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Id Subject Object Predicate Lexical cue
T24 0-119 Sentence denotes The Absence of Nrf2 Enhances NF-κB-Dependent Inflammation following Scratch Injury in Mouse Primary Cultured Astrocytes
T1 0-119 Sentence denotes The Absence of Nrf2 Enhances NF-κB-Dependent Inflammation following Scratch Injury in Mouse Primary Cultured Astrocytes
T2 121-129 Sentence denotes Abstract
T25 130-341 Sentence denotes It has been proved that Nrf2 depletion enhances inflammatory process through activation of NF-κB in the brain after TBI, but little is known about the relationship between Nrf2 and NF-κB in astrocytes after TBI.
T3 130-341 Sentence denotes It has been proved that Nrf2 depletion enhances inflammatory process through activation of NF-κB in the brain after TBI, but little is known about the relationship between Nrf2 and NF-κB in astrocytes after TBI.
T26 342-557 Sentence denotes Hence, we used primary cultured astrocytes from either Nrf2 wildtype or knockout mice to study the influence of Nrf2 on the activation of NF-κB and expression of proinflammatory cytokines in a model of TBI in vitro.
T4 342-557 Sentence denotes Hence, we used primary cultured astrocytes from either Nrf2 wildtype or knockout mice to study the influence of Nrf2 on the activation of NF-κB and expression of proinflammatory cytokines in a model of TBI in vitro.
T27 558-640 Sentence denotes Primary cultured astrocytes were scratched to mimic the traumatic injury in vitro.
T5 558-640 Sentence denotes Primary cultured astrocytes were scratched to mimic the traumatic injury in vitro.
T28 641-702 Sentence denotes Then the DNA-binding activity of NF-κB was evaluated by EMSA.
T6 641-702 Sentence denotes Then the DNA-binding activity of NF-κB was evaluated by EMSA.
T29 703-783 Sentence denotes The mRNA and protein levels of TNF-α, IL-1β, IL-6, and MMP9 were also evaluated.
T7 703-783 Sentence denotes The mRNA and protein levels of TNF-α, IL-1β, IL-6, and MMP9 were also evaluated.
T30 784-848 Sentence denotes Gelatin zymography was performed to detect the activity of MMP9.
T8 784-848 Sentence denotes Gelatin zymography was performed to detect the activity of MMP9.
T31 849-970 Sentence denotes The activity of NF-κB and expression of proinflammatory cytokines mentioned above were upregulated at 24 h after scratch.
T9 849-970 Sentence denotes The activity of NF-κB and expression of proinflammatory cytokines mentioned above were upregulated at 24 h after scratch.
T32 971-1026 Sentence denotes The expression and activity of MMP9 were also elevated.
T10 971-1026 Sentence denotes The expression and activity of MMP9 were also elevated.
T33 1027-1118 Sentence denotes And such tendency was much more prominent in Nrf2 KO astrocytes than that in WT astrocytes.
T11 1027-1118 Sentence denotes And such tendency was much more prominent in Nrf2 KO astrocytes than that in WT astrocytes.
T34 1119-1289 Sentence denotes These results suggest that the absence of Nrf2 may induce more aggressive inflammation through activation of NF-κB and downstream proinflammatory cytokines in astrocytes.
T12 1119-1289 Sentence denotes These results suggest that the absence of Nrf2 may induce more aggressive inflammation through activation of NF-κB and downstream proinflammatory cytokines in astrocytes.
T13 1291-1293 Sentence denotes 1.
T14 1294-1306 Sentence denotes Introduction
T620 1307-1440 Sentence denotes Brain damage following traumatic injury is a result of direct (primary injury) and indirect (secondary or delayed injury) mechanisms.
T15 1307-1440 Sentence denotes Brain damage following traumatic injury is a result of direct (primary injury) and indirect (secondary or delayed injury) mechanisms.
T621 1441-1785 Sentence denotes The secondary injury mechanism involves the initiation of an acute inflammatory response, including breakdown of blood-brain barrier (BBB), brain edema, infiltration of peripheral blood cells, and activation of resident immunocompetent cells, as well as the release of numerous immune mediators such as interleukins and chemotactic factors [1].
T16 1441-1785 Sentence denotes The secondary injury mechanism involves the initiation of an acute inflammatory response, including breakdown of blood-brain barrier (BBB), brain edema, infiltration of peripheral blood cells, and activation of resident immunocompetent cells, as well as the release of numerous immune mediators such as interleukins and chemotactic factors [1].
T622 1786-1893 Sentence denotes And the subsequent inflammation leads to secondary damage not only in brain but also in other organs [2–4].
T17 1786-1893 Sentence denotes And the subsequent inflammation leads to secondary damage not only in brain but also in other organs [2–4].
T623 1894-2017 Sentence denotes Nuclear factor erythroid 2-related factor2 (Nrf2) is a transcription factor that regulates many kinds of antioxidant genes.
T18 1894-2017 Sentence denotes Nuclear factor erythroid 2-related factor2 (Nrf2) is a transcription factor that regulates many kinds of antioxidant genes.
T624 2018-2137 Sentence denotes Several studies have demonstrated that Nrf2 regulates the inflammation in the brain after traumatic brain injury (TBI).
T19 2018-2137 Sentence denotes Several studies have demonstrated that Nrf2 regulates the inflammation in the brain after traumatic brain injury (TBI).
T625 2138-2250 Sentence denotes It was also showed that TBI could induce more aggravated damage in Nrf2 knockout mice than in wildtype mice [5].
T20 2138-2250 Sentence denotes It was also showed that TBI could induce more aggravated damage in Nrf2 knockout mice than in wildtype mice [5].
T626 2251-2388 Sentence denotes On the contrary, such inflammation can be extenuated through elevating the level of Nrf2 by its inducers, such as sulforaphane (SFN) [6].
T21 2251-2388 Sentence denotes On the contrary, such inflammation can be extenuated through elevating the level of Nrf2 by its inducers, such as sulforaphane (SFN) [6].
