Transcriptomic screening of novel targets of sericin in human hepatocellular carcinoma cells.

Sericin, a natural protein derived from Bombyx mori, is known to ameliorate liver tissue damage; however, its molecular mechanism remains unclear. Herein, we aimed to identify the possible novel targets of sericin in hepatocytes and related cellular pathways. RNA sequencing analysis indicated that a low dose of sericin resulted in 18 differentially expressed genes (DEGs) being upregulated and 68 DEGs being downregulated, while 61 DEGs were upregulated and 265 DEGs were downregulated in response to a high dose of sericin (FDR ≤ 0.05, fold change > 1.50). Functional analysis revealed that a low dose of sericin regulated pathways associated with the complement and coagulation cascade, metallothionine, and histone demethylate (HDMs), whereas a high dose of sericin was associated with pathways involved in lipid metabolism, mitogen-activated protein kinase (MAPK) signaling and autophagy. The gene network analysis highlighted twelve genes, A2M, SERPINA5, MT2A, MT1G, MT1E, ARID5B, POU2F1, APOB, TRAF6, HSPA8, FGFR1, and OGT, as novel targets of sericin. Network analysis of transcription factor activity revealed that sericin affects NFE2L2, TFAP2C, STAT1, GATA3, CREB1 and CEBPA. Additionally, the protective effects of sericin depended on the counterregulation of APOB, POU2F1, OGT, TRAF6, and HSPA5. These findings suggest that sericin exerts hepatoprotective effects through diverse pathways at different doses, providing novel potential targets for the treatment of liver diseases.

Exploring the apoptotic effects of sericin on HCT116 cells through comprehensive nanostring transcriptomics and proteomics analysis.

Sericin, a silk protein from Bombyx mori (silkworms), has many applications, including cosmetics, anti-inflammation, and anti-cancer. Sericin complexes with nanoparticles have shown promise for breast cancer cell lines. Apoptosis, a programmed cell death mechanism, stops cancer cell growth. This study found that Sericin urea extract significantly affected HCT116 cell viability (IC50 = 42.00 ± 0.002 µg/mL) and caused apoptosis in over 80% of treated cells. S-FTIR analysis showed significant changes in Sericin-treated cells' macromolecule composition, particularly in the lipid and nucleic acid areas, indicating major cellular modifications. A transcriptomics study found upregulation of the apoptotic signaling genes FASLG, TNFSF10, CASP3, CASP7, CASP8, and CASP10. Early apoptotic proteins also showed that BAD, AKT, CASP9, p53, and CASP8 were significantly upregulated. A proteomics study illuminated Sericin-treated cells' altered protein patterns. Our results show that Sericin activated the extrinsic apoptosis pathway via the caspase cascade (CASP8/10 and CASP3/7) and the death receptor pathway, involving TNFSF10 or FASLG, in HCT116 cells. Upregulation of p53 increases CASP8, which activates CASP3 and causes HCT116 cell death. This multi-omics study illuminates the molecular mechanisms of Sericin-induced apoptosis, sheds light on its potential cancer treatment applications, and helps us understand the complex relationship between silk-derived proteins and cellular processes.

Sericin coated thin polymeric films reduce keratinocyte proliferation via the mTOR pathway and epidermal inflammation through IL17 signaling in psoriasis rat model.

Therapeutic treatment forms can play significant roles in resolving psoriatic plaques or promoting wound repair in psoriatic skin. Considering the biocompatibility, mechanical strength, flexibility, and adhesive properties of silk fibroin sheets/films, it is useful to combine them with anti-psoriatic agents and healing stimulants, notably silk sericin. Here, we evaluate the curative properties of sericin-coated thin polymeric films (ScF) fabricated from silk fibroin, using an imiquimod-induced psoriasis rat model. The film biocompatibility and psoriatic wound improvement capacity was assessed. A proteomics study was performed to understand the disease resolving mechanisms. Skin-implantation study exhibited the non-irritation property of ScF films, which alleviate eczema histopathology. Immunohistochemical and gene expression revealed the depletion of ß-defensin, caspase-3 and -9, TNF-α, CCL-20, IL-1ß, IL-17, TGF-ß, and Wnt expressions and S100a14 mRNA level. The proteomics study suggested that ScF diminish keratinocyte proliferation via the mTOR pathway by downregulating mTOR protein, corresponding to the modulation of TNF-α, Wnt, and IL-1ß levels, leading to the enhancement of anti-inflammatory environment by IL-17 downregulation. Hematology data demonstrated the safety of using these biomaterials, which provide a potential therapeutic-option for psoriasis treatment due to desirable effects, especially anti-proliferation and anti-inflammation, functioning via the mTOR pathway and control of IL-17 signaling.

