| Part.1Title:The possible role of Nrf2-ARE pathway on the ubiquitin proteasome system following traumatic brain injury in miceBackground:Traumatic brain injury (TBI) is a main cause of brain damage in modern society, and it happened at high morbidity and mortality, leading to a serious threat to human health. The mechanism of neurons injury after TBI mainly includes primary injury and secondary brain injury. The primary injury is caused immediately by the external force, such as laceration, cerebral hemorrhage and diffuse axonal injury, which are inevitably and preventable. Secondary injury appears gradually after injury and the pathological processes including mitochondrial dysfunction and apoptosis, oxidative stress, calcium overload, neurotoxicity. How to active treat and prevent these secondary injury is the basic principle and significance in the treatment of traumatic brain injury. At present, there are no other recognized effective treatments in clinically apart from surgery. Research of secondary injury after TBI is always the focus of research on world, and it is important to find effective treatment, and the key pathological pathways or the core signal transduction factor from the intricate mechanism.Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor researched hot recently. Under physiological condition, Nrf2 is normally localized in the cytoplasm and is bound to its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), which transferred to the ubiquitin proteasome system for degradation and maintains a relative inhibition state. Under conditions of stress, infection, trauma or other types of stimulation such as with tert-butylhydroquinone (tBHQ), Nrf2 dissociates from Keap1 and translocates into the nucleus, where it binds to the antioxidant-response element (ARE) and induces the transcription of genes encoding protective factors, improving the resistance of cells to stimulation. Studies have shown that the pathway activation can be effective express phase Ⅱ detoxifying enzymes, antioxidant, anti-inflammatory protein downstream molecules, which covers many aspects of the secondary injury defensive mechanism after TBI, including anti-oxidative stress, reducing inflammation, anti-apoptosis.The ubiquitin-proteasome system (UPS) is a specificity protein degradation pathway in vivo, and has attracted more attention with its important role in maintaining the normal function of cells and proteins. The study found that the dysfunction of ubiquitin-proteasome system after injury might lead to abnormal ubiquitin protein aggregation, causing cell dysfunction and death. In the nervous system of Alzheimer’s, Parkinson’s disease, Huntington’s disease and many other diseases have been detected large numbers of abnormal ubiquitin proteins. Some scholars discovered that the dysfunction of ubiquitin-proteasome system in the brain leads to a large number of abnormal aggregation of ubiquitinated proteins, leading to the damage of brain function and affecting the prognosis of nerve function. At the early stage of our research, we also found that the quickly and significantly increase of the ubiquitination level in cerebrospinal fluid (CSF) and contusion tissues after TBI. These results show that ubiquitin-proteasome system is an early response to injury in the nervous system, and plays an important role in the secondary neural injury of TBI. Therefore, it is important to study the protein degradation pathway after TBI.Studies show that the cytosolic chaperonin of Nrf2, Keap1, is a ubiquitin protein ligase E3 essentially, and the interaction between Keapl and Nrf2 constitute the regulation of the center of Nrf2 signaling pathway. The pretreatment of the UPS inhibitor, lactacystin, in PC 12 cell line can enhance Nrf2 activation induced by manganese chloride, combining with DNA sequence and raising the expression of protective factors. These results suggest that there is a close relationship between Nrf2 and UPS system.Currently, there is less literature about effect of UPS pathway after TBI, and what factors in the regulation of UPS and effects on protein or gene in brain trauma will be discussed in this paper.For illustration of the drugs used in this paper:tert-butylhydroquinone (tBHQ), a classic Nrf2 agonist, used as the activation of Nrf2 expression; Nrf2 gene knock mice used as inhibition of Nrf2 expression; MG132, a proteasome inhibitors, used as inhibition