T627 2389-2523 Sentence denotes It is well documented that astrocytes play a critical role in maintaining normal brain physiology and responding to injury or disease.
T22 2389-2523 Sentence denotes It is well documented that astrocytes play a critical role in maintaining normal brain physiology and responding to injury or disease.
T628 2524-2586 Sentence denotes In fact, all the aforementioned studies are performed in vivo.
T23 2524-2586 Sentence denotes In fact, all the aforementioned studies are performed in vivo.
T629 2587-2680 Sentence denotes Little is known about the relationship between Nrf2 and inflammation in astrocytes after TBI.
T24 2587-2680 Sentence denotes Little is known about the relationship between Nrf2 and inflammation in astrocytes after TBI.
T630 2681-2851 Sentence denotes Therefore, here we analysed the influence of Nrf2 on the expression of proinflammatory cytokines in primary cultured astrocytes from transgenic mice after scratch injury.
T25 2681-2851 Sentence denotes Therefore, here we analysed the influence of Nrf2 on the expression of proinflammatory cytokines in primary cultured astrocytes from transgenic mice after scratch injury.
T26 2853-2855 Sentence denotes 2.
T27 2856-2877 Sentence denotes Methods and Materials
T1224 2879-2883 Sentence denotes 2.1.
T28 2879-2883 Sentence denotes 2.1.
T1225 2884-2938 Sentence denotes Primary Culture and Identification of Mouse Astrocytes
T29 2884-2938 Sentence denotes Primary Culture and Identification of Mouse Astrocytes
T30 2939-2999 Sentence denotes Nrf2 knockout ICR mice were kindly provided by Dr. Thomas W.
T1226 2939-3055 Sentence denotes Nrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA).
T31 3000-3055 Sentence denotes Kensler (Johns Hopkins University, Baltimore, MD, USA).
T1227 3056-3184 Sentence denotes Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype).
T32 3056-3184 Sentence denotes Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype).
T1228 3185-3301 Sentence denotes Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed.
T33 3185-3301 Sentence denotes Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed.
T1229 3302-3436 Sentence denotes The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes.
T34 3302-3436 Sentence denotes The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes.
T1230 3437-3637 Sentence denotes After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium).
T35 3437-3637 Sentence denotes After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium).
T1231 3638-3777 Sentence denotes After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells.
T36 3638-3777 Sentence denotes After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells.
T1232 3778-3829 Sentence denotes Then the cells were cultivated at 37°C with 5% CO2.
T37 3778-3829 Sentence denotes Then the cells were cultivated at 37°C with 5% CO2.
T1233 3830-3892 Sentence denotes And the complete culture medium was half-changed twice a week.
T38 3830-3892 Sentence denotes And the complete culture medium was half-changed twice a week.
T1234 3893-3950 Sentence denotes Astrocytes expanded for about 7 days to reach confluence.
T39 3893-3950 Sentence denotes Astrocytes expanded for about 7 days to reach confluence.
T1235 3951-4075 Sentence denotes Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures.
T40 3951-4075 Sentence denotes Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures.
T1236 4076-4236 Sentence denotes After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA).
T41 4076-4236 Sentence denotes After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA).
T1237 4237-4307 Sentence denotes Cells passaged for 2-3 generations were used in the following studies.
T42 4237-4307 Sentence denotes Cells passaged for 2-3 generations were used in the following studies.
T1813 4309-4313 Sentence denotes 2.2.
T43 4309-4313 Sentence denotes 2.2.
T1814 4314-4365 Sentence denotes In Vitro Model of TBI Established by Scratch Injury
T44 4314-4365 Sentence denotes In Vitro Model of TBI Established by Scratch Injury
T1815 4366-4433 Sentence denotes Astrocyte scratch injury was performed as in a previous report [7].
T45 4366-4433 Sentence denotes Astrocyte scratch injury was performed as in a previous report [7].
T1816 4434-4499 Sentence denotes Astrocytes were planted in 6-well plates and grown to confluence.
T46 4434-4499 Sentence denotes Astrocytes were planted in 6-well plates and grown to confluence.
T1817 4500-4616 Sentence denotes The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap.
T47 4500-4616 Sentence denotes The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap.
T1818 4617-4748 Sentence denotes Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group.
T48 4617-4748 Sentence denotes Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group.
T1819 4749-4839 Sentence denotes Cells, which did not received scratch, were used as blank control and named as sham group.
T49 4749-4839 Sentence denotes Cells, which did not received scratch, were used as blank control and named as sham group.
T2033 4841-4845 Sentence denotes 2.3.
T50 4841-4845 Sentence denotes 2.3.
T2034 4846-4883 Sentence denotes Cell Death Measurement by CCK-8 Assay
T51 4846-4883 Sentence denotes Cell Death Measurement by CCK-8 Assay
T2035 4884-4975 Sentence denotes 1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere.
T52 4884-4975 Sentence denotes 1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere.
T2036 4976-5021 Sentence denotes Then the cell was scrated as mentioned above.
T53 4976-5021 Sentence denotes Then the cell was scrated as mentioned above.
T2037 5022-5170 Sentence denotes Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol.
T54 5022-5170 Sentence denotes Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol.
T2038 5171-5240 Sentence denotes Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h.
T55 5171-5240 Sentence denotes Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h.
T2039 5241-5346 Sentence denotes Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA).
T56 5241-5346 Sentence denotes Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA).
T2040 5347-5395 Sentence denotes All measurements were performed in sextuplicate.
T57 5347-5395 Sentence denotes All measurements were performed in sextuplicate.
T2041 5396-5454 Sentence denotes Results were expressed as mean of OD value at 450 nm ± SD.
T58 5396-5454 Sentence denotes Results were expressed as mean of OD value at 450 nm ± SD.
T2309 5456-5460 Sentence denotes 2.4.
T59 5456-5460 Sentence denotes 2.4.
T2310 5461-5504 Sentence denotes Electrophoretic Mobility Shift Assay (EMSA)
T60 5461-5504 Sentence denotes Electrophoretic Mobility Shift Assay (EMSA)
T2311 5505-5591 Sentence denotes Monolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS.
