
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00285-4
10.1016/j.redox.2024.103307
103307
Research Paper
Unravelling the antioxidant behaviour of self-assembly β-Sheet in silk fibroin
Qian Zhiyong ab1
Sun Chang b1
Li Qianqian b
Xie Yafan c
Zhan Lingpeng d
Liu Xiangli b
Wang Guanbo d
Wei Yen weiyen@tsinghua.edu.cn
ef⁎
Qiu Juhui jhqiu@cqu.edu.cn
c⁎⁎
Peng Qin pengqin@szbl.ac.cn
b⁎⁎⁎
a Department of Anatomy the Basic Medicine College, Inner Mongolia Medical University, Hohhot, 010000, Inner Mongolia, China
b Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, 518132, China
c Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400030, China
d Institute for Chemical Biology, Shenzhen Bay Laboratory, Shenzhen, 518132, China
e The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing, 100084, China
f School of Materials Science and Engineering, North Minzu University, Yinchuan, 750021, China
⁎ Corresponding author. The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing, 750021, China. weiyen@tsinghua.edu.cn
⁎⁎ Corresponding author. Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400030, China. jhqiu@cqu.edu.cn
⁎⁎⁎ Corresponding author. pengqin@szbl.ac.cn
1 contributed equally to this report.

20 8 2024
10 2024
20 8 2024
76 10330717 7 2024
8 8 2024
© 2024 Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Local oxidative stress in diseases or injury severely hinders cell homeostasis and organ regeneration. Antioxidant therapy is an effective strategy for oxidative stress treatment. Biomaterials with good biocompatibility and reactive oxygen species (ROS) scavenging ability are good choices for antioxidant therapeutics. However, there are few natural biomaterials that are identified with both biocompatibility and strong antioxidant activity. Here, we show, for the first time, that silk fibroin (SF) is a strong antioxidant, which can eliminate ROS in both cells and zebrafish. We further demonstrate that the β-sheet structures turn into a random coiled structure when SF is treated with hydrogen peroxide. The content of β-sheet structures can be increased by heating, thus enhancing the antioxidation properties of SF. Therefore, SF can serve as a good antioxidant biomaterial for therapeutics, and its β-sheet structure-based antioxidation mechanism provides a novel theoretical basis, which could be a new cue for more antioxidant biomaterial discovery and identification.

Graphical abstract

Image 1

Highlights

• Silk fibroin solution is a strong antioxidant in vitro and in vivo.

• β-Sheet structures in silk fibroin are responsible for the antioxidant effect.

• The antioxidant property of silk fibroin is tunable by heating.

Keywords

Oxidative stress
Reactive oxygen species
Antioxidant activity
Silk fibroin
β-sheet structures
==== Body
pmc1 Introduction

Oxidative stress is the major problem leading to disease and ageing, so antioxidation is the key to the treatment of many diseases, such as neurodegeneration, atherosclerosis, ageing, and even cancer [1,2]. Oxidative stress depends mainly on the massive accumulation of reactive oxygen species (ROS) [3] and intensifies inflammatory responses, and then oxidative stress and inflammation form a vicious cycle. ROS are chemically reactive molecules containing oxygen, including hydrogen peroxide (H2O2), superoxide anion radical (O2−•) and hydroxyl radical (•OH) [4]. The irreversible oxidative damage of biological macromolecules, such as oxidized proteins, lipids, and nucleic acids, triggers a variety of cell dysfunctions and then induces diseases [5,6]. Antioxidants, such as glutathione (GSH), can maintain the original function of cells by improving the cell growth of the microenvironment or directly ameliorating the oxidative stress state of cells [7,8] and further boost the activity of cells for therapeutic applications [9].

The ideal goal of disease therapy and regenerative medicine is to achieve perfect tissue repair and functional recovery [10], and antioxidation is necessary for biomaterial-based tissue engineering products. At present, biomaterials have multiplex applications in many fields, such as vascular scaffolds [11,12], bone [13], and skin [10]. However, less attention has been given to the antioxidation of biomaterials. Collagen has good antioxidant function [14] but is accompanied by some side effects, such as allergies [15], which limits its extensive applications. Hence, biomaterials possessing both good antioxidation effects and good biocompatibility are still urgently needed in regenerative medicine [16].

Silk fibroin (SF) is a natural protein-based biopolymer with hierarchical mesoscopic structures containing secondary structures, β-crystallites and nanofibrils [17]. Because of its excellent mechanical properties and biocompatibility, SF has been widely used in biomedical fields, such as scaffolds in tissue engineering and regenerative medicine [18]. The β-sheet structure is necessary for machinability, biocompatibility, and biodegradability in SF and can be altered by heat treatment [19]. A recent report showed that SF can modulate the hypoxic tumour microenvironment, and the peptide brain factor-7 from SF can inhibit ROS generation in the brain [20]. Furthermore, SF-based biomaterials with rich β-sheet structures have a good repair effect [21,22], and all cysteines in SF form disulfide bonds to connect the heavy chain and the light chain [23]. However, whether SF has a potential function in alleviating ROS and the related mechanism is still elusive [24].

