PMC:3317373 / 11660-15415 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3317373","sourcedb":"PMC","sourceid":"3317373","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3317373","text":"3. Results\n\n3.1. Depletion of Nrf2 Aggravated the Cell Death in Astrocytes after Scratch Injury\nCell death was detected by microscope and CCK-8 analysis at 24 h after scratch. The detachment from culture plate, cell lost, and cytoplasmic process distortion were mostly observed in both sides of scratch line in group KO TBI. 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 \u003c 0.01), which also suggested more cell death in group KO TBI (Figure 1). There was no difference between two sham-operated groups.\n\n3.2. Disruption of Nrf2 Enhanced Upregulation of NF-κB DNA-Binding Activity after Scratch Injury\nIt has been reported that NF-κB is activated in brain after TBI [2]. 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. 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 \u003c 0.01) (Figure 2).\n\n3.3. Expression of Proinflammatory Cytokines Was Elevated in Nrf2 Knockout Astrocytes after Scratch Injury\nAs we all know, TNF-α, IL-1β, and IL-6 are regulated by NF-κB and reflect the endogenous activity of NF-κB. It has been revealed that these proinflammatory cytokines are increased after TBI in animals [2, 8]. 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]. But it is still ambiguous about the relationship between these cytokines and Nrf2 in astrocytes after TBI. 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. 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. 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. P \u003c 0.01) (Figures 3(a)–3(d)). 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. And it was also higher in group KO TBI than in group WT TBI (3.67 ± 0.156 versus 2.31 ± 0.087, P \u003c 0.01) (Figure 4(a)). Similar tendency was observed in protein level of IL-6 and IL-1β. For IL-6, it was 36.07 ± 0.786 for group KO TBI while 25.76 ± 0.536 for group WT TBI (P \u003c 0.01) (Figure 4(b)). For IL-1β, it was 190.75 ± 6.339 for group KO TBI while 154.50 ± 5.348 for group WT TBI (P \u003c 0.01) (Figure 4(c)).\n\n3.4. Expression and Activity of MMP9 Were Greatly Enhanced in Nrf2 Knockout Astrocytes after Scratch Injury\nMMP9 is an important gelatinase to induce or aggravate the inflammation process. 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. Expression of MMP9 was elevated after scratch injury in WT and KO astrocytes as compared with their sham counterparts. 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 \u003c 0.01) (Figures 3(a) and 3(e)). 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 \u003c 0.01) (Figure 5). 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 \u003c 0.01) (Figure 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