| BACKGROUD With the development of society, traumatic brain injury (TBI) has become one of the major causes of death or disailities among children and young adults. It is the main problem affecting the health in the current.15%-20%of those who died are in the age between5-35years old. The cause of the damage is mostly traffic accidents, followed by fall injury and fighting, and highway traffic accidents show an upward trend. There are10million people died in traffic accidents in china last year, and the mortality rate is the first place. Traumatic brain injury is the most common diseases in neurosurgery and has a high morbidity or motality. With the continuous progress of basic research on brain injury, many new methods, and new technologies, new theories have been applied to the clinical treatment of the work.Traumatic brain injury has only the primary injury at first, but after a few hours to a few days, there will be much secondary damage. The primary injuries mainly occurred in the external force at the same time, such as the scalp tear, skull fracture, and brain contusion and diffuse axonal injury; the secondary damage has more complex mechanisms of injury, including cerebral edema, oxidative stress injury, cell metabolic disorders, cerebral ischemia, brain swelling, inflammation, oxygen free radicals and the release of neurotransmitters. It shows a variety of pathological changes. In this paper, we will research the mechanisms of secondary injury.Cell metabolic disorders may be secondary to traumatic brain injury. Traumatic brain injury can cause glucose metabolism a short-term elevated at the beginning, then the brain tissue of sugar utilization begins to decline, following the accumulating of lactic acid in the cell and a series of pathological have changed, leading to cell poisoning, necrosis, apoptosis and cytotoxic brain edema. In this stress response process, ketone bodies become the main fuel of the brain tissue. Under normal circumstances, sugar is the material for brain tissue. However, inadequate or increased demand for energy, ketone bodies is the material for brain tissue to adapt to changes in cell metabolism. Different ages have different utilization of ketone bodies. The neuroprotective effect of ketone bodies is confirmed in neurodegenerative diseases, cerebral ischemia, epilepsy, traumatic brain injury and other pathological conditions. But there are few studies about the ketogenic diet after traumatic brain injury and the mechanism is unclear.NF-E2-related factor2(Nrf2) is the main regulator of a response to oxidative stress. It can induce a protective response gen, and activate the body’s own protective response. It is also able to withstand the broad-spctrumtoxic substances and pathogens on the cell injury. Under normal conditions, Nrf2is present in the cytoplasm together with Keapl and it has no biological activity. Nrf2separates from Keapl and transports to the nucleus when the cells subjected to stress, and then binding to the Antioxidant Response Element (ARE), starting the response to oxidative stress. The Nrf2-mediated cytoprotective responses have no cell-specific or organ-specific. It has been confirmed that Nrf2can protect multiple organs or tissues, such as lung, liver, gastrointestinal tract, nervous system and cardiovascular system. The protective effects to cerebral of Nrf2have been studied with traumatic brain injury model. Nrf2can activate the antioxidant response, reduce cerebral edema, anti-apoptosis and so on.AIMThere are no noticeable and effective ways to reduce the mortality rate of traumatic brain injury in clinical treatment at present. The ultimate goal of the treatment to traumatic brain injury is to reduce mortality and morbidity, improve patients’quality of life. To achieve this goal is not to rely on a single certain drugs or some kind of measures. It requires a comprehensive treatment. The omission of any link in the entire course of treatment may lead to poor final results. This experiment studies the activity of Nrf2and the level of HO-1in TBI model after feeding one week with the ketogenic diet, and to further clarify the mechanisms of the ketogenic diet in the neuroprotective effects after TBI. It may provide new ideas and theoretical basis for the treatment of TBI.METHODForty eight Sprague-Dawley rats with postnatal day (PND)35were randomly divided into four groups:normal animals with normal diet group (Sham+ND, n=12), normal animals with ketogenic diet group (Sham+KD, n=12), normal diet after traumatic brain injury (TBI+ND, n=12), ketogenic diet after traumatic brain injury (TBI+KD, n=12). Traumatic brain injury group rats were performed the right parietal bone drilling operation and applied the improved Feeney’s free-fall device to make a traumatic brain injury rat model. A hammer with40g weight falls free from10cm height. The other two sham operation groups were only performed the left parietal bone window without TBI. The two sham or TBI groups were treated with ketogenic diet and normal diet after24hours later. After feeding a week, the animals were killed by decapitation, and each animal was cut the5cm brain tissue around the lesion and stored at-80℃, for the detection of Nrf2activity and content, HO-1content and some for the detection of brain water content. 