In this study, we demonstrated that SF with β-sheet structures is a strong antioxidant in vitro and in vivo through systematic experimental investigations (Fig. 1). The antioxidant capacity of SF in a concentration-dependent manner is observed in both biochemical assays and intracellular ROS scavenging. Due to the high molecular weight of SF, representative polypeptides e.g. (GAGSGA)2 and GAGVGAGY from SF heavy chains were chosen for the mechanistic study of the antioxidant effect. The results showed that the β-sheet structures of SF and corresponding peptides are responsible for antioxidant activity, and the β-sheet structures turn into a random coiled structure upon oxidation. In addition, SF gains stronger antioxidant properties as the content of β-sheet structure is increased by heating. Therefore, this study revealed a new antioxidant mechanism of SF and laid a foundation for the application of SF in the future.Fig. 1 Schematic diagram of SF with an antioxidant function and its mechanism. (A) Natural SF can efficiently scavenge intracellular ROS. Further study found that the β-sheet structure in SF was transformed into a random coil structure after being oxidized. (B) SF can efficiently scavenge •OH, H2O2 and O2−•. (C) The β-sheet structure increased after heating and turned into a random coil structure after being oxidized. (D) The antioxidation of SF could reduce the ROS in living cells and animals.

Fig. 1

2 Materials and methods

2.1 Preparation of SF solution

The degummed silk fibrion was obtained via degumming Bombyx mori silk in 0.02 M Na2CO3 with 30 min extraction. This process was repeated three times to ensure that the sericin proteins were completely removed. After drying, the extracted silk fibroin was dissolved in 9.3 M LiBr solution at 80 °C for 1h, yielding a 10 % (w/v) solution. The solution was dialyzed with a dialysis bag for 72 h to remove salt ions. The desalted silk fibroin solution was collected and centrifuged at 9800 G/min for 20 min at 4 °C to remove silk aggregates [25]. The final concentration of silk solution was determined using a BCA protein assay kit.

To investigate the temperature effect on the self-assembly of SF at nanoscale level, the SF aqueous solutions (20 mg/mL) were incubated at 35 °C, 45 °C, 55 °C, 65 °C, 75 °C, 85 °C and 95 °C for 30 min [26].

2.2 •OH scavenging activity

OH was generated using the Fenton reaction between Fe2+ and H2O2, which can react with 3,3′,5,5′-tetramethylbenzidine (TMB) to form a soluble blue compound with a characteristic absorption at 652 nm. In this study, the TMB chromogenic method was used to detect •OH scavenging activity [27]. In detail, Fenton reagent containing 400 μM FeSO4 and 10 mM H2O2 at room temperature was prepared in the dark for 10 min. Next, 200 μL TMBLS, 200 μL Fenton reagent, and 200 μL different biomaterials, including SF, glycol chitosan (GCTS), hyaluronic acid (HA), collagen (COL), gelatine (GEL), sodium alginate (SA), hydroxypropyl methyl cellulose (HPMC), gellan gum (GUM) and GSH (the concentration was 1 mg/mL), were reacted at 37 °C for 30 min. Then, the absorbance peak of the solution at 652 nm was monitored with a microplate reader (Synergy h1, Biotek, Winooski, VT, USA).

OH scavenging activity for different concentrations of SF was evaluated by the TMB chromogenic method. In detail, 200 μL TMBLS, 200 μL Fenton reagent, and 200 μL different concentrations of SF (3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL, and 50 mg/mL) were reacted at 37 °C for 30 min. Then, the absorbance spectra from 450 nm to 750 nm were collected by a microplate reader.

2.3 H2O2 scavenging activity

The H2O2 scavenging capacity of different biomaterials was detected by the Hydrogen Peroxide Detection Kit [27]. H2O2 reacts with ammonium molybdate to form a yellow complex, which displays an absorbance peak at 405 nm. SF, GCTS, HA, COL, GEL, SA, HPMC, GUM and GSH (1 mg/mL) were incubated with 100 mM H2O2 at 37 °C for 1 h, respectively. After the reaction, the amount of residue was determined according to the kit instructions, and the H2O2 scavenging activity was calculated.

The H2O2 scavenging capacity for different concentrations of SF was tested by the Hydrogen Peroxide Detection Kit. In detail, different concentrations of SF (3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL, and 50 mg/mL) were incubated with 100 mM H2O2 at 37 °C for 1 h. After the reaction, the amount of residue was determined according to the kit instructions, and the H2O2 scavenging activity was calculated.

2.4 In cell ROS scavenging

The 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, MO, USA) is an oxidation-sensitive fluorescent probe that was used to detect the intracellular ROS level according to the published protocol [28]. Briefly, to investigate the ROS scavenging ability of different biomaterials, mouse embryonic fibroblasts NIH 3T3 cells were seeded into 6-well plates pretreated with 0.1 % gelatin overnight at a density of 5 × 105 cells per well. After 24 h of incubation, SF, GCTS, HA, COL, GEL, SA, HPMC, GUM and GSH (0.01 mg/mL) were added to each group of wells, and then, the cells were treated with 100 μM H2O2 and further incubated at 37 °C for 30 min. After the cells were rinsed two times with serum-free medium, each group of wells was cultured with a final concentration of 10 μM of DCFH-DA in serum-free medium in the dark at 37 °C for 20 min. Then, the cells were washed and imaged using a Confocal Microscope (Nikon A1, Tokyo, Japan), and then the intracellular ROS levels were quantified by using NIS-Element AR software (version 5.30.03). Cells without the addition of H2O2 served as the control group.

