Anemoside B4 inhibits enterovirus 71 propagation in mice through upregulating 14-3-3 expression and type I interferon responses.

Enterovirus 71 (EV71) is the major pathogens of human hand, foot, and mouth disease (HFMD). EV71 efficiently escapes innate immunity responses of the host to cause infection. At present, no effective antiviral drugs for EV71 are available. Anemoside B4 (B4) is a natural saponin isolated from the roots of Pulsatilla chinensis (Bunge) Regel. P. chinensis extracts that shows a wide variety of biological activities. In this study, we investigated the antiviral activities of B4 against EV71 both in cell culture and in suckling mice. We showed that B4 (12.5-200 µM) dose dependently increased the viability of EV71-infected RD cells with an IC50 value of 24.95 ± 0.05 µM against EV71. The antiviral activity of B4 was associated with enhanced interferon (IFN)-ß response, since knockdown of IFN-ß abolished its antiviral activity. We also confirmed that the enhanced IFN response was mediated via activation of retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) pathway, and it was executed by upregulation of 14-3-3 protein, which disrupted the interaction between yes-associated protein (YAP) and interferon regulatory factor 3 (IRF3). By using amino acids in cell culture (SILAC)-based proteomics profiling, we identified the Hippo pathway as the top-ranking functional cluster in B4-treated EV71-infected cells. In vivo experiments were conducted in suckling mice (2-day-old) infected with EV71 and subsequently B4 (200 mg · kg-1 · d-1, i.p.) was administered for 16 days. We showed that B4 administration effectively suppressed EV71 replication and improved muscle inflammation and limb activity. Meanwhile, B4 administration regulated the expressions of HFMD biomarkers IL-10 and IFN-γ, attenuating complications of EV71 infection. Collectively, our results suggest that B4 could enhance the antiviral effect of IFN-ß by orchestrating Hippo and RLRs pathway, and B4 would be a potential lead compound for developing an anti-EV71 drug.

Molecular signal networks and regulating mechanisms of the unfolded protein response.

Within the cell, several mechanisms exist to maintain homeostasis of the endoplasmic reticulum (ER). One of the primary mechanisms is the unfolded protein response (UPR). In this review, we primarily focus on the latest signal webs and regulation mechanisms of the UPR. The relationships among ER stress, apoptosis, and cancer are also discussed. Under the normal state, binding immunoglobulin protein (BiP) interacts with the three sensors (protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE1α)). Under ER stress, misfolded proteins interact with BiP, resulting in the release of BiP from the sensors. Subsequently, the three sensors dimerize and autophosphorylate to promote the signal cascades of ER stress. ER stress includes a series of positive and negative feedback signals, such as those regulating the stabilization of the sensors/BiP complex, activating and inactivating the sensors by autophosphorylation and dephosphorylation, activating specific transcription factors to enable selective transcription, and augmenting the ability to refold and export. Apart from the three basic pathways, vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR)-phospholipase C-γ (PLCγ)-mammalian target of rapamycin complex 1 (mTORC1) pathway, induced only in solid tumors, can also activate ATF6 and PERK signal cascades, and IRE1α also can be activated by activated RAC-alpha serine/threonine-protein kinase (AKT). A moderate UPR functions as a pro-survival signal to return the cell to its state of homeostasis. However, persistent ER stress will induce cells to undergo apoptosis in response to increasing reactive oxygen species (ROS), Ca2+ in the cytoplasmic matrix, and other apoptosis signal cascades, such as c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription 3 (STAT3), and P38, when cellular damage exceeds the capacity of this adaptive response.

A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization.

A novel serine protease with fibrinolytic activity named CSP was purified from the culture supernatant of the fungus Cordyceps sinensis, a kind of Chinese herbal medicine. Analysis of the purified enzyme by SDS-PAGE indicated that CSP was a single polypeptide chain with an apparent molecular weight of 31 kDa, and N-terminal sequencing revealed that the first ten amino acid residues of the enzyme were Ala-Leu-Ala-Thr-Gln-His-Gly-Ala-Pro-Trp-. When casein was used as a substrate, the proteolytic activity of CSP reached its maximum at pH 7.0 and 40 degrees C. The effect of chemical agents on the enzyme activity indicated that CSP is a serine protease with a free cysteine residue near the active site. It hydrolysed fibrinogen, fibrin and casein with a high efficiency, while hydrolysing bovine serum albumin (BSA) and human serum albumin (HSA) to a lesser extent. CSP was found to be a plasmin-like protease, but not a plasminogen activator, and it preferentially cleaved the A alpha chain of fibrinogen and the alpha-chain of fibrin. Therefore, the extracellular protein CSP may represent a potential new therapeutic agent for the treatment of thrombosis.