Inhibition of cytotoxic fibril formation of α-synuclein and human insulin by Silymarin from the Silybum marianum

Abstract

Silymarin (SIL), the extract obtained from the seeds of milk thistle (Silybum marianum), contains several flavonolignans with a broad range of therapeutic properties such as antioxidant, anti-inflammatory, and neuroprotective effects. Despite several studies indicating the neuroprotective effects of SIL in relating to neurodegenerative diseases (NDs), there is no report regarding the anti-amyloidogenic activity and the mechanism of action of SIL in vitro. Here, we have extracted SIL from the seeds of milk thistle (SIL A), followed by investigating its potential, in comparison with SIL purchased from Sigma company (SIL B), in modulating fibrillogenesis and cytotoxicity of human insulin and α-synuclein (α-syn) amyloid fibrils. The obtained results indicated the potency of both SIL A and SIL B in inhibiting the assembly process and related cytotoxicity of both proteins but via different mechanisms, including inhibition of amyloid fibrillation with the appearance of short fibrils for human insulin and redirecting the assembly process of α-syn toward the formation of small globular structures. The higher inhibitory effects of SIL B may be attributed to its higher silybin content, which is responsible for the most biological, including anti-amyloidogenic, activities of SIL B. Nanonization increased the capacity of both SILs to inhibit fibrillation and related cytotoxicity of both proteins. Taken together, these results may suggest SIL A as a potent candidate relating to NDs and highlight nanonization as a promising approach to increase its anti-amyloidogenic properties.

Fig 1. LC-MS profile of SIL A.

The presence of key SIL components, including (A) silybin A and B, silydianin, silychristin, and isosilybin A and B, and (B) taxifolin are indicated.

Fig 2. Spectroscopic characterization of SIL A, SIL B, and their respective nanoparticles.

(A and B) The UV − vis spectra of SIL A/Nano A and SIL B/Nano B, respectively, prepared in a final concentration of 20 μg/mL. (C and D) The fluorescence emission spectra of SIL A and SIL B, respectively, excited at different wavelengths. Further details are provided in the text.

Fig 3. Microscopic characterization of Nano A and Nano B.

(A and B) AFM images, and (C and D) HR-TEM images of Nano A and Nano B, respectively. In AFM images the scale bars represent 500 nm.

Fig 4. DPPH-based antioxidant activity of SIL A, SIL B, and their respective nanoparticles.

The results are calculated as a fraction of 25 µg/mL ascorbic acid.

Fig 5. The effect of (A) SIL A, (B) SIL B, (C) Nano A, and (D) Nano B on the kinetics of human insulin amyloid fibrillation monitored by increasing fluorescence intensity of ThT.

Protein samples were incubated at 57 °C while begin stirred at 250 rpm. The ThT fluorescence signal was recorded in 20 min intervals, but for simplifying the figure, signals in the plateau phase are indicated at 1 h intervals. The solid lines show fitting developed by AmyloFit [47].

Fig 6. The effect of SIL A/Nano A and SIL B/Nano B on (A) amyloid fibrillation and (B) surface hydrophobicity of human insulin monitored by ThT and NR fluorescence microscopies, respectively.

The protein samples were incubated under amyloidogenic condition either alone or with increasing concentrations of compounds for 15 h, followed by microscopy imaging. The scale bars represent 500 nm.

Fig 7. The effect of SIL A, SIL B, Nano A, and Nano B on the amyloid fibrillation of

α-syn. (A-D) The kinetics of α-syn amyloid fibril formation monitored by measuring fluorescence intensity of ThT in the presence of increasing concentrations of SIL A, SIL B, Nano A, and Nano B, respectively. The solid lines show fitting developed by AmyloFit [47]. (E) AFM images of α-syn samples incubated alone or with increasing concentrations of SIL A, SIL B, Nano A, and Nano B for 96 h. The scale bars represent 500 nm.

Fig 8. Protective effects of SIL A, SIL B, and their respective nanoparticles against cytotoxicity induced by human insulin and

α-syn aggregates measured by MTT assay. (A and B) Cytotoxicity evaluation of human insulin and (C and D) α-syn aggregates produced in the absence or presence of increasing concentrations of SIL A, SIL B, or their respective nanoparticles, respectively. ^#p < 0.01, significantly different from control cells. ^*p < 0.01, significantly different from cells exposed only to human insulin or α-syn amyloid fibrils.