Sericin-Based Poly(Vinyl) Alcohol Relieves Plaque and Epidermal Lesions in Psoriasis; a Chance for Dressing Development in a Specific Area.

The noncontagious immune-mediated skin disease known as psoriasis is regarded as a chronic skin condition with a 0.09-11.4% global prevalence. The main obstacle to the eradication of the disease continues to be insufficient treatment options. Sericin, a natural biopolymer from Bombyx mori cocoons, can improve skin conditions via its immunomodulatory effect. Many external therapeutic methods are currently used to treat psoriasis, but sericin-based hydrogel is not yet used to treat plaques of eczema. Through the use of an imiquimod rat model, this study sought to identify the physical and chemical characteristics of a silk sericin-based poly(vinyl) alcohol (SS/PVA) hydrogel and assess both its therapeutic and toxic effects on psoriasis. The cytokines, chemokines, and genes involved in the pathogenesis of psoriasis were investigated, focusing on the immuno-pathological relationships. We discovered that the SS/PVA had a stable fabrication and proper release. Additionally, the anti-inflammatory, antioxidant, and anti-apoptotic properties of SS/PVA reduced the severity of psoriasis in both gross and microscopic skin lesions. This was demonstrated by a decrease in the epidermal histopathology score, upregulation of nuclear factor erythroid 2-related factor 2 and interleukin (IL)-10, and a decrease in the expression of tumor necrosis factor (TNF)-α and IL-20. Moreover, the genes S100a7a and S100a14 were downregulated. Additionally, in rats given the SS/PVA treatment, blood urea nitrogen, creatinine, and serum glutamic oxaloacetic transaminase levels were within normal limits. Our findings indicate that SS/PVA is safe and may be potentiated to treat psoriasis in a variety of forms and locations of plaque because of its physical, chemical, and biological characteristics.

Crocetin suppresses the growth and migration in HCT-116 human colorectal cancer cells by activating the p-38 MAPK signaling pathway.

BACKGROUND AND PURPOSE: Crocetin is a natural antioxidant that is found in the crocus flower and Gardenia jasminoides (fruit). Previous studies have reported its anticancer activity both in vivo and in vitro. In addition, crocetin suppresses the growth and migration of human colorectal cancer cells, however, its mechanism of action remains to be elucidated. Therefore, the present study investigated the molecular mechanism of crocetin effect on colorectal cancer cells (HCT-116) in vitro. EXPERIMENTAL APPROACH: HCT-116 cells were treated with different concentrations (0, 200, 400, 600, and 800 µM) of crocetin for 24 h. The cell survival rate was measured by MTT assay. Cell migration capacity was evaluated using the wound healing assay. The expression levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP-9) was monitored by RT-PCR. Phosphorylation of focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) was determined using western blot. FINDINGS/RESULTS: The proliferation of HCT-116 was inhibited by crocetin at 800 µM (P < 0.001). Crocetin prevented migration of HCT-116 cells (P < 0.05) and suppressed VEGF and MMP-9 mRNA expression (P < 0.001) and increased phosphorylation of p38 (MAPK; P < 0.001). However, no significant change in the phosphorylation of FAK was observed. CONCLUSION AND IMPLICATION: These data suggested that crocetin-induced growth- and migration- suppressing effects on HCT-116 cells may partially depend on the regulation of the p38 (MAPK) signaling pathway.

Curcumin modulates the angiogenic potential of human endothelial cells via FAK/P-38 MAPK signaling pathway.

Many phyto-compounds are found to have anti-angiogenesis property. Curcumin, a natural polyphenol, has been used as medicinal plant for years with different biological activities. Here, we investigated the effect of curcumin on angiogenesis potential of human endothelial cells. Human Umbilical Vein Endothelial Cells (HUVECs) were treated with different concentration of curcumin over a period of 72 h. Cell survival rate was measured by MTT assay. Cell migration and tubulogenesis were studied by scratch and tubulogenesis assays. The expression level of VEGF was monitored by RT-PCR. We also monitored the phosphorylation of FAK and P-38 MAPK by western blotting. Compared to control group, curcumin decreased HUVECs survival rate after 72 h. We found that the migration of HUVECs was decreased after curcumin treatment compared to the control (p < 0.0001). Cell alignment and tubulogenesis activity were found to be inhibited compared to cells from the VEGF group (p < 0.05). The expression level of VEGF was increased in curcumin treated cells at first 24 h time period. Based on data from the current experiment, the protein level of p-FAK/FAK ratio was increased coincided with a decrease in p-P38/P38 ratio treatment with curcumin (p < 0.0001). These data demonstrated that curcumin inhibited HUVECs angiogenesis potential by modulation of FAK/P-38 MAPK signaling pathway.

