Oral Administration of Silybin Protects Against MPTP-Induced Neurotoxicity by Reducing Pro-inflammatory Cytokines and Preserving BDNF Levels in Mice

Abstract

Parkinson's disease (PD) is the second most frequent neurodegenerative disease associated with motor dysfunction secondary to the loss of dopaminergic neurons in the nigrostriatal axis. Actual therapy consists mainly of levodopa; however, its long-term use promotes secondary effects. Consequently, finding new therapeutic alternatives, such as neuroprotective molecules, is necessary. Among these alternatives is silybin (Sb), the major bioactive flavonolignan in silymarin. Both exert neuroprotective effects, preserving dopamine levels and dopaminergic neurons when administered in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse PD model, being probably Sb the potential therapeutic molecule behind this effect. To elucidate the role of Sb in the PD model, we determined the dose-dependent conservation of striatal dopamine content following Sb oral administration. Then, we evaluated motor deficit tests using the best dopamine conservative dose of Sb and determined a cytokine-dependent inflammatory profile status, malondialdehyde as an oxidative stress product, and neurotrophic factors content in the MPTP-induced mouse PD model. Our results show that oral Sb at 100 mg/kg dose conserved about 60% dopamine levels. Also, Sb improved motor deficits, preserved neurotrophic factors content and mitochondrial function, reduced lipid peroxidation, diminished proinflammatory cytokines to basal levels, enhanced fractalkine production in the striatum and substantia nigra, and increased IL-10 and IL-4 levels in the substantia nigra in the MPTP mice. Thus, oral Sb may be a potential pharmacological PD treatment alternative.

Keywords: BDNF; Fractalkine; Neuroinflammation; Neuroprotection; Parkinson’s disease; Silybin.

Fig. 1

Silybin treatment attenuates MPTP-induced dopamine loss in the striatum. a C57BL6/J male mice were treated with intraperitoneal MPTP 30 mg/Kg doses daily followed by silybin (Sb) administration orally 30 min after each MPTP exposition for five consecutive days. Damage parameters assays were performed three days after the last MPTP administration; behavior tests were evaluated six days after the MPTP scheme; last HPLC and ELISA assays were performed seven days after the MPTP treatment. Motor behavior studies consist of three tests: b pole test, which evaluates bradykinesia and gross motor skills; c traction test to measure muscle strength, balance, and traction skills; and d beam test, which evaluates fine motor skills. e Striatal dopamine content was measured from mice administered with vehicles (C), MPTP (M), or MPTP coadministered with five different oral Sb dosages ranging from 100 to 500 mg/kg (MS100, MS200, MS300, MS400, and MS500). Data represent mean ± SEM (n = 4-5) and were analyzed by ANOVA, followed by Bonferroni post hoc test. **** p < 0.0001, ** p ≤ 0.01, and * p ≤ 0.05 compared to the M group

Fig. 2

Sb improves motor behavior in MPTP-induced PD mice. A pole test was performed to evaluate bradykinesia and gross motor skills by measuring the time for turning down (T-turn; a, n = 6–8) and time for landing (T-LA; b, n = 6–8 ). A traction test was performed to evaluate balance, muscle strength, and fine motor skills by measuring escape latency (c, n = 7–8), Deacon’s scale (d, n = 7–8), escape ratio (e, n = 7–8), and traction ratio (f, n = 7–8). The beam test was performed to evaluate balance behavior and to confirm fine motor skills changes together with the traction test by measuring cross latency (g, n = 6–8), error count (h, n = 7–8), relative cross error (i, n = 7–8), and escape ratio (j, n = 7–8). Mice were treated with vehicles (C), silybin (S100), MPTP (M), or MPTP coadministered with silybin (MS100). Data (a, b, e, f, g, j) are presented as median with interquartile range and were analyzed by Kruskal-Wallis test with Dunn post hoc comparisons. Data (c, d, h, i) are presented as mean values ± SEM and were analyzed by One-way ANOVA with Tukey post hoc comparisons. **** p < 0.0001, *** p ≤ 0.001, and ** p ≤ 0.01, compared to M. # p ≤ 0.05 C compared to the MS100 group

Fig. 3

Sb treatment improves mitochondrial function, attenuates lipid peroxidation, and preserves neurotrophic factor levels in striatum and substantia nigra. Analysis of mitochondrial reduction power and availability in brain tissue by MTT Assay was performed in PD mice. Relative MTT-reduction percentages compared to the control group in the striatum (a, n = 8) and substantia nigra (b, n = 4) are shown. Quantification of malondialdehyde as a final lipid peroxidation product by the TBARS assay was also measured. Relative TBARS percentages compared to the control group in the striatum (c, n = 8) and substantia nigra (d, n = 4) were obtained. Neurotrophic factor levels were measured by ELISA from both neuronal areas. BDNF content in the striatum (e, n = 6–8) and substantia nigra (f, n = 7–8), and IGF-1 levels in the striatum (g, n = 5) and substantia nigra (h, n = 5) are presented. Mice were treated with vehicles (C), silybin (S100), MPTP (M), or MPTP coadministered with silybin (MS100). Data (a, b, c, d) are presented as means ± SEM and were analyzed by one-way ANOVA with Tukey post hoc comparisons. Data (e, f, g, h) are presented as median with interquartile range and were analyzed by Kruskal-Wallis test with Dunn post hoc comparisons. **** p < 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05 compared to the M group. ++++ p <0.0001, +++ p ≤ 0.001, and ++ p ≤ 0.01 compared to the MS100 group. ### p ≤ 0.001 compared to the S100 group

Fig. 4

Sb treatment reduces MPTP-induced neuroinflammation by TNFα, IL-1β, and IL-6 and enhances IL-4 and fractalkine levels in MPTP-induced PD mice. Cytokine profiles were measured in the striatum (a, c, e, g, i, k) and substantia nigra (b, d, f, h, j, l) of PD mice. TNFα (a, n = 7–8; b, n = 7–8), IL-6 (c, n = 6; d, n = 6), IL-1β (e, n = 5; f, n = 5), IL-10 (g, n = 6; h, n = 6), IL-4 (i, n = 6; j, n = 6), and fractalkine (k, n = 6; l, n = 6) were determined in homogenized brain tissues by capture ELISA. Mice were treated with vehicles (C), silybin (S100), MPTP (M), or MPTP co-administered with silybin (MS100). Data (a, b, c, d, e, f, g, h, i, j) are presented as means ± SEM and were analyzed by one-way ANOVA with Tukey post hoc comparisons. Data (k, l) are presented as median with interquartile range and were analyzed by Kruskal-Wallis test with Dunn post hoc comparisons. **** p < 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05 compared to the M group. ++ p ≤ 0.01, and + p ≤ 0.05 compared to the MS100 group

Fig. 5

Oral administered silybin alleviates PD motor and biochemical outcomes in the MPTP-induced PD mice model. Sb preserves striatal dopamine content and improves bradykinesia, fine and gross motor skills, muscle strength, balance behavior, and motor coordination, acting as a neuroprotective agent by enhancing mitochondrial reduction power, neutralizing oxidative stress detected by MDA levels, diminishing neuroinflammation directed by TNFα, IL-1β, and IL-6, enhancing IL-10, IL-4 and fractalkine responses and preserving BDNF, and IGF-1 content in PD model mice