Erguang Li

Houttuynia cordata blocks HSV infection through inhibition of NF-κB activation

Abstract Houttuynia cordata Thunb. is a medicinal plant widely used in folk medicine in several Asian countries. It has been reported that a water extract of H. cordata exhibits activity against herpes simplex virus (HSV) and the virus of severe acute respiratory syndrome (SARS), although the mechanisms are not fully understood yet. Previous studies have demonstrated absolute requirement of NF-κB activation for efficient replication of HSV-1 and HSV-2 and inhibition of NF-κB activation has been shown to suppress HSV infection. Here we show that a hot water extract of H. cordata (HCWE) inhibits HSV-2 infection through inhibition of NF-κB activation. The IC50 was estimated at 50μg/ml of lyophilized HCWE powder. At 150 and 450μg/ml, HCWE blocked infectious HSV-2 production by more than 3 and 4 logs, respectively. The inhibitory activity was concomitant with an inhibition of NF-κB activation by HSV-2 infection. Although activation of NF-κB and Erk MAPK has been implicated for HSV replication and growth, HCWE showed no effect on HSV-2-induced Erk activation. Furthermore, we show that treatment with quercetin, quercitrin or isoquercitrin, major water extractable flavonoids from H. cordata, significantly blocked HSV-2 infection. These results together demonstrated that H. cordata blocks HSV-2 infection through inhibition of NF-κB activation.

Glycyrrhizic acid as the antiviral component of Glycyrrhiza uralensis Fisch. against coxsackievirus A16 and enterovirus 71 of hand foot and mouth disease.

Abstract Ethnopharmacological relevance The radices of Glycyrrhiza uralensis Fisch. and herbal preparations containing Glycyrrhiza spp. have been used for thousands of years as an herbal medicine for the treatment of viral induced cough, viral hepatitis, and viral skin diseases like ulcers in China. Glycyrrhizic acid (GA) is considered the principal component in Glycyrrhiza spp. with a wide spectrum of antiviral activity. Aim The present study attempt to validate the medicinal use of Glycyrrhiza uralensis for hand, foot and mouth disease (HFMD) and further to verify whether GA is an active antiviral component in the water extract of Glycyrrhiza uralensis. Materials and methods Radices of Glycyrrhiza uralensis Fisch. were extracted with hot water. The chemical contents of the extract were profiled with HPLC analysis. The antiviral activity of the extract and the major components was evaluated against infection of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) on Vero cells. The cytopathic effect caused by the infection was measured with MTT assay. Infectious virion production was determined using secondary infection assays and viral protein expression by immunoblotting analysis. Results The extract at 1000μg/ml suppressed EV71 replication by 1.0 log and CVA16 by 1.5 logs. The antiviral activity was associated with the content of GA in the extract since selective depletion of GA from the extract by acid precipitation resulted in loss of antiviral activity. In contrast, the acid precipitant retained antiviral activity. The precipitant at a concentration of 200μg/ml inhibited EV71 and CVA16 replication by 1.7 and 2.2 logs, respectively. Furthermore, GA dose-dependently blocked viral replication of EV71 and CVA16. At 3mM, GA reduced infectious CVA16 and EV71 production by 3.5 and 2.2 logs, respectively. At 5mM, CVA16 production was reduced by 6.0 logs and EV71 by 4.0 logs. Both EV71 and CVA16 are members of Enterovirus genus, time-of-drug addition studies however showed that GA directly inactivated CVA16, while GA anti-EV71 effect was associated with an event(s) post virus cell entry. Conclusions This study validated the medicinal usefulness of radices Glycyrrhiza uralensis against the etiological agents of HFMD. In addition to the identification of GA as the antiviral component of Glycyrrhiza uralensis against EV71 and CVA16 infection, this study also reveals that GA inhibits EV71 and CVA16 with distinct mechanisms.

Inhibition of herpes simplex virus infection by oligomeric stilbenoids through ROS generation.

Stilbenoids including resveratrol contain the basic structural unit of 1,2-diphenylethylene. Naturally occurring stilbenoids have broad structural features due to oligomerization and modifications and some have demonstrated potent biological activities. In an effort to identify bioactive stilbenoids, we screened a group of dimeric and oligomeric stilbenoids against HSV-1 and HSV-2 infection. Several trimeric and tetrameric derivatives showed anti-herpetic activity at single digit micromolar concentrations. HSV-1 and HSV-2 replication requires for NF-κB and MAPK activation. The compounds showed no inhibitory activity against NF-κB and Erk/MAPK activation, instead those compounds promoted rapid and transient release of reactive oxygen species (ROS). Addition of N-acetylcysteine (NAC), a scavenger of ROS, reversed the inhibitory effect of those compounds against HSV replication. In addition to the identification of resveratrol derivatives with potent anti-HSV activity, our results uncover a mechanism of polyphenol-mediated anti-HSV response, linking anti-herpetic activity of oligomeric stilbenoids to innate immunity.

