| BACKGROUD:Traumatic brain injury (TBI) is a serious and common disease of central nervous system, and it happened at high incidence rate, morbidity, mortality, leading to a serious threat to human health. TBI can be divided into primary brain injury and secondary brain injury, and prognosis of patients is closely related to the secondary brain injury. The present study found that the mechanism of secondary brain injury mainly includes the damage of mitochondrial function, apoptosis, oxidative stress, inflammatory reaction, Calcium overload and glutamate excitotoxicity. It is increasing paid much attention to the mechanism of the damage of mitochondrial function and cell apoptosis after TBI. How to reduce the mitochondrial injury and inhibition of neural apoptosis after TBI has become an important method for the treatment of TBI. In recent years, the basic and clinical research on TBI has made great progress, but the treatment of TBI is still a lack of effective protective drugs in clinical medicine at present, and the study of neural protection following TBI is still no breakthrough. The main reason may be the metabolic status of the related molecules and related regulation mechanism is not yet clear after TBI. Therefore, positive further explorethe regulation mechanism related signaling molecules and signaling pathways, and to seek the therapeutic targets and drugs have become a hot and difficult research on TBI.Apoptosis also known as programmed cell death (PCD), refers to the body cells in the face of internal and external environment-related factors stimulation, by the start of gene regulation, and suicide protection process through multiple pathways of signaling, which involves induction of activation and genetic programming of a series of molecular mechanisms. The apoptosis signal pathway mainly includes:the mitochondrial pathway, death receptor pathway and endoplasmic reticulum pathway. And the mitochondrial apoptotic pathway is the main pathway of apoptosis. Mitochondria are important energy producing organelles within cells. Its main function is to generate energy result of the oxidation of organic compounds, and then convert into ATP, which can supply energy for the body. When the body suffered from the influence of stimulation, the mitochondria would change their inner membrane permeabilitybe through the related molecular signal regulation, causing mitochondrial membrane potential change and even dissipation, and then to adjust and control of programmed cell death. In addition, when the body is subjected to the stimulation of the adverse factors, will also produce a lot of high activematerial such as active oxygen free radicals increased. When the relative excess the amount generated beyond the antioxidative capacity, will lead to increase lipid peroxidation level. And then may cause the protein level expression anomaly and DNA oxidative damage damage, and lead to the body tissue damage eventually. The main ingredients of mitochondria including mitochondrial protein, lipids, nucleic acids and many enzymes, so mitochondrial equally susceptible to free radical attack, causing the damage of mitochondrial function.Alpha lipoic acid (ALA) is widely distributed in nature, which belongs to the vitamin B compounds, now considered as a astrong natural antioxidants. Lipoic acid is the keycoenzyme of mitochondrial pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, can improve mitochondrial function, enhance the oxidative phosphorylation, eliminate free radical, chelate metal ion, regenerate of endogenous antioxidants in the body and reduce cell apoptosis. Some studies also show that, ALA has strong protective effects on ischemic reperfusion injury of heart, brain and kidney, neurological complications of diabetes, neurodegenerative disease of the central nervous system and so on. Due to the antioxidant effects of ALA can reduce the injury of free radicals and reactive oxygen species on peripheral nerve and blood vessel has been used in the treatment of diabetic peripheral neuropathy, and obtained the certain effect. At home and abroad, however, research into how the ALA has effects against brain damage following a TBI is limited.The molecular mechanisms underlying these effects are not well understood.In this experiment, to investigate whether the ALA has a protective effect on mitochondria via its antioxidant effects by the establishment of TBI model in rats, which play a role in inhibiting apoptosis of neural cells, and provide protection of TBI. It will also provide new ideas and experimental data for future research and clinical treatment of TBI.Objective:By observing the effect of ALA on neurological function and brain edema in rats TBI model, and the brain tissue Malonaldehyde (MDA) content and Glutathione pweoxidase (GPx) activity were determined, western blot were detected in cytochrome c and Bcl-2 associated X protein (Bcl-2 Associated X Protein, Bax) expression in mitochondria and the cytosol of brain tissue, the expression of Caspase-3 was evaluated by immunohistochemistry and western blot. At the same time, the neurons survival and the apoptosis of neural cells in cortical area were detected by Nissl staining and the terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) assay, respectively. And then explore the possible mechanism of ALA in inhibiting the apoptosis of neural cells after TBI, and provide a theoretical basis and experimental evidence for future study of ALA treatment of TBI. It will provide new ideas and experimental data for future TBI research and clinical treatment.Methods:1. Experimental