Accordingly, autophagy activator rapamycin could antagonize the antiviral ability of eugenol in a concentration dependent manner determined by the SRB method using CPE reduction and by plaque inhibition assay. Figure S7. We speculated that the ability of eugenol inhibiting IAV replication was related to its inhibition of autophagy. B Eugenol antagonized the promotions of the activators on the dissociation of Beclin1-Bcl2 heterodimer determined by co-IP assay.
Here we investigated the influence of eugenol on the expression of Atg5, Atg7, Atg12, and Beclin1. As shown in Figure 8 , the expressions of Atg5, Atg7, Atg12 and Beclin1 were significantly increased after IAV infection, and significantly inhibited by ribavirin and eugenol at both mRNA and protein levels. B Western blot assay. The treatments of untreated, virus only, ribavirin and Eug groups were same with those in Figure 6.
Autophagy in Health and Disease | ScienceDirect
It is reported that autophagy can regulate the release of cytokines  , and cytokine storm is an important pathogenesis of IAV-induced acute lung injury. As shown in Figure 9 , after IAV infection, the levels of these cytokines were significantly increased, whereas both of ribavirin and eugenol significantly inhibited the releases of these cytokines. Up to now, there are two anti-IAV drug screening strategies: virus-based and cell-based drug screening models.
Influenza A virus possesses high genetic variability, virus-based drug screening models often lead to the inevitable selection of drug-resistant viral mutants and drug-resistant variants can rapidly generate. Hunan genetic variability is very low, so cell-based drug screening models often decrease these resistant mutants .
Now, targeting human protein which is essential for viral replication has become an important strategy for developing new antiviral drugs . In our research, we first established a screening model to screen novel autophagy inhibitors based on the inhibition of the dissociation of Beclin1-Bcl2 heterodimer, and according to the previous reports that autophagy is essential for IAV replication or induces autophagic cell death which results in acute lung injury  ,  ,  , we then detect the anti-IAV activity of these autophagy inhibitors.
Because autophagy is a highly conserved process in all eukaryotic cells, so our drug screening model may lead to the discovery of novel antiviral drugs that resist the development of drug-resistant virus strains. Though there are many reports that IAV infection can enhance autophagic flux, some researchers think that the accumulation of autophagosome after IAV infection is not the consequence of a general enhancement of autophagy, but is only the block of autophagosome maturation by the binding of M2 to Beclin1 .
We do not support this point. As aforementioned, IAV involves in autophagy by its M2 protein binding with Beclin1, but there are at least three kinds of Beclin1 complexes.
The Atg14L complex localizes to the autophagosome and ER, and functions in autophagosome formation. We think that IAV M2 can interact with all of these three complexes, it not only influences the autophagosome maturation, but also influences the autophagosome initiation and formation. In fact, there are many proteins and signal pathways can upregulate the expression of autophagic genes and increase autophagic flux. ROS can increase the expression of Atg5 and Beclin1 and enhance autophagic flux  , . IAV infection can activate these proteins and these signal pathways, so we think the increase of autophagic flux induced by IAV infection plays an important role in the IAV-induced accumulation of autophagosome, and effectively controlling the autophagic flux is important for inhibiting autophagic cell death and acute lung injury induced by IAV.
This is the reason that we select the interaction of Beclin1 and Bcl2, which can at least in part control the autophagic flux, as our drug screening target. We then pick out eugenol, detect its anti-IAV activity, explore its mechanism of action, and simultaneously display the reasonableness of the design of our screening model. This conversely displays the reasonableness of the design of our screening model Figure Eighty six traditional Chinese medicines were screened by our drug screening model which was based on the inhibition of the dissociation of Beclin1-Bcl2 heterodimer, and Syzygium aromaticum L.
We purchased eugenol, the major active compound of Syzygium aromaticum L.
We then detected the anti-autophagy and anti-IAV activity of eugenol. Next we explored the mechanism of action of eugenol. Moreover, eugenol also inhibited the expression of autophagic genes.
Eventually, eugenol inhibited autophagy and impaired IAV replication. In conclusion, we have established a drug screening model based on the inhibition of the dissociation of Beclin1-Bcl2 heterodimer, an important regulator of autophagy. Using this model, we screen 86 examples of traditional Chinese medicine and find 15 examples can significantly inhibit the dissociation of Beclin1-Bcl2 heterodimer. We then detect the anti-autophagy and anti-IAV activity, and explore the mechanism of action of eugenol, and show that eugenol is a promising inhibitor for autophagy and IAV infection.
