| Objective: This investigate established a rat middle cerebral arteryocclusion model successfully, by observation the neurological functionscores in rats after focal cerebral ischemia-reperfusion injury; Infarctvolume percentage of brain tissue in each group was detected by TTCstaining；the water content of brain tissue in each group was detected bywet and dry weight method; SOD content of brain tissue in each group wasdetected by xanthine oxidase technique, Nrf2mRNA and proteinexpression of brain tissue in each group at different time points wasdetected by RT-PCR and Western blot., To explore SOD, Nrf2after focalcerebral ischemia-reperfusion changes of brain tissue with the time andpaeoniflorin may can reduce the oxidative damage in brain tissues of rats with middle cerebral artery occlusion caused by free radicals by improvingthe capacity of body eliminating free radicals through increasing thecontent of SOD content and increasing the expression of Nrf2.Methods: In this investigate, Totally130healthy adult maleSprague-Dawley rats which weighing250~300g were randomized intosham operation3h,6h,12h,24h,48h,72h groups,n=3; model3h,6h,12h,24h,48h,72h groups, n=9;experimental3h,6h,12h,24h,48h,72h groups, n=9; Model groups through suture method on the right middlecerebral artery occlusion model, preoperative fasting12h, drinking freely,ischemic reperfusion90minutes, the flowing referred to as the MCAOgroups; Sham operation groups are the same as the model groups withoutinserting a suture operations, the flowing referred to as the Sham groups;Preoperative administration group at48h,24h by intraperitoneal injectionof20mg.kg-1*3ml PF every time, then establish the MCAO model, theflowing referred to as MCAO+PF groups; Sham groups and MCAOgroups was injected with saline at the same time3ml abdominal cavity.After the packet is established model, each animal at the correspondingtime points neurological score by6points method, from MCAO groupsand MCAO+PF groups were randomly selected six rats decapitatedimmediately remove fresh brain tissue for TTC staining Determination ofbrain water content and dry and wet observation infarct volume percentageweight method. The left three rats methods using saline perfused ischemic cortex take the same brain tissue after cold mashed, part of the directground to obtain brain tissue extracts, taking centrifuged supernatant weremeasured by xanthine oxidase in brain tissue the content of SOD, anotherpart of the total RNA extraction reagent were added, the total proteinextraction reagent and then ground to obtain brain tissue extracts, takingcentrifuged supernatant RT-PCR, Western Blot assay Nrf2in brain tissue ineach mRNA, protein expression levels, the results obtained and then ImageLab software processing, collection points for each absorbance bands(integral absorption, IA), with a target gene (Nrf2) the IA value/referencegene (β-actin) of IA value to represent the relative expression of the targetfragment. Neurological score, infarct volume percentage of brain watercontent, SOD levels, the results of Nrf2expression were performed usingSPSS12.0statistical software for statistical analysis, analysis ofsingle-factor analysis of variance (one-way ANOVA) and SNK-q test forcomparison between multiple groups. Inspection standards take α=0.05.Results:1The neurological score: Sham group had no obvious neurologicalsymptoms defect; MCAO group and MCAO+PF group at each time pointcan have significant neurological deficit symptoms, scores were higherthan the Sham group, the difference was significant (P <0.05); MCAOgroup with time,24h peaked; MCAO+PF group compared with thesame time point MCAO group, significantly improved symptoms of neurological deficit, score somewhat lower, the difference was significant(P <0.05), explanatory experiment successful modeling and PF cansignificantly reduce neurological scores MCAO rats, reducing itsneurological score, mitigate the damage caused by cerebral ischemia, it isshowed that PF has a protective effect to cerebral ischemia.2The infarct volume measurement: TTC staining showed normal braintissue was red, white infarction. Sham brain tissue uniformly red dye noobvious infarction, MCAO group and MCAO+PF group can be seen alarge area of infarction, and more in the right basal ganglia, parietal andtemporal cortex, the difference was statistically (P <0.05), indicating thatthe experiment successful modeling; MCAO group was significantlyhigher percentage of infarct volume at3h,24h at the peak,48h began aslow decline; MCAO+PF compared with the same time point MCAOgroup, the percentage of infarct volume was significantly reduced,,described PF can significantly reduce infarct volume percentage, tomitigate the damage caused by cerebral ischemia (P <0.05), It is indicatedthat PF have a significant protective effect to cerebral ischemia.3The brain water content determination: rat brain tissue water contentno significant increase in Sham group, MCAO group were significantlyhigher than the Sham group,3h began to rise significantly,24h peaked,48h slow decline (P <0.05), indicating that the experiment successfulmodeling; MCAO+PF group had no significant changes in brain water content,6h was significantly higher compared with the beginning of theSham group at3h,48h and reached the peak,72h began to decline, andat the same time point MCAO group compared brain water content wassignificantly decreased (P <0.05). Description PF can significantly reducebrain water content, to mitigate the damage caused by cerebral ischemia,It is indicated that PF have a significant protective effect to cerebralischemia.4The SOD activity was measured: Sham group the highest SODactivity in brain tissue, and with time, did not change much; MCAOgroup of brain tissue SOD activity was significantly lower than the shamgroup, a significant decrease in3h begin until72h slowly recovery, thedifference was significant (P <0.05); MCAO+PF brain tissue SODactivity was no significant change in3h compared with Sham group,decreased significantly at6h firstly (P <0.05), and at the same time pointMCAO compare SOD activity increased in different time (P <0.05).Description PF significantly increased activity of SOD, mitigate thedamage caused by cerebral ischemia, cerebral ischemia have a significantprotective effect. it is also showed that the PF have a significantprotective effect to cerebral ischemia.5The Nrf2mRNA expression: Sham group Nrf2mRNA was expressedat low levels, and with time, no significant change; MCAO group beganto increase at3h,6h, and no significant difference in72h with Sham group,12h start significantly increased,48h peaked; MCAO+PF groupswere significantly higher than Sham group, and with the increase of time,the expression continues to rise; compared with MCAO group, Nrf2mRNA expression after giving varying paeoniflorin level increased (P<0.05). Description PF can increase the expression of Nrf2mRNA, andmitigate the damage caused by cerebral ischemia, cerebral ischemia have asignificant protective effect. cerebral ischemia have a significant protectiveeffect.6The Nrf2protein expression: Sham group Nrf2protein was expressedat low levels, and with time, no significant change; MCAO group had nosignificant difference in3h,6h was significantly higher,48h to reach thepeak,72h there Nrf2protein compared with MCAO group after givingpaeoniflorin (P <0.05); MCAO+PF groups were significantly higher thanthe Sham group, and with the increase of time, the expression continues torise expressed varying degrees increased, the difference was statisticallysignificant (P <0.05). Description PF can increase the expression of Nrf2protein and reduce the damage caused by cerebral ischemia, cerebralischemia have a significant protective effect. cerebral ischemia have asignificant protective effect.Conclusion: This investigate showed the changes of percentage ofinfarct volume, brain water content and SOD and Nrf2variation with timeand the PF can significantly improve symptoms of neurological impairment, reduced infarct volume percentage decline in brain water content, SODactivity was increased, sustained increase in Nrf2expression of focalcerebral ischemic injury have significant protection against oxidative stress,the mechanism may be to inhibit cell by increasing the activity of SOD andsignaling pathways regulated Nrf2/ARE calcium overload, anti-freeradicals, ischemia, cerebrovascular contraction induced by hypoxia Shudysfunction have a good improvement on the blood-brain barrier aftercerebral ischemia has a protective effect during perfusion, and maycontribute to early reperfusion recovery of cerebral blood flow. |
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