Dissolution and processing of silk fibroin for materials science.

In recent decades, silk fibroin (SF) from silkworm Bombyx mori has been extensively researched and applied in several fields, including: cosmetics, biomedicine and biomaterials. The dissolution and regeneration of SF fibers is the key and prerequisite step for the application of silk protein-based materials. Various solvents and dissolving systems have been reported to dissolve SF fibers. However, the dissolution process directly affects the characteristics of SF and particularly impacts the mechanical properties of the resulting silk biomaterials in subsequent processing. The purpose of this review is to summarize the common solvents, the dissolution methods for silk protein, the properties of the resulting SF protein. The suitable use of SF dissolved in the corresponding solvent was also briefly introduced. Recent applications of SF in various biomaterials are also discussed.

Characterization of undegraded and degraded silk fibroin and its significant impact on the properties of the resulting silk biomaterials.

The silk fibroin (SF) regeneration process significantly affects the resulting biomaterials, unfortunately, there has been insufficient study regarding the most suitable regeneration method for SF. In this study, we prepared undegraded SF (uSF) and degraded SF (dSF) by common regeneration methods and studied their difference in detail. The results demonstrated that the degradation degree of SF peptide chain had little influence on the secondary structure and thermal stability of SF materials. While, uSF solution showed higher viscosity and surface tension than dSF solution. The uSF membrane (uSFM) could be elongated approximately 134%, 1.6 times the degraded SF membrane (dSFM). SEM implied that both uSF and dSF existed in aqueous solution as micelles with a diameter of approximately 30 nm. dSF could directly form SF nanoparticles (dSFNPs) when poured into acetone while uSF could only form nanoparticles (uSFNP) with the addition of SDS. Glucose oxidase embedded into dSFM and dSFNP showed high catalytic activities, but uSFNP demonstrated nearly no activity. In addition, the dSFM was more appropriate for L929 cell culture. Considering the obvious difference between the two SF proteins, our results are significant in guiding the application of appropriate SF proteins in tissue engineering materials, bioactive materials, bioink, etc.

Excess acetone extraction in silk protein solution greatly accelerates the regeneration progress of silk fibroin for desalting and purification.

The dissolution of silk fibroin in highly concentrated neutral salt solution and the subsequent long time-costing desalination have long inhibited silk regeneration. Here, a 4-fold volume of acetone was first added into a silk fibroin of 9.3 M LiBr solution (SF-LBS) to extract the dissolved silk protein. In fact, acetone extracted the salt and water from SF-LBS rather than silk protein. The extracted protein has less than half of the original amount of LiBr and could be redissolved easily in water. The dialysis time of the extracted protein could reduce the original dialysis time by approximately half. SDS-PAGE revealed that acetone extraction didn't induce any breakage to the peptide chains of the extracted silk fibroin. FTIR indicated that the structure of the extracted fibroin was very similar to that of the unextracted fibroin, and displayed predominantly a mixture of random coil and α-helices. X-ray diffraction also showed that acetone extraction had no effect on the crystal structure of the fibroin. In addition, both acetone and LiBr from the processing waste could easily be recycled for silk regeneration. This novel method can significantly enhance the desalination efficiency of highly concentrated LiBr solution containing silk protein without affecting its structure.

Dissolution and regeneration of silk from silkworm Bombyx mori in ionic liquids and its application to medical biomaterials.

The dissolution and regeneration of silk fibre have long been an issue for producers of silk. The high solubility of silk fibroin (SF) in ionic liquids (ILs) is a promising new avenue in silk dissolution and regeneration as it may allow for a significant reduction in operational steps required for SF regeneration and the subsequent formation of SF biomaterials. The regenerated SF solution can be prepared by dissolving directly SF in ILs without the tedious steps of degumming, dissolution and dialysis. Besides, the regenerated SF solution can simply form different SF biomaterials with the help of coagulant, and ILs can be separated from SF biomaterials easily during SF solidification. The goal of this work is to summarize the commonly used ILs for dissolving silk protein and their dissolution methods and to present the potential application of SF/ILs mixed solution in medical biomaterials.

Silk sericin has significantly hypoglycaemic effect in type 2 diabetic mice via anti-oxidation and anti-inflammation.

