| Section one The role of Nrf2during fluid percussion brain injuryresistance of neural cell apoptosisObjective: To observe the expression of Nrf2, the neural cell apoptosisand the change of its related factors after fluid percussion brain injury in rats,then to discuss the role of Nrf2in nerve cell apoptosis after fluid percussionbrain injury.Methods:56adult male SD rats, weighting300g±50g, were randomlydivided into2groups: Normal control group (NC group) and Traumatic braininjury group (TBI group). Traumatic brain injury (TBI group) were dividedinto6subgroups according to the time points of sacrifice such as1h,6h,12h,24h,3d and7d after fluid percussion. Each group included8rats. TBI groupanimal were given general anesthesia and craniotomy, and then establishedinto the models of fluid percussion brain injury. NC group were given generalanesthesia and craniotomy without fluid percussion.NC group after shock24h were made neurological score at correspondingpoints and those of TBI group were made neurological score at correspondingpoints, animals of two groups were over-anesthesed with10%chloralhydrateand decapitated. Animal brains were taken out and about100mg brain tissuesand taken from the front of the injury area. They were measured the watercontent by dry-wet weight method. And the brain injury area were cut intoapproximately3mm thick coronal slices, the examples were stained with HEfor the observation of morphology and the expression Nrf2, caspase-3andcaspase-12were detected by immunohistochemistry staining, and detected thechanges of neuronal apoptosis by TUNEL. The brain tissue behind the injuryarea was divided into two parts, one of which was used to detect theexpression of Nrf2nucleoprotein, Nrf2cytoplasm protein, caspase-3protein and caspase-12protein by Western Blot, and the other of which was to detectNrf2mRNA expression by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Results:1Neurological scoreCompared with the NC group, the neurological score of the TBI groupdecreased at the6th hour after shock (2.750±0.707，P<0.05)and showedslightly higher at the12th hour after shock(4.50±0.756, P<0.05), but it waslower than that of the NC group. The neurological score reduced again at the24th hour after shock (2.250±0.707, P<0.05), then gradely increased. Until theseventh day the score was still lower than the NC group (7.750±0.707,P<0.05).2Histological observationsCellular edema was observed at6h after shock. Brain edema becamemuch severe at12th hour after shock than before, and cellular edema was themost obvious visible at24th after shock, when significantly increased cellvolume, cytoplasm dye, and vacuolar degeneration were observed,accompanied by the proliferation of glial cells. Edema began to reduce at the3rd day and significantly decreased at the7th day, but the proliferation of glialcells gradually increased significantly.3Brain water contentThere was not significant difference at1st hour (P>0.05) between TBIgroup and NC group. At6th hour, brain water content ascended slightly(78.937±0.125, P<0.05), and rose significantly higher at12th hour(81.801±0.601, P<0.05), then came up to its peak at24th hour (83.234±0.321,P<0.05). Brain water content began to decease at the3rd day (82.143±0.304,P<0.05) and became lower significantly at7th day (81.290±0.305, P<0.05).4Detected Nrf2, Caspase-3and Caspase-12by ImmunohistochemistryThe expression of Nrf2: A little Nrf2expressed in the NC group, whichmainly expressed in the cytoplasm of neurons and glial cells. Compared withthat of NC group, Nrf2of TBI group increased significantly in injury side of the cortex. Nrf2began to increase at1hour (0.307±0.023, P<0.05), andreached its peak at24th hour (1.288±0.015, P<0.05), then deceased graduallyThe expression of caspase-3: The number of positive cells, that caspase-3was detected in, began to increase in brain tissue of TBI Group in6hour(0.560±0.014, P<0.05). The number of positive cells increased significantlyhigher at12th hour (0.795±0.019，P<0.05), then achieved the highest at24thhour (2.047±0.022, P<0.05), and maintained at a high level until the3rd day(1.757±0.023, P<0.05). The number began to decease at the7th day(0.920±0.031, P<0.05), but it was still higher than the NC group.The expression of caspase-12: The number of positive cells, thatcaspase-12was detected in, began to increase in brain tissue of TBI Group in6hour (0.585±0.021, P<0.05). The number of positive cells increasedsignificantly higher at12th hour (0.878±0.019, P<0.05) and achieved thehighest at24th hour (1.989±0.015, P<0.05), then began to decease.5Detected Nrf2nucleoprotein, Nrf2cytoplasm protein, caspase-3protein andcaspase-12protein by Western BlotThe nucleoprotein of Nrf2expression in the TBI group, compared withthat of NC group, increased at1hour (0.563±0.026, P<0.05), and continued toincrease at6th hour (0.814±0.039, P<0.05),12th hour (1.043±0.039, P<0.05),reached a peak at24th hour (1.408±0.031, P<0.05), decreased (1.111±0.045,P<0.05) at