Furthermore, the ROS scavenging ability of (GAGSGA)2 was investigated using the same protocol with the DCFH-DA fluorescent probe.

The ROS scavenging ability of (GAGSGA)2 was tested by flow cytometry. NIH 3T3 cells (5 × 105) were seeded overnight on pretreated 6-well plates. (GAGSGA)2 was then added to the cells with H2O2 at 37 °C for 30 min. After DCFH-DA staining, the cells were collected and resuspended in cold phosphate-buffered saline (PBS) and sent for flow cytometry analysis. For each group, at least 5 × 104 cells were analysed. All flow cytometry experiments were performed by the Flow Cytometer (Attune NxT, Thermo Fisher, Singapore) and analysed by using FlowJo software (version 10.0.6).

2.5 RNA-seq

For RNA-Seq studies, 106 NIH 3T3 cells were treated with or without SF for 6 h in 37 °C 5 % CO2 and then collected in cold PBS. Total RNA was isolated using an E.Z.N.A. Total RNA Kit I (Omega, Fairfax, VA, USA). Libraries were constructed by Geneplus company (Beijing, China). An FDR cut-off of 0.05 and fold change more than 2 were used to determine significantly differentially expressed genes.

2.6 Characterization of thioflavine T (ThT) emission spectra

Fluorescence emission spectra were detected using a microplate reader. ThT emission spectra were collected from 475 to 550 nm with excitation at 430 nm. The fluorophore concentration was 0.5 μM for ThT. The concentration of SF was diluted to 0.5 mg/mL, and the concentration of polypeptide was diluted to 1 mg/mL.

2.7 Characterization of circular dichroism (CD) spectra

CD spectra were recorded from 190 to 260 nm using a spectrometer (J-1500-150, JASCO, Tokyo, Japan). Each spectrum was averaged from three consecutive scans. Samples were diluted to 0.5 mg/mL for SF and 1 mg/mL for peptide, and measured in a 0.1 mm path length quartz cuvette (Starna Cells, Inc., Atascadero, CA, USA). The scanning speed was 100 nm/min. The measured CD spectra were corrected by subtracting the spectra of deionized water (buffer) prior to analysis.

2.8 Characterization of attenuated total reflectance-Fourier transform (ATR-FTIR) spectra

After CD spectra measurements, FTIR absorption spectra in the range from 4000 to 400 cm−1 of the liquid samples were acquired on a spectrometer (VERTEX 70 V, Bruker Optics, Billerica, MA, USA) with an attenuated total reflectance (ATR) accessory. A two-compartment CaF2 sample cell with a 50-μm thick Teflon spacer was used for simultaneously loading the sample and reference. Samples of a concentration were 50 mg/mL (for SF) and 5 mg/mL (for peptide). The measured ATR-FTIR spectra were corrected by subtracting the spectra of deionized water (buffer) prior to analysis. Second derivative analysis based on the absorption spectra provides a better spectral resolution for a Gaussian type of line shape [29], and the negative peaks in the second derivative spectrum (with its magnitude corresponding to the absorption intensity) were used for the assignment of absorption peaks in the FTIR spectrum.

2.9 Characterization of atomic force microscopy (AFM)

AFM images were obtained using an AFM (Cypher S, Asylum Research, Morrisville, NC, USA). Samples were diluted to approximately 750 μg/mL (for SF) and 40 μg/mL (for peptide) to obtain the optimal feature densities on the substrate for topography imaging. A 10-μL aliquot of diluted sample was dropped on a freshly cleaved mica surface (ϕ = 10 mm, Ted Pella, Redding, CA, USA) and dried before imaging. All morphological characterization was performed by tapping mode in air at a scan rate of 2.0 Hz and a resolution of 256 × 256 pixels per image using AC160TS-R3 probes (Olympus, Tpkyo, Japan).

2.10 Characterization of transmission electron microscopy (TEM)

Samples (negatively stained with 3 % phosphotungstic acid for 1 min) were diluted to approximately 40 μg/mL and were characterized using a TEM (JEM-1200EX, JEOL Ltd., accelerating voltage of 120 kV; Tokyo, Japan).

2.11 The antioxidation of SF in zebrafish

Caudal fin amputation was established to remove the end of the tail using a scalpel [30]. Zebrafish larvae were anaesthetized in 40 μg/mL tricaine in E3 water. The fin fold wounds were treated with SF at a final concentration of 1 mg/mL at 28.5 °C for 30 min. After washing with E3 water, each group of zebrafish larvae was cultured with a final concentration of 20 μM dihydroethidium (DHE, KeyGEN BioTECH, Jiang Su, China) in E3 water in the dark at 28.5 °C for 30 min. DHE is an oxidation-sensitive fluorescent probe that was used to detect the intracellular ROS level. After washing with E3 water, the larvae were imaged using fluorescence microscope (ECLIPSE TS2, Nikon, Japan), and the intracellular ROS levels were quantified by NIS-Element AR software (version 5.30.03). Silk fibroin solution was added into E3 water, where zebrafish embryos and those with caudal fin amputation were kept. The numbers of inflammatory cells were counted around the injured caudal fin. Those without injured caudal fins were regarded as the control group, and those with injured caudal fins were regarded as the positive group.

