Inhibition of BET Protein Function Suppressed the Overactivation of the Canonical NF-κB Signaling Pathway in 6-OHDA-Lesioned Rat Model of Levodopa-Induced Dyskinesia

Abstract

Background: Neuroinflammation is involved in the mechanisms of levodopa-induced dyskinesia (LID). The canonical NF-κB activation signaling pathway plays a critical role in the neuroinflammation development and BET protein-induced NF-κB-mediated neuroinflammation. The inhibition of the BET protein function has been reported to alleviate LID; however, its association with the canonical NF-κB signaling pathway in the 6-OHDA-lesioned striatum of the LID rat model remains unknown. Accordingly, we identified the status of the canonical NF-κB signaling pathway in the 6-OHDA-lesioned striatum of the LID rat model and whether the anti-dyskinetic effect of the BET inhibitor JQ1 was associated with its suppression on NF-κB-mediated neuroinflammation.

Methods: 6-OHDA PD rat models were treated with either L-dopa plus JQ1 or L-dopa alone. L-dopa treatment was given for 2 weeks, and the JQ1 treatment was given for 3 weeks and was initiated a week prior to L-dopa treatment. As a control, the sham rats were treated with JQ1 or Veh for 3 weeks. The ALO AIM assessment and cylinder test were performed during the treatment. Glial activation markers, pro-inflammatory substances, and critical proteins in the canonical NF-κB signaling pathway were tested in the lesioned striatum after the final treatment.

Results: JQ1 effectively alleviated LID without influencing motor improvement. In the lesioned striatum, L-dopa triggered an overactivation of the canonical NF-κB signaling pathway, with an increase in the phospho-IKKα/β, phospho-IκBα, and NF-κB nuclear translocation and its phosphorylation at Ser 536 and Ser 276 sites (p < 0.01 vs. sham group). L-dopa induced an overexpression of the pro-inflammatory substances of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase (iNOS), and the glial activation markers CD68 and GFAP. All the molecular changes were greatly inhibited by JQ1.

Conclusion: L-dopa triggered an overactivation of the canonical NF-κB signaling pathway, leading to an enhanced neuroinflammation response in the 6-OHDA-lesioned striatum of LID rat models. The inhibition of the BET protein function significantly suppressed the activation of the canonical NF-κB signaling pathway in the striatum, alleviating the neuroinflammation response and the severity of LID.

Keywords: NF-κB signaling; inhibition of BET protein function; intervention; levodopa induced dyskinesia; neuroinflammation.

FIGURE 1

Schematic summarizing the experimental design. At 21 days after surgery, each group was administered with different intervention. Saline-lesioned sham rats were, respectively, treated with corresponding Veh (i.p.) (Veh group) or JQ1 (25 mg kg^–1, i.p.) (JQ1 treatment group) once a day for 21 days; the 6-OHDA-lesioned PD rats were randomly divided into the L-dopa group and the L-dopa + JQ1 group. From day 1 to day 21, the two groups were correspondingly treated once daily (9 a.m.) with Veh or JQ1 (25 mg kg^–1, i.p.) for 21 days; from day 8 to day 21, both the two groups were immediately treated with L-dopa (L-dopa, 12 mg kg^–1, bensarazide, 6 mg kg^–1) (i.p.) immediately after Veh or JQ1 administration. About 1 h after drug administration on day 21, all rats were anesthetized for scarification for tissue collection. Veh: an equal amount of DMSO diluted in carrier solution; S: scarification.

FIGURE 2

Dyskinetic movement assessment of the 6-OHDA-lesioned PD rats during the treatment with L-dopa (L-dopa, 12 mg kg^–1, bensarazide, 6 mg kg^–1, i.p., once a day) (L-dopa group, n = 18) and L-dopa (L-dopa, 12 mg kg^–1, bensarazide, 6 mg kg^–1, i.p., once a day) plus JQ1 (25 mg kg^–1, i.p., once a day) (L-dopa + JQ1 treatment group, n = 18). Measures of axial (A), limb (B), orolingual (C), and the total ALO AIMs (D) in the rats treated with L-dopa and L-dopa + JQ1 on each assessment during the 21 days of treatment. During the period, the 6-OHDA-lesioned rats in the PD + L-dopa group showed a robust increase in the scores of the total ALO AIM assessment and each of the subitems, with apparent dyskinetic movements. By contrast, this phenomenon was apparently inhibited in 6-OHDA-lesioned PD rats treated with L-dopa plus JQ1 (L-dopa + JQ1 treatment group). The scores of total ALO AIM assessment and its subitems in the L-dopa treatment group were all significantly higher than in the L-dopa + JQ1 treatment group at each assessment (p < 0.001). Data are presented as mean ± SD; **p < 0.001 vs. PD + L-dopa group.

