Accelerated degradation of cellulose in silkworm excrement by the interaction of housefly larvae and cellulose-degrading bacteria.

The environmental pollution caused by silkworm (Bombyx mori) excrement is prominent, and rich in refractory cellulose is the bottleneck restricting the efficient recycling of silkworm excrement. This study was performed to investigate the effects of housefly larvae vermicomposting on the biodegradation of cellulose in silkworm excrement. After six days, a 58.90% reduction of cellulose content in treatment groups was observed, which was significantly higher than 11.5% of the control groups without housefly larvae. Three cellulose-degrading bacterial strains were isolated from silkworm excrement, which were identified as Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis based on 16S rRNA gene sequence analysis. These three bacterial stains had a high cellulose degradation index (HC value ranged to between 1.86 and 5.97 and FPase ranged from 5.07 U/mL to 7.31 U/mL). It was found that housefly larvae increased the abundance of cellulose-degrading bacterial genus (Bacillus and Pseudomonas) by regulating the external environmental conditions (temperature and pH). Carbohydrate metabolism was the bacterial communities' primary function during vermicomposting based on the PICRUSt. The results of Tax4Fun indicated that the abundance of endo-ß-1,4-glucanase and exo-ß-1,4-glucanase increased rapidly and maintained at a higher level in silkworm excrement due to the addition of housefly larvae, which contributed to the accelerated degradation of cellulose in silkworm excrement. The finding of this investigation showed that housefly larvae can significantly accelerate the degradation of cellulose in silkworm excrement by increasing the abundance of cellulose-degrading bacterial genera and cellulase.

Enhancement of Stability and Antioxidant Activity of Mulberry Anthocyanins Through Succinic Acid Acylation.

Research background: Anthocyanins possess valuable health-promoting activities with significant health benefits for humans. However, their instability is a limiting factor for their usage in functional foods and beverages. Experimental approach: In this work, a new method to enhance the stability of anthocyanins from mulberry fruit through acylation by using succinic acid as a selected acyl donor was explored. The Box-Behnken design of response surface methodology was applied to determine the optimized conditions for the acylation process. Results and conclusions: The highest acylation conversion rate was 79.04% at anthocyanins to succinic acid mass ratio 1:8.96, acylation duration 3 h and temperature 50 °C. Structural analysis of acylated anthocyanins revealed that succinic acid introduces a C-O-C bond and a hydroxyl group. The thermostability and light stability of mulberry anthocyanins were significantly improved after acylation, and the antioxidant activity expressed as total reducing power and Fe2+-chelating capacity of the acylated anthocyanins was also enhanced. Novelty and scientific contribution: Succinic acid acylation provides a novel method for stabilizing mulberry anthocyanins, as evidenced by the increased stability and antioxidant ability of anthocyanins, and thus facilitates its use in the food and nutraceutical industries.

Dual-Targeting Antiproliferation Hybrids Derived from 1-Deoxynojirimycin and Kaempferol Induce MCF-7 Cell Apoptosis through the Mitochondria-Mediated Pathway.

1-Deoxynojirimycin, an α-glucosidase inhibitor, possesses various biological activities such as antitumor, antidiabetic, and antiviral effects. However, the application of 1-deoxynojirimycin is restricted by its poor lipophilicity and low bioavailability. In this study, three 1-deoxynojirimycin derivatives (8-10) comprising 1-deoxynojirimycin and kaempferol were designed and synthesized to modify their pharmacokinetics and improve their antitumor efficacy. Among them, compound 10, a conjugate of 1-deoxynojirimycin and kaempferol linked through an undecane chain, exhibited excellent lipophilicity, antiproliferative effects, and α-glucosidase inhibitory activity. Compared with 1-deoxynojirimycin, kaempferol, and their combination, compound 10 downregulated cyclooxygenase-2 (COX-2) expression, arrested the cell cycle at the S phase, induced cellular apoptosis, and inhibited the migration of MCF-7 cells. Moreover, further investigation indicated that compound 10 induced MCF-7 cell apoptosis through a mitochondrial-mediated pathway via the loss of mitochondrial membrane potential. This led to increasing intracellular levels of reactive oxygen species (ROS) and Ca2+, the downregulation of Bcl-2 expression, and the upregulation of Bax levels.

