Low Molecular Weight Sericin Enhances the In Vitro of Immunological Modulation and Cell Migration

Abstract

Sericin, a waste product of the silk textile industry, has favorable physicochemical and biological properties. In this study, we extracted a low molecular weight (MW) sericin (LMW-sericin; below 10 kDa) by a performing high-temperature and high-pressure method and confirmed the MW using matrix-assisted laser desorption ionization-time of flight and liquid chromatography-mass spectrometry. Furthermore, we determined its biological effects on macrophages and human adipose stem cells (hASCs) as cell models to investigate the biocompatibility, immunomodulation behavior, and potential signaling pathway-related wound healing via analyses of gene expression of focal adhesion and human cytokines and chemokines using quantitative real-time polymerase chain reaction and cytokine assay. LMW-sericin showed good biocompatibility both in macrophages and hASCs. Macrophages cultured with 0.1 mg/ml LMW-sericin displayed an improved inflammatory response shown by the upregulation of CXCL9, IL12A, BMP7, and IL10, which developed Th1 and Th2 balance. LMW-sericin also improved the differentiation of macrophages toward the M2 phenotype by significantly enhancing the expression of Arg-1, which is conducive to the repair of the inflammatory environment. Moreover, the gene expression of hASCs showed that LMW-sericin promoted the secretion of beneficial adhesion molecules that potentially activate the gene transcription of differentiation and migration in hASCs, as well as significantly enhanced the levels of PKCβ1, RhoA, and RasGFR1 as fruitful molecules in wound healing. These findings provide insights into LMW-sericin application as a potential biomaterial for wound management.

Keywords: adipose stem cell; cell signaling pathway; immune response; inflammatory; low molecular weight; macrophage; sericin.

FIGURE 1

Molecular weight (MW) analysis of filtered sericin solution. (A) Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of low MW sericin solution. [z = 1; m/z = m (Da)]; (B,C) liquid chromatography–mass spectrometry (LC-MS) of low MW sericin solution at major peak result and minor peak result, respectively. [z = 1; m/z = m (Da)].

FIGURE 2

Fourier-transform infrared (FT-IR) spectroscopy low molecular weight of sericin solution. (A) IR spectra; (B) second-derivative and curve-fitted spectra, illustrated at the upper and the bottom of the figure, respectively. Ten Lorentzian curves (red solid line) were de-convoluted from the amide I band (dash line).

FIGURE 3

Cell viability of macrophage cultured with low molecular weight of sericin solution. (n = 3; *p < 0.05, **p < 0.01).

FIGURE 4

Heatmap of genes associated with inflammation of macrophages treated with low molecular weight of sericin solution for 24 and 72 h. This heatmap was built using DESeq 2 on normalized gene read counts. All the log values of the dispersion estimates were clustered using the R distance function (dist) to calculate the Euclidean distance between samples. Distance plot showing 24 and 72 h treatment with sericin-treated versions. Red: upregulated gene; blue: downregulated gene.

FIGURE 5

Macrophage polarization in response to LMW-sericin treatment after 24 h. (A) Immunofluorescence staining images of macrophages stained for DAPI (blue), M1 surface marker iNOS (red), and M2 surface marker Arg-1 (green); (B) Semi-quantitative analysis of macrophages expression by each marker. This analysis was automatically performed by ZEN 2.6 lite blue software; (C) Flow cytometry analysis of M1/M2 macrophages. (1 = PMA + RPMI-1640; 2 = PMA + sericin 0.1 mg/ml; 3 = PMA + sericin 1.0 mg/ml; 4 = PMA + LPS 10 ng/ml; scale bar = 20 μm; n = 6 per group per condition, six random fields per well; *p < 0.05).

FIGURE 6

Cell viability of human adipose stem cells (hASCs) cultured with low molecular weight of sericin solution. (n = 3; *p < 0.05, ***p < 0.001).

FIGURE 7

Heatmap of genes associated with adhesion molecule of human adipose stem cells treated with low molecular weight of sericin solution for 24 and 72 h. This heatmap was built using DESeq 2 on normalized gene read counts. All the log values of the dispersion estimates were clustered using the R distance function (dist) to calculate the Euclidean distance between samples. Distance plot showing 24 and 72 h treatment with sericin-treated versions. Red: upregulated gene; blue: downregulated gene.

FIGURE 8

mRNA expression of protein kinase enzymes including PKCβ1, RhoA, and RasGFR1 generated by human adipose stem cells (hASCs) cultured with low molecular weight of sericin solution for 72 h (*p < 0.05).

FIGURE 9

Human adipose stem cells (hASCs) in response to low molecular weight of sericin solution treatment after 72 h. (A) Immunocytochemistry staining images of hASCs stained for DAPI and PKCβ1, RhoA, and RasGFR1 markers, respectively (scale bar = 20 μm); (B) quantitative analysis of positive hASC expression by each marker; (C) cell migration assay (scale bar = 50 μm). (*p < 0.05, **p < 0.01, and ***p < 0.001; n = 6 per group per condition, six random fields per well).

FIGURE 10

Schematic illustration of the immune response according to the gene expression of macrophages cultured with 0.1 mg/ml low molecular weight of sericin solution for a 3-day incubation.