2.12 Statistical analysis

The experiments in this study were repeated three times, and the data were expressed as mean ± standard deviation. Statistical analysis was conducted using the t-test by GraphPad Prism 8.0.1 software.

Statistically significant differences were presented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3 Results

3.1 SF has a strong antioxidant capacity in vitro and in cells

Biomaterials have the potential to enhance tissue repair and regeneration, but their function in ROS scavenging is still unclear. To examine the hypothesis that most biomaterials have strong antioxidant capacity, three representative types of ROS, H2O2, •OH, and O2−•, were selected to investigate the ROS scavenging activities of nine different biomaterials, including SF, GCTS, HA, COL, GEL, SA, HPMC, GUM and GSH. GSH serves as a positive control. Different biomaterials exhibited different efficiencies of scavenging H2O2 and •OH (Fig. 2A). GSH had the strongest ability to scavenge •OH attributed to its sulfhydryl on cysteine [31], which allows GSH has the most antioxidant groups in the same mass compared to the other 8 types of biomaterials. Both GCTS and HA had strong activities in scavenging H2O2 and •OH, mainly due to their positively and negatively charged properties, respectively. The antioxidant functions of COL and GEL are determined by many antioxidant amino acids with almost 20 % cysteine [32]. Notably, SF showed the strongest ability to scavenge H2O2, while its ability to scavenge •OH and O2−• was relatively weak among the nine biomaterials (Fig. 2A and Fig. S1).Fig. 2 Investigations on the antioxidation of different biomaterials in vitro and in cells. (A) H2O2 and •OH scavenging of different biopolymers. (B) Observations of ROS scavenging in NIH3T3 cells with different polymers using confocal microscopy (Scale bar = 100 μm). (C) Statistical analysis of ROS levels in b. ****P < 0.0001, NS, no significance, t-test, data represent the mean ± standard deviation (SD).

Fig. 2

The ROS scavenging efficiency of these biomaterials was further compared in NIH 3T3 cells. As shown in Fig. 2B, the intracellular ROS level of NIH 3T3 cells increased dramatically upon treatment with 100 μM H2O2 for 30 min, while the intracellular ROS level was obviously decreased when the cells were pretreated with different biomaterials (Fig. 2B and Fig. S2). Statistical analysis of ROS levels confirmed that intracellular ROS levels in the SF and GCTS groups were significantly lower than the intracellular ROS levels in the other seven groups (Fig. 2C). Therefore, through screening nine types of biomaterials, we identified that only SF performed the best ability in scavenging H2O2 and eliminating ROS both in vitro and in vivo. Additionally, the excellent property of SF in H2O2 scavenging does not rely on charges and cysteine [33], indicating that there might be a new mechanism in SF antioxidantion.

3.2 High antioxidant capacity of SF causes transcriptomic profile changes and reduces ROS and inflammation in Zebrafish

The SF solution was dialyzed to collect unfolded SF molecular solutions after 9.3 M LiBr degumming at 80 °C for 1 h [34]. As shown in Fig. 3A and Fig. S3, SF exhibited high •OH and H2O2 scavenging activity in a concentration-dependent manner. To further confirm the antioxidative properties of SF, we verified that different concentrations of SF could scavenge intracellular ROS. The results indicated that the scavenging effect of SF on intracellular ROS increased as SF concentration increased in NIH 3T3 cells (Fig. S4). In addition, SF can also effectively clear excess ROS in the bone marrow mesenchymal stem cells (BMSCs) stimulated by H2O2 in Fig. S5.Fig. 3 The antioxidation of SF in vitro and in vivo. (A) Concentration dependence of SF in scavenging •OH was measured by Fenton reagent. Data represent the mean ± SD. (B) Diagrammatic sketch of three-group RNA-seq. (C) Venn diagram of three groups of RNA-seq data. (D) and (E) Heatmap showing obvious changes in the expression of 57 genes (D) and 75 genes (E) rescued by SF from (C) (Color bar = log2(TPM+1)). (F) Schematic of SF scavenging ROS and inflammatory cells infiltration in the Tg(mfap4:EGFP) transgenic zebrafish caudal fin amputation model, ROS was detected using dihydroethidium (DHE) fluorescent probe. (G) Detection of scavenging ROS in caudal fin amputation after SF treatment (Scale bar = 50 μm). (H) Statistical analysis of ROS levels and inflammatory cells in G. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

To determine the SF antioxidant potential at the molecular level, we performed RNA-seq analysis of three groups of NIH3T3 cells, including the phosphate-buffered saline (PBS) only group, the H2O2 treated group, and both the H2O2 and SF treated groups (named SF group) (Fig. 3B). Venn diagram revealed that the SF group showed a less different expression pattern than the PBS group compared with the H2O2 group (Fig. 3C, Fig. S6 and Fig. S7). Fifty-seven genes upregulated by H2O2 and rescued by SF and 75 genes downregulated by H2O2 and rescued by SF were analysed and displayed in the heatmap (expression change >2, false discovery rate (FDR) < 0.05) (Fig. 3C–E). Generally, the expression levels of these genes in the SF group were similar to the expression levels of these genes in the PBS group, but they were significantly different in the H2O2 group. Notably, most of these differentially expressed genes (DEGs) were critical for ROS regulation in cells, implying that SF can rescue H2O2-induced ROS-related gene expression.