Transcriptional response of detoxifying enzyme genes in Bombyx mori under chlorfenapyr exposure.

The silkworm, Bombyx mori (B. mori) is an important economic insect which ingests mulberry leaves and products the silk in industry. Chlorfenapyr is a new halogenated pyrrole insecticide which has been promoted for the control of mulberry insect pests in China. However, the detoxification mechanism of the silkworm to chlorfenapyr has not been investigated yet. In the present study, we first estimated the LC30 dose of chlorfenapyr for 3rd instar B. mori larvae, and then, in order to characterise the chlorfenapyr detoxification mechanism, the transcriptomes of chlorfenapyr-treated and untreated 3rd instar B. mori larvae were compared using RNA-sequencing. In total, 146, 533, 126 and 148, 957, 676 clean reads were obtained from insecticide-treated and control silkworm larvae, respectively, and these reads generated 10, 954 genes. The transcriptional profile of silkworm larvae was significantly influenced by chlorfenapyr treatment. A total of 1196 differentially expressed genes (DEGs) were identified in insecticide-treated and control B. mori larvae, in which 644 genes were upregulated and 552 genes were downregulated. Results showed that multiple DEGs were enriched in detoxication-related gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Eleven detoxifying enzyme genes which differentially expressed were screened, and their expression patterns were validated by qRT-PCR. Furthermore, we successfully knocked down all differentially upregulated detoxifying enzyme genes, and a bioassay showed that the mortality of chlorfenapyr-treated silkworm larvae was significantly higher after silencing these genes than in groups injected with dsGFP. The present study reveals the molecular basis of silkworm detoxification to chlorfenapyr exposure, and provides new insights into the management of insecticide damage in the silkworm.

Comparative transcriptome analysis of mulberry reveals anthocyanin biosynthesis mechanisms in black (Morus atropurpurea Roxb.) and white (Morus alba L.) fruit genotypes.

BACKGROUND: To gain a better understanding of anthocyanin biosynthesis in mulberry fruit, we analyzed the transcriptome of the mulberry varieties Da 10 (Morus atropurpurea Roxb., black fruit) and Baisang (Morus alba L., white fruit). RESULTS: We found that whereas Da 10 had high levels of cyanidin 3-O-glucoside (Cy), and pelargonidin 3-O-glucoside (Pg), Baisang contained only Cy, at low levels. Based on a comparative transcriptome analysis, we annotated more than 27,085 genes (including 1735 new genes). Genes that were differentially expressed between Da 10 and Baisang were detected at three stages of fruit development: S1 [4256 genes, 10 days post-anthesis (DPA)], S2 (5612 genes, 19 DPA), and S3 (5226 genes, 28 DPA). Anthocyanin biosynthesis was found to be associated with the expression of 15 core genes and 5 transcription factors. Relative to Baisang, Da 10 showed a significant upregulation of genes involved in the early stages (production of the intermediate compounds chalcone and dihydroflavonol) and late stages (production of Cy and Pg) of anthocyanin biosynthesis. Baisang showed a significant downregulation of the genes involved in the early stages of anthocyanin biosynthesis and overexpression of flavanone 3-hydroxylase (FLS), resulting in the generation of quercetin and/or myricetin but not anthocyanins. CONCLUSIONS: The biosynthesis of anthocyanin in mulberry fruit is initiated from the precursor, phenylalanine, and mediated by the upregulation of dihydroflavonol 4-reductase, anthocyanidin synthase, anthocyanidin 3-O-glucosyltransferase, and cyanidin-3-O-glucoside 2-O-glucuronosyltransferase, and downregulation of FLS to produce Cy and Pg.