Effect of urea-extracted sericin on melanogenesis: potential applications in post-inflammatory hyperpigmentation

BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-p were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.

Effect of animal products and extracts on wound healing promotion in topical applications: a review.

Wound healing is a natural process of body reaction to repair itself after injury. Nonetheless, many internal and external factors such as aging, comorbidity, stress, smoking, alcohol drinking, infections, malnutrition, or wound environment significantly affect the quality and speed of wound healing. The unsuitable conditions may delay wound healing process and cause chronic wound or scar formation. Therefore, many researches have attempted to search for agents that can accelerate wound healing with safety and biocompatibility to human body. Widely studied wound healing agents are those derived from either natural sources including plants and animals or chemical synthesis. The natural products seem to be safer and more biocompatible to human tissue. This review paper demonstrated various kinds of the animal-derived products including chitosan, collagen, honey, anabolic steroids, silk sericin, peptides, and proteoglycan in term of mechanisms of action, advantages, and disadvantages when applied as wound healing accelerator. The benefits of these animal-derived products are wound healing promotion, anti-inflammatory, antimicrobial activity, moisturizing effect, biocompatibility, and safety. However, the drawbacks such as allergy, low stability, batch-to-batch variability, and high extraction and purification costs could not be avoided in some products.

Nontraditional Methods to Evaluate Wound Healing.

BACKGROUND: Traditional evaluation of wound healing is sometimes subjective. It is necessary to develop qualitative and quantitative methods to enable more efficient and accurate evaluation of wounds. Recently, new techniques have been introduced and the correspondence between these techniques and clinician judgment is critical. OBJECTIVE: Some nontraditional techniques that analyze wound healing were reviewed, which include measurements of the wound area, tissue color, skin barrier function, skin humidity, and keratinocyte morphology. METHODS: This review article is based on medical research that focuses on the evaluation of wound healing. RESULTS: Software-based and advanced device-based techniques generally provide more accurate and precise results than traditional ones, such as the ruler-based technique. Measurement of tissue color can also help to identify the type of tissue. Evaluation of skin barrier function can assist clinicians to analyze functional restoration of skin, whereas skin humidity demonstrates the exudate production status of the wound. In addition, keratinocyte morphology in the wound bed indicates quality of wound healing and side effects of treatment. CONCLUSION: There is no gold standard method for qualitative and quantitative evaluation of wound healing. It is important to understand the type of wound, sample size, results obtained, advantages, and limitations of each technique.

The development of non-toxic ionic-crosslinked chitosan-based microspheres as carriers for the controlled release of silk sericin.

Silk sericin is recently shown to possess various biological activities for biomedical applications. While various sericin carriers were developed for drug delivery system, very few researches considered sericin as a bioactive molecule itself. In this study, sericin incorporated in the chitosan-based microspheres was introduced as a bioactive molecule and bioactive carrier at the same time. The chitosan/sericin (CH/SS) microspheres at different composition (80/20, 70/30, 60/40, and 50/50) were successfully fabricated using anhydroustri-polyphosphate (TPP) as a polyanionic crosslinker. The microspheres with an average size of 1-4 µm and narrow size distribution were obtained. From FT-IR spectra, the presence of both chitosan and sericin in the microspheres confirmed the occurrence of ionic interaction that crosslink them within the microspheres. We also found that the CH/SS microspheres prepared at 50/50 could encapsulate sericin at the highest percentage (37.28%) and release sericin in the most sustained behavior, possibly due to the strong ionic interaction of the positively charged chitosan and the negatively charged sericin. On the other hand, the composition of CH/SS had no effect on the degradation rate of microspheres. All microspheres continuously degraded and remained around 20% after 14 days of enzymatic degradation. This explained that the ionic crosslinkings between chitosan and sericin could be demolished by the enzyme and hydrolysis. Furthermore, we have verified that all CH/SS microspheres at any concentrations showed non-toxicity to L929 mouse fibroblast cells. Therefore, we suggested that the non-toxic ionic-crosslinked CH/SS microspheres could be incorporated in wound dressing material to achieve the sustained release of sericin for accelerated wound healing.

Improvement of Physical and Wound Adhesion Properties of Silk Sericin and Polyvinyl Alcohol Dressing Using Glycerin.

