The Role Of Cellular Prion Protein In Splenic CD4~+T Lymphocytes Subtype Activation And Differentiation In Cerebral Ischemic/Reperfusion Injury

Objective:To prove that cellular prion protein(PrP~C)participates in peripheral immuneinflammation after IS and PrP~C has a regulatory effect on the differentiation and function of CD4~+T cell subsets after cerebral ischemic/reperfusion as well as to detect the apoptosis of OGD/R HT22 murine hippocampal neurons after the co-culture with differentiated splenic lymphocytes of different genotypes.Methods:Wild-type(WT)FVB/N male mice were selected and the MCAO model on one side of the was established.Flow cytometry was used to detect PrP~C expression on the surface of splenic CD4~+T cells.2)Mice of PRNP gene knockout,wild type and PRNP gene overexpression were selected to establish the MCAO model.The fluctuation of the ratio of splenic CD4~+T cells Th1/2/17 subtypes as well as the expression of supernatant and peripheral blood inflammatory cytokines were detected by flow cytometry.3)OGD/R HT22 mouse hippocampal neurons were co-cultured with the splenic lymphocytes of MCAO mice.Then the neuronal apoptosis was detected by FITC Annexin V Apoptosis Detection.Results:1)Compared with the sham-operated group,the MCAO mice were graded higher Longa score after IS.TTC staining indicated that the model was successfully established and the PrP~C on the surface of splenic CD4~+T cells increased;2)PRNP K.O.mice had a significantly higher Longa score than WT mice(p<0.05),and there was no significant difference in the volume of the cerebral infarction(p>0.05);the Longa score of Tga20 mice was significantly lower than that of WT mice(p<0.05),and the infarction volume was significantly lower(p<0.05).3)Expression of splenic CD4~+T cell Th1/2/17 subtypes of all genotypes increased significantly(p<0.05).Comparing to WT mice,the ratio of Th1 and Th17 subtypes of PRNP K.O.increased significantly(p<0.05)while the ratio of Th2 subtype of Tga20 mice increased and the Th1 subtype decreased significantly(p<0.05).4)The level of IL-10、IL-2、IL-6、IL-4、IFN-γ in WT mice splenic lymphocyte supernatants were significantly higher than the sham-operated group(p<0.05).Comparing to WT mice;IL-10,IL-2,IL-6,IL-17 A,IFN-17 A,IFN-γ of PRNP K.O.group significantly increased(p<0.05)and IL-4decreased(p<0.05);IL-10,IL-2,IL-4,IFN-γ of Tga20 mice splenic lymphocytes significantly increased and IL-17 A significantly decreased(p<0.05).5)IL-10,IL-2,IL-6,IL-4,TNF,IFN-γ levels in WT mice peripheral blood serum were significantly higher than the sham-operated group(p<0.05).Comparing to WT mice,IL-2,IL-6,IL-17 A,TNF of PRNP K.O.mice increased with statistical differences(p<0.05).IL-10,IL-2,IL-4 of Tga20 mice increased significantly with statistical significance(p<0.05).Conclusion:1)The expression of PrP~C on peripheral CD4~+T cells increases after cerebral ischemia/reperfusion,suggesting that it involves in the peripheral immune response of IS;2)PrP~C affects the severity and the volume of infarction after IS by participating in the T cells inflammatory response in the peripheral immune organs after IS.PrP~C deficiency causes the Th proinflammatory subtypes differentiation as well as the rise of the pro-inflammatory cytokines,while the overexpression of PrP~C leads to the Th anti-inflammatory subtypes differentiation,and the anti-inflammatory cytokines grows as well;3)The splenic T lymphocytes injure the neurons after IS;the splenic CD4~+T cells with more PrP~C expression reduce the death of neurons after IS,playing a neuroprotective role.