| Part one: Regulation of Nrf2on the expression of inflammatory cytokinesafter fluid percussion brain injury in ratsObjective: To observe the expression of Nrf2and its regulatory effectwith inflammatory cytokines expression after fluid percussion brain injury inrats.Methods:56adult male SD rats, weighting250g-300g, were randomlydivided into2groups: Normal control group (NC group) and Traumatic braininjury group (TBI group). NC group (8rats) were given general anesthesia andcraniotomy without a shock. Animals in TBI group were made the models offluid percussion brain injury after given general anesthesia and craniotomy,and divided into6subgroups according to the time points of sacrifice such as1h,6h,12h,24h,3d and7d after fluid percussion brain injury, Each group ofwhich was composed of8rats.Neurological score were made when animals of NC group were given craniotomy, and before animals of TBI group were killed at according points, then animals of two groups were over-anesthesed with10%chloralhydrate and sacrificed. About100mg brain tissues of the front of the injury area were obtained to measure the brain water content by dry-wet weight method. Coronal slices of3mm thick were obtained in thebrain injury areas after brains of rats were taken out. After fixatived,dehydrated and embedded into paraffin, the examples were stained withHE for the observation of morphology and the expression of Nrf2andICAM-I which were detected by immunohistochemistry staining. The brain tissue behind the injury area was divided into three parts, one of which was used to detecte the expression of Nrf2protein by Western Blot,and the other one of which was to detect Nrf2mRNA expression by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and the lastwas used to detect IL-6,IL-1β and TNF-α protein expression by Enzyme Linked Immunosorbent Assay (ELISA).Results:1Neurological scoreCompared with those of the NC group, the neurological score in animalsof TBI group decreased at the6th hour after fluid percussion (2.50±0.53，P<0.05), and rose at the12th hour (3.63±0.52，P<0.05)after fluid percussion,but its was still lower than that of the NC group. The neurological score of the24th hour after fluid percussion became lower again (2.65±0.32，P<0.05),then gradually rosed. The score of the3rd and7rd day is still lower than theNC group (4.75±0.71，6.88±0.83,P <0.05).2Histological observationsNeurocytes edema was observed at6h after fluid percussion. Brainedema at12th hour after fluid percussion became more severe than before, andcellular edema was the most serious at24th after fluid percussion, when cellvolume increased significantly, cytoplasmic dye, and vacuolar degenerationwere observed, accompanied by the proliferation of glial cells. Edema beganto reduce at the3rd day and decreased significantly at the7th day, but theproliferation of glial cells gradually significantly inceased.3Brain water contentCompared with the NC group, Brain water content of the TBI group wasnot significant difference at1st hour after fluid percussion (74.87±2.31,P>0.05). At6th hour, its ascended slightly (80.23±1.11, P<0.05), and rosesignificantly higher at12th hour (81.91±0.83, P<0.05), then came up to itspeak at24th (84.01±0.99, P<0.05). Brain water content of the3rd day beganto decease（81.82±1.26，P<0.05）, and its becamed lower significantly at7thday after fluid percussion（80.65±1.32，P<0.05）.4ImmunohistochemistryNrf2protein mainly expressed in the nucleus of neuron and astrocytescell, were negative or weakly positive in normal brain tissue, After fluid percussion brain injury, its expression showed a trend of increase. Nrf2expression began to increase at1th hour（3.25±1.04,P<0.05）,At6-12h(4.50±0.93，5.00±1.07， P<0.05) after fluid percussion, gradually increased infollowing time, reached its peak at24h (5.75±0.71，P<0.05), but began todecease at3d.