| Part Ⅰ Expression of PK2、A1&A2 phenotype Astrocytes marker proteins After SAHPurpose:To study the expression levels of PK2 and the marker proteins of A1&A2 astrocytes in rats and human brain tissues at different time points after SAH.Methods:1.Experimental animal grouping:96 Sprague-Dawley rats were divided into Sham group and 7 time points after SAH,respectively 3 h,6 h,12 h,24 h,48 h,72 h,168 h,total 8 groups(12 rats in each group)2.The levels of PK2,A1 astrocytes marker proteins(C3,Serping1)and A2 astrocyte marker protein(PTX3,S100A10)in rat and human brain tissue were detected by Western blot,and the localization and level of PK2,C3 and PTX3 in brain tissue were observed by immunofluorescence.3.Brain tissue samples of 20 clinical patients were collected,including 10 cases of Non-SAH group and 10 cases of SAH group.Immunofluorescence was used to observe the localization and level of PK2 in neurons,C3 and PTX3 in astrocytes.Results:1.Although the protein level of PK2 in the brain tissue of rats after SAH fluctuates to a certain extent within 3h-6 h after SAH,there is no statistically significant level change.Until 12h after SAH(P<0.01),the protein level increased by about half of the level of the sham group,and then decreased;2.Although the level of C3(one of marker protein of astrocyte A1)in the brain tissue of rats after SAH fluctuates to a certain extent within 3h-6h after SAH,there is no statistically significant level change.The protein level increased by about twice the level of the sham group 12 hours after SAH(P<0.01),and maintained high expression to 168 hours after SAH(P<0.05).The level of Serpingl(one of marker protein of A1 astrocytes)increased and was statistically significant at 3h after SAH(P<0.05),and then decreased.There was no statistically significant change in the protein expression of PTX3 at 3h-6h after SAH in the brain tissue.The protein level increased by about 2 times than the sham group 12 hours after SAH(P<0.005),and then decreased.The level of S100A10 increased after SAH,reached the highest level at 24 hours after SAH(P<0.05),and then decreased.The results of immunostaining showed that marker proteins of A1 and A2 were expressed in astrocyte cells,and the changes of distribution level were consistent with the results of Western blot.3.The results of IF of human brain tissue showed that the fluorescence intensity of PK2 in the SAH group was significantly increased than the non-SAH group(P<0.05).The expression of A1 and A2 markers proteins on astrocytes was significantly higher in the SAH group than in the non-SAH group(P<0.05).Conclusions:1.After SAH,The protein expression level of PK2 in temporal neurons was increased,and the marker proteins of A1 and A2 increased as well,but the expression of A1 cells seemed to be more persistent.2.After SAH,the synthesis of PK2 in the neurons of human brain tissue was promoted,and the contents of C3 and PTX3 in astrocyte were significantly increased.Part Ⅱ Effects of PK2 Interventions on SAH-Induced EBI and Astrocytic ActivationPurpose:To explore the effect of PK2 on the phenotype transformation of reactive astrocytes in the early brain injury of subarachnoid hemorrhage model in rats.Methods:1.Experimental animal grouping:SD rats(n=108,weight:300-350g)were randomly divided into Sham),SAH,SAH+ Si-NC,SAH+Si-PK2,SAH+Vehicle-PK2 and rPK2group.There are 18 rats in each group.2.In each group,6 SD rats weighing 300-350g were randomly chosen to be sacrificed at 12h after SAH and temporal tissues were collected for immunofluorescence and Western blot.The remaining 12 rats were used for behavioral score at 48 hours after SAH,and were divided into two groups:6 rats were sacrificed and tested for FJC,TUNEL and Cleaved caspase3,and the remaining 6 rats continued to undergo rotarods and water maze test on the 4th-7th day.Results:1.In SAH group,The protein expression level of PK2 was increased compared with Sham group(P<0.01);The protein expression level of PK2 expression was decreased in SAH+Si-PK2 group(P<0.05);while increasing in SAH+rPK2 group(P<0.05).2.Compared with the sham group,the expression of A1 phenotype marker C3 and A2 phenotype marker PTX3 in SAH group increased significantly,while in SAH+ Si-PK2 group A1 phenotype marker C3 was significantly increased(P<0.05)and A2 phenotype marker PTX3 was significantly decreased(P<0.05).Compared with SAH+Vehicle group,SAH+ rPK2 group has a significant decrease in A1 phenotype marker C3(P<0.05),while the expression of A2 phenotype marker PTX3 was significantly increased(P<0.05).Meanwhile,the expression of p-STAT3 was increased in SAH group than Sham group(P<0.05).Compare to SAH+Si-NC group,the expression of p-STAT3 was decreased in SAH+Si-PK2 group(P<0.05);The expression of p-STAT3 was increased in SAH+rPK2 group than SAH+Vehicle group(P<0.05).3.Compared with SAH group,The protein expression level of Cleaved-caspase3 expression was increased in SAH+Si-PK2 group(P<0.05)while decreased in SAH+rPK2 group(P<0.05);Compared with Sham group,the number of FJC-positive cells was increased in SAH group(P<0.01).The number of FJC-positive cells was significantly greater in SAH+Si-PK2 group than SAH+Si-NC group(P<0.05).The number of FJC-positive cells was significantly decreased in SAH+rPK2 group than SAH+Vehicle group(P<0.05).The results of TUNEL test were consistent with that of FJC test.4.Compared with the sham group,rats in the SAH group had significantly higher neurobehavioral scores(P<0.001),significantly shorter exercise time on the rotating rod(P<0.05),and spend more time to reach the target in the water maze(P<0.01).While compared with the SAH+Si-NC group,the SAH+Si-PK2 group significantly aggravated the neurobehavioral score(P<0.05)and spent less time on the rotating rod(P<0.05),and the time required to reach the target in the water maze was significantly increased(P<0.01).Meanwhile,SAH+rPK2 group shows an obvious improvement the neurobehavioral score(P<0.05)and the time spent on the rotating rod was significantly extend(P<0.05)and the time required to reach the target in the water maze was significantly decreased.than SAH+Vehicle group(P<0.01).Conclusion:1.PK2 can improve neuronal apoptosis and neurological impairment after SAH,and promote the recovery of sensory,motor and learning ability in rats.2.PK2 may mediate the production of A2 astrocytes through STAT3 phosphorylation,reduce the release of inflammatory factors,and thus play a role in alleviating EBI after SAHPart Ⅲ Effects of TNF-α Interventions on Neuronal PK2 and Astrocytes Activation In VitroPurpose:To explore the expression of PK2 in TNF-α stimulated neurons and the mechanism of PK2 in promoting the phenotype transformation of astrocytes in vitro.Methods:1.Cultured neurons were divided into three groups:Control group,TNF-α+Vehicle group and TNF-α group.The expression of PK2 in TNF-α stimulated neurons were evaluated by immunostaining.And the PK2 concentration in culture medium was tested by ELISA.2.TNF-α was used to simulate the inflammation response after SAH to activate astrocytes in vitro,and the cultured astrocytes divided into 6 groups:Control group,TNF-α+Vehicle group,TNF-α group,TNF-α+Vehicle-PK2 group and TNF-α+rPK2 group.The protein of A1 phenotype marker C3 and A2 phenotype marker PTX3 were detected by Western blot and immunofluorescence staining.Results:1.After treated by TNF-α to stimulate inflammatory responses after SAH,PK2 derived from neurons increased(P<0.05),and the content of PK2 secreted in the culture medium also increases,which indicate PK2 is synthesized and released by neurons(P<0.001).2.After treat by TNF-α,cultured astrocytes were activated.The expression of A1 phenotype marker C3 and A2 phenotype marker PTX3 in the TNF-α group was significantly increased than the Vehicle-1 group;Compared with the TNF-α+Vehicle-PK2 group,the expression of A1 phenotype marker C3 in the TNF-α+rPK2 group was significantly reduced(P<0.05),and the expression of PTX3 was significantly increased(P<0.05).3.After treat by TNF-α,cultured astrocytes were activated.The expression of P65 and p-STAT3 protein in the TNF-α group was significantly higher than that in the Vehicle-1 group(P<0.05);Compared with the TNF-α group,the expression of P65 in the TNF-α+rPK2 group was significantly reduced(P<0.05),and the expression of p-STAT3 protein was significantly increased(P<0.05).Conclusions:1.The synthesis and secretion of PK2 by neuron can be stimulated by TNF-α.2.Astrocytes can be activated by TNF-α,while PK2 promotes the conversion from A1 phenotype astrocytes to A2 phenotype astrocytes.3.P65 and STAT3 may be involved in the conversion between astrocyte phenotype,p-STAT3 may be related to A2 phenotype astrocytes and p65 may be related to A1 phenotype astrocytes. |
PDF Full Text Request