To further examine the antioxidant capacity of SF in vivo, we performed an ROS scavenging experiment in zebrafish embryos. First, we found that SF reduced H2O2-induced ROS production in early embryos at 11 h postfertilization (hpf) (Fig. S8). Caudal fin amputation in zebrafish is an effective endogenous sustained H2O2 accumulation model [35], and we determined the antioxidant capacity of SF in Tg(mfap4:EGFP) transgenic zebrafish larvae (Fig. 3F). High levels of ROS existed in cells along the edge of the amputated fin, and the ROS were immediately eliminated upon SF treatment for 30 min (Fig. 3G and H). Intriguingly, the number of mfap4-positive inflammatory cells was also significantly decreased in the stump region of the SF group (Fig. 3H). These results demonstrated that SF is a highly efficient antioxidant in vivo.

3.3 β-sheet structures mediate the antioxidant effect in SF and SF-derived peptides

The antioxidant mechanism of SF is strongly attractive because of its good antioxidant performance. However, it is difficult to test the antioxidant mechanism with intact SF because SF has a large molecular weight of over 5000 amino acids. Typically, the heavy chain of Bombyx mori SF is divided into tyrosine-rich amorphous sequences and highly repetitive amino acid sequences, as shown in Fig. 4A. Hence, we chose the most representative sequences of SF to explore its antioxidant mechanism: one was an octapeptide GAGVGAGY from the region containing amorphous sequences, and the other was a dodecapeptide GAGSGAGAGSGA (labelled (GAGSGA)2) from the region with repetitive sequences. The self-association of proteins to form higher-order oligomer structures in solutions was reported to be dependent on the concentration of salt (NaCl) [36] and mechanical shaking (600 rpm) [36]. Thus, (GAGSGA)2 and GAGVGAGY were dissolved in 5 mM phosphoric acid buffer solution (named (GAGSGA)2-8 h and GAGVGAGY-8 h) to shake for 8 h, and the controls were (GAGSGA)2 and GAGVGAGY dissolved in deionized water (named (GAGSGA)2-0 h and GAGVGAGY-0 h) under quiescent conditions. Comparing the antioxidant effect of the four peptides, the antioxidant effect of (GAGSGA)2-8 h was found to be significantly stronger than the antioxidant effect of the other groups (Fig. S9). Therefore, we chose (GAGSGA)2-8 h as a representative polypeptide for studying the antioxidant mechanism on SF. As shown in Fig. 4B and Fig. S10A, compared with the H2O2 group, the intracellular ROS level decreased significantly in the (GAGSGA)2-8 h group. There was no significant difference between (GAGSGA)2-8 h and SF in scavenging capacity at 1 mg/mL, although the scavenging capacity of (GAGSGA)2-8 h was lower than the scavenging capacity of SF at 0.01 mg/mL. We further confirmed by flow cytometry that both (GAGSGA)2-8 h group and SF group reduced ROS levels at the cellular level (Fig. 4C and Fig. S10B).Fig. 4 Studies on the antioxidant effect and structure of (GAGSGA)2 in silk after H2O2 treatment. (A) Hydrophobicity index of the Bombyx mori SF heavy chain, which is composed of a tyrosine-rich amorphous sequence (purple, blue and green) and 12 highly repetitive amino acid sequences (orange). (B) Statistical analysis of ROS levels in NIH 3T3 cells treated with SF and peptide. **P < 0.01, NS, no significance, t-test. Data represent the mean ± SD. (C) Flow cytometry detection of ROS levels in untreated and (GAGSGA)2-treated NIH 3T3 cells incubated with H2O2. (D) CD characterization of (GAGSGA)2. (E) ATR-FTIR characterization of (GAGSGA)2. (F) ATR-FTIR characterization of (GAGSGA)2 after treatment with H2O2. (G) Statistical analysis of secondary structure in untreated and H2O2-treated (GAGSGA)2. (H) AFM characterization of (GAGSGA)2 self-assembled after shaking for 8 h at 37 °C in water (Scale bar = 400 nm). (I) AFM characterization of (GAGSGA)2 after treatment with H2O2. (J) Representative negative-stain TEM characterization of (GAGSGA)2 self-assembled after shaking for 8 h at 37 °C in water (Scale bar = 50 nm). (K) Representative negative-stain TEM characterization of (GAGSGA)2 after treatment with H2O2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

To reveal the antioxidant mechanism of (GAGSGA)2, based on previous reports [[37], [38], [39]], we put forwards two hypotheses: (1) the amino acid in the (GAGSGA)2 sequence was oxidized by H2O2, and (2) the antioxidant function was achieved through structural changes. To test the first hypothesis, we examined the molecular weight variation of (GAGSGA)2 before and after being oxidized by H2O2. The peak was observed in Fig. S11A with a mass/charge value of 819.4, which should be attributed to (GAGSGA)2. After oxidation with H2O2, (GAGSGA)2 with a mass/charge value was still 819.4 (Fig. S11B). GAGVGAGY had no change in mass/charge value of 651.3 either after oxidation (Fig. S12). The results proved that the antioxidant activity of peptides was not resulted from the oxidation of their amino acids.