FIGURE 3

Motor improvement of 6-OHDA-lesioned rat models of PD treated with L-dopa (L-dopa, 12 mg kg^–1, bensarazide, 6 mg kg^–1, i.p., once a day) (L-dopa treatment group, n = 18), L-dopa (L-dopa, 12 mg kg^–1, bensarazide, 6 mg kg^–1, i.p., once a day) plus JQ1 (25 mg kg^–1, i.p., once a day) (L-dopa + JQ1 treatment group, n = 18), or Veh (n = 6) was assessed by cylinder tests on days 10, 14, and 18 of the treatment. At each assessment, both the L-dopa group and the L-dopa + JQ1 group showed a better performance as the treated 6-OHA-lesioned PD rats preferred to touch the inner wall of the cylinder with the impaired forelimbs more frequently (p < 0.001 vs. Veh group). Meanwhile, no difference was found in the rats’ preference in touching the inner wall with the impaired forelimbs in cylinder tests between the L-dopa and L-dopa + JQ1 treatment groups (p > 0.05). Data are presented as mean ± SD; **p < 0.001 vs. Veh treatment.

FIGURE 4

Changes in the TH protein level extracted from the saline- or 6-OHDA-lesioned striatum after 21 days of administration. Both L-dopa treatment group and L-dopa + JQ1 treatment groups showed a significant reduction in the TH protein level in the rats’ ipsilateral striatum after 21 days of treatment (p < 0.001 vs. Veh group). No difference was found in the TH protein level between L-dopa and L-dopa + JQ1 treatment groups (p > 0.05). TH levels expression relative to α-tubulin levels. The data are expressed in terms of mean ± SD and as a percentage of the Veh group. **p < 0.001 vs. Veh group (n = 3 per group).

FIGURE 5

Changes in the expression of ERK1/2 and its phosphorylation extracted from the saline- or 6-OHDA-lesioned striatum after 21 days of administration. Both the L-dopa and L-dopa + JQ1 treatment groups showed an aberrant increase in the protein level of phoshpo-ERK1/2 in the 6-OHDA-lesioned striatum of rats after 21 days of treatment (p < 0.001 vs. Veh group). Moreover, the increase in phospho-ERK1/2 in the L-dopa treatment group was greater than that in the L-dopa + JQ1 treatment group (p < 0.01). In addition, JQ1 barely affected the expression of phospho-ERK1/2 in the saline-lesioned striatum in the JQ1 treatment group (p > 0.05 vs. Veh group). The ERK1/2 protein level remained unchanged among the four treatment groups. All the aforementioned protein levels expressed relative to α-tubulin levels. The data are expressed in terms of mean ± SD and as a percentage of the Veh group. **p < 0.001 vs. Veh group, ^#p < 0.01 vs. L-dopa treatment group (n = 3 per group).

FIGURE 6

Changes in the mRNA (A–D) and protein (E–H) levels of pro-inflammatory mediators extracted from the saline- or 6-OHDA-lesioned striatum after 21 days of administration. Both L-dopa and the L-dopa + JQ1 treatment groups showed an aberrant increase in both the mRNA and protein levels of the TNF-α, IL-1 β, IL-6, and iNOS in the 6-OHDA-lesioned striatum of rats after the 21 days of treatment (p < 0.001 vs. Veh group). Moreover, the expression level in the L-dopa treatment group was much higher than that in the L-dopa + JQ1 treatment group (p < 0.01). JQ1 barely affected the expression level of the four pro-inflammatory substances in the saline-lesioned striatum of the JQ1 treatment group (p > 0.05 vs. Veh group). The data are expressed in terms of mean ± SD and as a percentage of the Veh group, **p < 0.001 vs. Veh group; ^#p < 0.01 vs. L-dopa treatment group, ^##p < 0.001 vs. L-dopa treatment group (n = 4 per group for RT-PCR; n = 5 per group for ELISA).