T61 5505-5591 Sentence denotes Monolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS.
T2312 5592-5784 Sentence denotes The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride.
T62 5592-5784 Sentence denotes The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride.
T2313 5785-5886 Sentence denotes Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation.
T63 5785-5886 Sentence denotes Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation.
T2314 5887-6060 Sentence denotes The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride.
T64 5887-6060 Sentence denotes The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride.
T2315 6061-6206 Sentence denotes Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use.
T65 6061-6206 Sentence denotes Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use.
T2316 6207-6364 Sentence denotes Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA).
T66 6207-6364 Sentence denotes Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA).
T2317 6365-6441 Sentence denotes EMSA was performed using gel shift assay system (Promega, Madison, WI, USA).
T67 6365-6441 Sentence denotes EMSA was performed using gel shift assay system (Promega, Madison, WI, USA).
T2318 6442-6599 Sentence denotes Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China).
T68 6442-6599 Sentence denotes Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China).
T2319 6600-6806 Sentence denotes Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice.
T69 6600-6806 Sentence denotes Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice.
T2320 6807-6919 Sentence denotes After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice.
T70 6807-6919 Sentence denotes After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice.
T71 6920-7085 Sentence denotes The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C.
T2321 6920-7184 Sentence denotes The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan).
T72 7086-7184 Sentence denotes After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan).
T2322 7185-7257 Sentence denotes Levels of NF-κB DNA binding activity were quantified by software ImageJ.
T73 7185-7257 Sentence denotes Levels of NF-κB DNA binding activity were quantified by software ImageJ.
T3100 7259-7263 Sentence denotes 2.5.
T74 7259-7263 Sentence denotes 2.5.
T3101 7264-7270 Sentence denotes RT-PCR
T75 7264-7270 Sentence denotes RT-PCR
T3102 7271-7483 Sentence denotes Total RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol.
T76 7271-7483 Sentence denotes Total RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol.
T77 7484-7513 Sentence denotes The cDNA was stored in −20°C.
T3103 7484-7638 Sentence denotes The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol.
T78 7514-7638 Sentence denotes Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol.
T3104 7640-7685 Sentence denotes Table 1 shows the primers and PCR parameters.
T79 7640-7685 Sentence denotes Table 1 shows the primers and PCR parameters.
T3105 7686-7744 Sentence denotes PCR products were detected by agarose gel electrophoresis.
T80 7686-7744 Sentence denotes PCR products were detected by agarose gel electrophoresis.
T3106 7745-7803 Sentence denotes The intensity of the bands was analyzed by ImageJ program.
T81 7745-7803 Sentence denotes The intensity of the bands was analyzed by ImageJ program.
T3107 7804-7858 Sentence denotes The level of β-actin was used as an internal standard.
T82 7804-7858 Sentence denotes The level of β-actin was used as an internal standard.
T3374 7860-7864 Sentence denotes 2.6.
T83 7860-7864 Sentence denotes 2.6.
T3375 7865-7877 Sentence denotes Western Blot
T84 7865-7877 Sentence denotes Western Blot
T85 7878-8157 Sentence denotes To obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C.
T3376 7878-8254 Sentence denotes To obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA).
T86 8158-8254 Sentence denotes Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA).
T3377 8255-8496 Sentence denotes Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane.
T87 8255-8496 Sentence denotes Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane.
T3378 8497-8802 Sentence denotes For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa).
T88 8497-8802 Sentence denotes For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa).
T3379 8803-8926 Sentence denotes Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature.
T89 8803-8926 Sentence denotes Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature.
T3380 8927-9084 Sentence denotes The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h.
T90 8927-9084 Sentence denotes The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h.
T3381 9085-9259 Sentence denotes Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan).
T91 9085-9259 Sentence denotes Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan).
T3382 9260-9314 Sentence denotes ImageJ was used to analyze the intensity of the blots.
T92 9260-9314 Sentence denotes ImageJ was used to analyze the intensity of the blots.
T3383 9315-9366 Sentence denotes The level of β-actin was used as internal standard.
T93 9315-9366 Sentence denotes The level of β-actin was used as internal standard.
T4052 9368-9372 Sentence denotes 2.7.
T94 9368-9372 Sentence denotes 2.7.
T4053 9373-9414 Sentence denotes Enzyme-Linked Immunosorbent Assay (ELISA)
T95 9373-9414 Sentence denotes Enzyme-Linked Immunosorbent Assay (ELISA)
T4054 9415-9472 Sentence denotes Cells of four groups were homogenized as mentioned above.
T96 9415-9472 Sentence denotes Cells of four groups were homogenized as mentioned above.
T4055 9473-9595 Sentence denotes The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA).
T97 9473-9595 Sentence denotes The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA).
T98 9596-9824 Sentence denotes Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc.
T4056 9596-9834 Sentence denotes Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA).
T99 9825-9834 Sentence denotes CA, USA).
T4057 9835-9928 Sentence denotes Briefly, prepared the standard and created the standard dilution for building standard curve.
T100 9835-9928 Sentence denotes Briefly, prepared the standard and created the standard dilution for building standard curve.
T4058 9929-9993 Sentence denotes Then samples and biotinconjugate were added to microwell strips.
T101 9929-9993 Sentence denotes Then samples and biotinconjugate were added to microwell strips.
T4059 9994-10143 Sentence denotes After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells.
T102 9994-10143 Sentence denotes After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells.
T4060 10144-10239 Sentence denotes After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate.
T103 10144-10239 Sentence denotes After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate.
T4061 10240-10305 Sentence denotes After incubated for about 10–30 min, the stop solution was added.
T104 10240-10305 Sentence denotes After incubated for about 10–30 min, the stop solution was added.
T4062 10306-10422 Sentence denotes The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA).
T105 10306-10422 Sentence denotes The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA).
T4063 10423-10539 Sentence denotes The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.
T106 10423-10539 Sentence denotes The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.
T4378 10541-10545 Sentence denotes 2.8.
