Xiao-Xuan Chen

Drug screening for autophagy inhibitors based on the dissociation of Beclin1-Bcl2 complex using BiFC technique and mechanism of eugenol on anti-influenza A virus activity.

Autophagy is involved in many human diseases, such as cancer, cardiovascular disease and virus infection, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza A virus (IAV) and coxsackievirus B3/B4 (CVB3/B4), so a drug screening model targeting autophagy may be very useful for the therapy of these diseases. In our study, we established a drug screening model based on the inhibition of the dissociation of Beclin1-Bcl2 heterodimer, an important negative regulator of autophagy, using bimolecular fluorescence complementation (BiFC) technique for developing novel autophagy inhibitors and anti-IAV agents. From 86 examples of traditional Chinese medicines, we found Syzygium aromaticum L. had the best activity. We then determined the anti-autophagy and anti-IAV activity of eugenol, the major active compound of Syzygium aromaticum L., and explored its mechanism of action. Eugenol could inhibit autophagy and IAV replication, inhibited the activation of ERK, p38MAPK and IKK/NF-κB signal pathways and antagonized the effects of the activators of these pathways. Eugenol also ameliorated the oxidative stress and inhibited the expressions of autophagic genes. We speculated that the mechanism underlying might be that eugenol inhibited the oxidative stress and the activation of ERK1/2, p38MAPK and IKK/NF-κB pathways, subsequently inhibited the dissociation of Beclin1-Bcl2 heterodimer and autophagy, and finally impaired IAV replication. These results might conversely display the reasonableness of the design of our screening model. In conclusion, we have established a drug screening model for developing novel autophagy inhibitor, and find eugenol as a promising inhibitor for autophagy and IAV infection.

A Drug Screening Method Based on the Autophagy Pathway and Studies of the Mechanism of Evodiamine against Influenza A Virus

In this research, we have established a drug screening method based on the autophagy signal pathway using the bimolecular fluorescence complementation - fluorescence resonance energy transfer (BiFC-FRET) technique to develop novel anti-influenza A virus (IAV) drugs. We selected Evodia rutaecarpa Benth out of 83 examples of traditional Chinese medicine and explored the mechanisms of evodiamine, the major active component of Evodia rutaecarpa Benth, on anti-IAV activity. Our results showed that evodiamine could significantly inhibit IAV replication, as determined by a plaque inhibition assay, an IAV vRNA promoter luciferase reporter assay and the Sulforhodamine B method using cytopathic effect (CPE) reduction. Additionally, evodiamine could significantly inhibit the accumulation of LC3-II and p62, and the dot-like aggregation of EGFP-LC3. This compound also inhibited the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-α, IL-1β, IL-6 and IL-8 after IAV infection. Evodiamine inhibited IAV-induced autophagy was also dependent on its action on the AMPK/TSC2/mTOR signal pathway. In conclusion, we have established a new drug screening method, and selected evodiamine as a promising anti-IAV compound.

High-throughput screening for anti-influenza A virus drugs and study of the mechanism of procyanidin on influenza A virus-induced autophagy.

In this research, we have established a high-throughput screening (HTS) platform based on the influenza A virus (IAV) vRNA promoter. Using this HTS platform, we selected 35 medicinal plants out of 83 examples of traditional Chinese medicine and found that 7 examples had not been reported. After examining many previous reports, we found that Vaccinium angustifolium Ait., Vitis vinifera L, and Cinnamomum cassia Presl had a common active compound, procyanidin, and then determined the anti-IAV effect of procyanidin and explored its mechanism of action. With a plaque inhibition assay and a time-of-addition experiment, we found that procyanidin could inhibit the IAV replication at several stages of the life cycle. In the Western blot and EGFP-LC3 localization assays, we found that procyanidin could inhibit the accumulation of LC3II and the dot-like aggregation of EGFP-LC3. In the RT-PCR and Western blot assays, we found procyanidin could inhibit the expression of Atg7, Atg5, and Atg12. Finally, by the bimolecular fluorescence complementation–fluorescence resonance energy transfer and co-immunoprecipitation assays, we found that procyanidin could inhibit the formation of the Atg5-Atg12/Atg16 heterotrimer and the dissociation of the beclin1/bcl2 heterodimer. In conclusion, we have established an HTS platform and identified procyanidin as a novel and promising anti-IAV agent.