Bis-N-norgliovictin, a small-molecule compound from marine fungus, inhibits LPS-induced inflammation in macrophages and improves survival in sepsis.

Sepsis is a highly lethal disorder characterized by systemic inflammation, and Toll-like receptor 4 (TLR4) in macrophages plays a crucial role in modulating innate immune response and outcome of sepsis. During the screening of natural products against inflammation, we identified bis-N-norgliovictin, a small-molecule compound isolated from marine-derived fungus, significantly inhibited lipopolysaccharide (LPS, ligand of TLR4)-induced tumor necrosis factor-α (TNF-α) production in RAW264.7 cells. In this study, we evaluated the effect of bis-N-norgliovictin on TLR4-mediated inflammation in mouse macrophages and LPS-induced sepsis model. In RAW264.7 and mouse peritoneal macrophages, bis-N-norgliovictin dose-dependently inhibited LPS-induced production of TNF-α, interleukin-6 (IL-6), interferon-β (IFN-β) and monocyte chemoattractant protein (MCP-1), but without suppressing cell viability. The anti-inflammatory effect was attributed to the down-regulation of TLR4-triggered myeloid differentiation primary response protein 88 (MyD88)-dependent and TIR-containing adapter inducing interferon-β (TRIF)-dependent signaling pathways, including p38 and c-Jun N-terminal kinase (JNK) of mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) cascades. Importantly, bis-N-norgliovictin also protected mice against LPS-induced endotoxic shock. Intravenous injection of bis-N-norgliovictin 1h before LPS challenge dose-dependently inhibited LPS-induced increases in serum levels of TNF-α, IL-6, MCP-1 and IL-10, attenuated liver and lung injury and diminished M1 macrophage polarization in liver. Our results demonstrate that bis-N-norgliovictin exhibit potent anti-inflammatory effect both in vitro and in vivo. These findings suggest that bis-N-norgliovictin can be a useful therapeutic candidate for the treatment of sepsis and other inflammatory diseases.

A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the causative agents of hand, foot, and mouth disease (HFMD). During recent epidemics of HFMD in China, medicinal herbals and preparations containing herbal extracts have demonstrated therapeutic efficacy with relative safety profiles. There have been no microbiological studies to validate their usefulness for HFMD. We selected 12 commonly used herbs for HFMD from government recommended guidelines as well as published reports and tested for their antiviral activity and anti-inflammatory activity. A water extract of Houttuynia cordata Thunb. (HCT) inhibited EV71 infection significantly and was marginally active against CVA16 infection. The IC50 (concentration to have 50% inhibitory effect) values of HCT against a Fuyang strain and a BrCr strain of EV71 were determined at 8.9 μg/mL and 20.6 μg/mL, respectively. Mentha haplocalyx Briq. (MHB) water extract was active against CVA16, with an IC50 value of 70.3 μg/mL. The extract did not exhibit activity against EV71 infection. Although the majority of the extracts showed no activity against viral infection, several extracts demonstrated activity in blocking proinflammatory response by viral infection. This study therefore validates the effectiveness of Chinese herbs for HFMD since some formulations containing the correct combination of the herbs can block viral replication as well as proinflammatory response of HFMD.

Apigenin inhibits enterovirus-71 infection by disrupting viral RNA association with trans-acting factors.

Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5′-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.

A Novel Benzenediamine Derivate Rescued Mice from Experimental Sepsis by Attenuating Proinflammatory Mediators via IRAK4

We designed and synthesized a novel benzenediamine derivate, FC-99, that was tested for its ability to protect mice from experimental sepsis. Moreover, we sought to determine whether FC-99 could control a bacterial infection and to clarify the mechanism by which FC-99 inhibited LPS-activated macrophages. The effects of FC-99 on inflammation were evaluated in two experimental sepsis models and in cultured macrophages. Microarrays and docking and molecular dynamics simulations were used to determine the target of FC-99. Surface plasmon resonance and molecular detection were performed to confirm the direct interaction of FC-99 with its target. FC-99 protected mice from experimental sepsis. The mice that received FC-99 exhibited a diminished inflammatory response, had a lower local bacterial burden, and experienced a significantly improved survival rate. Genome-wide transcriptional profiling of FC-99-treated macrophages identified IRAK4 as a drug-regulated gene involved in LPS/TLR4 signaling. A computer search and calculations indicated that IRAK4 directly interacted with FC-99. Surface plasmon resonance, IRAK4-regulated signaling pathway analysis, and gene expression profiling of proinflammatory mediators confirmed the direct interaction between FC-99 and IRAK4. FC-99 is a potential therapeutic molecule for sepsis that alleviated experimental sepsis by directly inhibiting IRAK4 activation, which represents a novel target for sepsis therapy.

Curcumin is a biologically active copper chelator with antitumor activity.

