group,OGD/R group,OGD/R+empty virus group(OGD/R+LV-NC),OGD/R+ZNRF2overexpression lentivirus group(OGDR+LV-ZNRF2),OGD/R+negative control group(OGD/R+si NC),OGD/R+ZNRF2 small interferingRNA group(OGD/R+si R-ZNRF2),n=3.3.Detection indexes:cell viability was measured by MTT assay;apoptosis rate was determined by flow cytometry;q RT-PCR was applied to detect the expression of ZNRF2 mRNA;WB was used to detect the proteins expressions of ZNRF2,m TOR,p-m TOR(ser2448)LC3II/LC3I,p62 and Beclin1 in PC12 cells.Results:1.Compared with normal group,in the OGD/R group,ZNRF2 mRNA(P<0.01)and protein(P<0.01)were significantly reduced;PC12 cell viability was significantly reduced(P<0.01);cell apoptosis rate was markedly increased;p62(P<0.001),the relative ratio of p-m TOR(ser2448)/m TOR(P<0.001)were significantly reduced,Beclin1(P<0.001)and LC3II/LC3I(P<0.001)proteins were significantly increased.2.Compared with the OGD/R+LV-NC group,the PC12 cell viability in the LV-ZNRF2 group was markedly increased(P<0.01);the apoptosis rate was significantly reduced;ZNRF2(P<0.05),the relative ratio of p-m TOR(ser2448)/m TOR(P<0.01)and p62(P<0.05)proteins was significantly up-regulated,and Beclin1(P<0.001)and LC3II/LC3I(P<0.001)proteins was significantly reduced.3.Compared with the OGD/R+si NC group,the PC12 cell viability in the OGD/R+si R-ZNRF2 group was reduced(P<0.05);the apoptosis rate was increased;p62 protein expression was down-regulated(P<0.001),Beclin1(P<0.05)and LC3II/LC3I(P<0.01)proteins expressions increased significantly.Conclusion:1.Expressions of ZNRF2 mRNA and protein were decreased significantly in OGD/R-treated PC12 cells.2.Autophagy in PC12 cell was activated after OGD/R and PC12 cells was injuryed.3.ZNRF2 alleviated the cell injury induced by OGD/R in PC12 cells4.Its protective effect may be attribute to regulation of autophagy induced by OGD/R.PartⅣ Effect of ZNRF2 on the Primary Cortical Neurons Injury induced by OGD/RObjective:To observe the effect of ZNRF2 on OGD/R-treated cortical neurons injury,and to determine whether the effect is related to inhibition of m TORC1-mediated autophagy.Methods:1.OGD/R in primary cortical neurons:cortical neurons were extracted from fetal rats and cultured for 7 days.Cortical neurons were deprived of oxygen and glucose for 30 min and reoxygenated for 24 hours to establish an OGD/R model.2.Groups and treatments:(1)To detect the expressions of ZNRF2 mRNA and protein in cortical neurons after OGD/R,the neurons were divided into two groups:normal group,model group,n=3.(2)In order to explore its role and mechanism,the neurons were divided into:control,OGD/R,model+empty virus group+DMSO group(OGD/R+LV-NC+DMSO),model+ZNRF2 overexpression lentivirus group(OGD/R+LV-ZNRF2),model+ZNRF2 overexpression lentivirus group+rapamycin(200 n M)group(OGD/R+LV-ZNRF2+rapamycin),n=3.3.Detection indexes:MTT assay was used to detect cell viability;TUNEL staining was used to measure neuronal apoptosis rate;q RT-PCR was used to test the expression of ZNRF2 mRNA;WB was used to determine the proteins expressions of ZNRF2,m TOR,p-m TOR(ser2448),LC3II/LC3I,p62,Beclin1,BAX and Bcl-2 in cortical neurons.Results:1.Compared with normal group,ZNRF2 mRNA(P<0.01)and protein(P<0.001)in the OGD/R group were significantly reduced;neuron viability was significantly reduced(P<0.001);neuronal apoptosis rate was significantly increased(P<0.001);p62(P<0.01),m TOR(P<0.01),p-m TOR(ser2448)(P<0.01)and Bcl-2 proteins were significantly reduced(P<0.001),Beclin1(P<0.001),LC3II/LC3I(P<0.001)and Bax(P<0.01)proteins expressions increased significantly.2.Compared with the LV-NC+DMSO group,in the LV-ZNRF2 group,cell viability was significantly increased(P<0.05);apoptosis rate was significantly reduced(P<0.01);ZNRF2(P<0.05),the relative ratio of p-m TOR(ser2448)/m TOR(P<0.05),p62(P<0.001)and Bcl-2(P<0.01)proteins were significantly up-regulated,Beclin1(P<0.01),Bax(P<0.01)and LC3II/LC3I(P<0.01)proteins expressions were significantly reduced.3.Compared with the OGD/R+LV-ZNRF2 group,in the OGD/R+LV-ZNRF2+rapamycin group,neuron viability was significantly reduced(P<0.05);neuronal apoptosis rate was significantly increased(P<0.05);The p62(P<0.05)and Bcl-2(P<0.05)proteins were significantly reduced,and the proteins expressions of Beclin1(P<0.05),LC3II/LC3I(P<0.05),and Bax(P<0.05)were increased significantly.Conclusion:1.ZNRF2 mRNA and protein expressions were decreased in OGD/R-treated cortical neurons.2.Autophagy in cortical neurons was over-activated after OGD/R.3.ZNRF2 protected cortical neurons from OGD/R.4.Its mechanism may be related to the upregulation of m TORC1 to inhibit OGD/R-induced neuronal autophagy activation.