T107 10541-10545 Sentence denotes 2.8.
T4379 10546-10565 Sentence denotes Gelatine Zymography
T108 10546-10565 Sentence denotes Gelatine Zymography
T109 10566-10711 Sentence denotes Cells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton.
T4380 10566-10895 Sentence denotes Cells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA).
T110 10712-10782 Sentence denotes Soluble extracts were separated by centrifugation and stored at –20°C.
T111 10783-10895 Sentence denotes Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA).
T4381 10896-10972 Sentence denotes Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis.
T112 10896-10972 Sentence denotes Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis.
T4382 10973-11138 Sentence denotes Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin.
T113 10973-11138 Sentence denotes Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin.
T4383 11139-11242 Sentence denotes After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol.
T114 11139-11242 Sentence denotes After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol.
T4384 11243-11348 Sentence denotes Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate.
T115 11243-11348 Sentence denotes Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate.
T4385 11349-11434 Sentence denotes Quantification of MMP9 band density was performed with image analysis program ImageJ.
T116 11349-11434 Sentence denotes Quantification of MMP9 band density was performed with image analysis program ImageJ.
T4757 11436-11440 Sentence denotes 2.9.
T117 11436-11440 Sentence denotes 2.9.
T4758 11441-11461 Sentence denotes Statistical Analysis
T118 11441-11461 Sentence denotes Statistical Analysis
T4759 11462-11551 Sentence denotes Data were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test.
T119 11462-11551 Sentence denotes Data were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test.
T4760 11552-11601 Sentence denotes P values <0.05 were considered to be significant.
T120 11552-11601 Sentence denotes P values <0.05 were considered to be significant.
T4761 11602-11658 Sentence denotes All analyses were performed by using SPSS 18.0 software.
T121 11602-11658 Sentence denotes All analyses were performed by using SPSS 18.0 software.
T122 11660-11662 Sentence denotes 3.
T123 11663-11670 Sentence denotes Results
T4844 11672-11676 Sentence denotes 3.1.
T124 11672-11676 Sentence denotes 3.1.
T4845 11677-11755 Sentence denotes Depletion of Nrf2 Aggravated the Cell Death in Astrocytes after Scratch Injury
T125 11677-11755 Sentence denotes Depletion of Nrf2 Aggravated the Cell Death in Astrocytes after Scratch Injury
T4846 11756-11835 Sentence denotes Cell death was detected by microscope and CCK-8 analysis at 24 h after scratch.
T126 11756-11835 Sentence denotes Cell death was detected by microscope and CCK-8 analysis at 24 h after scratch.
T4847 11836-11984 Sentence denotes The detachment from culture plate, cell lost, and cytoplasmic process distortion were mostly observed in both sides of scratch line in group KO TBI.
T127 11836-11984 Sentence denotes The detachment from culture plate, cell lost, and cytoplasmic process distortion were mostly observed in both sides of scratch line in group KO TBI.
T4848 11985-12172 Sentence denotes CCK-8 assay also showed lower OD value of group KO TBI (0.38 ± 0.064) than that of group WT TBI (0.98 ± 0.098) (P < 0.01), which also suggested more cell death in group KO TBI (Figure 1).
T128 11985-12172 Sentence denotes CCK-8 assay also showed lower OD value of group KO TBI (0.38 ± 0.064) than that of group WT TBI (0.98 ± 0.098) (P < 0.01), which also suggested more cell death in group KO TBI (Figure 1).
T4849 12173-12230 Sentence denotes There was no difference between two sham-operated groups.
T129 12173-12230 Sentence denotes There was no difference between two sham-operated groups.
T5106 12232-12236 Sentence denotes 3.2.
T130 12232-12236 Sentence denotes 3.2.
T5107 12237-12328 Sentence denotes Disruption of Nrf2 Enhanced Upregulation of NF-κB DNA-Binding Activity after Scratch Injury
T131 12237-12328 Sentence denotes Disruption of Nrf2 Enhanced Upregulation of NF-κB DNA-Binding Activity after Scratch Injury
T5108 12329-12397 Sentence denotes It has been reported that NF-κB is activated in brain after TBI [2].
T132 12329-12397 Sentence denotes It has been reported that NF-κB is activated in brain after TBI [2].
T5109 12398-12524 Sentence denotes Here, we studied the DNA-binding activity of NF-κB of astrocytes from Nrf2 WT or KO mice by EMSA at 24 h after scratch injury.
T133 12398-12524 Sentence denotes Here, we studied the DNA-binding activity of NF-κB of astrocytes from Nrf2 WT or KO mice by EMSA at 24 h after scratch injury.
T5110 12525-12735 Sentence denotes Scratch injury induced activation of NF-κB in astrocytes of both genotypes, while higher NF-κB activity was observed in group KO TBI than in group WT TBI (2.67 ± 0.173 versus 2.28 ± 0.072, P < 0.01) (Figure 2).
T134 12525-12735 Sentence denotes Scratch injury induced activation of NF-κB in astrocytes of both genotypes, while higher NF-κB activity was observed in group KO TBI than in group WT TBI (2.67 ± 0.173 versus 2.28 ± 0.072, P < 0.01) (Figure 2).
T5417 12737-12741 Sentence denotes 3.3.
T135 12737-12741 Sentence denotes 3.3.
T5418 12742-12843 Sentence denotes Expression of Proinflammatory Cytokines Was Elevated in Nrf2 Knockout Astrocytes after Scratch Injury
T136 12742-12843 Sentence denotes Expression of Proinflammatory Cytokines Was Elevated in Nrf2 Knockout Astrocytes after Scratch Injury
T5419 12844-12951 Sentence denotes As we all know, TNF-α, IL-1β, and IL-6 are regulated by NF-κB and reflect the endogenous activity of NF-κB.
T137 12844-12951 Sentence denotes As we all know, TNF-α, IL-1β, and IL-6 are regulated by NF-κB and reflect the endogenous activity of NF-κB.
T5420 12952-13052 Sentence denotes It has been revealed that these proinflammatory cytokines are increased after TBI in animals [2, 8].