Effects of NS1 variants of H5N1 influenza virus on interferon induction, TNFα response and p53 activity

Non-structural protein 1 (NS1) is an important virulence factor of the highly pathogenic H5N1 avian influenza virus. A five-amino-acid (5 aa) deletion at position 80–84 and an aspartic acid to glutamic acid substitution at position 92 (D92E) are two major NS1 mutations that are highly correlated with enhanced virulence. To investigate the effect of these mutations in H5N1 virulence, three H5N1-NS1 variants were constructed: NS51 (lacking 5 aa at position 80–84), NS51(I) (carrying a 5-aa insertion at position 80–84) and NS51(IM) (carrying both the 5-aa insertion and the D92E mutation). We examined the effects of these mutations on interferon (IFN) induction, tumor-necrosis factor (TNF)α response, p53 activity and apoptosis. We found that the D92E mutation eliminated NS1s repressive effect on IFN induction, while the 5-aa deletion resulted in enhanced resistance to TNFα responses. We also observed that all three variants exhibited a similar suppressive effect on p53 transcriptional activity, although none of them significantly influenced apoptosis of host cells. Our findings shed new light on the role of NS1 in the pathogenicity of H5N1 virus.

The distinct binding properties between avian/human influenza A virus NS1 and Postsynaptic density protein-95 (PSD-95), and inhibition of nitric oxide production

BackgroundThe NS1 protein of influenza A virus is able to bind with many proteins that affect cellular signal transduction and protein synthesis in infected cells. The NS1 protein consists of approximately 230 amino acids and the last 4 amino acids of the NS1 C-terminal form a PDZ binding motif. Postsynaptic Density Protein-95 (PSD-95), which is mainly expressed in neurons, has 3 PDZ domains. We hypothesise that NS1 binds to PSD-95, and this binding is able to affect neuronal function.ResultWe conducted a yeast two-hybrid analysis, GST-pull down assays and co-immunoprecipitations to detect the interaction between NS1 and PSD-95. The results showed that NS1 of avian influenza virus H5N1 (A/chicken/Guangdong/1/2005) is able to bind to PSD-95, whereas NS1 of human influenza virus H1N1 (A/Shantou/169/2006) is unable to do so. The results also revealed that NS1 of H5N1 significantly reduces the production of nitric oxide (NO) in rat hippocampal neurons.ConclusionIn summary, our study indicates that NS1 of influenza A virus can bind with neuronal PSD-95, and the avian H5N1 and human H1N1 influenza A viruses possess distinct binding properties.

PI3K/Akt signaling pathway modulates influenza virus induced mouse alveolar macrophage polarization to M1/M2b.

Macrophages polarized to M1 (pro-inflammation) or M2 (anti-inflammation) phenotypes in response to environmental signals. In this study, we examined the polarization of alveolar macrophage (AM), following induction by different influenza virus strains (ST169 (H1N1), ST602 (H3N2) and HKG9 (H9N2)). Macrophages from other tissues or cell line exert alternative responding pattern, and AM is necessary for investigating the respiratory system. AM polarized toward the M1 phenotype after 4 hours of infection by all three virus strains, and AM to presented M2b phenotype after 8 hours induction, and immunosuppressive phenotype after 24 hours of induction. Protein expression assay showed similar results as the gene expression analysis for phenotype verification. The ELISA assay showed that TNF-α secretion was up-regulated after 4 and 8 hours of infection by influenza viruses, and it returned to basal levels after 24 hours of infection. IL-10 expression was elevated after 8 and 24 hours of infection. Immunofluorescence showed that iNOS expression was up-regulated but not Arg1 expression. Influenza virus notably increased phospho-Akt but not phospho-Erk1/2 or phospho-p38, and the AM polarization pattern have been changed by LY294002 (PI3K inhibitor). In conclusion, our results demonstrate the dynamic polarization of AM induced by influenza viruses, and suggested that PI3K/Akt signaling pathway modulates AM polarization to M1/M2b.