Genome-wide analysis of DNA methylation in subcultured Cordyceps militaris.

The entomopathogenic mushroom Cordyceps militaris is a storehouse of various medicinal compounds and pharmacological effects. However, the high frequency of strain degeneration during subculture and preservation severely limits the large-scale production of C. militaris. DNA methylation is an important epigenomic modification involved in gene regulation. In this study, we used bisulfite sequencing for DNA methylation profiling of wild-type and mutant C. militaris. The differentially methylated regions (DMRs) of the two types were analyzed using Gene Ontology (GO) clustering and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. DNA methylation levels of the wild-type and mutant-type C. militaris were 0.48% and 0.56%, respectively. Methylation appeared at CHH dinucleotides in 58.62% and 58.20% of all methylated cytosine sites in the wild and mutant types, respectively. In all, 188 DMRs were identified from the wild and mutant types. Most of the DMRs ranged from 200 to 350 bp in length. KEGG pathways of the expression of DMR-related genes, which are involved in pyruvate metabolism, glycerophospholipid metabolism, DNA replication, and N-glycan biosynthesis. This contributes to the knowledge and understanding of the possible mechanisms of C. militaris strain degeneration.

Histidine-Rich Cell-Penetrating Peptide for Cancer Drug Delivery and Its Uptake Mechanism.

In this work, we report a drug delivery system based on the pH-responsive self-assembly and -disassembly behaviors of peptides. Here, a systematically designed histidine-rich lipidated peptide (NP1) is presented to encapsulate and deliver an anticancer drug ellipticine (EPT) into two model cells: non-small-cell lung carcinoma and Chinese hamster ovary cells. The mechanism of pH-responsive peptide self-assembly and -disassembly involved in the drug encapsulation and release process are extensively investigated. We found that NP1 could self-assemble as a spherical nanocomplex (diameter = 34.43 nm) in a neutral pH environment with EPT encapsulated and positively charged arginine amino acids aligned outward and EPT is released in an acidic environment due to the pH-triggered disassembly. Furthermore, the EPT-encapsulating peptide could achieve a mass loading ability of 18% (mass of loaded-EPT/mass of NP1) with optimization. More importantly, it is revealed that the positively charged arginine on the periphery of the NP1 peptides could greatly facilitate their direct translocation through the negatively charged plasma membrane via electrostatic interaction, instead of via endocytosis, which provides a more efficient uptake pathway.

Identification and functional analysis of the cathepsin D gene promoter of Bombyx mori.

The gene encoding cathepsin D of silkworm, Bombyx mori (BmCatD) is specifically expressed in the larval fat body and pupal gut, and plays an important role in the programmed cell death during metamorphosis. To identify element involved in this transcription-dependent spatial restriction, truncation and deletion of the 5' terminal from the BmCatD promoter were conducted in vivo. The recombinant AcMNPV vector (Autographa californica multiple nucleopolyhedrovirus) with a dual-luciferase quantitative assay system was used as the transfer. A 289 bp DNA sequence (-1,214 to -925) upstream of the transcriptional start site is found to be responsible for promoting tissue-specific transcription. Further analysis of a series of deletion within the 289 bp region of overlapping deletion showed that a 33 bp region (-1,071 to -1,038) sequence suppresses the ectopic expression of the BmCatD promoter. These results suggest that this 33 bp region could function as a promoter element in the tissue-specificity expression.

Characterization of Cellulose-Degrading Bacteria Isolated from Silkworm Excrement and Optimization of Its Cellulase Production.