animals and groupsSelect 150 healthy adult Sprague-Dawley (SD) male rats, weight 250～280g, were provided by Nanjing General Hospital of Nanjing Military Area Command Department of comparative medicine. According to the random number table method, rats were randomly divided into 5 groups:Sham+vehicle, Sham+ALA (100 mg/kg), TBI+vehicle, TBI+ALA (20 mg/kg) and TBI+ALA (100 mg/kg). A total of 12 animals per group were used for western blotting; 6 animals per group were used for the analysis of brain water content and neurological function; 6 animals per group were used for biochemical analyses; and 6 animals per group were used for immunohistochemistry, Nissl staining, and the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end labeling (TUNEL) assay.2. Rat TBI modelThe TBI model used in this study is made according to the modified Feeney method. Rats were anesthetized, and then opened a small window of skull trauma group in the left parietal bone of rat head, TBI was induced by a 40 g weight dropped from a height of 25 cm along a stainless steel string. Sham rats were subjected to identical treatment but without injury. The whole process is carried out under sterile conditions, and pay attention to maintaining the integrity of the dura mater.3. Drug administrationALA was dissolved in the vehicle solution (10% dimethyl sulfoxide in corn oil), and administered by oral gavage once daily for 2 days starting 30 min after the induction of TBI. Animals in the sham+ALA group received ALA and those in the sham+ vehicle and TBI+vehicle groups received equivalent volumes of vehicle at the same time points.4. Sampling preparationAnimals were sacrificed 48 h after the surgery.The rats were anesthetized, the left ventricular infusion of isotonic saline (4℃),then the brain was collected after liver pale. The brain tissue around the injury focus of 3mm placed at-80℃ refrigerator, use for the detection of Western blot and MDA and GPx. Among them the heart perfusion with 6 rats in each group of 4℃ isotonic saline, by the same way perfusion of 4% paraformaldehyde again, then the brain was collected, fixed in 4% paraformaldehyde, and then embedded in paraffin, sliced, for the detection of immune tissue chemical detection group, Nissl staining and cells apoptosis of TUNEL.5. Neurological evaluationNeurological evaluation was performed by the balance beam experiment.Neurological function was evaluated 24 and 48 h successively in the TBI. The lower scores indicate more serious nerve function defect.6. Brain water contentThe brain water content was measured after the neurological function test 48 h after TBI, animals were sacrificed and their brains quickly dissected. After removal of the brain stem and cerebellum, the right and left cortical tissue was harvested. Each sample was immediately weighed to determine the wet weight, then dried for 72 h at 80℃ and weighed to obtain the dry weight. The brain water content was calculated with the formula:[(wet weight-dry weight)/wet weight] ×100%.7. Determination of malondiadehyde (MDA) content and glutathione peroxidase (GPx) activityMDA content and GPx activity were determined using commercial kits, according to the manufacturer’s instructions and measurements were made using a spectrophotometer. Total protein concentration was determined by the Bradford method. MDA level and GPx activity are expressed as nmol/mg protein and U/mg protein, respectively.8. Western blottingMitochondrial and cytosolic proteins were extracted from the fresh tissue samples(4℃) using the Tissue Mitochondria Isolation Kit for Tissue Protocols.The protein content of each sample was determined using a protein assay kit under the condition at 4℃. The extracted brain tissue proteinswere quantified. Take the right amount of extracted proteins, according to the volume ratio of 4/1 to add loading buffer,100℃ water bath for 10min. Then for Western blot analysis. equal amounts of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10%-12%SDS polyacrylamide gel electrophoresis (90V), then transferred to polyvinylidene difluoride (PVDF) membranes (100V,90min), which were blocked with nonfat dry milk buffer for 2 h and then incubated overnight at 4℃ with primary antibodies against cleaved caspase-3, Bax, cytochrome c, COX Ⅳ and β-actin. The membranes were washed three times in Tris-buffered saline with Triton X-100 (TBST) for 15 min and then incubated with HRP-conjugated secondary antibodies, for 2 h at room temperature. After three 15-min washes with TBST, protein signals were detected on X-ray film in the dark room with chemiluminescence liquid (ECL)and quantified using UN-SCAN-IT6.1 image analysis software to measure the average gray obtained target protein bands of color positive area values, and statistical analysis.9. Caspase-3 expression was evaluated by immunohistochemistry (IHC)With foci of injury asthecenter, brain tissue soaked in 4% paraformaldehyde was embedded in paraffin, and thenserial sections, slice thickness of 5 μm. The sectionswere deparaffinized, hydrated, antigen retrieval, blocking with 10%goat seruml h (37℃),then incubated with a rabbit monoclonal anti-caspase-3 antibody (dilution 1:300) overnight at 4℃. Followed by three 15-min washes in PBS, then blocked with 1.6% H2O2 in PBS for 10 min.followed by three 15-min washes in PBS and incubation with horseradish peroxidase (HRP)-conjugated IgG (1:500) for 60 min at room temperature. DAB was used as chromogen, and then rinsed with tap water, then with hematoxylin counterstain, thendehydrated, transparentize, and finally mounted. Under a microscope (×400), each