T138 12952-13052 Sentence denotes It has been revealed that these proinflammatory cytokines are increased after TBI in animals [2, 8].
T5421 13053-13204 Sentence denotes And our previous study has proved that higher levels of such proinflammatory cytokines were observed in the brain of Nrf2-deficient mice after TBI [5].
T139 13053-13204 Sentence denotes And our previous study has proved that higher levels of such proinflammatory cytokines were observed in the brain of Nrf2-deficient mice after TBI [5].
T5422 13205-13311 Sentence denotes But it is still ambiguous about the relationship between these cytokines and Nrf2 in astrocytes after TBI.
T140 13205-13311 Sentence denotes But it is still ambiguous about the relationship between these cytokines and Nrf2 in astrocytes after TBI.
T5423 13312-13437 Sentence denotes Here, we tested the levels of TNF-α, IL-1β, and IL-6 in cultured astrocytes at 24 h after scratch injury by RT-PCR and ELISA.
T141 13312-13437 Sentence denotes Here, we tested the levels of TNF-α, IL-1β, and IL-6 in cultured astrocytes at 24 h after scratch injury by RT-PCR and ELISA.
T5424 13438-13613 Sentence denotes It was shown that the mRNA levels of TNF-α, IL-1β, and IL-6 were increased after scratch in both WT and KO astrocytes when compared with their sham counterparts, respectively.
T142 13438-13613 Sentence denotes It was shown that the mRNA levels of TNF-α, IL-1β, and IL-6 were increased after scratch in both WT and KO astrocytes when compared with their sham counterparts, respectively.
T5425 13614-13854 Sentence denotes Moreover, the mRNA levels of these cytokines in group KO TBI were much higher than those in group WT TBI (1.66 ± 0.085 versus 1.39 ± 0.110 for TNF-α, 1.34 ± 0.064 versus 0.73 ± 0.088 for IL-1β, and 1.28 ± 0.102 versus 0.99 ± 0.073 for IL-6.
T143 13614-13854 Sentence denotes Moreover, the mRNA levels of these cytokines in group KO TBI were much higher than those in group WT TBI (1.66 ± 0.085 versus 1.39 ± 0.110 for TNF-α, 1.34 ± 0.064 versus 0.73 ± 0.088 for IL-1β, and 1.28 ± 0.102 versus 0.99 ± 0.073 for IL-6.
T5426 13855-13885 Sentence denotes P < 0.01) (Figures 3(a)–3(d)).
T144 13855-13885 Sentence denotes P < 0.01) (Figures 3(a)–3(d)).
T5427 13886-14052 Sentence denotes The results of ELISA revealed that the protein level of TNF-α was upregulated after scratch injury in WT and KO astrocytes when compared with their sham counterparts.
T145 13886-14052 Sentence denotes The results of ELISA revealed that the protein level of TNF-α was upregulated after scratch injury in WT and KO astrocytes when compared with their sham counterparts.
T5428 14053-14172 Sentence denotes And it was also higher in group KO TBI than in group WT TBI (3.67 ± 0.156 versus 2.31 ± 0.087, P < 0.01) (Figure 4(a)).
T146 14053-14172 Sentence denotes And it was also higher in group KO TBI than in group WT TBI (3.67 ± 0.156 versus 2.31 ± 0.087, P < 0.01) (Figure 4(a)).
T5429 14173-14238 Sentence denotes Similar tendency was observed in protein level of IL-6 and IL-1β.
T147 14173-14238 Sentence denotes Similar tendency was observed in protein level of IL-6 and IL-1β.
T5430 14239-14349 Sentence denotes For IL-6, it was 36.07 ± 0.786 for group KO TBI while 25.76 ± 0.536 for group WT TBI (P < 0.01) (Figure 4(b)).
T148 14239-14349 Sentence denotes For IL-6, it was 36.07 ± 0.786 for group KO TBI while 25.76 ± 0.536 for group WT TBI (P < 0.01) (Figure 4(b)).
T5431 14350-14463 Sentence denotes For IL-1β, it was 190.75 ± 6.339 for group KO TBI while 154.50 ± 5.348 for group WT TBI (P < 0.01) (Figure 4(c)).
T149 14350-14463 Sentence denotes For IL-1β, it was 190.75 ± 6.339 for group KO TBI while 154.50 ± 5.348 for group WT TBI (P < 0.01) (Figure 4(c)).
T6473 14465-14469 Sentence denotes 3.4.
T150 14465-14469 Sentence denotes 3.4.
T6474 14470-14572 Sentence denotes Expression and Activity of MMP9 Were Greatly Enhanced in Nrf2 Knockout Astrocytes after Scratch Injury
T151 14470-14572 Sentence denotes Expression and Activity of MMP9 Were Greatly Enhanced in Nrf2 Knockout Astrocytes after Scratch Injury
T6475 14573-14653 Sentence denotes MMP9 is an important gelatinase to induce or aggravate the inflammation process.
T152 14573-14653 Sentence denotes MMP9 is an important gelatinase to induce or aggravate the inflammation process.
T6476 14654-14827 Sentence denotes In the present study, we elevated the mRNA, protein levels, and activity of MMP9 in astrocytes at 24 h after scratch injury by RT-PCR, western blot, and gelatine zymography.
T153 14654-14827 Sentence denotes In the present study, we elevated the mRNA, protein levels, and activity of MMP9 in astrocytes at 24 h after scratch injury by RT-PCR, western blot, and gelatine zymography.
T6477 14828-14946 Sentence denotes Expression of MMP9 was elevated after scratch injury in WT and KO astrocytes as compared with their sham counterparts.
T154 14828-14946 Sentence denotes Expression of MMP9 was elevated after scratch injury in WT and KO astrocytes as compared with their sham counterparts.
T6478 14947-15087 Sentence denotes The mRNA level of MMP9 was higher in group KO TBI than in group WT TBI (1.36 ± 0.090 versus 1.01 ± 0.068, P < 0.01) (Figures 3(a) and 3(e)).
