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Pattern recognition receptors (PRRs) are employed in insects to defend against infectious pathogens by triggering various immune responses. Peptidoglycan recognition proteins (PGRPs), a vital family of PRRs, are widely distributed and highly conserved from vertebrates to invertebrates. To date, five PGRP genes have been identified in Antheraea pernyi, but their biochemical roles still remain unknown. In this study, we focused on the immune functions of PGRP-SA in A. pernyi (ApPGRP-SA), which was confirmed to be immune-related according to its significantly up-regulated expression level post microbial injection. In addition, the binding properties of ApPGRP-SA were investigated using a recombinant protein produced in a prokaryotic expression system, revealing that rApPGRP-SA displayed a multi-binding ability to various microbes, including the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and fungus Candida albicans, together with their surface pathogen associated molecular patterns (PAMPs). Further studies showed that after recognition, the mixture of rApPGRP-SA/PAMP remarkably stimulated prophenoloxidase (PPO) activation in the hemolymph of A. pernyi in vitro, while the ds-PGRP-SA-treated hemolymph exhibited a lower sensitivity to PAMPs in comparison to the native sample. Moreover, the transcriptional level of the three antimicrobial peptides was also decreased in PGRP-SA knock-down larvae in response to immune-challenge. In summary, we conclude that ApPGRP-SA is a novel identified PGRP in A. pernyi that might act as a broad-spectrum pattern recognition receptor and is involved in the PPO activation system as well as antimicrobial peptide production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Apolipoproteins are protein components of lipoprotein particles, and are increasingly recognized to be functioning in the innate immune systems of both insects and mammals. Mammalian apolipoprotein B (apoB) is associated with a diverse range of innate immune defenses including suppression of bacterial pathogenesis, virus toxicity neutralization, and inhibition of cytokine releases from immune cells. However, little is known about apoB homologous insect apolipophorin-II/I (apoLp-II/I) in controlling of specific pathogen-host encounters such as microbial infections. In the present study, we describe cDNA cloning and characterization of an apoLp-II/I from Chinese oak silk worm, Antheraea pernyi. The apoLp-II/I cDNA is 10237bp in length, which possesses an open reading frame encoding 3305 amino acids. A consensus cleavage site RGRR presenting from Arg 710 to Arg 713 implies posttranslational cleavage of this protein. Ap-apoLp-II/I shares high sequence identities with apoLp-II/I in lepidoptera and other insects. In addition, considerable similarities also exist between Ap-apoLp-II/I and human apoB, which basically positioned in first 1000 residues of the amino termini. Tissue distribution and time-course expression results demonstrate that Ap-apoLp-II/I transcripts were detected predominantly in the fat body, less in epidermis and rarely in midgut, while the synthetic apoLp-II/I protein was abundant in hemocytes and plasma instead of the fatbody. Expression of Ap-apoLp-II/I was stimulated in response to bacterial challenge. In addition, our preliminary studies established a novel role for Ap-apoLp-II/I in regulating prophenoloxidase activation system. Taken together, apoLp-II/I may play an essential role in innate responses of Antheraea pernyi., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Serpins are a superfamily of proteins engaged in various physiological processes in all kingdoms of life. To date, many striking results have demonstrated serpins are involved in the invertebrate immune system by regulating the proteolytic cascades. However, in most insect species, the immune functions of serpins in response against pathogen invasion remain obscure. In this study, we identified a full-length cDNA sequence of serpin, named serpin-3, from the Chinese oak silkworm Antheraea pernyi. Sequence alignments have indicated that Apserpin-3 might regulate the melanization reaction via inhibiting prophenoloxidases-activating protease(s) in plasma. Furthermore, it was detected to be primarily transcribed within the fat body, epidermis and hemocytes with significant induction following immune-challenge. Further studies have shown that the knockdown of serpin-3 up-regulated the prophenoloxidases cascade stimulated by pathogen in hemolymph, while the addition of recombinant serpin-3 along with the same elicitor led to the suppressed activation of prophenoloxidase. Besides, the injection of dsRNA of serpin-3 caused the elevated expression of antimicrobial peptides. Altogether, we arrived at a conclusion that serpin-3 might act as a negative-regulator in prophenoloxidases activation and inhibit the production of antimicrobial peptides in Antheraea pernyi larvae., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Apolipophorin-III (apoLp-III) is an exchangeable apolipoprotein found in many insect species and functions as a lipid transport vehicle. Recent studies have shown that apoLp-III is a multifunctional molecule involved in not only lipid transportation but also innate immune responses. In the present study, the pattern recognition properties of Antheraea pernyi apoLp-III were investigated. Recombinant Ap-apoLp-III was bound to different species of microbes and further study showed the rAp-apoLp-III is capable of interacting with pathogen associated molecular patterns (PAMPs) on the microbial cell surface. In addition, an Ap-apoLp-III/PAMP mixture stimulated the prophenoloxidase (PPO) activation of A. pernyi hemolymph in vitro, to a greater extent than PAMP alone while Ap-apoLp-III itself failed to activate the PPO system, indicating that Ap-apoLp-III up-regulates PPO activation by combining with PAMP. After pathogen invasion following an injection of Staphylococcus aureus, RNAi-mediated silencing of apoLp-III decreased the transcriptional abundance of three antimicrobial peptide genes. These data suggest that apoLp-III is a versatile pattern recognition receptor and may play important roles in the innate immune responses of Antheraea pernyi., (Copyright © 2016. Published by Elsevier Ltd.)