OBJECTIVE: This study aimed to use glycerin to improve physical and wound adhesion properties of a wound dressing made of silk sericin and polyvinyl alcohol (PVA). DESIGN: Glycerin of a natural-derived plasticizer was used to modify the properties of silk sericin/PVA scaffolds. Various concentrations of glycerin were mixed with silk sericin and PVA and then fabricated into the scaffolds by a freeze-drying technique. The control study was performed to examine the properties of the silk sericin/PVA scaffolds with and without glycerin. MAIN OUTCOME MEASURES: Physical, mechanical, wound adhesion properties, the release profile of silk sericin, and in vivo safety of the silk sericin/PVA scaffolds with and without glycerin were investigated. MAIN RESULTS: The silk sericin/PVA scaffolds with glycerin exhibited more homogenous structure, less compressive modulus, higher Young modulus and elongation percentage, and a higher degree of crosslinking compared with the scaffold without glycerin. The silk sericin/PVA scaffold with 2% wt/vol glycerin showed more controlled release of silk sericin than the other scaffolds. The sustained release of silk sericin from the scaffold with glycerin would be advantageous for long-term healing of wounds. The silk sericin/PVA scaffold with 2% (wt/vol) glycerin was less adhesive to the wound compared with the scaffold without glycerin. Furthermore, the implantation of silk sericin/PVA scaffolds with 2% (wt/vol) glycerin did not cause any irritation to the tissue. CONCLUSION: The silk sericin/PVA scaffolds with glycerin were introduced as a biocompatible, more flexible, and less adhesive wound dressing than the scaffold without glycerin.

Toxicity evaluation of cordycepin and its delivery system for sustained in vitro anti-lung cancer activity.

In the previous study, we have found that the cordycepin which was extracted from Cordyceps mycelia produced by growing Cordyceps militaris on the dead larva of Bombyx mori silkworms showed the anti-proliferative effect toward lung cancer cells without toxicity to non-cancer cells. In this work, the cordycepin was tested for its in vitro mutagenicity and in vivo toxicity. From the Ames test and subacute toxicity test using oral administration in a rat model, the cordycepin was proved to be a non-mutagenic and non-toxic compound. The hematology and blood chemistry as well as the microanatomical characteristic of the tissues of rats fed with cordycepin every day for consecutive 30 days were comparable to those of the normal ones. Then, the cordycepin was incorporated in gelatin type A (GA) and gelatin type B (GB) nanoparticles aimed to sustain its release and activity. The cordycepin incorporated in both GA and GB nanoparticles showed the sustained release profiles. GA nanoparticles could encapsulate cordycepin at higher encapsulation efficiency due to the attractive electrostatic interaction between the positive-charged GA and the negative-charged cordycepin. However, GA nanoparticles released cordycepin at the higher amount possibly because of the large surface area of small size nanoparticles. Comparing to GB nanoparticles, the higher amount of cordycepin released from GA nanoparticles showed the higher anti-proliferative and anti-migratory effects on A549 lung cancer cells. In conclusion, GA nanoparticles were suggested as a suitable carrier for the sustained release of cordycepin. The GA nanoparticles releasing cordycepin could be an effective and non-invasive material for the treatment of lung cancer cells.

Dialysate cancer antigen 125 in long-term peritoneal dialysis patients.

Structural and functional peritoneal membrane changes are associated with long-term peritoneal dialysis. These changes can lead to ultrafiltration failure and peritoneal fibrosis, reducing the efficacy of the peritoneal membrane to remove waste and balance fluid and electrolytes. The loss of mesothelial cells from the basement membrane is one of the major characteristics in peritoneal membrane structural change. Thus, if the reduction of peritoneal mesothelial cell mass in peritoneal dialysis patients is monitored, signs of ultrafiltration failure and peritoneal fibrosis can be detected early. One of biomarkers that can be used to indicate the change in peritoneal mesothelial cell mass is CA125, which is produced by mesothelial cells. In this article, we review the measurement and clinical use of CA125 in peritoneal dialysate effluent. Additionally, we address the data and studies on the association between dialysate CA125 levels and factors related to ultrafiltration failure and peritoneal fibrosis, including the parameters used to monitor the functional status of the peritoneal membrane. Our review shows that dialysate CA125 can be used to evaluate the peritoneal membrane in noninfected patients to predict peritoneal fibrosis, and it can also be used as a biomarker of biocompatible dialysis solutions.

Bioactivity and toxicity studies of amphotericin B incorporated in liquid crystals.

