Table 1.
Sequence of mouse mdr1a (Abcb1a; GenBank accession No.: NC_000071.5) promoter.
Fig 1.
Expression and distribution of HMGB1 in the KA-induced mouse epileptic brain tissues.
(A&B) Immunohistochemical study detecting HMGB1 in the CA1 (A) and CA3 (B) hippocampal regions from the NS group mice (n = 5). HMGB1 immunoreactivity was detected mostly in the nuclei of the pyramidal neurons. (C&D) Immunohistochemical study detecting HMGB1 in the CA1 (C) and CA3 (D) hippocampal regions from the EP group mice (n = 6). Cytoplasmic staining of HMGB1 was substantially increased in the pyramidal neurons compared with the NS group. Glial cells with both nuclear and cytoplasmic staining were also found. Scale bar: 50 μm. Red arrows: neurons with nuclear staining; Blue arrows: glial cells with nuclear staining; Red arrowheads: neurons with both nuclear and cytoplasmic staining; Blue arrowheads: glial cells with both nuclear and cytoplasmic staining. EP, KA-induced epileptic seizure group; HMGB1, high-mobility group box-1; KA, kainic acid; NS, normal saline control group.
Fig 2.
Pre-treatment with HMGB1 enhanced KA-induced up-regulation of P-gp in the epileptic brain.
(A) Immunohistochemical staining of P-gp in CA1 hippocampal areas of each group mice (NS group, n = 5; EP group, n = 6; EP+BoxA group, n = 6; EP+BoxA group, n = 6). HMGB1 injection enhanced the over-expression of P-gp in the EP group mice while BoxA attenuated it. Scale bar: 50 μm. NS: normal saline control group; EP: KA-induced epileptic seizure group; EP+HMGB1: EP group pretreated with HMGB1; EP+BoxA: EP group pretreated with BoxA. Green arrows: vascular endothelial cells; Red arrows: neurons; Blue arrows: glial cells; Yellow bidirectional arrows: Stratum pyramidale (sp). (B) Western blotting detects the protein levels of HMGB1 and P-gp in the mice brains of each group 24 h after seizure onset (B left panel, n = 6). Protein levels were quantified and normalized to that in the NS group (B right panel). Injection of HMGB1 and BoxA increased and decreased the protein levels of HMGB1 and P-gp in the EP group mice respectively. *P<0.05 vs. NS group; #P<0.05 vs. EP group. HMGB1, high-mobility group box-1; KA, kainic acid; P-gp, P-glycoprotein.
Table 2.
The staining intensity of P-gp in the CA1 regions from mice in different groups.
Fig 3.
HMGB1 had no significant effect on the viability of bEnd.3 cells.
Cells were treated with different concentrations of HMGB1 for the indicated time periods. Cell viability was determined using CCK8 assay. Data from three independent experiments was shown as mean±SD; n = 3. CCK8, cell counting kit; HMGB1, high-mobility group box-1; SD, standard deviation.
Fig 4.
HMGB1 upregulated the expression of P-gp, TLR4, and RAGE in bEnd.3 cells.
(A&B) The mRNA levels of mdr1a (A) and TLR4 (B) in bEnd.3 cells treated with different concentrations of HMGB1 were measured using q-PCR and normalized to the cells treated without HMGB1. HMGB1 at all tested concentrations (from 10 to 500 ng/mL) increased mdr1a mRNA and TLR4 mRNA levels in bEnd.3 cells as compared to cells treated without HMGB1. (C&D) The protein levels of P-gp (C), TLR4, and RAGE (D) in bEnd.3 cells treated with different concentrations of HMGB1 were analyzed using Western blotting with β-actin as a loading control. Protein levels were quantified and normalized to the cells treated without HMGB1. Treatment with HMGB1 at all tested concentrations increased P-gp, TLR4, and RAGE protein levels in bEnd.3 cells. Data were shown as mean±SD; n = 3. *P<0.05, **P<0.01 vs. cells treated with medium only. HMGB1, high-mobility group box-1; mdr1a, multidrug resistance1a; P-gp, P-glycoprotein; SD, standard deviation; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products.
Fig 5.
HMGB1 enhanced phosphorylation and nuclear translocation of NF-κB p65 in bEnd.3 cells.