of the ubiquitin-proteasome system.Objective:The major purpose of this study is to detect the expression of Nrf2 and UPS after TBI, and to observe the prognosis of mice used of MG132, exploring the possible regulation mechanism between them.Methods:1. Animals and groups:Male ICR mice (28-32 g) were randomly assigned to sham group, TBI group, TBI+tBHQ group. Male Nrf2 (-/-) mice (28-32 g) were randomly assigned to TBI+Nrf2 (-/-) group, TBI+Nrf2 (-/-)+MG132 group. The experimental mice were provided from department of Comparative Medicine of the General Hospital of Nanjing Military Region and the each group had 12.2. Mice TBI model:The TBI model used in this study is made according to the improved flierl free falling method. The mouse was placed on a stereotaxic instrument after prepared with disinfected of skin, and the gravity impact point positioning in 3mm after of hemisphere coronal suture,3 mm left of sagittal side seam, along cutting the mice head about 1 cm midline. The sham group cut the scalp only, and the TBI mice used 200 g weight on vertical positioning point from 2.5 cm height free fall impact. The whole process is carried out under aseptic conditions, and pay attention to maintaining the integrity of the dura mater.3. Drug administration:tBHQ was formulated as a concentration of 5mg/ml which dissolved in 1% DMSO solution, and administered by intraperitoneal injection a day before TBI in accordance with calculation 50mg/kg dose; MG132 is formulated as a concentration of 3mg/ml dissolved in a solution of 1% DMSO, and administered by intraperitoneal injection 6 hour before TBI in accordance with calculation 20mg/kg dose.4. Sampling preparation:In 24 hours after TBI, the mice were anesthetized and perfusion of isotonic saline (4℃) through the left ventricle until the liver white and then collected the brain. The brain tissue around the injury focus of 1mm3 placed in 4% glutaraldehyde, used for electron microscopy; the injury focus of 3mm placed at -80℃ refrigerator, used for western blot; after perfusion through the same way with 4% poly formaldehyde, collected the brain and placed in 4% poly formaldehyde for fixed, and then embedded in paraffin, sliced, for detection of immunohistochemistry and TUNEL apoptosis.5. Neurological evaluation:The mice were evaluated according to NSS at 1 and 3 day after TBI. The higher score represented the worse neural function defect.6. Brain water content:The mice were sacrificed at 24 hours after TBI, and removed from the brain quickly. After removal of brainstem and cerebellum, the left cortical tissue was obtained. The wet weight was measured for each sample immediately, and measured for dry weight after 72h dried at 80℃. The brain water content was calculated with the formula:[(wet weight-dry weight)/wet weight] X 100%7. TUNEL assay:Using TUNEL apoptosis kit on paraffin sections of brain tissue (5um). Randomly selected six view in high magnification field microscope (X 400) each slice, TUNEL positive cells were counted and calculated the average value (per 100 cells in TUNEL positive cells on the number of said positive), and then were statistically analyzed.8. Western blot:Detected the expression of Nrf2 in plasma protein and nuclear protein by 12% and 10% gel electrophoresis separation; detected the expression of free ubiquitin and ubiquitinated protein by 8% and 15% gel electrophoresis separation.9. Immunohistochemistry:tissue sections were made and detected the Nrf2 staining and localization in each group by immunohistochemistry operating instructions.10. Electrophoretic mobility shift assays (EMSA):using EMSA kit to detect Nrf2 DNA biotin labeled of mouse cerebral cortex tissues.11. Electron microscope:Tissue sections from experimental and control animals were stained by either 1% EPTA. Briefly, coronal brain sections were cut at a thickness of 200mm with a vibratome and post-fixed for 1 hr with 4% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4. For EPTA staining, sections were dehydrated in an ascending series of ethanol to 100% and stained for 50 min with 1% phosphotungstic acid (PTA) prepared by dissolving 0.1 gm of PTA in 10 ml of 100 ethanol and adding four drops of 95% ethanol. The EPTA solution was changed once after a 25 min interval during the staining. The sections were then further dehydrated in dry acetone and embedded in Durcupan ACM resin.12. Statistical analysis:The data were analyzed using SPSS 19.0 statistical software, all the data are presented as mean ± SEM. Differences between groups were assessed by one-way analysis of variance followed by Dunnett’s or Newman-Keuls Multiple Comparisons Test.