Article Figures & Data
Figures
- Figure 1.
Serum CORT levels produced by CORT/cholesterol pellets in rats treated with saline (SAL) or LPS. Results are presented as mean ± SEM (n = 6 animals per group). Bonferroni test: *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 2.
Influence of CORT levels on NF-κB activation induced by LPS or saline (SAL) in the frontal cortex and hippocampus. Top, Representative EMSA autoradiography of frontal cortex (A) and hippocampus (B). NF-κB-specific band is indicated by arrow. Representative autoradiography from Western blot assay of the translocation of p65-NF-κB subunit in frontal cortex (C) and hippocampus (D). Bottom, Densitometric analysis of the NF-κB-specific band (representing p65/p50 heterodimers) or cytosolic and nuclear ratio presented in the top (mean ± SEM; n = 5 animals per group). Bonferroni test: **p < 0.01; ***p < 0.001.
- Figure 3.
Competition studies and supershift assays were performed on nuclear extract from frontal cortex (A) and hippocampus (B) of rats implanted with CORT pellets and treated with saline or LPS (1 mg/kg, i.v., 2 h) in the absence or presence of unlabeled specific (NF-κB consensus sequence, 15-fold molar excess) or nonspecific (TFIID consensus sequence, 15-fold molar excess) oligonucleotide, as indicated. Supershift assays were performed with the same nuclear extract (10 μg) incubated in the absence and presence of antibodies against subunits p50 (1:20 dilution), p65 (1:20 dilution), and cRel (1:20 dilution), as indicated. The position of specific NF-κB/DNA binding complex p50/p65 is indicated. NS represents nonspecific binding. The localization of the probe is also indicated. Results are representative of three independent experiments.
- Figure 4.
Influence of CORT levels on proinflammatory genes (IL-1β and TNF-α) in the absence (SAL) or presence of LPS in the frontal cortex (A, C) and hippocampus (B, D). Real-time PCRs were performed in duplicate and results are represented as mean ± SEM; n = 6 animals per group. Bonferroni test: *p < 0.05; **p < 0.01; ***p < 0.001 vs INT/LPS.
- Figure 5.
Influence of CORT levels on anti-inflammatory genes (IκB-α, IL-1ra, and MKP-1) in the absence (SAL) or presence of LPS in the frontal cortex (A, C, E) and hippocampus (B, D, F). Real-time PCRs were performed in duplicate and results are represented as mean + SEM; n = 6 animals per group. Bonferroni test: *p < 0.05; **p < 0.01; ***p < 0.001. For MKP-1: ###p < 0.001 vs INT/SAL; **p < 0.05 vs INT/LPS.
- Figure 6.
Influence of CORT levels on phosphorylated forms of MAP kinases 42/44 MAP kinase (A), p38 MAP kinase (B), SAPK/JNK (C), and AKT (D) in the absence (SAL) or presence of LPS in the frontal cortex. Top, Representatives of Western blot autoradiographies. Bottom, Densitometric analysis of the specific bands of SAL- or LPS-treated groups presented in the top panels (mean ± SEM; n = 5 animals per group). Protein levels are presented as ratios of specific kinases to β-actin levels. Bonferroni test: *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 7.
Influence of CORT levels on phosphorylated forms of MAP kinases 42/44 MAP kinase (A), p38 MAP kinase (B), SAPK/JNK (C), and AKT (D) in the absence (SAL) or presence of LPS in the hippocampus. Top, Representatives of Western blot photographs. Bottom, Densitometric analysis of the specific bands of SAL- or LPS-treated groups presented in the top (mean ± SEM; n = 5 animals per group). Protein levels are presented as ratios of specific kinases to β-actin levels. Bonferroni test: *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 8.
Schematic of the proposed role of high CORT levels on the proinflammatory response triggered by LPS and related to MAP kinase–NF-κB pathway in rat brain. Normally, LPS binds to TLR4 and activates the MAP kinase pathway, thus activating NF-κB and increasing expression of proinflammatory cytokines. Basal levels of GCs inhibit this pathway through a number of steps, including (1) GR interacting with the p65 subunit of NF-κB, (2) increasing the expression of the inhibitory protein IκB-α, and (3) increasing expression of anti-inflammatory genes such as IL-1ra and MKP-1 (A). However, when GC exposure is elevated and prolonged, the hormone increases and potentiates the proinflammatory response related to MAP kinase–NF-κB pathway (B). Straight lines indicate activation and dotted lines indicate inhibition.
Tables
Primer Sequence TNF-α FW: TCTGCTTGGTGGTTTGCTACGAC RV: TGAGTACCTGGACTTGCAGAACG IL-1β FW: GGGTTCCATGGTGAAGTCAAC RV: AAGACCTTCTACCTGAGGAACAACC IκB-α FW: TGTAGATGCCTCTCCAAGGATGG RV: CTCATGGACTGATTATGGACAGGAC IL1ra FW: GCCCAAGAACACATTCCGAAAGTC RV: AAATGGGCTCCCTCTCATCAGTTC MKP-1 FW: ACTTCCCTGTCTCCATCCCT RV: ACGTGAAACTCCACACCTCC HPRT FW: CTCATGGACTGATTATGGACAGGAC RV: GCAGGTCAGCAAAGAACTTATAGCC FW, Forward; RV, reverse.
- Table 2.
Summary of the main CORT effects on frontal cortex (top) and hippocampus (bottom) of rats treated with saline (SAL) or LPS
SAL LPS PRO Mixed NO ANT PRO Mixed NO ANT Cortex NF-κ B ✓ ✓ IL-1β ✓ ✓ TNF-α ✓ ✓ Iκ B-α ✓ ✓ IL-1ra ✓ ✓ MKP-1 ✓ ✓ 42/44 MAPK ✓ ✓ SAPK/JNK ✓ ✓ p38 MAPK ✓ ✓ AKT ✓ ✓ Hippocampus NF-κ B ✓ ✓ IL-1β ✓ ✓ TNF-α ✓ ✓ Iκ B-α ✓ ✓ IL-1ra ✓ ✓ MKP-1 ✓ ✓ 42/44 MAPK ✓ ✓ SAPK/JNK ✓ ✓ p38 MAPK ✓ ✓ AKT ✓ ✓ PRO, Proinflammatory effects for all CORT levels; Mixed, pro- and anti-inflammatory effects dose-dependent; NO, no CORT effects in any level; ANTI, anti-inflammatory effects for all CORT levels.