T155 14947-15087 Sentence denotes The mRNA level of MMP9 was higher in group KO TBI than in group WT TBI (1.36 ± 0.090 versus 1.01 ± 0.068, P < 0.01) (Figures 3(a) and 3(e)).
T6479 15088-15259 Sentence denotes Significant difference was also discovered in MMP9 protein level by western blot, as 1.44 ± 0.076 for group KO TBI and 1.13 ± 0.048 for group WT TBI (P < 0.01) (Figure 5).
T156 15088-15259 Sentence denotes Significant difference was also discovered in MMP9 protein level by western blot, as 1.44 ± 0.076 for group KO TBI and 1.13 ± 0.048 for group WT TBI (P < 0.01) (Figure 5).
T6480 15260-15415 Sentence denotes Gelatine zymography revealed higher MMP9 activity in group KO TBI than that in group WT TBI too (101.76 ± 6.343 versus 76.32 ± 3.388, P < 0.01) (Figure 6).
T157 15260-15415 Sentence denotes Gelatine zymography revealed higher MMP9 activity in group KO TBI than that in group WT TBI too (101.76 ± 6.343 versus 76.32 ± 3.388, P < 0.01) (Figure 6).
T158 15417-15419 Sentence denotes 4.
T159 15420-15430 Sentence denotes Discussion
T7214 15431-15611 Sentence denotes The present study demonstrated that scratch injury induced the upregulation of NF-κB DNA binding activity and overexpression of TNF-α, IL-1β, IL-6, and MMP9 in cultured astrocytes.
T160 15431-15611 Sentence denotes The present study demonstrated that scratch injury induced the upregulation of NF-κB DNA binding activity and overexpression of TNF-α, IL-1β, IL-6, and MMP9 in cultured astrocytes.
T7215 15612-15825 Sentence denotes Also we revealed that scratch injury induced higher activity of NF-κB and enhanced expression of proinflammatory cytokines in Nrf2 knockout cultured astrocytes than those in wildtype astrocytes for the first time.
T161 15612-15825 Sentence denotes Also we revealed that scratch injury induced higher activity of NF-κB and enhanced expression of proinflammatory cytokines in Nrf2 knockout cultured astrocytes than those in wildtype astrocytes for the first time.
T7216 15826-15930 Sentence denotes It has been demonstrated that NF-κB is activated in brain and spinal cord after traumatic injury [2, 9].
T162 15826-15930 Sentence denotes It has been demonstrated that NF-κB is activated in brain and spinal cord after traumatic injury [2, 9].
T7217 15931-16149 Sentence denotes As a transcript factor, NF-κB binds with DNA once it was activated and induces transcription of MMP9 and a battery of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, in brain tissues after TBI [3, 10, 11].
T163 15931-16149 Sentence denotes As a transcript factor, NF-κB binds with DNA once it was activated and induces transcription of MMP9 and a battery of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, in brain tissues after TBI [3, 10, 11].
T7218 16150-16338 Sentence denotes Our results also revealed that scratch injury induced elevation of NF-κB DNA binding activity, overexpression of MMP9, and proinflammatory mediators mentioned above in cultured astrocytes.
T164 16150-16338 Sentence denotes Our results also revealed that scratch injury induced elevation of NF-κB DNA binding activity, overexpression of MMP9, and proinflammatory mediators mentioned above in cultured astrocytes.
T7219 16339-16439 Sentence denotes It has been demonstrated that there is an autoregulatory loop among these proinflammatory mediators.
T165 16339-16439 Sentence denotes It has been demonstrated that there is an autoregulatory loop among these proinflammatory mediators.
T7220 16440-16667 Sentence denotes For example, TNF-α and IL-1β are potent stimulators for MMP9 in astrocytes [12, 13], and IL-6 induces overexpression of MMP9 in human colon carcinoma cells [14], and TNF-α and IL-1β also can induce activation of NF-κB [15, 16].
T166 16440-16667 Sentence denotes For example, TNF-α and IL-1β are potent stimulators for MMP9 in astrocytes [12, 13], and IL-6 induces overexpression of MMP9 in human colon carcinoma cells [14], and TNF-α and IL-1β also can induce activation of NF-κB [15, 16].
T7221 16668-16788 Sentence denotes This autoregulatory loop extremely aggravates the damaging effect of inflammation and induces secondary injury to brain.
T167 16668-16788 Sentence denotes This autoregulatory loop extremely aggravates the damaging effect of inflammation and induces secondary injury to brain.
T7222 16789-16891 Sentence denotes It is a good choice for targeting on an upstream factor to prevent such autoregulatory loop after TBI.
T168 16789-16891 Sentence denotes It is a good choice for targeting on an upstream factor to prevent such autoregulatory loop after TBI.
T7223 16892-17069 Sentence denotes Nrf2-ARE pathway has been proved to be the key regulator in reducing oxidative stress, inflammatory damage, and accumulation of toxic metabolites, which are all involved in TBI.
T169 16892-17069 Sentence denotes Nrf2-ARE pathway has been proved to be the key regulator in reducing oxidative stress, inflammatory damage, and accumulation of toxic metabolites, which are all involved in TBI.
T7224 17070-17156 Sentence denotes Our previous study has proved the augmentation of Nrf2 in brain tissue after TBI [17].
T170 17070-17156 Sentence denotes Our previous study has proved the augmentation of Nrf2 in brain tissue after TBI [17].
T7225 17157-17373 Sentence denotes Enhanced level of Nrf2 activates transcription of a group of antioxidant genes, such as heme oxygenase-1 (HO-1) and NAD (P)H: quinone oxidoreductase-1 (NQO1), which would subsequently reduce the damage in brain [18].
T171 17157-17373 Sentence denotes Enhanced level of Nrf2 activates transcription of a group of antioxidant genes, such as heme oxygenase-1 (HO-1) and NAD (P)H: quinone oxidoreductase-1 (NQO1), which would subsequently reduce the damage in brain [18].