*P<0.05 was considered as statistically significant.Results:1. Nrf2-ARE pathway activation improves motor performance, inhibits neuronal apoptosis, and reduces cerebral edema after TBI. Compared with the TBI group, TBI+tBHQ group nuclear Nrf2 protein was significantly increased (P< 0.001), and neurological severity score (NSS) decreased (P<0.05), the brain tissue water content decreased (P<0.001), the number of apoptotic neurons reduced (P< 0.001).2. Nrf2-ARE pathway activation inhibits protein ubiquitination after TBI. Compared with the TBI group, ubiquitinated protein in the TBI+tBHQ group decreased (P<0.05), the relative free ubiquitin increased (P<0.05), which indicated that UPS activity increased.3. Nrf2-ARE pathway inhibits aggravates motor performance, promotes neuronal apoptosis, and increases cerebral edema after TBI. Compared with the TBI group, nuclear Nrf2 expression in the TBI+Nrf2 (-/-) group significantly decreased in (P<0.05), and NSS neurological function score increased (P<0.05), water content in brain tissue increased (P<0.05), the number of apoptotic neurons in more (P<0.05).4. Nrf2-ARE pathway inhibits increases protein ubiquitination after TBI. Compared with the TBI group, ubiquitinated proteins in the TBI+Nrf2 (-/-) group increased (P<0.05), relative free ubiquitin decreased (P<0.05), which indicated that UPS activity decreased.5. Effect of proteasome inhibition on neuronal apoptosis and encephaledema after TBI is negated in Nrf2 mutants. In Nrf2 (+/+) mice, administration with MG132 inhibited the UPS activity (P<0.01), TBI group has more serious apoptosis neurons than the sham group (P<0.001); in the Nrf2 (-/-) mice, there is no significant change whether administration with MG132 or not (P> 0.05), and the apoptosis level of those two groups was no statistical difference (P> 0.05).Conclusion:1. UPS changes along with Nrf2 after TBI.2. Nrf2 may play a neuroprotective role by regulating UPS.Part.2Title:The possible mechanism of Nrf2-ARE pathway regulate ubiquitin-proteasome system following traumatic brain injury in miceBackground:In the first part of the experiment, we detected the expression of Nrf2 and UPS after TBI, finding the activity of UPS increased with the increased expression of Nrf2, along with the activity of UPS inhibited with the decreased expression of Nrf2. And there is no significantly changed inhibited activity of UPS after using the inhibitor MG132 in Nrf2 gene knockout mice. Therefore, we have every reason to believe that Nrf2 may regulate UPS to play a neuroprotective effect ultimately, and then we will further explore the possible mechanism.Literature studies have shown that decreased glutathione (GSH) content, accompanied with high activity of reactive oxygen species (ROS) after the use of protease inhibitor MG132. As a downstream factor activated by Nrf2, GSH has a very close relationship with Nrf2 and studies confirmed that the dysfunction of Nrf2-GSH pathway influence the growth of type Ⅱ cells directly, increased sensitivity to oxidants, to be damage to cells. These results indicate that the redox homeostasis represented by GSH/ROS has a closely relationship with Nrf2 and UPS.For illustration of the drugs used in this paper:ferric ammonium citrate (FAC) used as an activator of ROS; N-acetyl cysteine (NAC) used as a suppression of the expression of ROS.Objective:Detect the levels of GSH/ROS by activating or inhibiting the expression of Nrf2; detect the levels of UPS activity by activating or inhibiting the expression of GSH/ROS at the same time, thus exploring the possible regulation mechanism between Nrf2 and UPS.Method1. Animals and groups:Male ICR mice (28-32 g) were randomly assigned to sham group, TBI group, TBI+tBHQ group, TBI+FAC group, TBI+NAC group. Male Nrf2 (-/-) mice (28-32 g) were randomly assigned to TBI+Nrf2 (-/-) group. The experimental mice were provided from department of Comparative Medicine of the General Hospital of Nanjing Military Region.2. Mice TBI model:The TBI model used in this study is made according to the improved flierl free falling method. The mouse was placed on a stereotaxic instrument after prepared with disinfected of skin, and the gravity impact point positioning in 