To test the other hypothesis, we analysed the secondary structures for both octapeptide and dodecapeptide: GAGVGAGY-0 h, GAGVGAGY-8 h, (GAGSGA)2-0 h and (GAGSGA)2-8 h. The high fluorescence intensity of ThT indicates the presence of a β-sheet structure [40]. The results showed that the high fluorescence intensity of ThT appeared at 495 nm in the (GAGSGA)2-8 h group. However, there was less fluorescence intensity of ThT in the (GAGSGA)2-0 h, GAGVGAGY-0 h and GAGVGAGY -8 h groups (Fig. S13A). The CD spectrum of (GAGSGA)2-8 h showed a positive band centred at 201 nm and a negative band centred at 220 nm (Fig. S13B), indicating a typical β-sheet structure [41]. However, GAGVGAGY-8 h exhibited a negative band centred at 197 nm, indicating a typical random coil structure [41].

To further reveal the β-sheet structure in antioxidation, the structure of (GAGSGA)2-8 h was investigated after treatment with H2O2 by using CD, ATR-FTIR spectroscopy, AFM and TEM. The CD spectra showed that the secondary structure of (GAGSGA)2 changed from a β-sheet structure to a random coil after oxidation (Fig. 4D). Structural characterization of (GAGSGA)2 exhibited β-sheet and β-antiparallel conformations (Fig. 4E and G), with the ATR-FTIR spectra depicting a sharp peak at 1619 cm−1 and 1700 cm−1 in the amide I band [42]. After oxidation by 500 mM H2O2 for 30 min, unexpected results showed that the β-sheet structure was reduced from 83 % to 76 %, and the β-antiparallel conformation was reduced from 17 % to 5 %, resulting in a random coil conformation of 19 % (Fig. 4F and G). The ATR-FTIR spectra were consistent with the spectra of the CD experiment. β-Sheet structures could form β-crystallite by orderly arrangement [43] and then form nanofibril structures [17]. The dodecapeptide (GAGSGA)2 assembled into regular nanowhisker-like superfibrils, as shown in Fig. 4H–J and Fig. S14A. The nanowhisker was changed from superfibrils to oligomers after oxidation (Fig. 4I–K and Fig. S14B). The results regarding the structure of the peptide are consistent with a previous report that the highly repetitive (GAGSGA)n sequence can form a pleated β-sheet structure [27]. The assembled β-sheet structure was further confirmed to have a strong antioxidation function, in which the intracellular ROS level of NIH 3T3 cells decreased significantly in the (GAGSGA)2-8 h group compared with the (GAGSGA)2-0 h group (Fig. S15). Putting together, (GAGSGA)2 plays an antioxidant role by changing its secondary structures, particularly β-sheet structure changes are responsible for its ROS scavenging rather than amino acid oxidation.

3.4 Polypeptides of SF repeats form folded structures with good antioxidant properties

To determine whether β-sheet structures were also responsible for the antioxidation of full-length SF, the H2O2-oxidized SF (labelled SF-H2O2) was examined by ThT assay, CD spectroscopy and ATR-FTIR spectra to confirm the changes at the secondary structure level. The ThT fluorescence intensity of SF, which is correlated with the contents of the β-sheet structure, decreased with an H2O2 concentration dependent manner from 0 M to 2 M (Fig. 5A and B and Fig. S16). SF exhibited more β-sheet structures, while SF-H2O2 showed a more random coil structure in the CD measurements (Fig. 5C). The secondary structure changes of SF were further characterized by ATR-FTIR spectra. The molecular structure of SF was composed of 24 % β-sheets, 35 % β-turns, 14 % β-antiparallel and 27 % random coil structures (Fig. 5D and F), while SF-H2O2 was composed of 19 % β-sheets, 21 % β-turns, 8 % β-antiparallel and 52 % random coil structures (Fig. 5E and F). Therefore, compared to SF, the β-sheet, β-turn and β-antiparallel structures in SF-H2O2 were reduced, while the random coil structure was increased up to 1.5 folds. The topological structure of SF displayed nanofibres with an orderly arrangement of β-sheet structures (Fig. 5G–I Fig. S17A and Fig. S18A), which changed from nanofibres to spherical oligomers after oxidation (Fig. 5H–J, Fig. S17B and Fig. S18B). Overall, our results demonstrated that β-sheet structures play the same role in SF antioxidant activity (Fig. 5K).Fig. 5 Study of the structural changes in SF antioxidants. (A) and (B) SF structure in a concentration-dependent manner with H2O2. Data represent the mean ± SD. (C) CD characterization of SF. The orange line shows a typical β-sheet structure of SF, and the blue line depicts a similar random coil secondary structure of SF after treatment with H2O2. (D) Characterization of the secondary structure of SF by ATR-FTIR spectra. (E) Characterization of the secondary structure of SF after oxidation by ATR-FTIR spectroscopy. (F) Histogram of SF secondary structure elements calculated from the amide I band in the ATR-FTIR spectra. (G) AFM characterization of SF, revealing a nanofibre-like structure (Scale bar = 400 nm). (H) AFM characterization of SF after oxidation using H2O2, showing the formation of microsphere-like oligomers. (I) and (J) Representative negative staining of the SF nanofibre-like and microsphere-like oligomers before and after oxidation, respectively (Scale bar = 50 nm). (K) Schematic diagram of secondary structure changes of SF after oxidation by H2O2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