Identification of 23-(S)-2-Amino-3-Phenylpropanoyl-Silybin as an Antiviral Agent for Influenza A Virus Infection In Vitro and In Vivo

ABSTRACT It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection.

Heterologous SH3-p85β inhibits influenza A virus replication

Phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway can support the replication of influenza A virus through binding of viral NS1 protein to the Src homology 3 (SH3) domain of p85β regulatory subunit of PI3K. Here we investigated the effect of heterologously overexpressed SH3 on the replication of different influenza A virus subtypes/strains, and on the phosphorylation of Akt in the virus-infected cells. We found that heterologous SH3 reduced replication of influenza A viruses at varying degrees in a subtype/strain-dependent manner and SH3 overexpression reduced the induction of the phosphorylation of Akt in the cells infected with PR8(H1N1) and ST364(H3N2), but not with ST1233(H1N1), Ph2246(H9N2), and Qa199(H9N2). Our results suggest that interference with the NS1-p85β interaction by heterologous SH3 can be served as a useful antiviral strategy against influenza A virus infection.

Differential suppressive effect of promyelocytic leukemia protein on the replication of different subtypes/strains of influenza A virus

Promyelocytic leukemia protein (PML) plays an important role in the defense against a number of viruses, including influenza A virus. However, the sensitivity of influenza A virus subtypes/strains to PML is unknown. We investigated the role of PML in the replication of different influenza A virus subtypes/strains using pan-PML knock-down A549 cells and PML-VI-overexpressed MDCK cells. We found that (i) depletion of pan-PML by siRNA rendered A549 cells more susceptible to influenza A virus strains PR8(H1N1) and ST364(H3N2), but not to strains ST1233(H1N1), Qa199(H9N2) and Ph2246(H9N2); (ii) overexpression of PML-VI in MDCK cells conferred potent resistance to PR8(H1N1) infection, while lacked inhibitory activity to ST1233(H1N1), ST364(H3N2), Qa199(H9N2) and Ph2246(H9N2). Our results suggest that the antiviral effect of PML on influenza A viruses is viral subtype/strain specific.

Inhibition of Tanshinone IIA, salvianolic acid A and salvianolic acid B on Areca nut extract-induced oral submucous fibrosis in vitro.

Salvia miltiorrhiza Bunge has been reported to possess excellent antifibrotic activity. In this study, we have investigated the effect and mechanism of tanshinone IIA1 (Tan-IIA1), salvianolic acid A (Sal-A) and salvianolic acid B (Sal-B), the important active compounds of Salvia miltiorrhiza Bunge, on areca nut extract (ANE)-induced oral submucous fibrosis (OSF) in vitro. Through human procollagen gene promoter luciferase reporter plasmid assay, hydroxyproline assay, gelatin zymography assay, qRT-PCR, ELISA and Western blot assay, the influence of these three compounds on ANE-stimulated cell viability, collagen accumulation, procollagen gene transcription, MMP-2/-9 activity, MMP-1/-13 and TIMP-1/-2 expression, cytokine secretion and the activation of PI3K/AKT, ERK/JNK/p38 MAPK and TGF-β/Smads pathways were detected. The results showed that Tan-IIA1, Sal-A and Sal-B could significantly inhibit the ANE-stimulated abnormal viability and collagen accumulation of mice oral mucosal fibroblasts (MOMFs), inhibit the transcription of procollagen gene COL1A1 and COL3A1, increase MMP-2/-9 activity, decrease TIMP-1/-2 expression and inhibit the transcription and release of CTGF, TGF-β1, IL-6 and TNF-α; Tan-IIA1, Sal-A and Sal-B also inhibited the ANE-induced activation of AKT and ERK MAPK pathways in MOMFs and the activation of TGF-β/Smads pathway in HaCaT cells. In conclusion, Tan-IIA1, Sal-A and Sal-B possess excellent antifibrotic activity in vitro and can possibly be used to promote the rehabilitation of OSF patients.