| Objective: On the basis of the previous research work,the expression of Cdk5 signal pathway was observed after cerebral ischemia 2h and reperfusion for 22 h.Whether NRG1? inhibits neuronal apoptosis through Cdk5 pathway,and what's its specific mechanism,to provide a theoretical basis for the future application of NRG1? in the prevention and treatment of ischemic cerebrovascular disease.Methods:(1)The MCAO/R models were established by inserting a monofilament threads into middle cerebral artery in 360 Wistar rats.These rats were divided into five groups according to the random number table method.Each 55 rats were in sham group,model(MCAO/R)group,treatment(NRG1?)group,inhibitor+treatment(Ros+NRG1?)group and inhibitor(Ros)group.(2)Sham group was not inserted into the craniotomy,MCAO model was not established,5?l was injected with 0.1mol/L PBS after 2h;MCAO/R group established MCAO/R model 2h after the removal of bolt,0.1ml/L PBS injection of 5?l;In the treatment group,the rats were sacrificed 2 hours after ischemia,and 5?l(2?g/kg)of 8% NRG1? was injected into ICA with ECA stump;In the Ros+NRG1? group,5 ?l of Roscovitine was injected into the ICA by ECA,and then reperfusion was performed after 2h of MCAO administration,and 5 ?l of 8% NRG1? was given immediately.In the inhibitor group,Roscovitine was injected into the Ros+NRG1? group,but after 2h occlusion,5?l of 0.1mol/l PBS was given.(3)After treatment(22h after reperfusion),the neurological function of the animals was measured by the modified neurological severity score points(m NSS).The water content of the brain tissue after MCAO injury was measured by dry and wet weight method.The volume of cerebral infarction was measured by staining with triphenyl tetrazolium chloride(TTC).The morphological changes of neurons were observed by HE staining and toluidine blue(Nissl)staining.The ultrastructural changes of neurons were observed by transmission electron microscopy(TEM).Apoptosis was detected by in situ terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL)and flow cytometry(FCM).Immunohistochemistry(IHC)and Western Blot were used to detect the expression of Calpain1,p35/p25,Cdk5 and phosphorylated tau(p-Tau)proteins in neurons.Results:(1)The effect of NRG1? on neurological function: the m NSS score of MCAO/R group,NRG1? group,Ros+NRG1? group and Ros group was significantly higher than that of Sham group(P<0.05).The m NSS scores of NRG1? group,Ros+NRG1? group,Ros group decreased to varying degrees than those of MCAO/R group(P<0.05).The m NSS score of Ros+NRG1? group was the lowest(P<0.05),and the difference was statistically significant(P<0.05).Compared with other groups,the scores of neurological function were significantly lower than those of MCAO/R group(P<0.05).(2)The effects of NRG1? on the water content of brain tissue: the water content of brain tissue in Sham group was significantly lower than that in MCAO/R group,NRG1? group,Ros+NRG1? group and Ros group,the difference was statistically significant(P<0.05).Compared with the Ros group,the water content of the NRG1? group,Ros+NRG1? group and Ros group was significantly lower than that of the MCAO/R group(P<0.05).There was no significant difference in the water content of brain tissue between NRG1? group and Ros group(P>0.05).The water content of Ros+NRG1? group was significantly lower than that of MCAO/R group,NRG1? group and Ros group(P<0.05).(3)The effects of NRG1? on cerebral infarction volume: Sham group had no cerebral infarction;The volume of cerebral infarction increased significantly in MCAO/R group,NRG1? group,Ros+NRG1? group and Ros group compared with Sham group,the difference was significant(P<0.05).The infarct size of MCAO/R group was significantly higher than that of NRG1? group,Ros group and Ros+NRG1? group(P<0.05).The volume of cerebral infarction in NRG1? group and Ros+NRG1? group was significantly lower than that in Ros group(P<0.05),while the volume of cerebral infarction in Ros+NRG1? group was the smallest,compared with other groups,the difference was statistically significant(P<0.05).(4)The effects of NRG1? on the Morphological Structure of Neurons: Both HE staining and Nissl staining showed that Sham group morphological structure was normal,no significant degeneration and necrosis of neuronal cells,the Denatured cell index(DCI)was low;DCI in MCAO/R group,NRG1? group,Ros group and Ros+NRG1? group was significantly higher than Sham group(P<0.05),there were different degrees of neuronal cell damage,such as cell arrhythmias,shrinkage,cell rupture,disappearance of nuclei,vacuolar changes,and so on;the levels of DCI in NRG1? group,Ros group and Ros+NRG1? group were significantly lower than those in MCAO/R group,the morphological structure and neuronal cell damage were between Sham group and MCAO/R group,and the difference was significant(P<0.05);Compared with NRG1? group and Ros group,DCI decreased in Ros+NRG1? group,degeneration and necrosis of neuronal cells is more reduced,the difference was statistically significant(P<0.05).(5)The effects of NRG1? on the ultrastructure were as follows: The ultrastructure of neurons in Sham group was normal,the nucleus was large and round,the chromatin was distributed uniformly,the organelles were abundant,the cell membrane and nuclear membrane were intact,neuronal axons were normal,microtubules were normal,occasionally apoptotic neurons;MCAO/R group compared with Sham group,neuronal damage,cell shrinkage,the number of organelles decreased or even disappeared,vacuolar degeneration,the formation of apoptotic bodies,neuronal apoptosis significantly;In NRG1? group,Ros+NRG1? group,Ros group: nerve cell injury was between Sham group and MCAO/R Group;NRG1? group was closed to the ultrastructure of neurons in Ros group;the neuronal apoptosis of Ros+NRG1? group was lighter than NRG1? group and Ros group,and in this group,there were part of the vacuoles,clear edge,and organelles damage is also reduced.