slice randomly selected six views and positive cells were counted. The mean number of the positive cells in six views was finally used forstatistical analysis (expressed as per 100 cells in the number of positive cells).10. Nissl stainingThe paraffin sections of brain tissue (5 m) were stained in strict accordance with the method of Nissl staining solution. In the view of high magnification (X 400), each slice randomly selected six views and positive cells were counted. The mean number of the positive cells in six views was finally used for statistical analysis (the survival rate was expressed as per 100 cells in the number of neuronal survival cells).11. TUNEL assayParaffin-embedded brain tissue sections (5 μm thick) were assessed for apoptotic cells using a TUNEL detection kit. In the same field at high magnification (X 400)under, each section of 6 randomly selected view count of TUNEL positive cells and calculated the average value (positive rate based on the number of TUNEL positive cells per 100 cells), and then the positive rate used for statistics analysis.12. Statistical analysisThe data were analyzed using SPSS 16.0 statistical software, all the data are expressed as the mean ±SEM. Statistical significance was analyzed by one-way analysis for multiple groups analysis, But the neurobehavioral scores using non parameter Mann-Whitney test. Statistical significance was inferred at P<0.05.Results:1. General observationIn this experiment, rats were from the same batch, there was no significantly difference between experimental groups in body weight of rats. The production of TBI model and the operation steps in the process of drug or vehicle administration are same in each rat. The rats in each experimental group survived.There was no significant difference between the sham+vehicle group and sham+ALA (100 mg/kg) group (P<0.05). In this experiment, compared with the dose of 20mg/kg, the dose of 100mg/kg of ALA was given to the rats after TBI was better.2. ALA improves neurological function and reduces cerebral edema after TBINeurological function was tested by beam-walking performance 24 and 48 h after TBI. Within 48 h of injury, the neurological score was lower in the TBI+vehicle group than in the sham+vehicle or the sham+ALA groups (P< 0.001). Although lower than those of the sham+vehicle and sham+ALA groups, the neurological score was significantly improved by ALA treatment relative to the TBI+vehicle group (P< 0.01), with a dose of 100 mg/kg showing the greatest effect (P< 0.01). The brain water content measured 48 h after brain injury was increased in the TBI+vehicle group as compared to the sham+vehicle or sham+ALA groups (P< 0.001). Rats treated with ALA showed reduced brain edema compared to the TBI+vehicle group (P< 0.05), with the greatest effect observed with a dose of 100 mg/kg.3. ALA reduces oxidative stress following TBIMDA level and GPx activity-indicators of lipid peroxidation and antioxidant activity, respectively-were assessed. MDA level was increased in the TBI+vehicle group relative to sham-operated controls (P< 0.01); this effect was mitigated by the administration of 100 mg/kg ALA (P< 0.01 vs. TBI+vehicle). GPx is an antioxidant enzyme responsible for scavenging metabolites generated by free radicals; its activity was decreased after TBI (P< 0.01), while ALA treatment increased GPx activity at a dose of 100 mg/kg (P< 0.05 vs. TBI+vehicle).4. Neural apoptosis is suppressed by ALA following TBITo investigate the protective effects of ALA, neuronmorphology was examined by Nissl staining.In TBI+vehicle group can see the survival of neurons decreased significantly (P< 0.01), whereas in ALA treatment group the neuron survival rate compared with TBI+vehicle group were increased (P< 0.01).To investigate the mechanistic basis for the effects of ALA, TUNEL staining was used to examine neural cell in injured brain tissue. TUNEL-positive cells were detected at a low frequency in the brains of rats in the sham+vehicle and sham+ ALA groups. The apoptotic index was increased in the TBI+vehicle group as compared to sham animals (P< 0.001), but was reduced in the TBI+ALA group (P< 0.01 vs. TBI+ vehicle).These results indicate that ALA blocks neural apoptosis induced by TBI.5. ALA treatment resulted in a downregulation of Caspase-3 expression after TBIThe Caspase-3 expression was examined by immunohistochemistry and western blotting of rat brain tissue, compared with the sham groups, the higher expression of Caspase-3 in TBI+vehicle group (P< 0.01). ALA treatment after TBI resulted in the number of Caspase-3 positive cells decreased, while its protein expression decreased (P<0.01).6. Effect of ALA on mitochondria following TBIThe levels of Bax protein in mitochondria and cytosol were increased and decreased, respectively, after TBI as compared to the sham+vehicle and sham+ALA groups (P < 0.01), whereas mitochondrial and cytosolic cytochrome c levels were decreased and increased, respectively, relative to sham-operated animals (P<0.05). These effects were reversed in TBI rats treated with ALA, in which mitochondrial translocation of Bax and subsequent cytosolic release of cytochrome c were inhibited (P<0.05).Conclusion:The experimental research demonstrated that ALA exerts neuroprotective effects against TBI by mitigating oxidative stress and suppressing the mitochondrial apoptotic pathway via an inhibition of Bax translocation and cytochrome c release from the mitochondria. These findings indicate that ALA has potential therapeutic applications for preventing secondary brain damage following TBI. |
PDF Full Text Request