Medicinal plants were collected from the Yulin medicinal market Guangxi, China. The specimens were deposited in our lab. The virus stocks were prepared in MDCK cells or day-old embryonating eggs. The virus titers were determined by a plaque assay . The maximal concentration without cytotoxicity was used as the optimal concentration. All constructs were verified by double enzyme digestion and DNA sequencing.
To observe the results of BiFC assay, A cells were seeded onto glass cover slip in a well plate, and visualized under an upright fluorescence microscope Nikon Eclipse 90i. Plaque inhibition assay was performed on MDCK cells . Multiplicity of infection MOI was 0. The incubation time after absorption was 48 h. The supernatants were collected and the virus titers were determined by a plaque assay . Briefly, MDCK cells were seeded in well plate. OD was read at nm. Three wells were used each for the negative virus-infected non-drug-treated and the mock controls non-infected non-drug-treated.
The Western blotting assay was performed as previously reported . The influence of drugs on the dissociation of Beclin1-Bcl2 heterodimer and of Beclin1-IAV M2 heterodimer were detect by co-IP assay, A cells were seeded into a 6-well plate for 24 h, after cotransfection with corresponding plasmids for 6 h, the drugs were added, and the cells were collected after 24 h. The interactions were determined following the instrument of the Co-Immunoprecipitation Kit Thermo scientific, Normal rabbit IgG was used as a control.
Statistical significance was determined using the SPSS The regulation of autophagy and the involvement of IAV. In autophagy induction, the kinase mTOR is a critical negative regulator. Downstream of mTOR, Beclin1 is a very important protein required for autophagy which exists in three kinds of complexes. Among Beclin1-binding proteins, Bcl2 functions as an important autophagy inhibitor. A Reconstitution of RFP. A cells were cotransfected with pMN-Bcl2 and pMC-Beclin1, after 8 h, the cotransfected cells appeared a lot of red fluorescence.
These graphs were corresponding to Figure 1B c in text. Beclin1-binding proteins HMGB1 and MyD88 were expected to disrupt the Beclin1-Bcl2 heterodimer, after cotransfection for 8 h, the cells were visualized, These graphs were corresponding to Figure 1C a , b and c in text. After cotransfection, A cells were treated with these inhibitors and activators, after 8 h, the cells were visualized, These graphs were corresponding to Figure 1D a, b, c, d, e and f in text.
The ratios of RFP-positive cells were calculated in 5 fields chosen at random from three independent experiments. Rapamycin antagonized the anti-IAV activity of eugenol. In another experiment, the supernatants were collected and the virus titers were determined by a plaque assay on MDCK cells B.blog.am.mlsit.ru/wp-includes/baranov/reshebnik-po-russkomu.html
Macropinocytosis and autophagy crosstalk in nutrient scavenging
Additionally, rapamycin could promote the replication of IAV C. Influence of plant extracts on the dissociation of the Beclin1-Bcl2 heterodimer fold change. The FI was determined at nm after excitation at nm using a microplate reader Tecan infinite M All numbers were expressed as fold change relative to the negative control NC, virus only.
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Download: PPT. Description Autophagy in Health and Disease offers an overview of the latest research in autophagy with a translational emphasis. Key Features Discusses current understanding of the roles of autophagy in health and disease Covers the background of autophagy, the development of tools and therapeutics to measure and modulate autophagy, and autophagy in tissues and disease processes. Discusses current understanding of the roles of autophagy in health and disease Covers the background of autophagy, the development of tools and therapeutics to measure and modulate autophagy, and autophagy in tissues and disease processes.
Details ISBN All rights reserved. Editors Roberta A. This process generally leads to the destruction of the invasive microorganism , although some bacteria can block the maturation of phagosomes into degradative organelles called phagolysosomes. Vesicular stomatitis virus is believed to be taken up by the autophagosome from the cytosol and translocated to the endosomes where detection takes place by a pattern recognition receptor called toll-like receptor 7 , detecting single stranded RNA.
Following activation of the toll-like receptor, intracellular signaling cascades are initiated, leading to induction of interferon and other antiviral cytokines. A subset of viruses and bacteria subvert the autophagic pathway to promote their own replication. When galectin-8 binds to a damaged vacuole , it recruits an autophagy adaptor such as NDP52 leading to the formation of an autophagosome and bacterial degradation.