T7226 17374-17635 Sentence denotes Postinjury administration of SFN, an inducer of Nrf2, significantly improves spatial memory of rat and decreases the immunoreactivity for 4-Hydroxynonenal (4-HNE), a marker of lipid peroxidation, in the cortex and the CA3 subfield of hippocampus after TBI [19].
T172 17374-17635 Sentence denotes Postinjury administration of SFN, an inducer of Nrf2, significantly improves spatial memory of rat and decreases the immunoreactivity for 4-Hydroxynonenal (4-HNE), a marker of lipid peroxidation, in the cortex and the CA3 subfield of hippocampus after TBI [19].
T7227 17636-17706 Sentence denotes On the other hand, Nrf2-deficient mice appear more susceptible to TBI.
T173 17636-17706 Sentence denotes On the other hand, Nrf2-deficient mice appear more susceptible to TBI.
T7228 17707-17804 Sentence denotes Depletion of Nrf2 induces higher expression of proinflammatory mediators in brain after TBI [20].
T174 17707-17804 Sentence denotes Depletion of Nrf2 induces higher expression of proinflammatory mediators in brain after TBI [20].
T7229 17805-18095 Sentence denotes Here, our results revealed that overexpression of TNF-α, IL-1β, IL-6, and MMP9 after scratch injury was more aggravated in cultured Nrf2 knockout astrocytes than in wildtype astrocyetes for the first time, and overexpression of these proinflammatory mediators led to more astrocytes deaths.
T175 17805-18095 Sentence denotes Here, our results revealed that overexpression of TNF-α, IL-1β, IL-6, and MMP9 after scratch injury was more aggravated in cultured Nrf2 knockout astrocytes than in wildtype astrocyetes for the first time, and overexpression of these proinflammatory mediators led to more astrocytes deaths.
T7230 18096-18283 Sentence denotes Data obtained from animal studies suggest the possibility that antioxidant effect of Nrf2 may be achieved by suppression of proinflammatory pathways which are mediated by NF-κB signaling.
T176 18096-18283 Sentence denotes Data obtained from animal studies suggest the possibility that antioxidant effect of Nrf2 may be achieved by suppression of proinflammatory pathways which are mediated by NF-κB signaling.
T7231 18284-18452 Sentence denotes Administration of SFN is found to be able to inhibit IKK/IκB phosphorylation and p65 NF-κB subunit nuclear translocation, consequently alleviating NF-κB signaling [21].
T177 18284-18452 Sentence denotes Administration of SFN is found to be able to inhibit IKK/IκB phosphorylation and p65 NF-κB subunit nuclear translocation, consequently alleviating NF-κB signaling [21].
T7232 18453-18594 Sentence denotes And NF-κB activation induced by lipopolysaccharide (LPS) could be attenuated by diverse Nrf2 activators, such as SFN and curcumin (CUR) [22].
T178 18453-18594 Sentence denotes And NF-κB activation induced by lipopolysaccharide (LPS) could be attenuated by diverse Nrf2 activators, such as SFN and curcumin (CUR) [22].
T7233 18595-18777 Sentence denotes Furthermore, our previous studies indicate that depletion of Nrf2 induces augmentation of NF-κB activity and inflammatory response in lung, brain, and intestine after TBI [4, 5, 23].
T179 18595-18777 Sentence denotes Furthermore, our previous studies indicate that depletion of Nrf2 induces augmentation of NF-κB activity and inflammatory response in lung, brain, and intestine after TBI [4, 5, 23].
T7234 18778-18890 Sentence denotes Results from this study further confirmed such relationship existed in cultured astrocytes after scratch injury.
T180 18778-18890 Sentence denotes Results from this study further confirmed such relationship existed in cultured astrocytes after scratch injury.
T7235 18891-19047 Sentence denotes Enhanced activation of NF-κB is also discovered in lung, macrophages, and mouse embryonic fibroblasts of Nrf2-deficient mice after experimental sepsis [24].
T181 18891-19047 Sentence denotes Enhanced activation of NF-κB is also discovered in lung, macrophages, and mouse embryonic fibroblasts of Nrf2-deficient mice after experimental sepsis [24].
T7236 19048-19131 Sentence denotes Interestingly, it is reported that NF-κB can inhibit Nrf2 at transcriptional level.
T182 19048-19131 Sentence denotes Interestingly, it is reported that NF-κB can inhibit Nrf2 at transcriptional level.
T7237 19132-19302 Sentence denotes NF-κB p65 subunit repressed the Nrf2-ARE pathway at transcriptional level by competitive interaction with the CH1-KIX domain of CBP or local histone hypoacetylation [25].
T183 19132-19302 Sentence denotes NF-κB p65 subunit repressed the Nrf2-ARE pathway at transcriptional level by competitive interaction with the CH1-KIX domain of CBP or local histone hypoacetylation [25].
T7238 19303-19483 Sentence denotes All these findings indicate the potential complicate crosstalk between NF-κB and Nrf2, which may be regulated by the upstream mitogen-activated protein kinase (MAPKs) pathway [26].
T184 19303-19483 Sentence denotes All these findings indicate the potential complicate crosstalk between NF-κB and Nrf2, which may be regulated by the upstream mitogen-activated protein kinase (MAPKs) pathway [26].
T7239 19484-19575 Sentence denotes It has been confirmed that Nrf2 is mainly detected in nucleus of astrocytes after TBI [18].
T185 19484-19575 Sentence denotes It has been confirmed that Nrf2 is mainly detected in nucleus of astrocytes after TBI [18].
T7240 19576-19793 Sentence denotes In view of the fact that Nrf2 is a transcription factor which should take function mainly in nucleus, it can be reasoned that astrocytes may be one kind of respondent cells in activation of Nrf2-ARE pathway after TBI.
T186 19576-19793 Sentence denotes In view of the fact that Nrf2 is a transcription factor which should take function mainly in nucleus, it can be reasoned that astrocytes may be one kind of respondent cells in activation of Nrf2-ARE pathway after TBI.
