| ObjectiveTo explore the brain protective effect of Fingolimod(FTY720)on rats with focal cerebral ischemia reperfusion injury and its possible regulatory mechanism.Method75 male Sprague-Dawley(SD)rats of SPF grade were randomly divided into sham operation group(Sham group),model group(MCAO/R group),fengomod low-dose group(0.5mg/kg,FTY-L group),fengomod medium-dose group(1mg/kg,FTY-M group)and fengomod high-dose group(2mg/kg,FTY-H group),with 15 rats in each group.Except for Sham group,which only stripped blood vessels,the remaining four groups were prepared by modified Longa suture method.The rats were reperfusion after two hours of ischemia.After ischemia induction,the rats in each group were intraperitoneally injected with the same volume of normal saline and the corresponding dose of FTY720,and then administered again after 24 hours,and the materials were taken after 24 hours of reperfusion.The neurological deficit score,brain water content and TTC staining were used to observe the overall effect of drugs on the brain;HE staining was used to observe the histopathological changes;Detection of serum inflammatory factor IL-1β、 IL-6 and TNF-ɑ by ELISA.The target pathway proteins HMGB1,TLR4,NF-κ B were detected by immunohistochemistry and Western blotting for testing.Result1.The neurological deficit score,brain water content test and TTC staining showed that there was no neurological deficit in Sham group,and the HE staining cells were normal in morphology and orderly in arrangement.Compared with Sham group,MCAO/R group had significantly higher scores of nerve function defect;The cerebral tissue on the surgical side showed ischemia and edema of the middle cerebral artery;The color of ischemic area is whiter than that of normal brain tissue.TTC staining has a large area of gray-white infarction.HE results show that the cells in the CA1 area of the rat hippocampus are ischemic,anoxic,loosely arranged,with a large number of vacuoles,and the nucleus is deformed and displaced,indicating that the MCAO/R model is successful.Compared with MCAO/R group,the scores of nerve function defect and brain water content of rats in FTY-M group and FTY-H group decreased after the intervention of FTY720.The volume of infarcted area of rats in each dose group was significantly reduced by TTC staining(P<0.05,P<0.01),and the difference was statistically significant(P<0.05,P<0.01).The HE staining results showed that the cell damage of each dose group was improved to different degrees,and the improvement effect of FTY-H group was the most obvious.2.ELISA results showed that compared with Sham group,MCAO/R group IL-1 β、 IL-6 and TNF-ɑ.The levels were significantly higher;Compared with MCAO/R group,IL-1β、IL-6 and TNF-ɑ in each dose group after FTY720 intervention The levels were decreased to different degrees,and the difference was statistically significant(P<0.05,P<0.01),especially in FTY-H group.3.Immunohistochemical results showed that HMGB1 and TLR4 were mainly localized in cytoplasm,p NF-κ Bp65 is located in the nucleus.Compared with Sham group,HMGB1,TLR4 and p NF in CA1 region of hippocampus in MCAO/R group-κ The expression of Bp65 was significantly increased;Compared with MCAO/R group,the expression of HMGB1 and TLR4 in FTY-M group and FTY-H group decreased significantly,and the p NF in each dose group-κ The expression level of Bp65 decreased significantly(P<0.05,P<0.01).4.Immunoblotting results showed that compared with Sham group,HMGB1,TLR4 and p NF-κBp65 in MCAO/R group The expression of pathway protein increased significantly;Compared with MCAO/R group,HMGB1,TLR4 and p NF-κBp65 in FTY-M group and FTY-H group after treatment The expression of pathway protein decreased significantly(P<0.05,P<0.01).ConclusionFingolimod can improve the degree of brain injury in rats with focal cerebral ischemia and reperfusion injury,and has certain brain protective effect.Its mechanism may be related to reducing inflammatory reaction and inhibiting HMGB1/TLR4/NFκB pathway,high dose group has the best comprehensive effect. |
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