the3rd day, significantly deceased (0.803±0.027) at the7th day,but was still higher than that of NC group (P<0.05).Nrf2cytoplasm protein expression each point in time in TBI group hasno significant difference compared with that of NC group (P>0.05).Compared with that of NC group, caspase-3expression in TBI groupincreased at the6th hour (0.801±0.029, P<0.05), and continued to rise at the12h (1.142±0.029, P<0.05), reached a peak at the24th hour after injury(1.413±0.042, P<0.05), decreased at the3rd day (1.151±0.100, P<0.05), andsignificantly deceased (0.834±0.022) at the7th day, but was still higher thanthat of NC group (P<0.05).Compared with that of NC group, caspase-3expression in TBI group increased at the6th hour (0.801±0.029, P<0.05), and continued to rise at the12h (1.098±0.051, P<0.05), reached a peak at the24th hour after injury(1.382±0.110, P<0.05), decreased at the3rd day (0.814±0.041, P<0.05), andsignificantly deceased at the7th day(0.567±0.017), but was still higher thanthat of NC group (P<0.05).6Detected the expression of Nrf2mRNA by RT-PCRThere was a little Nrf2mRNA expression in NC group. There was noobvious change in each time point after injury about Nrf2mRNA expressionand compared with NC group it had no significant difference (P>0.05).7Measure apoptotic cells by TUNELThe positive cells which stained by TUNEL were found in the two groupsof postoperative time points. There was a small number of positive cells in NCgroup. The Apoptotic cells began to increase at the6th hour (27.385±0.158,P<0.05) after injury in TBI group, then continue to increase at the12h(42.366±0.303, P<0.05), reached a peak at the24th hour (55.222±0.124,P<0.05), decreased at the3rd day (48.207±0.149, P<0.05), and significantlydeceased at the7th day (22.226±0.273, P<0.05), but was still higher than thatof NC group (P<0.05).8The correlation analysis in the nerve cell apoptosis, Nrf2, caspase-3andcaspase-12.Caspase-3and12expression were positively correlated with nerve cell apoptosis in TBI group,(rcaspase-3=0.871, rcaspase-12=0.916, P<0.01).Caspase-3and12expression were positively correlated with Nrf2nucleoprotein expression in TBI group,(rcaspase-3=0.918, rcaspase-12=0.961, P<0.01).Conclusions:1Nrf2protein, nerve cells apoptosis, caspase-3and caspase-12are highexpression after fluid percussion brain injury.2Nrf2is activated after TBI, probably by inhibiting the caspase-3andcaspase-12expression, and play a role of nerve protective by inhibiting cellapoptosis. Section two: Curcumin influence of Nrf2on Resistance to apoptosis ofnerve cells during fluid percussion brain injuryObjective: To observe the impact of different doses of curcumin on fluidpercussion brain injury Nrf2and nerve cell apoptosis, and to explore itsmechanism.Methods:144health adult male SD rats, weighting300g±50g, wererandomly divided into three groups: Traumatic brain injury group (TBI group),Low doses of curcumin group (LC group) and High doses of curcumin group(HC group).The TBI group, LC group and HC group were divided into sixsubgroups according to1h,6h,12h,24h,3d and7d after shock. Eachsubgroup was made up with8rats. LC and HC group after establishingmodels were injected curcumin by abdominal cavity. The concentration wererespectively50㎎/㎏and100㎎/㎏.The TBI groups, LC group and HC group were respectively acceptedfluid percussion. According to the corresponding time points after shock,neurological score were valued. All animals were over-anesthesed by10%chloralhydrate and decapitated. Animal brains were taken out and about100mg brain tissues and taken from the front of the injury area. They weremeasured the water content by dry-wet weight method. And the brain injuryarea were cut into approximately3mm thick coronal slices, the examples werestained with HE for the observation of morphology and the expression Nrf2,caspase-3and caspase-12were detected by immunohistochemistry staining,and detected the changes of neuronal apoptosis by TUNEL. The brain tissuebehind the injury area was divided into two parts, one of which was used todetect the expression of Nrf2nucleoprotein, Nrf2cytoplasm protein, caspase-3protein and caspase-12protein by Western Blot, and the other of which was todetect Nrf2mRNA expression by Reverse Transcription-Polymerase ChainReaction (RT-PCR). Results:1Neurological function scoresCompared with that of TBI group, the neurological function scores of LCgroup and HC group at all points increased and was statistically significantbetween them (P<0.05). Between LC group and HC group, only at12h(4.750±0.886,4.875±0.835),24h (2.500±0.926,2.875±0.991),3d(6.125±1.126,6.375±1.685), the difference of neurological function scoreswas statistically significance (P<0.05).2Histological observationsCompared with TBI group, the degree of brain edema in LC group andHC group reduced at each time point, and the range of brain edema reducedtoo. Inflammatory cell exudation, proliferation of glial cells and the gaparound neurons in LC group and HC group arrived at the