T7241 19794-19935 Sentence denotes Previous study identified the expression of TNF-α, IL-1β, and IL-6 in cultured astrocytes after treatment with LPS or oxyhemoglobin [27, 28].
T187 19794-19935 Sentence denotes Previous study identified the expression of TNF-α, IL-1β, and IL-6 in cultured astrocytes after treatment with LPS or oxyhemoglobin [27, 28].
T7242 19936-20089 Sentence denotes Another study revealed that after middle cerebral artery occlusion, MMP9-positive astrocytes were observed in brain tissues by immunohistochemistry [29].
T188 19936-20089 Sentence denotes Another study revealed that after middle cerebral artery occlusion, MMP9-positive astrocytes were observed in brain tissues by immunohistochemistry [29].
T7243 20090-20179 Sentence denotes Those results indicate the role of astrocytes in inflammatory process after brain injury.
T189 20090-20179 Sentence denotes Those results indicate the role of astrocytes in inflammatory process after brain injury.
T7244 20180-20304 Sentence denotes But till now, there is no study focused on the relationship among astrocytes, Nrf2, and proinflammatory mediators after TBI.
T190 20180-20304 Sentence denotes But till now, there is no study focused on the relationship among astrocytes, Nrf2, and proinflammatory mediators after TBI.
T7245 20305-20457 Sentence denotes Our results demonstrated the upregulated expression of TNF-α, IL-1β, IL-6, and MMP9 in Nrf2 knockout astrocytes after scratch injury for the first time.
T191 20305-20457 Sentence denotes Our results demonstrated the upregulated expression of TNF-α, IL-1β, IL-6, and MMP9 in Nrf2 knockout astrocytes after scratch injury for the first time.
T7246 20458-20639 Sentence denotes In conclusion, depletion of Nrf2 induced the activation of NF-κB and the expression of TNF-α, IL-1β, IL-6, and MMP9 resulting in more cell deaths in astrocytes after scratch injury.
T192 20458-20639 Sentence denotes In conclusion, depletion of Nrf2 induced the activation of NF-κB and the expression of TNF-α, IL-1β, IL-6, and MMP9 resulting in more cell deaths in astrocytes after scratch injury.
T7247 20640-20739 Sentence denotes These results suggest that Nrf2 may be an important target for anti-inflammatory therapy after TBI.
T193 20640-20739 Sentence denotes These results suggest that Nrf2 may be an important target for anti-inflammatory therapy after TBI.
T194 20741-20878 Sentence denotes Figure 1 Loss of Nrf2 enhanced cell death after scratch. (a) Cultured astrocytes scratched or not were observed under microscope (×100).
T195 20879-21000 Sentence denotes There was more cell death in group KO TBI. (b) CCK-8 analysis revealed lower OD value in scratched astrocytes (P < 0.01).
T196 21001-21156 Sentence denotes Group KO TBI showed significant lower OD value than group WT TBI (P < 0.01). (*P < 0.01, compared with sham counterpart; # P < 0.01, compared with WT TBI).
T197 21157-21404 Sentence denotes Figure 2 EMSA analysis for DNA-binding activity of NF-κB. (a) Representative film shows DNA-binding activity of NF-κB in Nrf2 WT or KO astrocytes 24 h after scratch or not. (b) Quantification of NF-κB DNA-binding activity was performed by ImageJ.
T198 21405-21636 Sentence denotes NF-κB DNA-binding activity in astrocytes increased after scratch (P < 0.01) and was prominently greater in group KO TBI than in group WT TBI (P < 0.01). (*P < 0.01, compared with sham counterpart; # P < 0.01, compared with WT TBI).
T199 21637-22072 Sentence denotes Figure 3 RT-PCR analysis of mRNA level of proinflammatory cytokines and MMP9. (a) RT-PCR showed that mRNA level of TNF-α, IL-1β, IL-6, and MMP9 was elevated in astrocytes after scratch and was significantly higher in group KO TBI than in group WT TBI. (b–e) Quantitative analysis of RT-PCR results showed that relative mRNA level of proinflammatory cytokines mentioned above and MMP9 was higher in astrocytes after scratch (P < 0.01).
T200 22073-22232 Sentence denotes Such elevation was more severe in group KO TBI than in group WT TBI (P < 0.01).  (*P < 0.01, compared with sham counterpart; # P < 0.01, compared with WT TBI).
T201 22233-22304 Sentence denotes Figure 4 ELISA analysis of protein level of proinflammatory cytokines.
T202 22305-22444 Sentence denotes ELISA results revealed protein level of these cytokines were elevated in astrocytes after scratch as compared with sham control (P < 0.01).
T203 22445-22645 Sentence denotes And they were much higher in Nrf2 KO astrocytes than in their WT counterparts (P < 0.01). (a) TNF-α. (b) IL-6. (c) IL-1β  (*P < 0.01, compared with sham counterpart; # P < 0.01, compared with WT TBI).
T204 22646-23002 Sentence denotes Figure 5 Western blot analysis of MMP9 protein level. (a) Western blot showed that protein level of MMP9 was elevated in astrocytes after scratch and was significantly higher in group KO TBI than in group WT TBI. (b) Quantitative analysis of western blot result showed that relative protein level of MMP9 was higher in astrocytes after scratch (P < 0.01).
T205 23003-23161 Sentence denotes Such elevation was much severe in group KO TBI than in group WT TBI (P < 0.01). (*P < 0.01, compared with sham counterpart; # P < 0.01, compared with WT TBI).
T206 23162-23409 Sentence denotes Figure 6 Gelatin zymography of MMP9 activity. (a) Gelatin zymography analysis showed higher MMP9 activity in group KO TBI than in group WT TBI. (b) Quantitative analysis showed that MMP9 activity was higher in astrocytes after scratch (P < 0.01).
T207 23410-23568 Sentence denotes Such elevation was much severe in group KO TBI than in group WT TBI (P < 0.01). (*P < 0.01, compared with sham counterpart; # P < 0.01, compared with WT TBI).
T208 23569-23617 Sentence denotes Table 1 Primers and parameters used in RT-PCR.