Epigallocatechin-3-Gallate: A potential amyloid Fibril Disaggregator of Serum amyloid A1

Abstract

Serum amyloid A1 (SAA1) is a 122-amino acid protein that, after cleavage, matures into a 104-amino acid form. Its N-terminus is responsible for binding high-density lipoprotein (HDL), while the C-terminus maintains its structural integrity. As an acute-phase protein, SAA1 is produced by the liver in response to acute inflammation. SAA1 is also a precursor to amyloid A (AA), and its accumulation can lead to AA amyloidosis-a condition secondary to chronic inflammation that causes tissue damage and organ dysfunction. Our study explores methods to disaggregate SAA1 fibrils isolated from the cat spleen, chicken liver, and cow liver. Specifically, we investigate the use of epigallocatechin-3-gallate (EGCG), a polyphenolic flavonoid extracted from green tea known for its anti-inflammatory and antioxidant properties, to disaggregate these fibrils. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to analyze these fibrils after treatment with 1 % DMSO and 400 μM of EGCG in 10 mM PBS (pH 7.4). The results demonstrated that EGCG effectively reduced fibril size, as confirmed by DLS characterization, with the disappearance or diminished prominence of the 10^3-4 nm peak. Additional TEM results confirmed that EGCG disaggregated amyloid-beta fibrils isolated from Alzheimer's disease brains. These findings suggest that compounds like EGCG could be valuable in treating inflammatory and neurodegenerative conditions by disaggregating amyloid fibrils.

Keywords: AA amyloidosis; Amyloid A (AA); Dynamic light scattering (DLS); Epigallocatechin-3-gallate (EGCG); Serum amyloid A1 (SAA1); Transmission electron microscopy (TEM).

Graphical abstract

Fig. 1

Photomicrographs of the cat spleen, chicken liver, and cow liver histological sections stained with hematoxylin & eosin (H&E), Congo red (CR, polarized light) and SAA immunohistochemistry confirmed the presence and nature of amyloid deposits. Pictures were acquired at a magnification of 20X. Black scale bar: 100 μm; White scale bar: 500 μm.

Fig. 2

Dynamic light scattering (DLS) analysis of SAA1 fibril samples extracted from post-mortem specimens: cat spleen, chicken liver, and cow liver. The samples were incubated at 37 °C for seven (cat), six (chicken), and five (cow) days in either 1 % DMSO or 400 μM EGCG, prepared in 10 mM PBS (pH 7.4). The samples were centrifuged at 150,000 rpm for 15 min, and the supernatant was analyzed with the Zetasizer Pro (Malvern). DLS graphs of the intensity (percent) in function of the size of particles (diameter, nm) resulting from the supernatant of the A) DMSO-treated cat spleen SAA1 fibrils; B) EGCG-treated cat spleen SAA1 fibrils; C) DMSO-treated chicken liver SAA1 fibrils; D) EGCG-treated chicken liver SAA1 fibrils; E) DMSO-treated cow liver SAA1 fibrils; F) EGCG-treated cow liver SAA1 fibrils.

Fig. 3

Transmission electron microscopy (TEM) images of chicken and cow liver SAA1 fibrils after incubation with 1 % DMSO or 400 μM EGCG in 10 mM PBS (pH 7.4) at 37 °C for 5 days, using a magnification of 40k. The samples were centrifuged for 15 min at 150,000 rpm, and the supernatant was analyzed. High resolution images (40k magnification) of the A) DMSO-treated chicken liver SAA1 fibrils; B) EGCG-treated chicken liver SAA1 fibrils; C) DMSO-treated cow liver SAA1 fibrils; D) DMSO-treated cow liver SAA1 fibrils; E) EGCG-treated cow liver SAA1 fibrils; F) EGCG-treated cow liver SAA1 fibrils. Scale bar: 200 nm.

Fig. 4

Transmission electron microscopy (TEM) images of SAA1 fibrils of the cat spleen, chicken liver, and cow liver after incubation with 1 % DMSO or 400 μM EGCG in 10 mM PBS (pH 7.4) at 37 °C for 5 days, using a magnification of 40k. Samples were not centrifuged. Total protein concentration for samples analyzed are as follows: cat spleen – 178.8 μg/mL; cow liver – 252.5 μg/mL; chicken liver – 140.6 μg/mL. A) TEM image of the DMSO-treated cat spleen SAA1 fibrils. B) TEM image of the EGCG-treated cat spleen SAA1 fibrils. C) TEM image of the DMSO-treated chicken liver SAA1 fibrils. D) TEM image of the EGCG-treated chicken liver SAA1 fibrils. E) TEM image of the DMSO-treated cow liver SAA1 fibrils. F) TEM image of the EGCG-treated cow liver SAA1 fibrils. The arrows indicate examples of amyloid fibrils. Scale bar: 200 nm.

Fig. 5

Measurement of SAA1 fibrils of the cat spleen, chicken liver, and cow liver in 1 % DMSO or 400 μM EGCG in 10 mM PBS (pH 7.4) after 5 days incubation at 37 °C. The samples were not centrifuged. Total protein concentration for samples analyzed are as follows: cat spleen – 178.8 μg/mL; chicken liver – 140.6 μg/mL; cow liver – 252.5 μg/mL. The surface areas of the cat spleen, chicken liver, and cow liver aggregates were analyzed using ImageJ and TEM images at a magnification of 20k (A–C). In addition, the surface areas of the aggregates for the cat, chicken, and cow samples were analyzed using TEM images at 2500 magnification (D–F). The histograms allow for comparison of the surface areas as measured by ImageJ of the SAA1 aggregates resulting from DMSO- and EGCG-treated A) cat spleen SAA1 fibrils (high magnification); B) chicken liver SAA1 fibrils (high magnification); C) cow liver SAA1 fibrils (high magnification); D) cat spleen SAA1 fibrils (high magnification); E) chicken liver SAA1 fibrils (high magnification); F) cow liver SAA1 fibrils (low magnification). ∗p < 0.05 by unpaired one-tailed t-test.

Fig. 6

Transmission electron microscopy (TEM) images of amyloid plaques isolated from Alzheimer's disease (AD) brains after incubation in 0.25 % DMSO or 50 μM EGCG in 10 mM PBS (pH 7.4) at 37 °C for five days. The samples were not centrifuged prior to grid preparation. The extraction consisted of 0.5 ± 0.1 mg/mL of proteins. A) TEM image of DMSO-treated Aβ dense aggregates isolated from AD brains at high magnification (40k). Scale bar 200 nm. B) TEM image of EGCG-treated amyloid plaques isolated from the AD brain at high magnification (40k). Scale bar 200 nm. C) TEM image of DMSO-treated Aβ dense aggregates isolated from AD brains at low magnification (2500). Scale bar 2 μm. D) TEM image of EGCG-treated Aβ dense aggregates isolated from the AD brain at low magnification (2500). Scale bar: 2 μm.