lighter state than thatin TBI group. The nerve cells of the HC group at each time point becamemilder edema than those of the LC group.3Brain water contentCompared with TBI group, brain water content of LC group and HCgroup were indiscrimination at the1h (78.204±0.318,78.154±0.244,78.105±0.263) after shock(P>0.05), and was significantly lower at the6h(78.937±0.125,78.553±0.179,78.121±0.327). The difference of brain watercontent between them was statistically significant (P<0.05) but had nodifference between the LC group and HC group (P>0.05). At the12h, brainwater content in three groups (81.801±0.601,80.763±0.159,79.449±0.798)were statistically different (P<0.05). At the24h, the brain water content inthree groups reached the highest (83.234±0.321,82.218±0.257,80.945±0.084),and was statistically different (P<0.05). At the3rd day, they began to decline,and the same statistical differences existed in three groups (82.143±0.304,81.178±0.181,79.939±0.025, P<0.05). At the7th day, brain water contentbecame significantly lower (81.290±0.305,80.344±0.158,79.702±0.194) inthree groups, but the LC group and HC group was still slightly higher than thatof TBI group (P<0.05).The difference between the LC group and the HC group was not statistically significant (P>0.05).4Detected Nrf2, Caspase-3and Caspase-12by ImmunohistochemistryCompared with that of TBI group, Nrf2protein expression in LC groupand HC group was statistically significant (P<0.05) at all points,12h(0.742±0.074,1.198±0.151,0.581±0.061),24h (1.532±0.052,1.278±0.051,2.058±0.045),3d (1.301±0.025,1.102±0.071,1.980±0.214, P<0.05). Therewere statistically significance difference between LC group and HC groupeach point in time (P<0.05), the HC group is significantly higher than LCgroup.Compared with the TBI group, the expression of caspase-3protein, inaddition to the1h(0.353±0.026，0.350±0.028，0.347±0.021，P>0.05) werelower at other point in time LC and HC group. At12h (0.565±0.025,0.423±0.025,0.802±0.029),24h (1.597±0.165,1.202±0.306,1.997±0.051),3d(1.285±0.023,1.040±0.031,1.801±0.034) there were most obvious (P<0.05).The HC group is significantly lower than LC group.Compared with the TBI group, the expression of caspase-12protein, inaddition to the1h (0.451±0.033,0.432±0.106,0.443±0.102, P>0.05) werelower at other point in time LC and HC group. At12h (0.881±0.026,0.671±0.034,0.585±0.106),24h (1.990±0.045,1.548±0.036,1.058±0.027),3d(1.779±0.037,1.333±0.026,0.938±0.027) there were most obvious (P<0.05).The HC group is significantly lower than LC group.5Detected Nrf2nucleoprotein, Nrf2cytoplasm protein, caspase-3protein andcaspase-12protein by Western BlotNrf2nucleoprotein express in three groups at each time point weresignificant differences, and at12h (1.056±0.027,1.173±0.018,1.287±0.018),24h (1.415±0.041,1.619±0.029,1.840±0.028),3d (1.087±0.038,1.257±0.196,1.427±0.021) were more obvious (P<0.05). The HC group is higher than LCgroup and TBI group (P<0.05).Nrf2cytoplasm protein express in three groups at each time point wereno significant differences (P>0.05).Caspase-3protein expression among the three groups in addition to1h (0.557±0.031,0.555±0.024,0.547±0.102) had no obvious difference (P<0.05),but at6h(1.242±0.039,1.139±0.103,1.033±0.005) had difference. At12h(1.662±0.121,1.385±0.028,1.237±0.018),24h (2.113±0.041,1.625±0.029,1.269±0.028,3d (2.017±0.038,1.625±0.196,1.251±0.021) there were moreobvious (P<0.05). The TBI group is higher than LC group and HC group andLC group is higher than the HC group. The differences were statisticallysignificant (P<0.05).Caspase-12protein expression among the three groups in addition to1h(0.563±0.027,0.536±0.120,0.543±0.024) had no obvious difference (P<0.05),but at6h (1.024±0.101,0.823±0.054,0.648±0.051) had difference, At12h(1.180±0.042,1.037±0.161,0.931±0.005),24h (1.359±0.045,1.225±0.024,1.147±0.057),3d (1.276±0.101,1.015±0.101,0.937±0.103) there were moreobvious (P<0.05). The TBI group is higher than LC group and HC group andLC group is higher than the HC group. The differences were statisticallysignificant (P<0.05).6Detected the expression of Nrf2mRNA by RT-PCRNrf2mRNA expression has no obvious difference compared with TBIgroup, LC group and HC group at each point in time (P>0.05).7Measure apoptotic cells by TUNELApoptosis results showed that LC and HC group in addition to1h(11.611±0.075,11.571±0.345,11.308±0.155) had no obvious differencecompared with TBI group(P<0.05). At12h (42.310±0.024,39.658±0.241,36.376±1.024),24h (54.149±0.126,52.391±0.241,47.945±0.255),3d(48.307±0.260,45.319±0.132,42.486±0.321) there were more obvious(P<0.05). LC group is higher than HC group (P<0.05).Conclusion:1The early application of curcumin can activate Nrf2high expression,play nerve protective effect by inhibiting cell apoptosis after fluid percussionbrain injury. 2High doses of curcumin (100㎎/kg) intervention on hydraulic impactbrain injury of nerve protective effect is more apparent. |
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