3.5 Heat treatment of SF solution can increase its β-sheet structure and oxidation resistance

Since oxidation induced secondary structure changes in SF from β-sheets into random coils, we wondered whether the increase in β-sheet structures can promote stronger antioxidants in SF solution. There are some reported methods that can increase the β-sheet structure in the SF solution, such as heating and organic solvent treatment [[44], [45], [46]]. Heating was chosen here because of the low toxicity. SF was heated to 95 °C, which was named SF-95 °C. We first characterized the secondary structure changes of SF after oxidation by H2O2. The fluorescence intensity of ThT assay, which is correlated with the content of β-sheet structures on SF molecules, increased as the temperature increased from 4 °C to 95 °C (Fig. 6A). CD spectra further showed that more β-sheet structure in SF solution with higher temperature (Fig. 6B). ATR-FTIR spectra showed that the content of the β-sheet structure in SF-95 °C group reached as high as 60 %, more than twice that without heating, and could be oxidized into random coil and α-helix structures (Fig. 6C and Fig. S19B). The topological structure of SF was also changed after heating. Compared to unheated SF, the nanofibres in SF-95 °C were thinner, longer and denser (Fig. 6E, Fig. S20B and Fig. S21B). After oxidization, the topological structure of SF changed from dense fibres to ellipsoidal oligomers (Fig. 6F, Fig. S20C and Fig. S21C).Fig. 6 The increased β-Sheet in SF treated by heating induces strong antioxidant function. (A) Fluorescence spectra of ThT bound to the β-sheet structure of SF. The increase in the fluorescence intensity of ThT indicates a positive correlation between temperature and the content of β-sheet structure in SF solution. Data represent the mean ± SD. (B) CD spectra of SF among different temperature treatments, showing an increase in the β-sheet structure content with increasing temperature. (C) Histogram of SF secondary structure elements calculated from the amide I band in the ATR-FTIR spectra under different conditions. (D) AFM characterization of SF, revealing a nanofibre-like structure (Scale bar = 400 nm). (E) AFM characterization of SF-95 °C, revealing dense nanofibres. (F) AFM characterization of SF-95 °C after oxidation using H2O2, showing the formation of dense microsphere oligomers. (G) Schematic diagram of secondary structure change: the β-sheet structure in SF solution increased after heating at 95 °C, and the β-sheet structure in SF solution decreased after oxidation by H2O2.

Fig. 6

The ability of SF-95 °C to scavenge •OH was further tested using the Fenton reagent. Compared to SF without heating, the ability of SF-95 °C to scavenge •OH was significantly increased in all the groups as long as SF concentration was above 1 mg/mL (Figs. S22A–D). To further explain the enhanced effect of SF-95 °C on antioxidation, we compared the ability of SF and SF-95 °C to scavenge ROS in living cells. The ability of SF-95 °C to scavenge ROS was significantly increased, no matter the protein concentration was 10 mg/mL or 0.1 mg/mL (Fig. S23). Overall, the results showed that the antioxidant properties of SF are controllable by heating. Higher temperatures promote more antioxidant capacity of SF through increasing the content of β-sheet structures (Fig. 6G).

4 Discussion

The oxidative stress reaction, combined with ROS-related chronic inflammation, can seriously destroy cellular homeostasis to hinder organ regeneration and repair [47], and there is no direct and effective strategy for ROS inhibition in the clinic. Biomaterials with a good capacity for ROS scavenging and biocompatibility are a potential way to treat ROS-related diseases [48]. In this study, we proved that SF is a good natural biomaterial for ROS scavenging, and discovered for the first time that β-sheet structures in SF are responsible for and positively correlated with ROS scavenging. Furthermore, SF gains a stronger ROS scavenging capacity through a heat-induced β-sheet increase (Fig. 1).

The antioxidant effects of nine potential antioxidant natural biomaterials were tested in this study, and the results showed that SF, GCTs, GSH, COL, and GEL had a good antioxidant effect in scavenging •OH and H2O2. The scavenging capacity of intracellular ROS in the SF and GCT groups was then found to be significantly different from the scavenging capacity of intracellular ROS in the other groups. GCTS has an antioxidant function due to the presence of a positively charged amino group in its polymeric framework [49,50]. There are only six cysteines on both the heavy chain and the light chain in SF, which are connected by disulfide bonds to form a whole without a separate cysteine [23]. Only SF performed the best ability in scavenging H2O2 and eliminated ROS both in vitro and in vivo in nine biomaterials (Fig. 2).