An abundance of refractory cellulose is the key limiting factor restricting the resource utilization efficiency of silkworm (Bombyx mori) excrement via composting. Screening for cellulose-degrading bacteria is likely to provide high-quality strains for the safe and rapid decomposition of silkworm excrement. In this study, bacteria capable of degrading cellulose with a high efficiency were isolated from silkworm excrement and the conditions for cellulase production were optimized. The strains were preliminarily screened via sodium carboxymethyl cellulose culture and staining with Congo red, rescreened via a filter paper enzyme activity test, and identified via morphological observation, physiological and biochemical tests, and phylogenetic analysis of the 16S rDNA sequence. Enzyme activity assay was performed using the 3,5-dinitrosalicylic acid method. DC-11, a highly cellulolytic strain, was identified as Bacillus subtilis. The optimum temperature and pH of this strain were 55 °C and 6, respectively, and the filter paper enzyme activity (FPase), endoglucanase activity (CMCase), and exoglucanase activity (CXase) reached 15.40 U/mL, 11.91 U/mL, and 20.61 U/mL. In addition, the cellulose degradation rate of the treatment group treated with DC-11 was 39.57% in the bioaugmentation test, which was significantly higher than that of the control group without DC-11 (10.01%). Strain DC-11 was shown to be an acid-resistant and heat-resistant cellulose-degrading strain, with high cellulase activity. This strain can exert a bioaugmentation effect on cellulose degradation and has the potential for use in preparing microbial inocula that can be applied for the safe and rapid composting of silkworm excrement.

Studies on 1-deoxynojirimycin biosynthesis in mulberry (Morus alba L.) seeds through comparative transcriptomics.

Mulberry (Morus alba L.) plants are rich in 1-deoxynojirimycin (DNJ), which is a potential α-glucosidase inhibitor exhibiting various physiological activities. Compared to other tissues, Morus alba L. seeds contain the highest DNJ content, however, the DNJ biosynthesis mechanisms are unclear. In this study, we examined fruits of 27 mulberry varieties and found that variety MS02 had the highest DNJ levels (22.28 mg/g), whereas variety MS15 contained the lowest DNJ levels (0.37 mg/g). Through comparative transcriptomics, 1,719 differentially expressed genes (DEGs) were identified, 1,170 of which were upregulated, and 549 were downregulated in MS02 compared to MS15. DEGs were associated with cellular processes, metabolic processes, and catalytic activity. Specifically, nine DEGs were identified to be involved in alkaloid biosynthesis pathways, according to Kyoto Encyclopaedia of Genes and Genomes enrichment analysis, and four enzymes, i.e. polyphenol oxidase, tyrosine aminotransferase, aromatic-L-amino-acid decarboxylase, and tropinone reductase, are proposed to play important roles in DNJ biosynthesis. In conclusion, DNJ biosynthesis in mulberry seeds appears to be mediated by upregulation of polyphenol oxidase, tyrosine aminotransferase, aromatic-L-amino-acid decarboxylase, and tropinone reductase.

Identification of ecdysone response elements (EcREs) in the Bombyx mori cathepsin D promoter.

Bombyx mori Cathepsin D (BmCatD) is specifically expressed in the fat body, and plays a critical role for the programmed cell death of the larval fat body and pupal gut during metamorphosis. To better understand the transcriptional control of BmCatD expression, we conducted this study to identify the ecdysone response elements (EcREs) in the BmCatD promoter and clarify their regulational functions. We inserted EcREs into a recombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector and performed luciferase assay with a dual-luciferase quantitative assay system. Three putative EcREs were located at positions -109 to -99, -836 to -826 and -856 to -846 relative to the transcription start site. Overlapping deletion studies of this EcRE region showed that the three EcREs could suppress the ectopic expression of the BmCatD promoter. EcRE mutations resulted in the loss of the fat body-specific expression of the BmCatD gene. These results suggest that the EcREs are vital for activation of the promoter by 20-hydroxyecdysone (20E) in the larval fat body and further support the crucial role of ecdysone signaling to control cathepsin D gene transcription. It may suggest that the heterodimeric complex EcR/USP mediates the activation of ecdysone-dependent BmCatD transcription in the larval fat body of B. mori.

Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media.