The sericin protein from silk-processing waste added to the normal diet at 0.8% (g%) level was administered orally to type 2 diabetic (T2D) mice to investigate its hypoglycaemic effects and mechanism. The oral protein is in the form of silk sericin hydrolysate, obtained from a boiling treatment of 0.025% calcium hydroxide solution. The protein significantly decreased fasting blood glucose, fasting plasma insulin, and glycosylated serum protein levels; improved oral glucose tolerance and insulin tolerance, and enhanced antioxidative activities. The protein could ameliorate the pathological damage in pancreatic ß-cells and the liver tissue. It enhanced the expression of key proteins and enzymes, including insulin receptor, insulin receptor substrate, PI3K, phosphorylated-AKT, hepatic kinase, GLUT4, glycogen synthase, GSK3ß, GLK, PFK1, PKM2, and AMPKα, which are related to insulin metabolism and glycolysis. The protein also reduced the expression of G6Pase, PCK, and ACC, which are related to gluconeogenesis and lipid metabolism in the liver, and decreased the expression of TNF-α, IL-6, P65, and IKKß related to inflammation. In general, sericin could maintain normal glucose levels and regulate insulin secretion, insulin and lipid metabolism, and inhibition of inflammation. Therefore, sericin protein could be developed into a novel functional health food with significantly hypoglycaemic effect.

The renal protection of flavonoid-rich ethanolic extract from silkworm green cocoon involves in inhibiting TNF-α-p38 MAP kinase signalling pathway in type 2 diabetic mice.

BACKGROUND: Diabetic nephropathy (DN) is one of the major complications of diabetes. Sericin and flavonoids are two bioactive substances which have been demonstrated to have some therapeutical effect on diabetic nephropathy. The aim of this paper is to investigate the effect of ethanolic extract (EE) rich in quercetin and kaempferol and their glucosides from the green cocoon of silkworm Bombyx mori on DN in type 2 diabetic (T2D) mice induced by high-fat and streptozotocin (STZ). METHODS: Ethanolic extract from the green cocoon shell of silkworm Bombyx mori was used as material. Diabetic mice were orally treated with three doses (150, 250 and 350 mg/kg) of the extract for 7 weeks, then the levels of oxidases and related inflammatory factors were measured in kidneys. RESULTS: Compared with the negative control, EE administration resulted in an evident reduction in blood glucose levels and an improvement of the body weight in diabetic mice. Oral EE could inhibit the expressions of renal tumour necrosis factor TNF-α, monocyte chemoattractant protein-1 (MCP-1), fibronectin (FN) and P38 mitogen-activated protein kinase (p38 MAPK) in T2D mice. Moreover, the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were significantly elevated in T2D mice treated with EE. CONCLUSIONS: The activities of anti-fibrosis and anti-inflammation contributed to the renal protective effects by reducing oxidative stress, which might be mediated by the inhibition of the TNF-α-p38 MAP kinase signalling pathway. It is suggested that the ethanolic extract from silkworm green cocoon might be potential as an herbal medicine for DN.

Therapeutic effects of ethanolic extract from the green cocoon shell of silkworm Bombyx mori on type 2 diabetic mice and its hypoglycaemic mechanism.

Diabetes mellitus is a clinically complex disease characterized by hyperglycaemia with disturbances in carbohydrate, fat and protein metabolism. The aim of this study was to determine the therapeutic effect of ethanolic extract (EE) from the green cocoon sericin layer of silkworm Bombyx mori on mice with type 2 diabetes mellitus (T2DM) and its hypoglycaemic mechanisms. The results showed that oral EE for 7 weeks had significant ameliorative effects on all the biochemical parameters studied in vivo. The levels of oral glucose tolerance and insulin tolerance were significantly improved. The hypoglycaemic rate in the 350 mg kg-1 high dosage group was 39.38%. The levels of nuclear factor kappa B (NFκB), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) in the high dosage EE-treated group were significantly reduced, while activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were obviously increased. The islet area and the number of insulin-positive beta cells increased significantly in the high dose group. Furthermore, expression levels of insulin receptor (IR), insulin receptor substrate (IRS), phosphatidylinositide 3-kinase (PI3K), p-Akt and phospho-glycogen synthase kinase-3ß (p-GSK3ß) involved in insulin signalling were increased. Adenosine 5'-monophosphate-activated protein kinase (AMPK) and glucose transporter 4 (GLUT4) also were activated to regulate glucose metabolism in EE-treated groups. The levels of glucose 6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PEPCK) decreased, while the glucokinase (GK) level increased to promote glycolysis. The results clearly indicated that oral EE, especially at a high dose, could improve the glucose metabolism of T2DM by reducing inflammatory reactions, enhancing the antioxidant capacity and insulin sensitivity, and regulating the balance between glycolysis and gluconeogenesis, which means that EE has potential ameliorative effects on T2DM mice.