3mm after of hemisphere coronal suture,3 mm left of sagittal side seam, along cutting the mice head about 1 cm midline. The sham group cut the scalp only, and the TBI mice used 200 g weight on vertical positioning point from 2.5 cm height free fall impact. The whole process is carried out under aseptic conditions, and pay attention to maintaining the integrity of the dura mater.3. Drug administration:tBHQ was formulated as a concentration of 5mg/ml which dissolved in 1% DMSO solution, and administered by intraperitoneal injection a day before TBI in accordance with calculation 50mg/kg dose; FAC was formulated as a concentration of lug/ul which dissolved in 0.9% saline and administered by lateral ventricle injection before the model of TBI in accordance to 2.5ul/each mouse; NAC was formulated as concentration of 30mg/ml which dissolved in 0.9% saline and administered by intraperitoneal injection twice at 5 minutes,6 hours after TBI in accordance with calculation 150 mg/kg dose.4. Sampling preparation:In 24 hours after TBI, the mice were anesthetized and perfusion of isotonic saline (4℃) through the left ventricle until the liver white and then collected the brain. The brain tissue around the injury focus of 3mm placed at -80℃ refrigerator, used for detect of GSH, ROS and western blot; after perfusion through the same way with 4% poly formaldehyde, collected the brain and placed in 4% poly formaldehyde for fixed, and then embedded in paraffin, sliced, for detection of immunohistochemistry; after perfusion through the same way with isotonic saline (4℃), collected the brain and placed at -80℃ refrigerator, and then sucrose gradient dehydration, embedding frozen sections for immunofluorescence.5. Determination of GSH and ROS in brain tissue:Detected the GSH and ROS according to the kit and used the enzyme labeling instrument; determination of total protein with Bradford method. The content of GSH was used by umol/L and the fluorescence of ROS was used by intensity/mg. protein.6. Immunofluorescence:Frozen tissue sections were made and detected the expression of ROS in neurons and nucleus by immunofluorescence double staining method.7. Western blot:Detect the expression of ubiquitinated proteins by 8% gel electrophoresis in total protein.8. Immunohistochemistry:Tissue sections were made and detected the expression and localization of ubiquitinated protein in each group by immunohistochemistry operating instructions.9. Statistical analysis:The data were analyzed using SPSS 19.0 statistical software, all the data are presented as mean ± SEM. Differences between groups were assessed by one-way analysis of variance followed by Dunnett’s or Newman-Keuls Multiple Comparisons Test.*P< 0.05 was considered as statistically significant.Results:1. The content of GSH increased with the activation of Nrf2 after TBI, while the intensity of ROS became weaker. Compared with the TBI group, the content of GSH was significantly increased (P<0.05) after the activation of Nrf2 administrated of tBHQ and the intensity of ROS weakened (P<0.0001).2. The content of GSH decreased with the inhibited of Nrf2 after TBI, while the intensity of ROS became stronger. Compared with the TBI group, the content of GSH was significantly decreased (P<0.05) in the group of TBI+Nrf2 (-/-) and the intensity of ROS stronger (P<0.0001).3. The activity of UPS decreased with the activation of ROS after TBI, while the content of GSH dropped and ubiquitin protein increased. Compared with the TBI group, the conjugated ubiquitin protein increased (P<0.01) after stimulate the production of ROS (P<0.0001) administrated of FAC, which indicates that the activity of UPS decreased; while the content of GSH dropped (P<0.05).4. The activity of UPS increased with the inhibited of ROS after TBI, while the content of GSH rose and ubiquitin protein decreased. Compared with the TBI group, the conjugated ubiquitin protein decreased (P<0.05) after inhibited the production of ROS (P<0.0001) administrated of NAC, which indicates that the activity of UPS increased; while the content of GSH enhanced (P<0.05).Conclusion:1. Nrf2 can regulate the expression of GSH/ROS.2. GSH/ROS can regulate the activity of UPS.3. Nrf2 can regulate the activity of UPS through influence the redox homeostasis.Part.3Title:Further verify the hypothesis of Nrf2-ARE regulate ubiquitin-proteasome system through influence redox homeostasis in vitroBackground:In above