Two acidic polysaccharide fractions, CM-jd-CPS2 and CM-jd(Y)-CPS2, were isolated from the fruiting bodies of cultured Cordyceps militaris grown on solid rice medium and silkworm pupa, respectively, by hot-water extraction, ethanol precipitation and fractionation using ion-exchange column (DEAE-cellulose-52) and gel-filtration column (Sephadex G-100) chromatography. Their structural characterizations were performed by gas chromatography and fourier-transform infrared spectroscopy. Some differences existed between their structures, which indicated that culture media could influence the structure of polysaccharides of C. militaris. The antioxidant activities of CM-jd-CPS2 and CM-jd(Y)-CPS2 were evaluated by various methods in vitro. They had strong 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferrous ion-chelating capacity, but moderate reducing power. The antioxidant activities of CM-jd(Y)-CPS2 were slightly higher than those of CM-jd-CPS2. These two acidic fractions were evaluated for proliferation of mouse splenocyte activity in vitro. They both possessed does-dependent mitogenic effects on mouse splenocytes, and could synergistically promote murine T- and B-lymphocytes induced by Con A and LPS. CM-jd(Y)-CPS2 exhibited stronger stimulatory activities upon immunomodulation than CM-jd-CPS2. These results are beneficial for the interpretation of the connection between polysaccharide structures and their biological activities.

[Isolation, identification and biodegradation characterization of a novel monensin-degrading bacterial strain].

Biodegradation of antibiotic pollutants by microorganisms has received widespread attention, to which the identification of microorganisms capable of efficiently degrading antibiotics is a key. In this study, a strain DM-1 with high degradation capability was successfully isolated from monensin-contaminated chicken manure by using monensin as the sole carbon source. The strain was further identified basing on morphological, physiological and biochemical characteristics and 16S rRNA gene sequence-based phylogenetic analysis. The degradation efficiency of DM-1 for monensin was determined by HPLC post-column derivatization, and then the degradation conditions of DM-1 were optimized. DM-1 was identified as a strain of Acinetobacter and named as Acinetobacter baumannii DM-1. The optimal conditions for monensin degradation by strain DM-1 were pH 7.0, 30 ℃, and initial monensin concentration of 50 mg/L. The strain DM-1 degraded more than 87.51% of monensin at an initial concentration of 10 mg/L in 28 days, while only a slight decrease in monensin concentration was observed in the control without monensin-degrading strain. This study indicates that the strain DM-1 has a promising application prospect in the bioremediation of monensin-contaminated environment.

Expression profiles of glutathione S-transferase genes in larval midgut of Bombyx mori exposed to insect hormones.

Glutathione S-transferases (GSTs) are believed to play a role in the detoxification of xenobiotics, resistance to insect viruses and pesticides, intracellular transport, biosynthesis of hormones and protection against oxidative stress. In this study, we used quantitative real time RT-PCR to examine expression profiles of the silkworm Bombyx mori GST-Sigma (BmGSTS2) and GST-Delta (BmGSTD2) genes in the larval midgut of the silkworm after exposure to 2-hydroxyecdysone (20E) and juvenile hormone analog (JHA). In concentration-course study, 20E at higher concentrations (1.0 and 2.0 µg/µl) caused significant upregulation of BmGSTD2, and all concentrations (0.5-2.0 µg/µl) of 20E caused significant upregulation of BmGSTS2. However, JHA in all concentrations downregulated the expression of BmGSTD2 and BmGSTS2. When exposed to either 20E (2.0 µg/µl) or JHA (2.0 µg/µl) on the third day of the fifth instar, the silkworm had higher BmGSTD2 at later time points: 15, 18, and 24 h for 20E and 24 h for JHA. BmGSTS2 expression was downregulated within 24 h after exposure to JHA and showed a time-dependent response after exposure to 20E. We also did a stage-dependent study, in which JHA downregulated BmGSTD2 expression and upregulated BmGSTS2 expression significantly at both day 1 and day 3 of the fifth instar. 20E upregulated the expression of BmGSTD2 and BmGSTS2 at the two stages. These findings imply that hormones have an important role in the regulation of basal GST expression. However, further validation and field trials should be carried out on the regulatory elements relevant to BmGSTD2 and BmGSTS2 gene expression.