Silk Sericin Hydrolysate is a Potential Candidate as a Serum-Substitute in the Culture of Chinese Hamster Ovary and Henrietta Lacks Cells.

The silk sericin hydrolysate (SSH) from the waste of silk processing as a substitute of fetal bovine serum (FBS) was used for the culture of Chinese hamster ovary (CHO) cells and Henrietta Lacks (Hela) strain of human cervical cancer cells. The survival ratio of these cells cultured in SSH media were similar to or higher than those in FBS media. Especially after the serum was replaced by low concentration of SSH at 15.0 µg/ml for 5 d, the proliferation of both cells was also similar to or higher than that of FBS group; the percentages of CHO and Hela cells in S-phase were 28.9 and 28.0%, respectively. The former is nearly two times that of FBS group, the latter is also higher than the control group. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that among the differentially expressed genes, the relative expression of CXCL12 gene of CHO cells in SSH group increased, was three times that of serum group, and the relative expression of LCN2 gene of Hela cells increased 2.8 times, indicating that these related genes were activated to promote cell growth and proliferation. These results fully illustrated the hydrolysated sericin has a potential use as serum substitutes in cell culture.

Promoter analysis and RNA interference of CYP6ab4 in the silkworm Bombyx mori.

In insects, cytochrome P450 monooxygenases (P450s) are involved in the metabolism of endogenous compounds such as steroid hormones and lipids. In this study, we measured the 20-hydroxyecdysone (20E)-induced transcriptional level of the CYP6ab4 gene using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) with a dual spike-in strategy. We then probed possible physiological functions using RNAi experiments in the silkworm Bombyx mori. The activity of the CYP6ab4 promoter in various silkworm tissues was measured by firefly luciferase activity and normalized by Renilla luciferase activity. Our results showed that the activity of the CYP6ab4 promoter was highest in the malpighian tubule, followed by the fat body, the silk gland, the midgut, the epidermis, and the hemocyte. The essential region for basal and 20E-induced transcriptional activity was between -908 and -456 bp from the transcription start site. Through promoter truncation analysis using a dual-luciferase reporter assay in B. mori ovary cells (BmN), we showed that the region between -827 and -722 bp was essential for basal and 20E-induced transcriptional activity. Sequence analysis of this region revealed several potential transcriptional regulatory elements such as Hunchback (Hb) and BR-C Z. Mutation of the core bases of the BR-C Z binding site demonstrated that BR-C Z induces 20E-mediated CYP6ab4 transcription. Further identification of cis- and trans-elements and their roles in the upregulation of CYP6ab4 may be useful for elucidating the contribution of P450 to the response mechanism to 20E.

The expression profile and promoter analysis of ultraspiracle gene in the silkworm Bombyx mori.

The nuclear receptor, ultraspiracle protein (USP), is a transcription factor and an essential component of a heterodimeric receptor complex with ecdysone receptor. However, the mechanisms underlying the transcriptional regulation of USP in silkworm are unknown. In this study, using dual-spike-in qPCR method, we examined the expression of Bombyx ultraspiracle gene (BmUSP) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. The results showed that the expression levels of BmUSP gene varied in different tissues and were increased 2 h after exposure to ecdysone. To identify the molecular mechanism underlying the regulation of USP gene expression in silkworm Bombyx mori, promoter truncation analyses were performed using the luciferase reporter assay and Bac-to-Bac expression system in several tissues of B. mori. BmUSP gene promoter with 5' end serial deletions showed different levels of activity in various tissues, higher in fat body and Malpighian tubule. Deletion of the region from -485 to -445 and -307 to -281 upstream of BmUSP gene abolished and increased its promoter activity, respectively. This region contains AP-1, Dfd transcription factor binding sites. These results indicate that BmUSP are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. This study would provide an important foundation for investigating the mechanism underlying the transcriptional regulation of BmUSP in the silkworm.

The expression profile and promoter analysis of ß-N-acetylglucosaminidases in the silkworm Bombyx mori.