BACKGROUND Curcumin is a natural product with antitumor activity. The compound targets multiple cell signaling pathways, including cell survival and proliferation, caspase activation and oncogene expression. As a β-diketone, curcumin also exists as a keto-enol tautomer that chelates transition metal ions with high affinity. PURPOSE Copper has an integral role in promoting tumor growth and angiogenesis. This study aims to investigate whether curcumin exerts its antitumor activity through copper chelation. METHODS Copper chelation ability of curcumin was validated by measuring US/VIS spectrum. The antitumor activity and in vivo copper removal ability of curcumin was determined in a murine xenograft model. The effect of curcumin on copper-induced MAPK activation and cell proliferation was determined in cell culture system. RESULTS Administration of curcumin to tumor-bearing animals resulted in suppression of A549 xenograft growth, an effect that was also observed in animals treated with ammonium tetrathiomolybdate (TM), a metal chelator used for copper storage disorders clinically. The inhibition on tumor growth was associated with reduction of copper concentrations in the serum of treated groups. In cell culture studies, we showed that copper promoted cell proliferation through Erk/MAPK activation. Treatment with curcumin or U0126, a specific MAPK inhibitor, or suppression of cellular uptake of copper by siRNA knockdown of copper transporter protein 1 (CTR1) blocked copper-induced cell proliferation. CONCLUSIONS This study therefore demonstrates curcumin antitumor effect to its copper chelation capability. These results also implicate copper chelation as a general mechanism for their action of some biologically active polyphenols like flavonoids.

Antiviral activity of Paulownia tomentosa against enterovirus 71 of hand, foot, and mouth disease.

The bark, leaves, and flowers of Paulownia trees have been used in traditional Chinese medicine to treat infectious and inflammatory diseases. We investigated the antiviral effects of Paulownia tomentosa flowers, an herbal medicine used in some provinces of P. R. China for the treatment of skin rashes and blisters. Dried flowers of P. tomentosa were extracted with methanol and tested for antiviral activity against enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), the predominant etiologic agents of hand, foot, and mouth disease in P. R. China. The extract inhibited EV71 infection, although no effect was detected against CAV16 infection. Bioactivity-guided fractionation was performed to identify apigenin as an active component of the flowers. The EC50 value for apigenin to block EV71 infection was 11.0 µM, with a selectivity index of approximately 9.3. Although it is a common dietary flavonoid, only apigenin, and not similar compounds like naringenin and quercetin, were active against EV71 infection. As an RNA virus, the genome of EV71 has an internal ribosome entry site that interacts with heterogeneous nuclear ribonucleoproteins (hnRNPs) and regulates viral translation. Cross-linking followed by immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that EV71 RNA was associated with hnRNPs A1 and A2. Apigenin treatment disrupted this association, indicating that apigenin suppressed EV71 replication through a novel mechanism by targeting the trans-acting factors. This study therefore validates the effects of Paulownia against EV71 infection. It also yielded mechanistic insights on apigenin as an active compound for the antiviral activity of P. tomentosa against EV71 infection.

Respiratory Syncytial Virus Infection Upregulates NLRC5 and Major Histocompatibility Complex Class I Expression through RIG-I Induction in Airway Epithelial Cells

ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of acute respiratory tract viral infection in infants, causing bronchiolitis and pneumonia. The host antiviral response to RSV acts via retinoic acid-inducible gene I (RIG-I). We show here that RSV infection upregulates major histocompatibility complex class I (MHC-I) expression through the induction of NLRC5, a NOD-like, CARD domain-containing intracellular protein that has recently been identified as a class I MHC transactivator (CITA). RSV infection of A549 cells promotes upregulation of NLRC5 via beta interferon (IFN-β) production, since the NLRC5-inducing activity in a conditioned medium from RSV-infected A549 cells was removed by antibody to IFN-β, but not by antibody to IFN-γ. RSV infection resulted in RIG-I upregulation and induction of NLRC5 and MHC-I. Suppression of RIG-I induction significantly blocked NLRC5, as well as MHC-I, upregulation and diminished IRF3 activation. Importantly, Vero cells deficient in interferon production still upregulated MHC-I following introduction of the RSV genome by infection or transfection, further supporting a key role for RIG-I. A model is therefore proposed in which the host upregulates MHC-I expression during RSV infection directly via the induction of RIG-I and NLRC5 expression. Since elevated expression of MHC-I molecules can sensitize host cells to T lymphocyte-mediated cytotoxicity or immunopathologic damage, the results have significant implications for the modification of immunity in RSV disease. IMPORTANCE Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and young children worldwide. Infection early in life is linked to persistent wheezing and allergic asthma in later life, possibly related to upregulation of major histocompatibility class I (MHC-I) on the cell surface, which facilitates cytotoxic T cell activation and antiviral immunity. Here, we show that RSV infection of lung epithelial cells induces expression of RIG-I, resulting in induction of a class I MHC transactivator, NLRC5, and subsequent upregulation of MHC-I. Suppression of RIG-I induction blocked RSV-induced NLRC5 expression and MHC-I upregulation. Increased MHC-I expression may exacerbate the RSV disease condition due to immunopathologic damage, linking the innate immune response to RSV disease.