（5.00±1.07，P<0.05）. At7d（3.75±1.28, P<0.05）after fluidpercussion, Nrf2protein deceased significantly but still higher than that ofNC group.ICAM-1of TBI group began to increase at1th hour after fluid percussion（3.38±1.41,P<0.05）, gradually increased at6-12h (4.50±0.93,5.38±1.92，P<0.05), reached its peak at24h (7.25±1.39，P<0.05), but began to decease at3d（4.00±1.51，P<0.05）. At7d（3.75±1.04, P<0.05）after fluid percussionICAM-1deceased significantly but still higher than NC group.5Western BlotCompared with that of NC group, Nrf2protein in those of TBI groupincreased at1th hour(1.19±0.09), continued to rise at6th hour and12th hour(1.38±0.08,1.51±0.09, P<0.05), reached its peak at24th hour (1.59±0.08,P<0.05), decreased(1.41±0.09, P<0.05) at the3rd day, significantly deceased(1.24±0.10) at the7th day，but was still higher than that of NC group(P<0.05).6RT-PCRThere was no significant difference in Nrf2mRNA expression betweenanimals of TBI subgroups with those of NC group.7ELISACompared with that of NC group, IL-1β expression in those of TBI groupincreased at6th hour(41.72±2.13,P<0.05), continued to rise at12th hour(42.17±3.30, P<0.05), reached its peak at24th hour (44.22±3.97, P<0.05),decreased(41.71±3.09, P<0.05) at the3rd day, significantly deceased(39.94±2.98) at the7th day，but was still higher than that of NC group(P<0.05).Compared with that of NC group, IL-6expression of TBI groupincreased at6th hour(103.59±3.67, P<0.05), continued to rise at12th hour (109.25±5.68, P<0.05,), reached its peak at24th hour (120.21±10.00, P<0.05),decreased(112.60±6.71, P<0.05) at the3rd day, significantly deceased(101.25±5.00) at the7th day，but was still higher than that of NC group(P<0.05).Compared with that of NC group, TNF-α expression in those of TBIgroup increased at1th hour(36.61±4.01, P<0.05), continued to rise at6th hourand12th hour (38.98±1.07, P<0.05), reached its peak at24th hour(41.02±1.49, P<0.05), decreased(45.74±3.48, P<0.05) at the3rd day,significantly deceased (41.15±1.20) at the7th day，but was still higher thanthat of NC group (P<0.05).Conclusions:1Nrf2protein tends to increase in edemous brain after fluid percussionbrain injury in rats, but its mRNA show no change.The expression ofinflammatory cytokines tend to increase too.2Up-regulation of Nrf2protein may be a kind of defensive mechanism toresisit to secondary brain injury such as brain edema and inflammatorycytokines after fluid percussion brain injury in rats.3Up-regulation of Nrf2may be a new target in the treatment ofsecondary brain injury after TBI. Part two Effects of Curcumin activating Nrf2on inflammatory cytokinesexpression after fluid percussion brain injury in ratsObjective: To observe effects of Curcumin activating Nrf2oninflammatory cytokines expression after fluid percussion brain injury in rats.Methods:144adult male SD rats, weighting250g-350g, were randomlydivided into3groups: Traumatic brain injury group（TBI group）, Curcumin Igroup (CI group) and Curcumin II group(CII group), each group were dividedinto six subgroups which was made up with8rats according to1h,6h,12h, 24h,3d and7d after fluid percussion fluid percussion. Curcumin wasadministered peritoneal injection with the concentration of50mg/kg/d and100mg/kg/d respectively in CI group and CII group after fluid percussionbrain injury in rats.Neurological score were made before animals were killed at accordingpoints, then were over-anesthesed with10%chloralhydrate and sacrificed.About100mg brain tissues of the front of the injury area were obtained tomeasure the brain water content by dry-wet weight method. Coronal slices of3mm thick were obtained in the brain injury areas after brains of rats weretaken out. After fixatived, dehydrated and embedded into paraffin, theexamples were stained with HE for the observation of morphology and theexpression of Nrf2and ICAM-I which were detected byimmunohistochemistry staining. The brain tissue behind the injury area wasdivided into three parts, one of which was used to detecte the expression ofNRF2protein by Western Blot, and the other one of which was to detectNRF2mRNA expression by Reverse Transcription-Polymerase ChainReaction (RT-PCR), and the last was used to detect IL-6,IL-1β and TNF-αprotein expression by Enzyme Linked Immunosorbent Assay (ELISA)..Results:1Neurological scoreCompared with that of TBI group, the neurological function scores inanimals of CI group and CII group increased and was statistically significant(P<0.05) except at1h. There was statistical significance in neurological functionscores between animals of CI group and CII group at12h (4.875±0.835,5.125±0.835),24h (2.500±0.926,2.75±0.707),3d (6.125±0.996.38±0.744)(P <0.05), but was no statistical significance between TBI group, CIgroup and CII group at1h (9.000±0.00,8.375±0.744,8.500±0.535,8.625±0.518)(P>0.05).2Histological observationNeurons and neurogliocytes edema in CI group and CII group reduced