The main objective of the present work was to evaluate the bioactivity and safety of amphotericin B-liquid crystal mixtures (AmB-LC). The effects of liquid crystal (LC) materials and AmB-LC ratios on bioactivity and toxicity to respiratory cell lines were investigated. The formation of AmB-LC mixtures did not change the physical properties of LC when the AmB loading was not more than a 1:3 mole ratio (25%). The exposure of respiratory cell lines to LC did not generate any toxicity (2.5-80 µg/mL). The inhibitory activity of AmB in all liquid crystal formulations (cholesteryl palmityl carbonate: CPC, dicholesteryl carbonate: DCC and sodium cholesteryl carbonate: SCC) on fungi was significantly enhanced when compared to that of the same amount of pure AmB. However, their toxicity to respiratory related cells and red blood cells was significantly decreased. This could be a huge advantage in clinical applications as there is more possibility for dose adjustments. The exposure of small airway epithelial cells (SAEC) and alveolar macrophages (AMs NR8383) to liquid crystals had no significant detrimental effects at doses of between 2.5 and 80 µg/mL (viability was always over 80%). The production of toxic cytokines and inflammatory mediators, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß) and nitric oxide (NO), after treatment with AmB in liquid crystals at concentrations of between 2 and 32 µg/mL was significantly reduced by about a 1000-fold compared to that generated by lipopolysaccharide (LPS).

The effect of sericin with variable amino-acid content from different silk strains on the production of collagen and nitric oxide.

Although silk sericin (SS) enhances the growth and attachment of fibroblast cells, its toxicity remains questionable. We investigated the effect of SS extracted by heat with variable amino-acid content on in vitro collagen promotion and nitric oxide synthesis. After 24 h of incubation, SS, especially from the Chul 1/1 strain which has the most methionine and cysteine content, enhanced fibroblast growth. The molecular mass of heat-extracted SS from these three strains showed a slightly different range, but within 20-200 kDa, which were all identified as sericin. SS from all strains promoted type-I collagen production in a concentration-dependent manner, while SS from Chul 1/1 strain could induce the highest amount of collagen synthesis when compared to SS from other strains. Nitric oxide was found in the culture medium after activation by SS from the Chul 1/1 strain but reached a level that was not toxic to the cells. We conclude that SS is not toxic to fibroblast cells. Moreover, methionine and cysteine content in SS are important factors to promote cell growth and collagen synthesis.

Monitoring of inflammatory mediators induced by silk sericin.

Silk proteins have been shown to be good candidates for biomedical materials. However, there have been some reports regarding immunological and allergic responses to silk sericin. Our objective was to investigate the inflammatory mediators induced by sericin both in vitro and in vivo. Mouse monocyte and alveolar macrophage cell lines were used for monitoring levels of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha generated after activation by sericin at concentrations of 0.2-1.0 mg/mL. The amounts of TNF-alpha and IL-1beta produced by both cell lines corresponded, in a dose-dependent manner, with the sericin concentration in the culture medium. The levels of TNF-alpha and IL-1beta generated after sericin activation by macrophage cells were higher than those generated by monocytes. However, these cytokine levels would not cascade to other inflammatory effects. Inflammatory mediators were also monitored from sericin-treated, cream base-treated and normal saline-soaked full-thickness rat excisions. Using wound size measurements and ELISA assays, sericin-treated wounds were shown to heal faster and had lower levels of inflammatory mediators, as compared with the cream base-treated and normal saline-soaked wounds. It can be concluded that sericin promotes the wound healing process without causing inflammation.

In vitro plasma compatibility study of a nanosuspension formulation.

Lyophilized nanosuspension formulation intended for intravenous administration has been developed. The in-vitro plasma compatibility of the formulation was evaluated under conditions that mimic intravenous injection. After reconstitution of the lyophilized nanosuspension formulation with sterile water for injection, the drug was then further diluted with 0.9 % w/v NaCl or 5% w/v dextrose to make concentrations of 1.5 and 5.0 mg/mL with each diluent. The 1.5-mg/mL solutions were diluted with plasma in the ratio of 1:1 and 1:2, and the 5.0-mg/mL solutions were diluted with plasma in the ratio of 1:2 and 1:10, to simulate different intravenous infusion rates. Different contributing factors for particle aggregation upon mixing with plasma-such as the concentration of active ingredient, the types of diluents, the formulation/plasma ratio dependent upon infusion rate and the plasma flow rate, and the incubation time-were evaluated with respect to particle aggregation. It was found that aggregation occurred very rapidly in all conditions and that more aggregation takes place with higher drug concentrations in the mixture with plasma. It was also found that there was minimum aggregation at the concentration of 1.5 mg/mL when delivered at an infusion rate of 2.5 mL/min. This combination is recommended for further toxicological and clinical evaluation.