A study on effects of glutathione s-transferase from silkworm on CCL4-induced mouse liver injury.

To assess the hepatoprotective activity of Glutathione S-transferase(GSTsw), extracted and purified from silkworm, in experimental acute mice liver injury and explore mechanisms. Mice were divided into five groups: control group, carbon tetrachloride (CCl4) group, and three treatment groups that received CCl4 and GSTsw at doses of 0.083 mg•g(-1), 0.0415 mg•g(-1) and 0.0207 mg•g(-1) for 3 days. ALT in serum, GST, SOD and T-AOC in liver tissue homogenate, and changes in liver pathology in the five groups were studied. CCl4 administration led to pathological and biochemical evidence of liver injury as compared to untreated controls. GSTsw administration led to significant protection against CCl4-induced changes in liver pathology. It was also associatedwith significantly lower serum ALT levels, higher GST-SOD and T-AOC level in live tissue homogenate. Thus, GSTsw showed protective activity against CCl4-induced hepatotoxicity in mice.

Chemical synthesis, inhibitory activity and molecular mechanism of 1-deoxynojirimycin-chrysin as a potent α-glucosidase inhibitor.

Hyperglycemia can be efficaciously regulated by inhibiting α-glucosidase activity and this is regarded as an effective strategy to treat type 2 diabetes. 1-Deoxynojimycin, an α-glucosidase inhibitor, can penetrate cells rapidly to potently inhibit α-glucosidase in a competitive manner. However, the application of 1-deoxynojimycin is limited by its poor lipophilicity and low bioavailability. Herein, three 1-deoxynojimycin derivatives 4-6 were designed and synthesized by linking 1-deoxynojimycin and chrysin to ameliorate the limitations of 1-deoxynojimycin. Among them, compound 6, a conjugate of 1-deoxynojimycin and chrysin linked by an undecane chain, could better bind to the α-glucosidase catalytic site, thereby exhibiting excellent α-glucosidase inhibitory activity (IC50 = 0.51 ± 0.02 µM). Kinetics analyses revealed that compound 6 inhibited the activity of α-glucosidase in a reversible and mixed competitive manner. Fluorescence quenching and UV-Vis spectra showed that compound 6 changed the conformation of the α-glucosidase via complex formation, which triggered a static fluorescence quenching of the enzyme protein.

Effect of the silkworm pupa protein-glucose conjugate on the thermal stability and antioxidant activity of anthocyanins.

Anthocyanin (cyanidin-3-O-glucose) is a natural water-soluble pigment with a robust antioxidant capacity. However, its poor stability and bioavailability limits its application as a functional food ingredient. This study explored the ability of the silkworm pupa protein-glucose (Spp-Glu) conjugate, developed under wet-heating conditions, to improve the thermal stability and antioxidant activity of cyanidin-3-O-glucose (C3G) at pH 3.0 and 6.8. The characterization experiments suggested that C3G complexed with the Spp-Glu conjugate could modify the protein's microenvironment and cause unfolding of the protein's secondary structures under varied pH conditions. Spectroscopic techniques further revealed the formation of complexes via hydrophobic interactions and static quenching processes when C3G was bound to Spp or Spp-Glu. The formation of these complexes effectively attenuated C3G degradation, thereby enhancing its stability under heat treatment over a range of pH values, and the experiments measuring antioxidant activity suggested that the Spp-Glu conjugate formed does not affect the efficacy of C3G after complexation. Therefore, our study suggests that Spp-Glu has the potential to effectively protect and deliver anthocyanins during industrial application for functional food formulation.

Studies on the interactional characterization of preheated silkworm pupae protein (SPP) with anthocyanins (C3G) and their effect on anthocyanin stability.