(A) NF-κB p65 subcellular distribution in bEnd.3 cells treated with HMGB1 for indicated durations was observed using immunofluorescence staining. HMGB1 promoted cytoplasmic to nuclear translocation of NF-κB p65 in bEnd.3 cells. Representative pictures are shown. Scale bar: 100 μm. Red arrows: cells with nuclear NF-κB p65 staining. (B) Protein levels of phosphor-NF-κB p65 and NF-κB p65 in the cytoplasm and nuclear fractions from bEnd.3 cells treated with HMGB1 for 60 min. H3 and β-actin were utilized as loading controls for nuclear and cytoplasmic proteins, respectively. Protein levels were normalized to the cells treated without HMGB1. HMGB1 increased cytoplasmic p-NF-κB p65 and nuclear NF-κB p65 levels in bEnd.3 cells. Data were shown as mean±SD; n = 3. *P<0.05 vs. cells treated with medium only. HMGB1, high-mobility group box-1; NF-κB, nuclear factor-kappa B; SD, standard deviation.
Fig 6.
Pre-treatment with LPS-RS, FPS-ZM1, or SN50 attenuated HMGB1-induced up-regulation of P-gp in bEnd.3 cells.
(A) The mRNA levels of mdr1a in bEnd.3 cells treated with HMGB1 plus different inhibitors. Inhibition of TLR4, RAGE, and NF-κB attenuated HMGB1-induced up-regulation of mdr1a mRNA in bEnd.3 cells. LPS-RS: TLR4 antagonist; FPS-ZM1: RAGE inhibitor; SN50: NF-κB nuclear translocation inhibitor. (B&C) The protein levels of P-gp in bEnd.3 cells treated with HMGB1 plus different inhibitors. Representative examples are shown (B) and protein levels were normalized to the cells treated without HMGB1 (C). Inhibition of TLR4, RAGE, and NF-κB attenuated HMGB1-induced up-regulation of P-gp protein in bEnd.3 cells. Data were shown as mean±SD; n = 3. *P<0.05, **P<0.01. HMGB1, high-mobility group box-1; LPS-RS, lipopolysaccharide from Rhodobacter sphaeroides; mdr1a, multidrug resistance1a; NF-κB, nuclear factor-kappa B; P-gp, P-glycoprotein; SD, standard deviation; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products.
Fig 7.
TLR4 and RAGE were involved in HMGB1-induced activation of NF-κB in bEnd.3 cells.
(A) Nuclear NF-κB p65 protein levels in bEnd.3 cells treated with HMGB1 plus different inhibitors. Inhibition of TLR4 and RAGE resulted in a significant decrease in HMGB1-induced NF-κB p65 translocation to nuclei. (B) NF-κB DNA-binding activity in bEnd.3 cells treated with HMGB1 plus different inhibitors detected by EMSA. HMGB1 increased NF-κB p65 binding activity to DNA in bEnd.3 cells while inhibition of TLR4 or SN50 attenuated this effect of HMGB1. Data were shown as mean±SD; n = 3. **P<0.01. HMGB1, high-mobility group box-1; EMSA, electrophoretic mobility shift assay; LPS-RS, lipopolysaccharide from Rhodobacter sphaeroides; NF-κB, nuclear factor-kappa B; SD, standard deviation; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products.
Fig 8.
Exogenous expression of NF-κB p65 activated the mdr1a promoter in bEnd.3 cells and the predictive NF-κB binding site was essentially involved in this process.
(A) Promoter constructs used in the study. -1 represents the first nucleotide upstream the transcription start site. Plasmid pGL3-mdr1a-luc included wild-type mdr1a promoter fragment and pGL3-NFκBmut-mdr1a-luc included an eleven-nucleotide mutation in the putative NF-κB binding site in mdr1a promoter. (B) Luciferase activity in transfected bEnd.3 cells was measured 72 h after the cotransfection of reporter plasmids and p65 expression vector. Overexpression of p65 in bEnd.3 cells led to a significant increase in mdr1a promoter activity as compared to its counterpart control. Mutation of the potential NF-κB binding site in mdr1a promoter abolished the promotor activation. Data were shown as mean±SD; n = 3. **P<0.01, compared with promotorless pGL3.0-Basic vector; #P<0.05, compared with pcDNA3.1 negative control. NF-κB, nuclear factor-kappa B.
Fig 9.
A proposed model of P-gp up-regulation by HMGB1 in epileptic brain.