Amyloid protein was reported to change from a β-sheet structure to a random coil structure after oxidation [51]. This result inspired us to conclude that the β-sheet structure of SF may be the key factor in its antioxidant function. Representative repeat polypeptides from SF molecules were introduced to indirectly study the antioxidant mechanism of SF. We tested our conjecture by self-assembly into β-sheet structure (GAGSGA)2 polypeptides. The β-sheet structure of (GAGSGA)2 was found to be high, consistent with a recent report [52]. After oxidation, the β-sheet changed into a random coil structure by hydrogen peroxide. The β-sheet structure of (GAGSGA)2 was further proven to directly scavenge intracellular ROS in this study (Fig. 4). We can also predict the SF and the peptide have catalytic properties, but the results showed the H2O2 scavenging ability was linear correlation with the SF concentration, this phenomenon of SF property was different with the intrinsic enzymatic activity of enzyme or nanozyme. Structural characterization of (GAGSGA)2 exhibited β-sheet conformations. β-Sheet structures could form β-crystallite by orderly arrangement. β-crystallite exhibited ROS in response when it was used as a drug carrier. Drug was released when β-crystallite was changed from β-sheet to random coil after oxidation by ROS.

To confirm our proposal, the β-sheet structure in SF showed an antioxidant effect. ATR-FTIR spectra showed that 24 % of β-sheet and 14 % of β-antiparallel in SF were reduced to 19 % of β-sheet and 8 % of β-antiparallel after oxidation. The β-sheet, β-turn and β-antiparallel structures decreased; in contrast, the random coil conformation increased 1.5 folds. We also confirm that the β-sheet has a 96 % correlation with the oxidative capability.

We then wanted to ask whether increasing the β-sheet structure in SF can induce oxidative capability. Many methods can increase the β-sheet structure in SF solution, such as heating treatment, ultraviolet ray irradiation, organic solvent treatment, etc. [46]. To avoid introducing chemicals into SF, heating treatment was chosen to increase the β-sheet structure content in the SF-95 °C solution. With increasing temperature, the β-sheet structure content increased in the SF-95 °C solution, consistent with a recent report [53]. Compared with the SF-95 °C solution, the β-sheet structure was reduced by nearly a factor of two, exhibiting random coil and α-helix conformations after oxidation. The scavenging capacity of intracellular ROS in the SF-95 °C group was significantly different from the scavenging capacity of intracellular ROS in the SF group, and the expression of antioxidant defence genes was significantly upregulated after SF-95 °C treatment compared with SF treatment.

SF has been widely used in tissue engineering, such as bone tissue engineering, skin tissue function, vascular scaffolding and other fields, because of its good biocompatibility, mechanical properties and degradation performance. In the zebrafish tail injury model, ROS and inflammatory infiltration were alleviated at the injury site treated with SF, indicating the future clinic utilization of silk fibroin as an antioxidant biomaterial.

5 Conclusion

Finding a biomaterial with both strong antioxidation and good biocompatibility is challenging, and we screened out a new antioxidation biomaterial, SF, and clarified its antioxidation mechanisms in this study. We first determined that silk fibroin is a strong antioxidant and could eliminate excessive ROS in living cells and zebrafish. We revealed that the β-sheet structure of SF is responsible for antioxidation through β-sheets turning into a random coil structure after being oxidized. The antioxidant activity of SF can be further enhanced by physical and chemical methods accompanying with increasing of β-sheet content, e.g. heating, suggesting a new type of tunable antioxidant biomaterial for broad-demand therapeutics. This paper establishes a new theoretical basis for the future utilization of silk fibroin as an antioxidant biomaterial and paves the way for the discovery and identification of more antioxidative biomaterials.

Funding

This work was financially supported by the National Natural Science Foundation of China (32100450 and 32471370 to Q.P., U2241214, 62373008, 62203017, T2121002 and 21788102), Guangdong Pearl River Talent Program (2021QN02Y781 to Q.P.), the National Key R&D Program of China (2022ZD0116401 and 2022-ZD0116400), Inner Mongolia Natural Science Foundation (2024SHZR0982), Introduction of high-level talents for scientific research support of Inner Mongolia Autonomous Region in 2023 (DC2400000887), and Ulanqab Basic research projects.

Data availability

The authors declare that all data supporting the results in this study are available within the paper and its Supplementary information.

CRediT authorship contribution statement

Zhiyong Qian: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft. Chang Sun: Investigation, Methodology. Qianqian Li: Conceptualization, Methodology. Yafan Xie: Investigation, Methodology. Lingpeng Zhan: Data curation, Formal analysis. Xiangli Liu: Data curation, Investigation. Guanbo Wang: Investigation. Yen Wei: Conceptualization, Data curation, Supervision. Juhui Qiu: Funding acquisition, Methodology, Resources, Writing – review & editing. Qin Peng: Funding acquisition, Investigation, Methodology, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix ASupplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgements

We thank the mass spectrometry core facility and biochemical analysis core facility in Shenzhen Bay Laboratory for their assistance in running samples. We thank Bioimaging Core of 10.13039/501100021177 Shenzhen Bay Laboratory for providing imaging support. We also would like to acknowledge Bioimaging Core engineer Shan Liu for assistance with the atomic force microscopy (AFM).

Appendix Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103307.
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