1. Measure the brain water contentEdema was determined using the wet/dry method as previously described where%brain water=[(wet weight-dry weight)/wet weight]×100%.2. Electrophoretic mobility shift assay (EMSA) for Nrf2activity.In brief,1.75%pmol/μl Nrf2consensus obigonucleotide probe (5’-TTTTATGC TGTGTCATGGTT-3’and5’-AACCATGACACAGCATAAAA-3’) was end-labeled with [y-32P]-ATP and T4-polynucleotide kinase. Nuclear protein (60μg) was added to7μl with freewater, and then incubated in2μl binding buffer and the32p-labeled oligonucleotide probe1μl. This incubation continued for20min at4℃. The mixture was subjected to non-denaturing4%polyacrylamide gel electrophoresis in a TBE buffer, constant voltage of120v for two hours. The gel was vacuum-dried and exposed to x-ray film at-80℃till an intensifying screen. Using the Image J analysis software measure the average gray value.3. Western blot analysis for Nrf2and HO-1protein content100mg specimens were grinded and then extracted nuclear protein and cytoplasmic protein. The protein concentration was determined using Bradford method. The protein was sampled to the prepared polyacrylamide gel after boiled for three minutes, then transferred to the PVDF membrane, added to3%blocking buffer, rocked gently for at least1hour, pour off3%blocking buffer and rinse briefly with TBS buffer three times, add first antibody at appropriate dilution in10ml0.5%blocking buffer, rock gently for at least1hour, pour off first antibody solution from membrane and wash for three time with TTBS buffer, add second antibody at appropriate dilution in5ml0.5%blocking buffer, rock gently for30minutes, pour off second antibody and wash three times, add developing reagent. The results were analyzed using Image J.4. RT-PCR detection for HO-1mRNA expression Incubate the homogenized samples for5minutes to permit the complete dissociation of nucleoprotein complexes, add0.2ml of chloroform per1ml Trizol, shake tubes vigorously by hand for15seconds and incubate them at15to30℃for2to15minutes, centrifuge the samples at no more than12,000×g for15minutes, the mixture separates, and get the colorless upper aqueous phase, transfer the aqueous phase to a clean tube, add0.5ml of isopropyl alcohol, incubate samples at15to30℃for10minutes and centrifuge at no more than12,000×g for10minutes, the RNA precipitate on the side and the bottom of the tube, wash the RNA pellet with1ml of75%ethanol, at the end of the procedure, briefly dry the RNA pellet. The RNA transcribed into cDNA by reverse transcription kit, add primers and the polymerase, put into PCR instrument and start reaction, reaction products added in agarose gel, at last place the gel in imager, using Image J to analysis the results.5. Statistical analysisAll data were presented as mean±S.D. SPSS13.0was used for statistical analysis of the data. Groups were compared using ANOVA analysis, homogeneity of variance using LSD test at the same time, when heterogeneity of variance with Tamhane T2test. Statistically significance was inferred at P<0.05.RESULTS1. Changes in brain water content:The ketogenic diet group of the brain edema was significantly higher than the normal diet group,(79.55±0.41)%and (81.15±1.16)%at one week after brain injury, using single factor analysis of variance, the difference was statistically significant(P=0.001).2. Changes of the activity of Nrf2in the nucleus:Normal rat were fed with normal diet and ketogenic diet for one week, the Nrf2activity was higher in ketogenic diet group compared with the normal diet group(P<0.001); it was also higher after traumatic brain injury(P<0.001). 3. Brain tissue content of Nrf2protein and HO-1protein:Rats in each group were treated with ketogenic diet and normal diet for one week, the Nrf2content in nucleus of the two ketogenic diet groups were higher than the other two normal diet groups, and the content of HO-1protein was also increased(P<0.001,P=0.002).4. Brain tissue changes in HO-1mRNA:Four groups of rats were given one week of the ketogenic diet or normal diet. The HO-1mRNA expression of the two ketogenic diet groups were higher compared with the other two normal diet groups(P=0.001, P<0.001).CONCLUSION Oxidative stress injury is the important reason for the secondary brain damage after traumatic brain injury. So it plays an important role to against oxidative stress in neuroprotective effects. The ketogenic diet can reduce cerebral edema; inhibit apotosis, anti-oxidation and so on. Our results suggest that the ketogenic diet can activate the Nrf2pathway, and then transported into the nucleus, followed by the expression of the downstream antioxidant protein HO-1. Therefor, we suggest that ketogenic diet plays a significant neuroprotective effect by activating the Nrf2pathway after traumatic brain injury. |
PDF Full Text Request