In this paper, the interaction of silkworm pupae protein (SPP) with cyanidin-3-O-glucoside (C3G) was studied the protective anthocyanins stability. Characterization experiments suggested that C3G-SPP complexes mainly through hydrophobic interactions, with a decrease in the α-helix content and increases in the ß-sheet and ß-turn contents. Fluorescence results revealed that C3G quenched the intrinsic fluorescence of SPP by static quenching. The highest quenching constant, Kq, was recorded to be1.26 × 1012 M-1s-1 for the SPP preheated at 80 °C. Following the C3G-SPP complexes, the degradation rate constant decreased, and the half-life of C3G was prolonged from 64.81 ± 1.07 to 261.99 ± 13.32 min at 80 °C (p < 0.05). The SPP preheated at 80 °C exhibited the highest binding affinity towards C3G and also effectively increased the thermal and oxidative stability of the C3G. The obtained results suggest that the novel protein proposed in this study could expand the application of anthocyanins as stable, functional food ingredients.

Effects of insect viruses and pesticides on glutathione S-transferase activity and gene expression in Bombyx mori.

Glutathione S-transferase (GST) is a gene family generally associated with detoxification and plays an important role in detoxifying exogenous compounds. The silkworm Bombyx mori is an important economic animal for silk production. However, it is liable to infection by a number of viruses and chemical agents that can contaminate its food and growing environment. Here we conducted a comparative study of strain- and tissue-specific expressions of GST in the silkworm under infections by Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori densonucleosis virus (BmDNV) and under the stress of pesticides (insecticide and herbicide). BmDNV induced an increase of GST at 24 h and a decrease at 48 and 72 h in the midgut of the DNV-susceptible strain and a decrease at 24 h and increase at 48 and 72 h in the midgut of the DNV-tolerant strain. BmDNV induced a significant increase of GST from 24 to 72 h in the fat body of both DNV-susceptible and DNV-tolerant strains. In contrast, BmNPV induced a significant decrease of GST in both the fat body and midgut of the NPV-susceptible strain and induced increase of GST from 24 to 48 h in the midgut and at 72 h in the fat body of the NPV-tolerant strain. All of these results suggest that BmGST activity varies with the strain, tissue, feeding behavior, and developmental stage of the silkworm. On treating silkworm larvae with pesticides (insecticide and herbicide), expression of the BmGSTS2 gene increased noticeably in the midgut and reached a peak at 6 to 12 h. The mRNA level of BmGSTS2 in the midgut increased during administration of the chemicals, suggesting that the induction of BmGSTS2 is part of the defense mechanism against exogenous chemicals.

Effect of silkworm peptide on inducting M1 type polarization and Th1 activation via TLR2-induced MyD88-dependent pathway.

The aim of this study was to explore immune activity and molecular mechanism of silkworm peptide. The cell subsets induced by silkworm peptides were detected by flow cytometry. The IFN-γ and IL-4 levels in CD4+ cells were measured by ELISA. TLR2 mRNA expression in mouse CD4+ T cells was detected by qRT-PCR. Western blot was used to detect the protein expression levels of MyD88 and p-IκB. The growth rate of Lewis lung cancer xenografts in mice of the medium-dose group was significantly reduced, and the tumor volume was significantly smaller than that of the control group on the 14th day. The relative vitality values of spleen lymphocytes in the medium-dose and high-dose groups were higher than the control group. The IFN-γ levels in the medium-dose and high-dose groups were significantly higher than the control group. The levels of IL-4 were no significant change among different groups. Compared with the control group, different doses of silkworm peptide groups could increase the levels of NO, IL-6, IL-12, and IL-1ß. Compared with the control group, the protein expression levels of MyD88 and p-IκB in 10 µg/ml group and 20 µg/ml groups were significantly increased compared with the control group. Silkworm peptide could induce Th1 activation and M1 type polarization, which was dose-dependent and was relative to the effect of silkworm peptide on inhibiting tumor growth. Silkworm peptide could directly induce M1 type polarization and Th1 activation via TLR2-induced MyD88-dependent pathway in vitro.