(6)The effects of NRG1? on Apoptosis:(1)TUNEL method showed: parietal cortical neurons of Sham group shallow staining of light cells,the vast majority of cell structure is complete,neatly arranged,cell membrane smooth,rich cytoplasm,the nucleus is clearly visible,interstitial no edema,occassionally cell apoptosis;MCAO/R group showed a large number of neuronal apoptosis,cell arrangement disorder,shrinkage,cell membrane shrinkage,cytoplasm was dyed brown,nuclei were dyed brown or tan,or shrink,fragmentation,and even disintegration,The formation of vacuoles;NRG1? group,Ros + NRG1? group,Ros group,neuronal cell morphology improved,arranged neatly,the structure is relatively complete,part of the nerve cell nuclear condensation,fragmentation,the formation of vacuoles;Ros+NRG1? group,compared with NRG1? group,Ros group was significantly improved,degeneration and necrosis of neurons decreased.(2)both FCM and TUNEL showed that the early apoptosis cell index(EACI)and the apoptotic cell index(ACI)were significantly increased in the group of Ros+NRG1?,NRG1?,Ros group and MCAO/R group,compared with the Sham group(P<0.05),the levels of EACI and ACI decreased in the Ros+NRG1? group,NRG1? group and Ros group than that in MCAO/R group,the difference was statistically significant(P<0.05).Compared with NRG1? group and Ros group,the levels of EACI and ACI were significantly decreased in Ros+NRG1? group(P<0.05).(7)The effects of NRG1? on Protein Localization:(1)Calpain 1: The expression of positive cell index(PCI)in MCAO/R group was significantly higher than that in that in Sham group(P<0.05).The expression of NRG1? group and Ros+NRG1? group was significantly lower than that of MCAO/R group(P<0.05),but there was no significant difference between the two groups(P>0.05).The expression of Calpain 1 in Ros group was not significantly different from that in MCAO/R group(P<0.05),but between Sham group,MCAO/R group and NRG1? group,Ros+NRG1? group,the levels of Calpain1 were significantly different,statistically significant(P<0.05).(2)Cdk5: Compared with Sham group,the levels of PCI in MCAO/R group,NRG1? group,Ros+NRG1? group and Ros group were significantly higher than those in control group(P<0.05),while Compare with each other,the difference in MCAO/R group,NRG1? group,Ros+NRG1? group,Ros group was not significant,and there is no statistically significant(P>0.05).(3)p35/p25: The expression of p35/p25 in MCAO/R group and Ros group was significantly higher than that in Sham group,and the difference was significant(P<0.05),but there was no significant difference between the two groups(P>0.05).The expression of p35/p25 in NRG1? group was significantly lower than that in MCAO/R group and Ros group(P<0.05),but there was no significant difference between NRG1? group and Ros+NRG1? group(P>0.05).(4)p-Tau: Compared with Sham group,the expression of protein in MCAO/R group,NRG1? group,Ros+NRG1? group and Ros group was significantly higher than that in Sham group(P<0.05);NRG1? group,Ros group,Ros+NRG1? group,were lower than that in MCAO/R group(P<0.05).When compared Ros+NRG1? group with NRG1? group and Ros group,the difference was statistically significant(P<0.05);the expression of Calpain1 protein in NRG1? group was lower than that in Ros group,and the difference was statistically significant(P<0.05).(8)The effects of NRG1? on Protein Quantitative Expression:(1)Calpain 1: The relative protein content of MCAO/R group was significantly higher than that of Sham group(P<0.05).The content of relative protein in NRG1? group and Ros+NRG1? group was significantly lower than that in MCAO/R group(P<0.05),but there was no significant difference between the two groups(P>0.05).The expression of Calpain 1 in Ros group was not significantly different from that in MCAO/R group(P>0.05),and there was significant difference between NRG1? group and Ros+NRG1? group(P<0.05).(2)Cdk5:Compared with Sham group,the relative protein contents of MCAO/R group,NRG1? group,Ros+NRG1? group and Ros group were significantly increased(P<0.05),while MCAO/R group,NRG1? group,Ros+NRG1? Group,Ros group between the two pairs,the difference was not statistically significant(P>0.05).(3)p35/p25: the relative protein contents of MCAO/R group and Ros group were significantly higher than those of Sham group(P<0.05),but there was no significant difference between the two groups(P>0.05).NRG1? group and Ros+NRG1? group were significantly lower than those in MCAO/R group and Ros group(P<0.05),but there was no significant difference between NRG1? group and Ros+NRG1? group(P>0.05).(4)p-Tau: The expression of protein in MCAO/R group,NRG1? group,Ros+NRG1? group,Ros group was significantly increased than that in Sham group,and the relative protein content was significantly increased(P<0.05).The expression of p-Tau in the NRG1? group,Ros group and Ros+NRG1? group was significantly lower than that in the MCAO/R group(P<0.05).Ros+NRG1? group had the lowest relative protein content,which was statistically significant(P<0.05)compared with NRG1? group and Ros group.Compared with Ros group,the expression of Calpain1 protein in NRG1? group was lower than that in Ros group,the difference was statistically significant(P<0.05).Conclusions: After cerebral ischemia-reperfusion injury,Cdk5 signal pathway is over-activated,leading to neuronal cell apoptosis;its specific inhibitor roscovitine can block its induction of apoptosis,is conducive to cerebral ischemia-reperfusion injury neurological function protection.Neuregulin 1? inhibits the expression of Calpain1,p35/p25 and p-Tau in the Cdk5 signal pathway,improves neurological function,reduces neuronal apoptosis,and plays a neuroprotective role after cerebral ischemia reperfusion injury. |
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