ß-N-acetylglucosaminidase (GlcNAcase) is a key enzyme in the chitin decomposition process. In this study, we investigated the gene expression profile of GlcNAcases and the regulation mechanism for one of these genes, BmGlcNAcase1, in the silkworm. We performed sequence analysis of GlcNAcase. Using dual-spike-in qPCR method, we examined the expression of Bombyx ß-N-acetylglucosaminidases (BmGlcNAcases) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. Using Bac-to-Bac system and luciferase reporter vectors, we further analyzed the promoter sequence of BmGlcNAcase1. The results showed that these proteins have a highly conserved catalytic domain. The expression levels of the BmGlcNAcase genes varied in different tissues, and were increased 48 h after exposure to ecdysone. BmGlcNAcase1 gene promoter with 5'-end serial deletions showed different levels of activity in various tissues, higher in the blood, skin and fat body. Deletion of the region from -347 to -223 upstream of BmGlcNAcase-1 gene abolished its promoter activity. This region contains the binding sites for key transcription factors including Hb, BR-C Z, the HSF and the typical TATA-box element. These results indicate that BmGlcNAcases are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. BmGlcNAcase1 promoter analysis has paved a foundation for further study of the gene expression patterns.

Expression analysis and RNA interference of BmCarE-10 gene from Bombyx mori.

Carboxylesterase (CarE) is a multifunctional superfamily, and it plays important roles in xenobiotic detoxification, pheromone degradation, neurogenesis and regulating development. In this research, firstly, we measured the rutin-induced transcriptional level of BmCarE-10 gene by using real-time quantitative RT-PCR method, and dual spike-in strategy. Several possible physiological functions were certified preliminarily by RNAi experiments in silkworm. Promoter truncation analysis using a dual-luciferase reporter assay in Bombyx mori ovary cells (BmN) showed that the region -705 to -625 for BmCarE-10 gene was essential for basal and rutin-induced transcriptional activity. Sequence analysis of this region revealed several potential transcriptional regulatory elements such as Croc and Dfd. The activities of the BmCarE-10 promoter in various tissues of silkworm were also measured by firefly luciferase activity and normalized by the Renilla luciferase activity. Results showed that the activity of the BmCarE-10 promoter were highest in the Malpighian tubule, followed by fat body, silk gland, midgut, epidermis, and hemocyte. The essential region for basal and rutin-induced transcriptional activity was also -894 to -502 in Malpighian tubule and fat body of silkworm. The potential core promoters of BmCarE-10 gene in B. mori are reported for the first time in this research. Further identification of cis- and trans-elements and their role in upregulation of BmCarE-10 gene may be useful for elucidating the contribution of CarE protein to the response mechanism to rutin.

Analyzing the promoters of two CYP9A genes in the silkworm Bombyx mori by dual-luciferase reporter assay.

Cytochrome P450s (CYPs) are widespread proteins that interact with exogenous chemicals from the diet or the environment. CYP9A subfamily genes are important in the silkworm Bombyx mori. We previously reported transcriptional levels of two CYP9A genes in different tissues and their responses to sodium fluoride (NaF). In this study, promoter truncation analysis using a dual-luciferase reporter assay in B. mori ovary cells (BmN) showed that the regions -1,496 to -1,102 bp for CYP9A19, and -1,630 to -1,210 bp for CYP9A22 were essential for basal transcriptional activity. Sequence analysis of these regions revealed several transcriptional regulatory elements but no typical promoter elements. Promoter activities were regulated after NaF induction and with an obvious dose effect. Although the dual-luciferase assay has been widely used to determine the activity of a given promoter in cell lines, problems with it still exist. Our results indicate that both plasmid size and construct protocols affect the experimental results.

Quantitative analysis of expression of six BmGST genes in silkworm, Bombyx mori.

Glutathione S-transferases (GSTs) are a multifunctional super gene family, some of which play an important role in insecticide resistance. In this research, we used a real-time quantitative RT-PCR method, and a novel strategy, to measure the transcriptional level per gene copy using an exogenous RNA reference and DNA reference. The transcription levels of six BmGST genes in different tissues of fifth instar Bombyx mori larvae and their responses to insecticide and fluoride were investigated. The results show different levels and patterns of expression of the different BmGSTs in the various tissues observed. The BmGSTs can be induced by insecticide and fluoride, but their responses to each are different. The results of this research are helpful in studying the tissue-specific expression of BmGSTs in Bombyx mori, and in developing new pesticide resistant silkworm varieties.