FIGURE 7

Changes in the CD68 and GFAP expressions in the saline- or 6-OHDA-lesioned striatum after 21 days of administration. Both L-dopa and L-dopa + JQ1 treatment groups showed an aberrant increase in the protein level of CD68 and GFAP in the 6-OHDA-lesioned striatum of rats after 21 days of treatment (p < 0.01 vs. Veh group). The protein level of CD68 and GFAP in the L-dopa treatment group was much higher than that in the L-dopa + JQ1 treatment group (p < 0.01). JQ1 barely affected the CD68 and GFAP protein expressions in the saline-lesioned striatum of the JQ1 treatment group (p > 0.05 vs. Veh group). The protein levels expressed relative to α-tubulin levels. The data are expressed in terms of mean ± SD and as a percentage of the Veh group. **p < 0.001 vs. Veh group, *p < 0.01 vs. Veh group, ^#p < 0.01 vs. L-dopa treatment group (n = 3 per group).

FIGURE 8

Changes in the expression of IKKα/β, phospho-IKKα/β, IκBα, and phospho-IκBα in the saline- or 6-OHDA-lesioned striatum after 21 days of administration. Intermittent L-dopa stimulation induced a significant increase in the protein level of phospho-IKKα/β and phospho-IκBα in the 6-OHDA-lesioned striatum in both L-dopa and L-dopa + JQ1 treatment groups (p < 0.001 vs. Veh group). The protein level of phospho-IKKα/β and phospho-IκBα in the L-dopa treatment group was much higher than that in the L-dopa + JQ1 treatment group (p < 0.01). Notably, intermittent L-dopa stimulation induced an apparent reduction in the protein level of IκBα in both the L-dopa and L-dopa + JQ1 treatment groups (p < 0.001 vs. Veh group). The protein level of IκBα in the L-dopa treatment group was statistically lower than that in the L-dopa + JQ1 treatment group (p < 0.01). JQ1 barely affected the expression of phospho-IKKα/β, phospho-IκBα, and IκBα in the saline-lesioned striatum of JQ1 treatment group. The IKKα/β protein level remained unchanged among the four treatment groups. The protein levels expressed relative to α-tubulin levels. The data are expressed in terms of mean ± SD and as a percentage of the Veh group, **p < 0.001 vs. Veh group, ^#p < 0.01 vs. L-dopa treatment group (n = 3 per group).

FIGURE 9

Changes in the ratio of nuclear NF-κB p65 to cytoplasmic NF-κB p65 in the saline- or 6-OHDA-lesioned striatum after 21 days of administration. Intermittent L-dopa stimulation induced a significant increase in the ratio of nuclear NF-κB p65 to cytoplasmic NF-κB p65 in the 6-OHDA-lesioned striatum from both L-dopa and L-dopa + JQ1 treatment groups (p < 0.001 vs. Veh group). Moreover, the ratio of nuclear NF-κB p65 to cytoplasmic NF-κB p65 in L-dopa treatment group was statistically higher than that in the L-dopa + JQ1 treatment group (p < 0.001). JQ1 barely affected the ratio in the saline-lesioned striatum in the JQ1 treatment group. The protein levels expressed relative to α-tubulin levels. The data are expressed in terms of mean ± SD and as a percentage of the Veh group. **p < 0.001 vs. Veh group; ^#p < 0.01 vs. L-dopa treatment group (n = 6 per group, the repetition no. = 3).

FIGURE 10

Changes in the expression of NF-κB p65 and its phosphorylation (Ser 536, Ser 276) extracted from the saline- or 6-OHDA-lesioned striatum after 21 days of administration. Both the L-dopa group and the L-dopa + JQ1 treatment groups showed an aberrant increase in the protein level of phospho-NF-κB p65 (Ser 536 and Ser276) in the 6-OHDA-lesioned striatum of rats after 21 days of treatment (p < 0.001 vs. Veh group). Moreover, the increase in phospho-NF-κB p65 at the two sites in the L-dopa treatment group was greater than in the L-dopa + JQ1 treatment group (p < 0.01). In addition, JQ1 barely affected the expression of phospho-NF-κB p65 (Ser 536 and Ser276) in the saline-lesioned striatum in JQ1 treatment group (p > 0.05 vs. Veh group). The NF-κB p65 protein level remained unchanged among the four treatment groups. All the aforementioned protein levels expressed relative to α-tubulin levels. The data are expressed in terms of mean ± SD and as a percentage of the Veh group, **p < 0.001 vs. Veh group, ^#p < 0.01 vs. L-dopa treatment group (n = 3 per group).