Gangliosides, sialic acid-containing glycosphingolipids, have been considered to be involved in the development, differentiation, and function of nervous systems in vertebrates. However, the mechanisms for anti-inflammation caused by gangliosides are not clear. In this paper, we investigated the anti-inflammation effects of ganglioside GD1a by using RAW264.7 macrophages. Our data demonstrated that treatment of macrophages with lipopolysaccharide significantly increased the production of NO and pro-inflammatory cytokines. GD1a suppressed the induction of iNOS and COX-2 mRNA and protein expression and secretory pro-inflammatory cytokines in culture medium, such as TNFα, IL-1α and IL-1β. In addition, LPS-induced phosphorylation of mitogen-activating protein kinases and IκBα degradation followed by translocation of the NF-κB from the cytoplasm to the nucleus were attenuated after GD1a treatment. Furthermore, GD1a probably inhibited LPS binding to macrophages and LPS-induced accumulation between TLR4 and MyD88. Taken together, the results demonstrated that ganglioside GD1a inhibited LPS-induced inflammation in RAW 264.7 macrophages by suppressing phosphorylation of mitogen-activating protein kinases and activation of NF-κB through repressing the Toll-like receptor 4 signaling pathway., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of the study is to investigate the direct angiogenic activities of SMND-309, a novel metabolite of salvianolic acid B, on human umbilical vein endothelial cells (HUVEC) in vitro and its potential molecular mechanisms. Effects of SMND-309 on proliferation and adhesion of HUVEC were measured using sulforhodamine B assay and cell adhesion assay kit, respectively. Effects of SMND-309 on migration and differentiation of HUVEC were examined through wound-healing assay and tube formation on matrigel method, respectively. Expressions of erythropoietin (EPO), EPO receptor, phosphorylated EPO receptor, signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 and vascular endothelial growth factor (VEGF) were detected by Western blot. Knocking down EPO receptor gene and blocking the epidermal growth factor (EGF) receptor/Janus kinase 2 (JAK2) pathways were used to explore the potential mechanisms in SMND-309 induced angiogenesis. SMND-309 strongly induced the proliferation of HUVEC in a concentration-dependent manner within the concentrations of 1-30 μg/ml and significantly promoted the adhesion of HUVEC to different extracellular matrix at 30μg/ml. SMND-309 at doses of 3, 10, 30 μg/ml significantly enhanced the migration, capillary-like structure formation, and the levels of VEGF, phosphorylated EPO receptor and phosphorylated STAT3. Results from further experiments using HUVEC(EPO receptor-) and AG-490 showed that SMND-309 activated EPO receptor first, and then stimulated JAK2/STAT3, which up-regulated the expression of VEGF, and resulted in the angiogenesis. These results clearly show that SMND-309 has powerful angiogenic activity on HUVEC, which is mostly correlated with the up-regulation of VEGF through EPO receptor/STAT3 signal pathways., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, uniform mesoporous carbon spheres (UMCS) with 3-D pore system and fibrous ordered mesoporous carbon (FOMC) with 2-dimensional hexagonal mesoporous structure were studied as drug carriers for oral drug delivery system. Lovastatin (LOV), which has low water solubility, was chosen as a model drug. Drug release rate and degree of drug loading of UMCS and FOMC were compared. The effects of different pore channel structures and pore sizes on LOV uptake and release were systematically investigated. Cytotoxicity of UMCS and FOMC on human colon carcinoma (Caco-2) cells were also studied. The results indicate that UMCS has a higher degree of drug loading (up to 36.26% drug weight/total weight) compared with FOMC. The dissolution rate of LOV from UMCS was found to be markedly increased compared with pure crystalline LOV, and the dissolution rate of LOV from FOMC was relatively sustained compared with UMCS, and both UMCS and FOMC exhibited a weak cytotoxicity at tested concentrations (10-800 μg/ml)., (Copyright © 2011 Elsevier B.V. All rights reserved.)