ateach time point except1h compared with that of TBI group, and the range of neurons and neurogliocytes edema also reduced. Inflammatory cell invasions,,proliferation of neurogliocytes and the gap around neurons and neurogliocytesin CI group and CII group arrived at the lighter level than that in TBI group.The neurons and neurogliocytes of the CII group at each time point except1hat the low level compared with those of the CI group.3Brain water contentCompared with the TBI group, brain water content of CI group and CIIgroup were slightly lower at1h after shock(P>0.05), was significantly lower at6h(P<0.05), but there was no statistically significant difference between CIgroup and CII group (P>0.05). At12h, statistically significant difference(82.15±2.27,79.25±2.65,76.70±2.27) existed in brain water contentbetween three groups (P<0.05). At24h, brain water content of three groupsreached their highest (83.23±2.24,80.37±2.31,77.06±2.27), and havestatistically difference between them (P <0.05). At the3rd day, they began todecline and the same statistical differences existed among three groups (82.76±2.52,79.39±3.28,76.23±2.51, P <0.05). At7d, brain water contentbecame significantly lower in TBI group, CI group and CII group(80.38±1.89，76.92±2.73,76.13±1.92), but which of the later two group was stillslightly higher than that of TBI group (P<0.05), and there was not statisticallysignificant difference between CI group and CII group (P>0.05).4ImmunohistochemistryNrf2protein expression in TBI group, CI group and the CII group allshowed rising trend. Compared with that of TBI group, Nrf2protein in CIgroup and CII group was higher at12h （5.88±1.55，7.88±1.55，4.50±0.93）,24h（4.75±1.04，6.13±1.36，8.25±1.39） and3d（6.00±0.00，7.00±1.07，4.50±0.93）except at1h. There were statistically significancedifference between CI group and CII group at12h,24h,3d (P <0.05).C IIgroup were higher than C I group.Compared with that of TBI group, ICAM-1in CI group and the CIIgroup were lower There was statistically significant difference of ICAM-1expression between TBI group, CI group and CII group at the rest time except at1h（3.50±0.93，3.25±0.71，3.75±1.28）. Except at1h（3.25±0.71，3.50±0.93）,6h（3.88±0.99，4.25±0.89）,7d（1.88±0.83，2.13±0.99）,therewere statistically significance difference between CI group and CII group.5Western BlotCompared with that of TBI group, Nrf2protein expression in CI groupand CII group were higher, and there were statistically significant differencesbetween TBI group and CI group, TBI group and CII group at all time poitsexcept at1h. Nrf2protein expression in CII group were higher than that of CIgroup at12h(1.86±0.09，2.17±0.17),24h(2.17±0.10，2.76±0.10)and at3d（1.91±0.12，2.49±0.16）(P <0.05), had statistically significant differencebetween them.6ELISACompared with that of TBI group, IL-1β expression in CI group and CIIgroup was lower. There showed statistically significant difference in IL-1βexpression between TBI group and CI group, TBI group and CII groupexcept at1h（32.12±2.74，36.05±2.63，36.16±3.69，P>0.05). IL-1βexpression in CII group were lower than that of CI group at all points except at1h（32.12±2.74，36.05±2.63）,6h（32.20±2.53，37.94±3.28).Compared with that of TBI group, IL-6expression in CI group and CIIgroup was lower. There showed statistically significant difference in IL-1βexpression between TBI group and CI group, TBI group and CII group exceptat1h（86.88±6.09，90.06±4.14，92.27±1.53,P>0.05) and7d（86.51±7.86，94.03±9.33，95.24±3.30，P>0.05）. IL-6expression in CII group werelower than that of CI group at all points except at1h,6h and7d.Compared with that of TBI group, TNF-α expression in CI group and CIIgroup was lower. There showed statistically significant difference in TNF-αexpression between TBI group and CI group, TBI group and CII group exceptat1h（32.90±2.28，34.37±2.81，35.57±1.01，P>0.05) and7d（32.90±2.28，34.37±2.81，35.57±1.01，P>0.05). TNF-α expression in CII groupwere lower than that of CI group at all points except at1h and6h. Conclusions:1Curcumin can activate the expression of Nrf2protein, depress theexpression of inflammatory cytokines and then alleviate secondary braininjury with fluid percussion brain injury in rats.2There is amount-effect relationship between effect of Curcumin onNrf2activation. Within certain extent, there is greater effect on Nrf2with themore amount of Curcumin. |
PDF Full Text Request