The Role Of Cyclophilin A/CD147 Signal Pathway On Early Brain Injury After Experimental Subarachnoid Hemorrhage And Its Molecular Mechanism Research

Part Ⅰ: The expression of Cyclophilin A and CD147 in brain following SAH in rats Objective To investigate the expression of Cyclophilin A and CD147 in brain following SAH in rats and to study the relationships with early brain injury(EBI).Methods1. Animal groups : 72 health male Sprague-dawley(SD) rats were assigned randomly into 6 groups of 12 rats each, a sham group and five SAH groups arranged by time: 2, 12,24, 48, and 72 hours after SAH.2.Animal model making : a rat SAH model was induced by injection of 0.3ml fresh arterial, nonheparinized blood into the prechiasmatic cistern in 20 s. All the rats in the experiment were killed at the indicated time point after SAH and cerebral tissue samples were taken for analysis.3. Detection methods and indicators : The brain cortex of six rats in each group was extracted and used for double immunofluorescence analysis,and partial brain samples of the other six were frozen in liquid nitrogen for Western blot analysis. By immunofluorescence and western blo method to evaluate the expression of Cyclophilin A and CD147 in brain tissue following SAH in rats at different time points.Results1. The western blot analysis showed that levels of Cy PA and CD147 expression in neurons were higher than in the sham group at 12 hours after SAH and peaked at 24 hour,and then decreased. However, at 72 hours after SAH,the protein levels of Cy PA and CD147 were still visibly higherthan that in sham group.2. The double immunofluorescence staining results demonstrated that the expressiontrend was similar to that of Western blot analysis.Conclusions1. Our findingssuggested that Cy PA/CD147 may participate in the pathologic progress of EBI after SAH.2. 24 hours following SAH may be a suitable time point for the next experement to research EBI.Part II: Experimental study of the effect of intervention in the regulation of Cy PA/CD147 on EBI after SAH and its possible mechanism ObjectiveTo observe the effect of intervention in the regulation of Cy PA/CD147 on EBI after SAH in rats and to investigate its possible mechanism.Methods1.Animal groups : 162 rats were randomly divided into nine groups: sham group,SAH group, SAH + recombinant humancyclophilin A(rh Cy PA) group, SAH + si Cy PA group, SAH + Ctrsi group, SAH + anti-CD147 group, SAH + anti-CD147 + rh Cy PA group,SAH + anti-CD147 + si Cy PA group, and SAH + anti-CD147 + Ctrsi group,each group with 18 rats.2.Animal model making:SAH model was produced by injecting fresh arterial,nonheparinized blood into the prechiasmatic cistern.3. Drug Administration : 1 μg/20 μL rh Cy PA was infused into the ventricle 15 minutes after blood injection in the rh Cy PA group. In the SAH + si Cy PAgroup or the SAH+ Ctrsigroup,500 pmol/20 μL Cy PA si RNA or controlsi RNA was injected intracerebroventricularly using a Hamilton microsyringe under the guidance of a stereotaxy instrument 24 hours before the SAH model was established. Monoclonal antibody of CD147(anti-CD147)was injectd into the vein at 3 mg/kg immediately after the SAH model was established in the SAH + anti-CD147 group. Drug Administration was the same with above in the SAH + anti-CD147 + rh Cy PA group, the SAH + anti-CD147 + si Cy PA group and the SAH + anti-CD147 + Ctrsi group.4. Detection methods and indicators :At 24 hours after SAH, behavioral activity was examined in all groups, and blood samples were used to detect the concentration of Cy PA.The brain cortexes of six rats were extracted for terminal deoxynucleotidyltransferase-mediated d UTP nick end labeling(TUNEL) staining, fluoro-jade B staining,and immunofluorescence analysis. Another six rats were exsanguinated and decollated and partial brain samples were frozen in liquid nitrogen for Western blot analysis,electrophoretic mobility shift assay(EMSA), and immunoprecipitation analysis. The remaining six rats were subjected to brain edema evaluation.Results1.Compared with the sham group: the expression of Cy PA and CD147 in the brain tissue was significantly higher than which in sham group 24 hours after SAH in rats(P <0.01),the expression of Cy PA increasedin serum(P < 0.05),neuroethology damage increased(P < 0.01), cerebral edema and albumin levels in brain tissue increased significantly(P < 0.01),TUNEL and FJB positive cells increased significantly(P <0.01),the expression of phosphorylation of ERK 1/2 increased obviously(P<0.01), the combination activity of the NF-kappa B significantly increased(P < 0.01) and the expression of P53 and caspase 3 increased inbrain tissue and neurons(P < 0.01).2. By Cy PA si RNA and anti-CD147 treatments, compared with the control group in SAH, Cy PA or CD147 expression in the brain tissue significantly decreased(P < 0.05),Cy PAsi RNA can reduce serum Cy PA expression(P < 0.05),rats neurobehavioral damage relief(P < 0.01), cerebral edema and brain tissue albumin levels reduced(P <0.05),TUNEL and FJB positive cells decreased significantly(P<0.01), phosphorylation of ERK 1/2 expression level in brain tissue and neurons decreased significantly(P < 0.05),the combination activity of the NF-kappa B significantly decreased(P < 0.05), p53 and caspase 3 expression level decreased inbrain tissue and neurons(P < 0.05).3. Rh Cy PA intervention induced the opposite results:Cy PA and CD147 expression in the brain tissue increased(P < 0.05), the expression of Cy PA increased in serum(P <0.05),rats neurobehavioral damage significantly increased(P < 0.01),brain edema increased(P < 0.05) and brain tissue albumin content increased(P < 0.01),TUNEL and FJB positive cells increased significantly(P < 0.05),brain tissue and neurons in the phosphorylation of ERK 1/2 expression level increased significantly(P < 0.01), the combination activity of the NF-kappa B increased significantly(P < 0.05), P53 and caspase 3 expression level increased significantly(P < 0.01).4. Use Cy PA si RNA and anti- CD147 intervention together,more than a separate application CD147 intervention, can significantly enhance resistance to cell death effect(P< 0.01), reduce brain edema(P < 0.01), reduce the the expression level of phosphorylation of ERK 1/2(P < 0.05), P53 and caspase 3(P < 0.05).5. Immunofluorescence staining results also showed that compared with SAH in the control group, Cy PA si RNA intervention reduced the expression of CD147 in brain neurons(P < 0.05). But with CD147 intervention, the expression Cy PA in brain neurons has no significant influence(P > 0.05). The results prompted that Cy PA might be upstream signal factor to CD147.6. Immunoprecipitation test results showed the interaction of Cy PA and CD147 increased that 24 hours after SAH.conclution1. Cyclophilin A/CD147 interactions may participate in subarachnoid hemorrhage–induced early brain injury via increasing neuronal apoptosis pathway, at least partly through the ERK1/2-nuclear factor-κB pathway.2. Cy PAsi RNA and CD147 monoclonal antibody therapy has protective effect on early brain injury after SAH.3.Rh Cy PA worsen early brain injury after SAH.4. Cy PA through combining with its receptor CD147 participate in the early of the pathological process of brain injury after SAH and it may be a upstream signal transduction factors of CD147.5. Cyclophilin A/CD147 may be a suitable therapeutic target for subarachnoid hemorrhage.