experiments, we initially investigated Nrf2-ARE played a neuroprotective effect in the mouse model of TBI, may through regulate ubiquitin-proteasome system via influence redox homeostasis. But in vitro environment this pathway has yet to be further verified.Objective:Validate the relationship of Nrf2, redox homeostasis, and UPS of the three in vitro.Methods:1. Cultured neurons:Cortical neuronal cells were isolated from embryos of gestating mice, between the 17th and 18th day of pregnancy (Nrf2+/+ and Nrf2-/-), and cultured onto poly-D-lysine-coated 6 hole plates that contain neurobasal medium supplemented with 2% B27,1% glutamate,0.75% HEPES and 0.125% double antibiotics as previously described. All of the above cell culture reagents were from Gibco, Thermo Fisher Scientific. Cells were maintained in growth medium at 37℃ in 5% CO2/95% air. Half of the culture medium was replaced with fresh medium every 3 days. All experiments were performed after 7 days in vitro.2. The model of TBI in neurons and groups:Cultured mature ICR neurons were randomly divided into sham group, TBI group, TBI+tBHQ group, TBI+FAC group, TBI+NAC group; cultured mature Nrf2 (-/-) neurons divided into TBI+Nrf2 (-/-); Injury models of neuronal cells were established as described. Briefly, each well of the six-well plates was manually scratched with a sterile plastic pipette tip within a 9×9-square grid (with 4-mm spacing between the lines). After applying injury to the neuronal cells, the cells were placed in an incubator at until the final treatment.3. Drug administration:tBHQ was formulated as a final concentration of 10um in culture medium, joining in 6-well plates 2 hours before TBI, and each hole added 2ml; FAC was formulated as a final concentration of 200um in culture medium, joining in 6-well plates 4 hours before TBI, and each hole added 2ml; NAC was formulated as a final concentration of 200um in culture medium, joining in 6-well plates 6 hours before TBI, and each hole added 2ml.4. Western blot:Detected the expression of Nrf2 in plasma protein and nuclear protein by 12% and 10% gel electrophoresis separation; detected the expression ubiquitinated protein by 8% gel electrophoresis separation.5. Real time fluorescence quantitative PCR (q-PCR):Detected the target genes Nrf2 according to the kit instructions.6. Fluorescence detection of reactive oxygen species (ROS):Firstly situ loaded probe and incubated for 20 minutes, then part of neurons used to a fluorescence microscope observation and then other parts used to determine the fluorescence intensity by a flow cytometry following washed three times.488 nm excitation wavelength and 525nm emission wavelength, examined the intensity of fluorescence on real-time.7. Statistical analysis:The data were analyzed using SPSS 19.0 statistical software, all the data are presented as mean ± SEM. Differences between groups were assessed by one-way analysis of variance followed by Dunnett’s or Newman-Keuls Multiple Comparisons Test.*P< 0.05 was considered as statistically significant.Results:1. The expression of Nrf2 activated after administration of tBHQ, while the intensity of ROS decreased. The expression of Nrf2 increased after administrated tBHQ after TBI, and compared with TBI group, the nuclear transfer of Nrf2 increased (P<0.05), while the fluorescence intensity of ROS weakened (P<0.001).2. The intensity of ROS became stronger with the inhibition expression of Nrf2 in Nrf2 (-/-) after TBI. The expression of Nrf2 (-/-) decreased significantly after TBI, and compared with TBI group, the nuclear transfer of Nrf2 decreased (P<0.001), while the fluorescence intensity of ROS stronger (P<0.01).3. The expression of ubiquitin protein increased with the activation of ROS, while the activity of UPS reduced. Compared with the TBI group, the ubiquitin protein increased with the use of FAC (P<0.01), suggesting the activity of UPS decreased, while the fluorescence intensity of ROS stronger (P<0.01).4. The expression of ubiquitin protein decreased with the inhibition of ROS, while the activity of UPS increased. Compared with the TBI group, the ubiquitin protein decreased with the use of NAC (P<0.05), suggesting the activity of UPS increased, while the fluorescence intensity of ROS weakened (P<0.001).Conclusion:1. Nrf2 can regulate the expression of ROS in vitro TBI model.2. ROS can regulate the activity of UPS in vitro TBI model. |
PDF Full Text Request