The Mechanism In The Protective Effect Of Adenovirus-mediated PPAR-γ1 Gene Transfection On Cerebral Ischemia-reperfusion Injury In Rats

OBJECTIVE:To explore the expression pattern of PPAR-γ1 mRNA and protein in the ischemia-reperfusion brain tissues, to analyze the relationship between PPAR-yl and cerebral ischemia based on the previous studies.METHODS:Adult male SD rats were subjected to middle cerebral artery occlusion 90min and followed reperfusion by monofilament method. The experimental groups included control group, sham group and ischemia-reperfusion group. The ischemia-reperfusion rats were sacrificed at 12h,24h and 48h after ischemia. The expression of PPAR-γ1, mRNA and protein were measured by RT-PCR and Western blotting respectively.RESULTS:The ischemia-reperfusion group displayed obvious downregulation of PPAR-γ1 mRNA and protein compared with the control group and the sham group. The level of PPAR-γ1 mRNA and protein might reach the lowest point in the 12h after ischemia onset, and increased gradually following the post-ischemic time.The expression of PPAR-γ1 mRNA and protein were still lower on 48h after ischemia onset.CONCLUSIONS:The ischemia-reperfusion injury downregulated PPAR-γ1 mRNA and protein expression. The PPAR-γ1 may be a valuable target and the experiment that make an intervention on it may be very important to improve the brain ischemia injury. OBJECTIVE:1.To explore the feasibility of PPAR-γ1 gene transfection via cerebral ventricle and the protective effect against cerebral ischemia reperfusion injury.2.To observe if the effection against cerebral ischemia reperfusion injury is enhanced when transfecting of PPAR-γ1 gene via cerebral ventricle association with the Pioglitazone which is PPAR-y's agonist.3.To explore the the expression pattern of IL-1β,ICAM-1,Bcl-2,Bax,MMP-9 and AQP-4 protein in the ischemia-reperfusion brain tissues and to investigate the potential mechanisms.METHODS:1.Construction of the replication-deficient recombinant adenovirus:Recombinant plasmids PDC-EGFP (containing enhanced green fluorescence protein EGFP gene), PDC-PPAR-γ1 (containing PPAR-γ1 gene). Recombinant adenovirus Adv-EGFP, Adv-PPAR-γ1 were produced by homologous recombinant in 293 cells, amplified also in 293 cells on a large scale, and purified by ultracentrifugation in CsCl step gradient solutions. The concentration of recombinant adenovirus Adv-EGFP purified by ultracentrifugation in CsCl step gradient solutions is 2.0×109Pfu/ml; that of Adv-PPAR-γ1 is 1.0×1011Pfu/ml.2.95 S-D rats were randomly divided into 6 groups.Each group was treated with corresponding agent via cerebral ventricle.In group I andⅡwere injected by normal saline.In groupⅢ,rat was injected with 0.1ml Adv-PPAR-γ1 and the groupⅣwas Adv-EGFP. The groupⅤwas intragastric administered with Pioglitazone. The groupⅥwas administered with PPAR-γ1 via cerebral ventricle and intragastric administered with Pioglitazone at the same time.3. After 3 days middle cerebral artery occlusion model was established.The groupⅠwas sham-operation group.In groupⅡ-Ⅵmiddle cerebral artery was obstructed for 90min and followed reperfusion for 24h.4.24 hours after operation sample was collected and cerebral infarction volume was measured by TTC staining, blood brain barrier permeability by Evan's blue dye Perfusion,brain moisture capacity by W-D weight method. To observe the pathomorphology by light microscope and electron microscope. Measuring the activity of MPO. To observe the expression of IL-1β,ICAM-1,Bcl-2,Bax,AQP-4 and MMP-9 protein by Western Blotting.RESULTS:After transfecting Adv-EGFP plasmid,the fluorescence for EGFP was positive in brain tissue suggesting the successful transfection of virus gene.In response to I/R injury, cerebral infarction was occurred,Blood brain barrier permeability,brain moisture capacity,the activity of MPO were dramatically incresed and the expression of IL-1β,ICAM-1,Bax,AQP-4,MMP-9 protein were upregulated while Bcl-2 was downregulated.After injected Adv-PPAR-γ1 via cerebral ventricle or intragastric administered with Pioglitazone could inhibited the above parameters of injured brain as well as upregulated the Bcl-2.The improvement by adv-PPAR-γ1 gene and Pioglitazone was enhanced by administration together.CONCLUSIONS:1.Transfection of PPAR-γ1 gene via cerebral ventricle was feasible and demostrated the protectiv effect against cerebral ischemia reperfusion injury.2. Combined transefction of PPAR-γ1 gene and intragastric administration with Pioglitazone could enhance the protectiv effect against cerebral ischemia reperfusion injury.3. The infiltrations of neutrophilic leukocytes could be reduced by PPAR-γ1 and the expreeion of IL-1β,ICAM-1,AQP-4,MMP-9 protein were downregulated, Bcl-2 was upregulated. These results suggests that the protective effect of PPAR-γ1 on ischemia-reperfusion injury is through regulating the inflammatory reaction and apoptosis.